Integrating molecular microbial methods to improve faecal pollution management in rivers with designated bathing waters.

Rivers are at risk from a variety of pollution sources. Faecal pollution is of particular concern since it disperses pathogenic microorganisms in the aquatic environment. Currently, faecal pollution levels in rivers is monitored using faecal indicator bacteria (FIB) that do not offer information about pollution sources and associated risks. This study used a combined molecular approach, along with measurements of water quality, to gain information on pollution sources, and risk levels, in a newly designated recreational bathing site in the River Wharfe (UK). Physico-chemical parameters were monitored in situ, with water quality multiparameter monitoring sondes installed during the 2021 bathing season. The molecular approach was based on quantitative PCR (qPCR)-aided Microbial Source Tracking (MST) and 16S rRNA gene metabarcoding to obtain a fingerprint of bacterial communities and identify potential bioindicators. The analysis from the water quality sondes showed that ammonium was the main parameter determining the distribution of FIB values. Lower faecal pollution levels were detected in the main river when compared to tributaries, except for samples in the river located downstream of a wastewater treatment plant. The faecal pollution type (anthropogenic vs. zoogenic) changed the diversity and the structure of bacterial communities, giving a distinctive fingerprint that can be used to inform source. DNA-based methods showed that the presence of human-derived bacteria was associated with Escherichia coli spikes, coinciding with higher bacterial diversity and the presence of potential pathogenic bacteria mainly of the genus Mycobacterium, Aeromonas and Clostridium. Samples collected after a heavy rainfall event were associated with an increase in Bacteroidales, which are markers of faecal pollution, including Bacteroides graminisolvens, a ruminant marker associated with surface run-off from agricultural sources. The combined use of qPCR and 16S rRNA sequencing was able to identify pollution sources, and novel bacterial indicators, thereby aiding decision-making and management strategies in recreational bathing rivers.

Intermittent Water Supply Impacts on Distribution System Biofilms and Water Quality.

Intermittent water supplies (IWS) are routinely experienced by drinking water distribution systems around the world, either due to ongoing operational practices or due to one off interruptions. During IWS events changing conditions may impact the endemic biofilms leading to hydraulic mobilisation of organic and inorganic materials attached to pipes walls with a resulting degradation in water quality. To study the impact of IWS on the microbiological and physico-chemical characteristics of drinking water, an experimental full-scale chlorinated pipe facility was operated over 60 days under realistic hydraulic conditions to allow for biofilm growth and to investigate flow resumption behaviour post-IWS events of 6, 48 and 144 hours. Turbidity and metal concentrations showed significant responses to flow restarting, indicating biofilm changes, with events greater than 6 hours generating more turbidity responses and hence discolouration risk. The increase in pressure when the system was restarted showed a substantial increase in total cell counts, while the subsequent increases in flow led to elevated turbidity and metals concentrations. SUVA254 monitoring indicated that shorter times of non-water supply increased the risk of aromatic organic compounds and hence risk of disinfection-by-products formation. DNA sequencing indicated that increasing IWS times resulted in increased relative abundance of potential pathogenic microorganisms, such as Mycobacterium, Sphingomonas, and the fungi Penicillium and Cladosporium. Overall findings indicate that shorter IWS result in a higher proportion of aromatic organic compounds, which can potentially react with chlorine and increase risk of disinfection-by-products formation. However, by minimising IWS times, biofilm-associated impacts can be reduced, yet these are complex ecosystems and much remains to be understood about how microbial interactions can be managed to best ensure continued water safe supply.

Implications of Climate Change: How Does Increased Water Temperature Influence Biofilm and Water Quality of Chlorinated Drinking Water Distribution Systems?

Temperature variation can promote physico-chemical and microbial changes in the water transported through distribution systems and influence the dynamics of biofilms attached to pipes, thus contributing to the release of pathogens into the bulk drinking water. An experimental real-scale chlorinated DWDS was used to study the effect of increasing temperature from 16 to 24°C on specific pathogens, bacterial-fungal communities (biofilm and water samples) and determine the risk of material accumulation and mobilisation from the pipes into the bulk water. Biofilm was developed for 30 days at both temperatures in the pipe walls, and after this growth phase, a flushing was performed applying 4 gradual steps by increasing the shear stress. The fungal-bacterial community characterised by Illumina MiSeq sequencing, and specific pathogens were studied using qPCR: Mycobacterium spp., Mycobacterium avium complex, Acanthamoeba spp., Pseudomonas aeruginosa, Legionella pneumophilia, and Stenotrophomonas maltophilia. Sequencing data showed that temperature variation significantly modified the structure of biofilm microbial communities from the early stages of biofilm development. Regarding bacteria, Pseudomonas increased its relative abundance in biofilms developed at 24°C, while fungal communities showed loss of diversity and richness, and the increase in dominance of Fusarium genus. After the mobilisation phase, Pseudomonas continued being the most abundant genus at 24°C, followed by Sphingobium and Sphingomonas. For biofilm fungal communities after the mobilisation phase, Helotiales incertae sedis and Fusarium were the most abundant taxa. Results from qPCR showed a higher relative abundance of Mycobacterium spp. on day 30 and M. avium complex throughout the growth phase within the biofilms at higher temperatures. The temperature impacts were not only microbial, with physical mobilisation showing higher discolouration response and metals release due to the increased temperature. While material accumulation was accelerated by temperature, it was not preferentially to either stronger or weaker biofilm layers, as turbidity results during the flushing steps showed. This research yields new understanding on microbial challenges that chlorinated DWDS will undergo as global temperature rises, this information is needed in order to protect drinking water quality and safety while travelling through distribution systems.

The microbial ecology of a Mediterranean chlorinated drinking water distribution systems in the city of Valencia (Spain).

Drinking water distribution systems host extensive microbiomes with diverse biofilm communities regardless of treatment, disinfection, or operational practices. In Mediterranean countries higher temperatures can accelerate reactions and microbial growth that may increase aesthetic water quality issues, particularly where material deposits can develop as a result of net zero flows within looped urban networks. This study investigated the use of flow and turbidity monitoring to hydraulically manage mobilisation of pipe wall biofilms and associated material from the Mediterranean city of Valencia (Spain). Pipe sections of different properties were subjected to controlled incremental flushing with monitoring and sample collection for physico-chemical and DNA analysis with Illumina sequencing of bacterial and fungal communities. A core microbial community was detected throughout the network with microorganisms like Pseudomonas, Aspergillus or Alternaria increasing during flushing, indicating greater abundance in underlying and more consolidated material layers. Bacterial and fungal communities were found to be highly correlated, with bacteria more diverse and dynamic during flushing whilst fungi were more dominant and less variable between sampling sites. Results highlight that water quality management can be achieved through hydraulic strategies yet understanding community dynamics, including the fungal component, will be key to maintaining safe and ultimately beneficial microbiomes in drinking water distribution systems.

Influence of phosphate dosing on biofilms development on lead in chlorinated drinking water bioreactors.

Phosphate dosing is used by water utilities to prevent plumbosolvency in water supply networks. However, there is a lack of knowledge regarding biofilm formation on lead and plastic materials when phosphate concentrations are modified in drinking water systems. In this study, biofilms were grown over lead coupons and PVC tubes in bioreactors supplied with local drinking water treated to provide different phosphate doses (below 1, 1 and 2 mg/L) over a period of 28 days. A range of commercial iron pellets (GEH104 and WARP) were tested aiming to maintain phosphate levels below the average 1 mg/L found in drinking water. Changes in biofilm community structure in response to three different phosphate treatments were characterised by Illumina sequencing of the 16S rRNA gene for bacteria and the ITS2 gene for fungi. Scanning electron microscopy was used to visualise physical differences in biofilm development in two types of materials, lead and PVC. The experimental results from the kinetics of phosphate absorption showed that the GEH104 pellets were the best option to, in the long term, reduce phosphate levels while preventing undesirable turbidity increases in drinking water. Phosphate-enrichment promoted a reduction of bacterial diversity but increased that of fungi in biofilms. Overall, higher phosphate levels selected for microorganisms with enhanced capabilities related to phosphorus metabolism and heavy metal resistance. This research brings new insights regarding the influence of different phosphate concentrations on mixed-species biofilms formation and drinking water quality, which are relevant to inform best management practices in drinking water treatment.

Phosphate Dosing in Drinking Water Distribution Systems Promotes Changes in Biofilm Structure and Functional Genetic Diversity.

Water utilities treat drinking water by adding phosphate to prevent metal dissolution from water pipe work systems and particularly lead poisoning. Phosphate can be a limiting nutrient for microbial biofilms in DWDS, yet its effects on these microbial consortia are not well understood. This research presents results from phosphate dosing experiments using a real scale chlorinated DWDS, comparing standard phosphate concentrations of United Kingdom drinking water (1 mgP/L) with a double dose (2 mgP/L) commonly used in plumbosolvency treatment. Biofilm development during phosphate treatment experiments was monitored using a holistic approach by combining metagenomics analysis, flow cytometry and SEM characterisation. The increase of phosphate levels in drinking water, reduced biofilm cell numbers and promoted the presence of poorly distributed biofilms on inner pipe surfaces. Metagenomics analysis using genetic markers (16S rRNA and ITS2) showed that phosphate influenced biofilm community structure, particularly fungal composition. Whole metagenome sequencing showed that phosphate enrichment favoured the presence of sequencing reads associated to ATPases, ion transporters and DNA-interacting proteins, whilst reads associated to nitrogen metabolism were predominant in control samples. This research brings new knowledge regarding the influence of phosphate treatment on the composition and structure of biofilms within DWDS, and the implications that this might have for the management of these systems.

Effects of phosphate and hydrogen peroxide on the performance of a biological activated carbon filter for enhanced biofiltration.

Biofilm formation on biofilters can influence their hydraulic performance, thereby leading to head loss and an increase in energy use and costs for water utilities. The effects of a range of factors, including hydrogen peroxide and phosphate, on the performance of biological activated carbon (BAC) and biofilm formation were investigated using laboratory-scale columns. Head loss, total carbohydrates, and proteins were reduced in the nutrient-enhanced, oxidant-enhanced, and nutrient + oxidant-enhanced BAC filters. However, there were no changes in the removal of dissolved organic matter, trihalomethane formation potential, or selected trace organic contaminants. The biofilm formation on polyvinyl chloride and stainless steel coupons using the laboratory biofilm reactor system was lower when the effluent from a nutrient-enhanced column was used, which indicated that there was less biofilm formation in the distribution systems. This may have been because the effluent from the nutrient-enhanced column was more biologically stable. Therefore, enhanced biofiltration could be used not only to reduce head loss in biofilters, but also to delay biofilm formation in distribution systems.

Bacterial growth through microfiltration membranes and NOM characteristics in an MF-RO integrated membrane system: Lab-scale and full-scale studies.

Biofilm formation on membrane surfaces causes many operational problems such as a decrease in permeate flux and an increase in hydraulic resistance. In this study, the ability of bacteria to pass through microfiltration (MF) membranes and the growth potential of microfilterable bacteria were investigated in order to understand biofouling in MF-reverse osmosis (RO) integrated membrane systems. Growth of microfilterable bacteria in MF permeate was observed, indicating that not all MF membranes can guarantee the total rejection of bacteria. Changes in natural organic matter (NOM) characteristics and growth potential of bacteria during the treatment process are important factors in the occurrence of biofilm development in water treatment systems. Analysis of protein-like and humic-like substances in NOM of two successive RO stages revealed an increase in the concentrations of both biopolymers and humic substances of RO concentrates. Unexpectedly, the use of antiscalants was seen to enhance the growth of bacteria in the RO feed water in this study. Bacterial 16s rRNA pyrosequencing revealed that passing source water through the MF membranes dramatically changed bacterial community structure. The bacterial communities that passed through the MF steps primarily belonged to the family Comamonadaceae. However, several bacteria groups including Flavobacteriaceae, Sphingobacteriaceae and Sphingomonadaceae selectively composed the biofilm community formed on the RO membranes. Thus, understanding the selectivity and filterability of MF towards microorganisms involved in biofouling on RO membrane surfaces is crucial for the improvement of membrane-related operational processes.

Field assessment of bacterial communities and total trihalomethanes: Implications for drinking water networks.

Operation and maintenance (O&M) of drinking water distribution networks (DWDNs) in tropical countries simultaneously face the control of acute and chronic risks due to the presence of microorganisms and disinfection by-products, respectively. In this study, results from a detailed field characterization of microbiological, chemical and infrastructural parameters of a tropical-climate DWDN are presented. Water physicochemical parameters and the characteristics of the network were assessed to evaluate the relationship between abiotic and microbiological factors and their association with the presence of total trihalomethanes (TTHMs). Illumina sequencing of the bacterial 16s rRNA gene revealed significant differences in the composition of biofilm and planktonic communities. The highly diverse biofilm communities showed the presence of methylotrophic bacteria, which suggest the presence of methyl radicals such as THMs within this habitat. Microbiological parameters correlated with water age, pH, temperature and free residual chlorine. The results from this study are necessary to increase the awareness of O&M practices in DWDNs required to reduce biofilm formation and maintain appropriate microbiological and chemical water quality, in relation to biofilm detachment and DBP formation.

Microbial analysis of in situ biofilm formation in drinking water distribution systems: implications for monitoring and control of drinking water quality.

Biofilm formation in drinking water distribution systems (DWDS) is influenced by the source water, the supply infrastructure and the operation of the system. A holistic approach was used to advance knowledge on the development of mixed species biofilms in situ, by using biofilm sampling devices installed in chlorinated networks. Key physico-chemical parameters and conventional microbial indicators for drinking water quality were analysed. Biofilm coverage on pipes was evaluated by scanning electron microscopy (SEM) and confocal laser scanning microscopy (CLSM). The microbial community structure, bacteria and fungi, of water and biofilms was assessed using pyrosequencing. Conventional wisdom leads to an expectation for less microbial diversity in groundwater supplied systems. However, the analysis of bulk water showed higher microbial diversity in groundwater site samples compared with the surface water site. Conversely, higher diversity and richness were detected in biofilms from the surface water site. The average biofilm coverage was similar among sites. Disinfection residual and other key variables were similar between the two sites, other than nitrates, alkalinity and the hydraulic conditions which were extremely low at the groundwater site. Thus, the unexpected result of an exceptionally low diversity with few dominant genera (Pseudomonas and Basidiobolus) in groundwater biofilm samples, despite the more diverse community in the bulk water, is attributed to the low-flow hydraulic conditions. This finding evidences that the local environmental conditions are shaping biofilm formation, composition and amount, and hence managing these is critical for the best operation of DWDS to safeguard water quality.

Characterisation of the physical composition and microbial community structure of biofilms within a model full-scale drinking water distribution system.

Within drinking water distribution systems (DWDS), microorganisms form multi-species biofilms on internal pipe surfaces. A matrix of extracellular polymeric substances (EPS) is produced by the attached community and provides structure and stability for the biofilm. If the EPS adhesive strength deteriorates or is overcome by external shear forces, biofilm is mobilised into the water potentially leading to degradation of water quality. However, little is known about the EPS within DWDS biofilms or how this is influenced by community composition or environmental parameters, because of the complications in obtaining biofilm samples and the difficulties in analysing EPS. Additionally, although biofilms may contain various microbial groups, research commonly focuses solely upon bacteria. This research applies an EPS analysis method based upon fluorescent confocal laser scanning microscopy (CLSM) in combination with digital image analysis (DIA), to concurrently characterize cells and EPS (carbohydrates and proteins) within drinking water biofilms from a full-scale DWDS experimental pipe loop facility with representative hydraulic conditions. Application of the EPS analysis method, alongside DNA fingerprinting of bacterial, archaeal and fungal communities, was demonstrated for biofilms sampled from different positions around the pipeline, after 28 days growth within the DWDS experimental facility. The volume of EPS was 4.9 times greater than that of the cells within biofilms, with carbohydrates present as the dominant component. Additionally, the greatest proportion of EPS was located above that of the cells. Fungi and archaea were established as important components of the biofilm community, although bacteria were more diverse. Moreover, biofilms from different positions were similar with respect to community structure and the quantity, composition and three-dimensional distribution of cells and EPS, indicating that active colonisation of the pipe wall is an important driver in material accumulation within the DWDS.

Methodological approaches for studying the microbial ecology of drinking water distribution systems.

The study of the microbial ecology of drinking water distribution systems (DWDS) has traditionally been based on culturing organisms from bulk water samples. The development and application of molecular methods has supplied new tools for examining the microbial diversity and activity of environmental samples, yielding new insights into the microbial community and its diversity within these engineered ecosystems. In this review, the currently available methods and emerging approaches for characterising microbial communities, including both planktonic and biofilm ways of life, are critically evaluated. The study of biofilms is considered particularly important as it plays a critical role in the processes and interactions occurring at the pipe wall and bulk water interface. The advantages, limitations and usefulness of methods that can be used to detect and assess microbial abundance, community composition and function are discussed in a DWDS context. This review will assist hydraulic engineers and microbial ecologists in choosing the most appropriate tools to assess drinking water microbiology and related aspects.