Biofilm formation in drinking water distribution systems is primarily managed by disinfectants such as free chlorine (FC) and monochloramine (MC). However, there is limited understanding of their long-term and dynamic effects on biofilm development. To address this, a 56-week study was conducted to comprehensively assess biofilm development in terms of microbial quantity and community under different disinfection regimes: no chlorine (NC), FC (0.1 mg/L), and MC (0.4 mg/L). The results showed that both FC and MC significantly inhibited biofilm growth compared to the NC condition while shaping distinct biofilm communities. Notably, FC drastically reduced biofilm biomass and community diversity, resulting in a more uniform biofilm community predominantly composed of Proteobacteria (e.g., Rhizobacter spp., Pseudomonas spp., and Hyphomicrobium spp.), indicating stronger selection pressures on the microbial population. In contrast, though MC effectively reduced the biofilm biomass to a level comparable to that of FC, it maintained a high diversity comparable to that of NC (dominated by Sphingobium spp. and Nocardioides spp.), reflecting weaker selection pressure on bacterial community. Temporally, biofilm communities under all conditions started from nearly identical states. From week-19 and week-36 onwards, deterministic processes predominantly governed biofilm formation under FC and NC conditions, signifying that these biofilms reached a stable state. Differently, under MC condition, the community assembly was continually influenced by stochastic processes, with the biofilm not achieving stability until week-56. Overall, this study provides valuable insights into the long-term dynamics of biofilm development and evidenced that FC is better than MC in controlling biofilm formation, particularly from the community diversity perspective. This challenges classical views that MC is more effective than FC in penetrating and controlling biofilm, which may change the popularity of MC as a disinfectant in water utilities.

Organic micropollutants (OMPs) facilitate the spread of antibiotic resistance genes (ARGs). Ammonia-oxidizing microorganisms (AOMs) are crucial for OMP degradation during riverbank filtration (RBF) and significantly influenced by NH4+-N concentrations. However, the effect of NH4+-N on OMP removal and ARG occurrence in RBF remains unclear. This study aimed to examine the effects of low (∼0.1 mg/L) and high (∼2.2 mg/L) NH4+-N concentrations on OMP removal, ARG occurrence, and microbial communities. NH4+-N addition had no significant effect on the removal of 108 out of 128 OMPs, suggesting that other factors primarily govern the removal process. Notably, NH4+-N addition enhanced the removal of 20 OMPs by 3–70%, including three quinolones (e.g., flumequine), indicating its promotion of specific OMP removals. This effect may primarily result from NH4+-N enhancing OMP biotransformation through the stimulation of AOMs (particularly AOA and comammox) and heterotrophs (e.g., Bradyrhizobium). Furthermore, NH4+-N addition significantly reduced the abundance of eight ARGs, including quinolone ARGs, likely due to its inhibition of antibiotic-resistant bacteria. Additionally, we hypothesize that NH4+-N alleviates OMP selective pressure on microorganisms by promoting OMP conversion through AOMs. This study enhances the understanding of microbe-mediated OMP removal in the presence of NH4+-N and its impact on ARG occurrence during RBF.

Shower systems create conditions conducive to the growth of opportunistic pathogens, but the timing and location of associated risks are poorly understood. In this study, we constructed 48 full size shower units with six incubation periods (4, 10, 16, 22, 30, and 40 weeks) and four water heater temperature (39, 45, 51, and 58 °C) to examine the dynamics of microbial growth and pathogen distribution. Results showed that during the initial stage (4 weeks), peak biomass was observed for all biofilms, ranked as shower hose (SHE) > cold-water pipe (CWP) > hot-water pipe (HWP), followed by a sharp decline by the 10th-week. At the 4th-week, the biofilm was loose and easily detached into the water, possibly promoted by leached organic carbon from plastic material, fostering the growth of specific microorganisms. The impacts of stagnation and temperature became more pronounced in CWP and HWP over time. Legionella pneumophila appeared in biofilms at the 4th-week, disappeared, and reappeared in large numbers since the 22nd-week. Differently, Mycobacterium spp. emerged in large numbers after 30 weeks. Both pathogens were notably enriched in showerheads and shower hoses. This study highlights critical periods of higher risk in shower systems, particularly in the early stages (4 weeks) and after 22 weeks, suggesting that risks can be mitigated by pre-soaking pipes or regularly cleaning (e.g., heat shock flushing) and replacing showerheads and hoses.

Drinking water distribution systems are increasingly vulnerable to sewage intrusion due to aging water infrastructure and intensifying water stress. While the health risks associated with sewage intrusion have been extensively studied, little is known about the impacts of intruded bacteria and dissolved organic matter (DOM) on microbiology in drinking water. In this dynamic study, we demonstrate that the intrusion of 1 % sewage into tap water resulted in immediate contamination, including an 8-fold increase in biomass (TCC), a 48.9 % increase in bacterial species (ASVs), a 12.5 % increase in organic carbon content (DOC), and a 13.5 % increase in unique DOM molecular formulae. Over time, sewage intrusion altered tap water microbiology by accelerating bacterial growth rates (5-fold faster), selectively promoting ASVs in community succession, and producing 998 more unique DOM formulae. More significantly, statistical analysis revealed that the intrusion of 1 % sewage shifted the driving force of bacterial and DOM composition covariance from a DOM-dependent process in tap water to a bacterial-governed process post-intrusion. Our results clearly demonstrate the disruptive effects of sewage intrusion into tap water, emphasizing the urgent need to consider the long-lasting impacts of sewage intrusion in drinking water distribution systems, in addition to its immediate health risks.

Drinking water biosafety has become an increasing concern for public health. Chlorination is widely used as the main disinfection strategy worldwide but has clear and well-known byproduct issues. The Netherlands has successfully demonstrated unchlorinated approach for almost 20 years but has not been widely adopted by other countries. To chlorine or not chlorine is becoming a critical question in front of all the water utilities. This review aims to provide a good overview of current biosafety management strategies, their disadvantages, as well as the latest developments and future trends. Firstly, the advantages and deficiencies of conventional disinfection and non-disinfection were discussed. Secondly, the commonly used and promising methods for biostability assessment are described. Finally, critical views on the strategy selection for ensuring drinking water biosafety were discussed. It is recommended to achieve both biological and chemical balance by removing pathogens while minimizing the organic matter and dosing a minimum level of disinfectants, which would represent the compromise choice between the current chlorine-based disinfection and chlorine-free strategy. It's worth noting that the complexity of ensuring biosafety lies in the variations among different regions, the selection of suitable methods should be tailored to specific situations on a case-by-case basis.

Treated drinking water is delivered to customers through drinking water distribution systems (DWDSs). Although studies have focused on exploring the microbial ecology of DWDSs, knowledge about the effects of different water treatments on the bacterial community of biofilm and loose deposits in DWDS is limited. This study assessed the effects of additional treatments on the bacterial communities developed in 10 months’ old pilot DWDSs. The results showed a similar bacterial community in the pipe-wall biofilm, which was dominated by Novosphingobium spp. (20–82 %) and Sphingomonas spp. (11–53 %), regardless of the treatment applied. The bacterial communities that were retained in the distribution systems (including pipe-wall biofilm and loose deposits) were similar to the particle-associated bacteria (PAB) in the corresponding supply water. The additional treatments showed clear effects of the removal and/or introduction of particles. The genera Aeromonas spp., Clostridium spp., Legionella spp., and Pseudomonas spp., which contain opportunistic pathogenic species, were only detected among the PAB in ion exchange system. Our study demonstrated that the biofilm community is consistent across treatments, and the contribution from bacteria in loose deposits is important but can be controlled by removing particles. These findings offer more insight into the origin and development of microbial ecology in DWDSs and suggest paths for further research on the possibility of managing the microbial ecology in distribution systems.

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.

This study assessed the evolution of wastewater systems during the rapid urbanization of Beijing, with special focuses on the carbon footprints and growing underground WWTPs (u-WWTPs). Specifically, the Bishui plant (in situ constructed u-WWTP) was assessed in detail regarding eco-environmental benefits. Our results showed that, the direct emission intensity of 65 WWTPs decreased from 0.47 to 0.24 kg CO_2eq/m³, when the electricity intensity increased from 0.22 to 0.39 kWh/m³ from 2010 to 2020. Bishui u-WWTP emitted 36.6 kt CO_2eq/year (0.09 kg CO_2eq/m³), with electricity intensity of 0.43 kg CO_2eq/m³. Additionally, compare to the hypothetical relocating scenario, it saved 6.67 × 10⁴ m² land and 33.0 kt CO_2eq/year, and the created urban river carries 6.5 × 10¹³ J/year heat outside town. The evaluation and balance of choice for conventional or underground WWTP should be made case by case. However, this study demonstrated that u-WWTP is not only a construction manner, but a sustainable management model with positive eco-environment effects, algin with future city expansion, and circular economy visions.

Although simulated studies have provided valuable knowledge regarding the communities of planktonic bacteria and biofilms, the lack of systematic field studies have hampered the understanding of microbiology in real-world service lines and premise plumbing. In this study, the bacterial communities of water and biofilm were explored, with a special focus on the lifetime development of biofilm communities and their key influencing factors. The 16S rRNA gene sequencing results showed that both the planktonic bacteria and biofilm were dominated by Proteobacteria. Among the 15,084 observed amplicon sequence variants (ASVs), the 33 core ASVs covered 72.8 %, while the 12 shared core ASVs accounted for 62.2 % of the total sequences. Remarkably, it was found that the species richness and diversity of biofilm communities correlated with pipe age. The relative abundance of ASV2 (f_Sphingomonadaceae) was lower for pipe ages 40–50 years (7.9 %) than for pipe ages 10–20 years (59.3 %), while the relative abundance of ASV10 (f_Hyphomonadaceae) was higher for pipe ages 40–50 years (19.5 %) than its presence at pipe ages 20–30 years (1.9 %). The community of the premise plumbing biofilm had significantly higher species richness and diversity than that of the service line, while the steel-plastics composite pipe interior lined with polyethylene (S-PE) harbored significantly more diverse biofilm than the galvanized steel pipes (S-Zn). Interestingly, S-PE was enriched with ASV27 (g_Mycobacterium), while S-Zn pipes were enriched with ASV13 (g_Pseudomonas). Moreover, the network analysis showed that five rare ASVs, not core ASVs, were keystone members in biofilm communities, indicating the importance of rare members in the function and stability of biofilm communities. This manuscript provides novel insights into real-world service lines and premise plumbing microbiology, regarding lifetime dynamics (pipe age 10–50 years), and the influences of pipe types (premise plumbing vs. service line) and pipe materials (S-Zn vs. S-PE).

Premise plumbing plays an essential role in determining the final quality of drinking water consumed by customers. However, little is known about the influences of plumbing configuration on water quality changes. This study selected parallel premise plumbing in the same building with different configurations, i.e., laboratory and toilet plumbing. Water quality deteriorations induced by premise plumbing under regular and interrupted water supply were investigated. The results showed that most of the water quality parameters did not vary under regular supply, except Zn, which was significantly increased by laboratory plumbing (78.2 to 260.7 µg/l). For the bacterial community, the Chao1 index was significantly increased by both plumbing types to a similar level (52 to 104). Laboratory plumbing significantly changed the bacterial community, but toilet plumbing did not. Remarkably, water supply interruption/restoration led to serious water quality deterioration in both plumbing types but resulted in different changes. Physiochemically, discoloration was observed only in laboratory plumbing, along with sharp increases in Mn and Zn. Microbiologically, the increase in ATP was sharper in toilet plumbing than in laboratory plumbing. Some opportunistic pathogen-containing genera, e.g., Legionella spp. and Pseudomonas spp., were present in both plumbing types but only in disturbed samples. This study highlighted the esthetic, chemical, and microbiological risks associated with premise plumbing, for which system configuration plays an important role. Attention should be given to optimizing premise plumbing design for managing building water quality.

Biofilm detachment contributes to water quality deterioration. However, the contributions of biofilm detachment from different pipes have not been quantified or compared. Following the introduction of partial reverse osmosis (RO) in drinking water production, this study analyzed particles at customers’ ends and tracked their origins to water distribution mains and service lines. For doing so, filter bags were installed in front of water meters to capture upstream detached particles, while biofilm from water main and service line were sampled by cutting pipe specimens. The results showed that elemental concentrations of the biofilm in mains were higher than those of service lines (54.3–268.5 vs. 27.1–44.4 μg/cm2), both dominated by Ca. Differently, filter bags were dominated by Fe/Mn (77.5–98.1%). After introducing RO, Ca significantly decreased in biofilms of mains but not service lines, but the released Fe/Mn rather than Ca arrived at customers’ ends. The ATP concentrations of service lines were higher than mains, which decreased on mains but increased in service lines after introducing RO. For the core ASVs, 13/24 were shared by service lines (17), mains (21), and filter bags (17), which were assigned mainly to Nitrospira spp., Methylomagnum spp., Methylocytis spp., and IheB2–23 spp. According to source tracking results, service lines contributed more than mains to the particulate material collected by filter bags (57.6 ± 13.2% vs. 13.0 ± 11.6%). To the best of our knowledge, the present study provides the first evidence of service lines’ direct and quantitative contributions to potential water quality deterioration at customers’ ends. This highlights the need for the appropriate management of long-neglected service line pipes, e.g., regarding material selection, length optimization, and proper regulation.

The presence of newly emerging pollutants in the aquatic environment poses great challenges for drinking water treatment plants. Due to their low concentrations and unknown characteristics, emerging pollutants cannot be efficiently removed by conventional water treatment processes, making technically, economically, and environmentally friendly water purification technologies increasingly important. This article introduces a one-step reverse osmosis (OSRO) concept consisting of riverbank filtration (RBF) and reverse osmosis (RO) for drinking water treatment. The OSRO concept combines the relatively low-cost natural pretreatment of river water with an advanced engineered purification system. RBF provides a continuous natural source of water with stable water quality and a robust barrier for contaminants. With the pre-removal of particles, organic matter, organic micro-pollutants (OMPs), and microbes, RBF becomes an ideal source for a purification system based on RO membranes, in comparison with the direct intake of surface water. OSRO treatment removes almost 99.9% of the particles, pathogens, viruses, and OMPs, as well as the vast majority of nutrients, and thus meets the requirements for the chlorine-free delivery of drinking water with high biostability. The OSRO treatment is cost effective compared with the standard conventional series of purification steps involving sprinkling filters, softening, and activated carbon. Artificial bank filtration (ABF), which functions as an artificial recharge in combination with a sand filtration system, is proposed as an alternative for RBF in the OSRO concept to supply drinking water from locally available resources. It is also suggested that the OSRO concept be implemented with wind power as an alternative energy source in order to be more sustainable and renewable. An OSRO-based decentralized water system is proposed for water reclaiming and reuse. It is suggested that future water treatment focus on the combination of natural and engineered systems to provide drinking water through technically efficient, financially feasible, resource reusable, and environmentally relevant means.

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.

The potential of membrane scaling control by a real-time optimization algorithm was investigated. The effect of antiscalant dosing was evaluated from the induction time measured in glass batch-reactors, and from the operational performance of a lab-scale reverse osmosis (RO) unit and two pilot-scale RO units. Step changes in the antiscalant dosing demonstrated that the accumulation of scaling is ‘paused’ during periods when the optimum dose is applied. This is paramount for the application of a dynamic dosing strategy that may briefly underdose, while searching for the optimum dose. It was found that antiscalant underdose and overdose were both detrimental to RO operation since underdose resulted in membrane scaling, while overdose led to membrane fouling due to calcium-antiscalant deposits. The dosing algorithm was used to minimize antiscalant consumption in two pilot RO units. The algorithm was able to lower the antiscalant doses to 0.2 mg/L and 0.6 mg/L, while the supplier's recommended antiscalant doses were 2.0 mg/L and 4.5 mg/L, respectively. As a result, the algorithm could reduce antiscalant consumption by up to 85–90% for the plants mentioned.

The role of phosphate and humic substances (HS) in preventing calcium carbonate scaling and their impact on antiscalant dose was investigated for a reverse osmosis (RO) system treating anaerobic groundwater (GW) (containing 2.1 mg/L orthophosphate and 6-8 mg/L HS). Experiments were conducted with the RO unit (treating anaerobic GW), and with a once-through lab-scale RO system (operating with artificial feedwater). Additionally, (batch) induction time (IT) measurements were performed with, i) real RO concentrate, and ii) artificial RO concentrates in the presence and absence of phosphate and HS. It was found that at 80% recovery (Langelier saturation index (LSI) 1.7), calcium carbonate scaling did not occur in the RO unit when the antiscalant dose was lowered from 2.2 mg/L (supplier's recommended dose) to 0 mg/L. The IT of the real RO concentrate, without antiscalant, was longer than 168 h, while, at the same supersaturation level, the IT of the artificial concentrate was approximately 1 h. The IT of the artificial concentrate increased to 168 h with the addition of 10 mg/L of phosphate, humic acid (HA), and fulvic acid (FA). Furthermore, in the lab-scale RO tests, the normalized permeability (Kw) of the membrane decreased by 20% in 2 h period when fed with artificial concentrate of 80% recovery containing no phosphate, whereas, with phosphate, no decrease in Kw was observed in 10 h period. These results indicate that phosphate and HS present in the GW prevented calcium carbonate scaling in the RO unit and reduced the use of commercial (synthetic) antiscalants.

The bacterial growth potential (BGP) of drinking water is widely assessed either by flow cytometric intact cell count (BGP_ICC) or adenosine triphosphate (BGP_ATP) based methods. Combining BGP_ICC and BGP_ATP measurements has been previously applied for various types of drinking water having high to low growth potential. However, this has not been applied for water with ultra-low nutrient content, such as remineralised RO permeate. To conduct a sound comparison, conventionally treated drinking water was included in this study, which was also used as an inoculum source. BGP_ICC, BGP_ATP, intact cell-yield (Y_ICC), and ATP-yield (Y_ATP) were determined for conventionally treated drinking water (Tap-water) and remineralised RO permeate (RO-water). In addition, both BGP_ICC and BGP_ATP methods were used to identify the growth-limiting nutrient in each water type. The results showed that the BGP_ICC ratio between Tap-water/RO-water was ∼7.5, whereas the BGP_ATP ratio was only ∼4.5. Moreover, the Y_ICC ratio between Tap-water/RO-water was ∼2 (9.8 ± 0.6 × 10⁶ vs. 4.6 ± 0.8 × 10⁶ cells/µg-C), whereas the Y_ATP ratio was ∼1 (0.39 ± 0.12 vs. 0.42 ± 0.06 ng ATP/µg-C), resulting in a consistently higher ATP per cell in RO-water than that of Tap-water. Both BGP_ICC and BGP_ATP methods revealed that carbon was the growth-limiting nutrient in the two types of water. However, with the addition of extra carbon, phosphate limitation was detected only with the BGP_ICC method, whereas BGP_ATP was not affected, suggesting that a combination of carbon and phosphate is essential for biomass synthesis, whereas carbon is probably utilised for cellular activities other than cell synthesis when phosphate is limited. It was estimated that the intact cell-yield growing on phosphate would be 0.70 ± 0.05 × 10⁹ cells/µg PO₄-P.

Antiscalants are well known to prevent the precipitation of carbonate and sulphate scales of calcium in reverse osmosis (RO) applications, but according to literature their inhibitory ability against calcium phosphate is not clear. The objective of this study was to investigate if antiscalants, without acid addition, can prevent calcium phosphate scaling in RO systems. Eight calcium phosphate antiscalants from different manufacturers spanning a range of concentrations were tested in batch (in glass reactors) experiments to inhibit the formation of calcium phosphate in synthetic concentrate corresponding to 85% recovery (Ca²⁺ = 765 mg/L, PO₄³⁻ = 13–15 mg/L and pH = 7.6) of a groundwater RO in the Netherlands. Additionally, once-through lab-scale RO tests were conducted where an RO element was fed with synthetic concentrate and the performance of antiscalants was evaluated from the rate of flux-decline in the RO element. Without antiscalant addition, a substantial flux-decline was observed due to the deposition of amorphous calcium phosphate (ACP) on the RO membrane. The tested antiscalants were unable to inhibit the formation of ACP and were incapable of preventing the deposition of the formed ACP particles, since with each antiscalant, the flux of the RO element decreased at least 15% in a 3-h period. Briefly, the available antiscalants, tested in this study, did not provide acceptable inhibition of calcium phosphate scaling in RO applications.

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.

Measuring bacterial growth potential (BGP) involves sample pre-treatment and inoculation, both of which may introduce contaminants in ultra-low nutrient water (e.g., remineralized RO permeate). Pasteurization pre-treatment may lead to denaturing of nutrients, and membrane filtration may leach/remove nutrients into/from water samples. Inoculating remineralized RO permeate samples with natural bacteria from conventional drinking water leads to undesired nutrient addition, which could be avoided by using the remineralized RO permeate itself as inoculum. Therefore, this study examined the effect of pasteurization and membrane filtration on the BGP of remineralized RO permeate. In addition, the possibility of using bacteria from remineralized RO permeate as inoculum was investigated by evaluating their ability to utilize organic carbon that is readily available (acetate, glucose) or complex (laminarin, gelatin, and natural dissolved organic carbon), as compared with bacteria from conventional drinking water. The results showed that membrane filtration pre-treatment increased (140–320%) the BGP of remineralized RO permeate despite the extensive soaking and flushing of filters (>350 h), whereas no effect was observed on the BGP of conventional drinking water owing to its high nutrient content. Pasteurization pre-treatment had insignificant effects on the BGP of both water types. Remineralized RO permeate bacteria showed limitations in utilizing complex organic carbon compared with bacteria from conventional drinking water. In conclusion, the BGP bioassay for ultra-low nutrient water (e.g., remineralized RO permeate) should consider pasteurization pre-treatment. However, an inoculum comprising bacteria from remineralized RO permeate is not recommended as the bacterial consortium was shown to be limited in terms of the compounds they could utilize for growth.

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.