Formation of natural biofilms during chlorine dioxide and u.v. disinfection in a public drinking water distribution system

AIMS: The influence of two disinfection techniques on natural biofilm development during drinking water treatment and subsequent distribution is compared with regard to the supply of a high-quality drinking water. METHODS AND RESULTS: The growth of biofilms was studied using the biofilm device technique in a real public technical drinking water asset. Different pipe materials which are commonly used in drinking water facilities (hardened polyethylene, polyvinyl chloride, steel and copper) were used as substrates for biofilm formation. Apart from young biofilms, several months old biofilms were compared in terms of material dependence, biomass and physiological state. Vital staining of biofilms with 5-cyano-2,3-ditolyl tetrazolium chloride (CTC) and the DNA-specific 4',6-diamidino-2-phenylindole (DAPI) staining resulted in a significant difference in physiological behaviour of biofilm populations depending on the disinfection technique. Compared with chlorine dioxide disinfection (0.12-0.16 mg l-1), the respiratory activities of the micro-organisms were increased on all materials during u.v. disinfection (u.v.254; 400 J m-2). The biofilm biocoenosis was analysed by in situ hybridization with labelled oligonucleotides specific for some subclasses of Proteobacteria. Using PCR and additional hybridization techniques, the biofilms were also tested for the presence of Legionella spp., atypical mycobacteria and enterococci. The results of the molecular-biological experiments in combination with cultivation tests showed that enterococci were able to pass the u.v. disinfection barrier and persist in biofilms of the distribution system, but not after chlorine dioxide disinfection. CONCLUSIONS: The results indicated that bacteria are able to regenerate and proliferate more effectively after u.v. irradiation at the waterworks, and chlorine dioxide disinfection appears to be more applicative to maintain a biological stable drinking water. SIGNIFICANCE AND IMPACT OF THE STUDY: As far as the application of u.v. disinfection is used for conditioning of critical water sources for drinking water, the efficiency of u.v. irradiation in natural systems should reach a high standard to avoid adverse impacts on human health.     

Influence of pipe materials and VBNC cells on culturable bacteria in a chlorinated drinking water model system

To elucidate the influence of pipe materials on the VBNC (viable but nonculturable) state and bacterial numbers in drinking water, biofilm and effluent from stainless steel, galvanized iron, and polyvinyl chloride pipe wafers were analyzed. Although no HPC (heterotrophic plate count) was detected in the chlorinated influent of the model system, a DVC (direct viable count) still existed in the range between 3- and 4-log cells/ml. Significantly high numbers of HPC and DVC were found both in biofilm and in the effluent of the model system. The pipe material, exposure time, and the season were all relevant to the concentrations of VBNC and HPC bacteria detected. These findings indicate the importance of determining the number of VBNC cells and the type of pipe materials to estimate the HPC concentration in water distribution systems and thus the need of determining a DVC in evaluating disinfection efficiency.     

Annual cancer risks from chemicals in North Carolina community water systems

Motivated partly by concerns about cancer, the U.S. Congress in 1986 amended the Safe Drinking Water Act (SDWA) by requiring that community water systems monitor 81 chemicals and remove those detected at concentrations above health-based standards. No prior research has used the resulting 30 years of monitoring data to analyze cancer risks from chemicals in US drinking water. To fill this gap, this paper uses chemical monitoring data from North Carolina's (NC's) 2,120 community water systems along with a risk assessment approach commonly applied in global burden of disease studies to quantify cancer risks of regulated chemicals in drinking water. The results indicate that 0.30% of NC cancer deaths are attributable to regulated drinking water contaminants and that the average annual individual risk is 7.2 × 10^?6. More than 99% of this risk arises from disinfection by-products, with the remaining risk mostly attributable to arsenic and alpha particle radiation. In no water system does the combined risk from chemicals other than disinfection by-products, arsenic, or alpha particles exceed 10^?4. The results suggest that regulated chemicals pose very low cancer risks and that risks from chemicals other than disinfection by-products, arsenic, and alpha particles are negligible in NC community water systems.     

Formation of biofilms in drinking water distribution networks,a case study in two cities in Finland and Latvia

The formation of biofilms in drinking water distribution networks is a significant technical, aesthetic and hygienic problem. In this study, the effects of assimilable organic carbon, microbially available phosphorus (MAP), residual chlorine, temperature and corrosion products on the formation of biofilms were studied in two full-scale water supply systems in Finland and Latvia. Biofilm collectors consisting of polyvinyl chloride pipes were installed in several waterworks and distribution networks, which were supplied with chemically precipitated surface waters and groundwater from different sources. During a 1-year study, the biofilm density was measured by heterotrophic plate counts on R2A-agar, acridine orange direct counting and ATP-analyses. A moderate level of residual chorine decreased biofilm density, whereas an increase of MAP in water and accumulated cast iron corrosion products significantly increased biofilm density. This work confirms, in a full-scale distribution system in Finland and Latvia, our earlier in vitro finding that biofilm formation is affected by the availability of phosphorus in drinking water.     

Identification of bacteria in biofilm and bulk water samples from a nonchlorinated model drinking water distribution system: detection of a large nitrite-oxidizing population associated with Nitrospira spp

In a model drinking water distribution system characterized by a low assimilable organic carbon content (<10 microg/liter) and no disinfection, the bacterial community was identified by a phylogenetic analysis of rRNA genes amplified from directly extracted DNA and colonies formed on R2A plates. Biofilms of defined periods of age (14 days to 3 years) and bulk water samples were investigated. Culturable bacteria were associated with Proteobacteria and Bacteriodetes, whereas independently of cultivation, bacteria from 12 phyla were detected in this system. These included Acidobacteria, Nitrospirae, Planctomycetes, and Verrucomicrobia, some of which have never been identified in drinking water previously. A cluster analysis of the population profiles from the individual samples divided biofilms and bulk water samples into separate clusters (P = 0.027). Bacteria associated with Nitrospira moscoviensis were found in all samples and encompassed 39% of the sequenced clones in the bulk water and 25% of the biofilm community. The close association with Nitrospira suggested that a large part of the population had an autotrophic metabolism using nitrite as an electron donor. To test this hypothesis, nitrite was added to biofilm and bulk water samples, and the utilization was monitored during 15 days. A first-order decrease in nitrite concentration was observed for all samples with a rate corresponding to 0.5 x 10(5) to 2 x 10(5) nitrifying cells/ml in the bulk water and 3 x 10(5) cells/cm(2) on the pipe surface. The finding of an abundant nitrite-oxidizing microbial population suggests that nitrite is an important substrate in this system, potentially as a result of the low assimilable organic carbon concentration. This finding implies that microbial communities in water distribution systems may control against elevated nitrite concentrations but also contain large indigenous populations that are capable of assisting the depletion of disinfection agents like chloramines.     

Chlorination-mediated EPS excretion shapes early-stage biofilm formation in drinking water systems

Microbial surface adhesion to surfaces and subsequent biofilm establishment are ubiquitous in drinking water systems, which often contribute to deteriorated water quality. Disinfectants are common agents applied to drinking water controlling microbial propagation, yet the underlying mechanisms of how disinfectants function to regulate microbial activity and thereby biofilm development remains elusive. We experimentally studied the effects of chlorination on extracellular polymeric substance (EPS) production, and its impacts on early-stage biofilm formation in a model drinking water system. Results showed that low-level chlorine (≤ 1.0 mg/L) stimulated microbial EPS (especially of proteins) excretion that favored early-stage biofilm formation. Microbes experiencing higher chlorination (>1.0 mg/L) exhibited clearly suppressed growth associated with reduced EPS release, consequently yielding less biofilm formation. Removal of cell-attached proteins and polysaccharides diminished biofilm formation, which highlighted the critical role of EPS (especially protein components) in biofilm development. A negative correlation between chlorination-mediated microbial protein production and cell surface charge suggested that chlorine disinfection may modify cell surface properties through regulation of microbial EPS excretion and thereby mediate biofilm formation. With these quantitative estimations, this study provides novel insights into how chlorination-mediated EPS excretion shapes early-stage biofilm formation, which is essential for practical functioning of drinking water systems.     

Biological control of microbial attachment: a promising alternative for mitigating membrane biofouling

Microbial attachment to a solid surface is a universal phenomenon occurring in both natural and engineering systems and is responsible for various types of biofouling. Membrane systems have been widely applied in drinking water production, wastewater reuse, and seawater desalination. However, membrane biofouling is the bottleneck that limits the development of membrane systems. In this review, some biological control strategies of microbial attachment which would have great potential in alleviating membrane biofouling are discussed, including inhibition of quorum sensing system, nitric oxide-induced biofilm dispersal, enzymatic disruption of extracellular polysaccharides, proteins, and DNA, inhibition of microbial attachment by energy uncoupling, use of cell wall hydrolases, and disruption of biofilm by bacteriophage. It appears that biological control of microbial attachment would be a novel and promising alternative for mitigating membrane biofouling and would be a new research niche that deserves further study.     

Microbial ecology of drinking water distribution systems

The supply of clean drinking water is a major, and relatively recent, public health milestone. Control of microbial growth in drinking water distribution systems, often achieved through the addition of disinfectants, is essential to limiting waterborne illness, particularly in immunocompromised subpopulations. Recent inquiries into the microbial ecology of distribution systems have found that pathogen resistance to chlorination is affected by microbial community diversity and interspecies relationships. Research indicates that multispecies biofilms are generally more resistant to disinfection than single-species biofilms. Other recent findings are the increased survival of the bacterial pathogen Legionella pneumophila when present inside its protozoan host Hartmannella vermiformis and the depletion of chloramine disinfectant residuals by nitrifying bacteria, leading to increased overall microbial growth. Interactions such as these are unaccounted for in current disinfection models. An understanding of the microbial ecology of distribution systems is necessary to design innovative and effective control strategies that will ensure safe and high-quality drinking water.     

The role of biofilms and protozoa in Legionella pathogenesis: implications for drinking water

Current models to study Legionella pathogenesis include the use of primary macrophages and monocyte cell lines, various free-living protozoan species and murine models of pneumonia. However, there are very few studies of Legionella spp. pathogenesis aimed at associating the role of biofilm colonization and parasitization of biofilm microbiota and release of virulent bacterial cell/vacuoles in drinking water distribution systems. Moreover, the implications of these environmental niches for drinking water exposure to pathogenic legionellae are poorly understood. This review summarizes the known mechanisms of Legionella spp. proliferation within Acanthamoeba and mammalian cells and advocates the use of the amoeba model to study Legionella pathogenicity because of their close association with Legionella spp. in the aquatic environment. The putative role of biofilms and amoebae in the proliferation, development and dissemination of potentially pathogenic Legionella spp. is also discussed. Elucidating the mechanisms of Legionella pathogenicity development in our drinking water systems will aid in elimination strategies and procedural designs for drinking water systems and in controlling exposure to Legionella spp. and similar pathogens.     

Colonization and Disinfection of Biofilms Hosting Coliform-Colonized Carbon Fines

The documented release of carbon fines from granular activated carbon filters is a concern for drinking water utilities, since these particles may carry coliform and even pathogenic bacteria through the disinfection barrier. Such a breakthrough could have an impact on distribution system biofilms. Using total cell counts, specific monoclonal antibody staining, and computerized image analysis, we monitored the colonization of introduced Klebsiella pneumoniae associated with carbon fines in mixed-population biofilms. The particles transported the coliforms to the biofilms and allowed successful colonization. Chlorine (0.5 mg/liter) was then applied as a disinfectant. Most K. pneumoniae along with the carbon fines left the biofilm under these conditions. The impact of chlorine was greater on the coliform bacteria and carbon fines than on the general fixed bacterial population. However, 10% of the introduced coliforms and 20% of the fines remained in the biofilm. The possibility that this represents a mechanism for bacteria of public health concern to be involved in regrowth events is discussed.     

Ammonia- and nitrite-oxidizing bacterial communities in a pilot-scale chloraminated drinking water distribution system.

Nitrification in drinking water distribution systems is a common operational problem for many utilities that use chloramines for secondary disinfection. The diversity of ammonia-oxidizing bacteria (AOB) and nitrite-oxidizing bacteria (NOB) in the distribution systems of a pilot-scale chloraminated drinking water treatment system was characterized using terminal restriction fragment length polymorphism (T-RFLP) analysis and 16S rRNA gene (ribosomal DNA [rDNA]) cloning and sequencing. For ammonia oxidizers, 16S rDNA-targeted T-RFLP indicated the presence of Nitrosomonas in each of the distribution systems, with a considerably smaller peak attributable to Nitrosospira-like AOB. Sequences of AOB amplification products aligned within the Nitrosomonas oligotropha cluster and were closely related to N. oligotropha and Nitrosomonas ureae. The nitrite-oxidizing communities were comprised primarily of Nitrospira, although Nitrobacter was detected in some samples. These results suggest a possible selection of AOB related to N. oligotropha and N. ureae in chloraminated systems and demonstrate the presence of NOB, indicating a biological mechanism for nitrite loss that contributes to a reduction in nitrite-associated chloramine decay.     

Protection of waterborne pathogens by higher organisms in drinking water: a review

Higher organisms are ubiquitous in surface waters, and some species can proliferate in granular filters of water treatment plants and colonize distribution systems. Meanwhile, some waterborne pathogens are known to maintain viability inside amoebae or nematodes. The well-documented case of Legionella replication within amoebae is only one example of a bacterial pathogen that can be amplified inside the vacuoles of protozoa and then benefit from the protection of a resistant structure that favours its transport and persistence through water systems. Yet the role of most zooplankton organisms (rotifers, copepods, cladocerans) in pathogen transmission through drinking water remains poorly understood, since their capacity to digest waterborne pathogens has not been well characterized to date. This review aims at (i) evaluating the scientific observations of diverse associations between superior organisms and pathogenic microorganisms in a drinking water perspective and (ii) identifying the missing data that impede the establishment of cause-and-effect relationships that would permit a better appreciation of the sanitary risk arising from such associations. Additional studies are needed to (i) document the occurrence of invertebrate-associated pathogens in relevant field conditions, such as distribution systems; (ii) assess the fate of microorganisms ingested by higher organisms in terms of viability and (or) infectivity; and (iii) study the impact of internalization by zooplankton on pathogen resistance to water disinfection processes, including advanced treatments such as UV disinfection.     

Chlorine disinfection of Francisella tularensis

Aims: To determine the range of free available chlorine (FAC) required for disinfection of the live vaccine strain (LVS) and wild‐type strains of Francisella tularensis. Methods and Results: Seven strains of planktonic F. tularensis were exposed to 0·5 mg·l^?1 FAC for two pH values, 7 and 8, at 5 and 25°C. LVS was inactivated 2 to 4 times more quickly than any of the wild‐type F. tularensis strains at pH 8 and 5°C. Conclusions: Free available chlorine residual concentrations routinely maintained in drinking water distribution systems would require up to two hours to reduce all F. tularensis strains by 4 log₁₀. LVS was inactivated most quickly of the tested strains. Significance and Impact of the Study: This work provides contact time (CT) values that are useful for drinking water risk assessment and also suggests that LVS may not be a good surrogate in disinfection studies.     

Shift in the Microbial Ecology of a Hospital Hot Water System following the Introduction of an On-Site Monochloramine Disinfection System

Drinking water distribution systems, including premise plumbing, contain a diverse microbiological community that may include opportunistic pathogens. On-site supplemental disinfection systems have been proposed as a control method for opportunistic pathogens in premise plumbing. The majority of on-site disinfection systems to date have been installed in hospitals due to the high concentration of opportunistic pathogen susceptible occupants. The installation of on-site supplemental disinfection systems in hospitals allows for evaluation of the impact of on-site disinfection systems on drinking water system microbial ecology prior to widespread application. This study evaluated the impact of supplemental monochloramine on the microbial ecology of a hospital’s hot water system. Samples were taken three months and immediately prior to monochloramine treatment and monthly for the first six months of treatment, and all samples were subjected to high throughput Illumina 16S rRNA region sequencing. The microbial community composition of monochloramine treated samples was dramatically different than the baseline months. There was an immediate shift towards decreased relative abundance of Betaproteobacteria, and increased relative abundance of Firmicutes, Alphaproteobacteria, Gammaproteobacteria, Cyanobacteria and Actinobacteria. Following treatment, microbial populations grouped by sampling location rather than sampling time. Over the course of treatment the relative abundance of certain genera containing opportunistic pathogens and genera containing denitrifying bacteria increased. The results demonstrate the driving influence of supplemental disinfection on premise plumbing microbial ecology and suggest the value of further investigation into the overall effects of premise plumbing disinfection strategies on microbial ecology and not solely specific target microorganisms.     

Bacteriology of drinking water distribution systems: an integral and multidimensional review

A drinking water distribution system (DWDS) is the final and essential step to supply safe and high-quality drinking water to customers. Biological processes, such as biofilm formation and detachment, microbial growth in bulk water, and the formation of loose deposits, may occur. These processes will lead to deterioration of the water quality during distribution. In extreme conditions, pathogens and opportunistic pathogens may proliferate and pose a health risk to consumers. It is, therefore, necessary to understand the bacteriology of DWDSs to develop effective strategies that can ensure the water quality at consumers' taps. The bacteriology of DWDSs, both the quantitative growth and the qualitative bacterial community, has attracted considerable research attention. However, the researchers have focused mainly on the pipe wall biofilm. In this review, DWDS bacteriology has been reviewed multidimensionally, including both the bacterial quantification and identification. For the first time, the available literature was reviewed with an emphasis on the subdivision of DWDS into four phases: bulk water, suspended solids, loose deposits, and pipe wall biofilm. Special concentration has been given to potential contribution of particulate matter: suspended particles and loose deposits. Two highlighted questions were reviewed and discussed: (1) where does most of the growth occur? And (2) what is the contribution of particle-associated bacteria to DWDS bacteriology and ecology? At the end of this review, recommendations were given based on the conclusion of this review to better understand the integral DWDS bacteriology.     

Assessment of an anti-scale low-frequency electromagnetic field device on drinking water biofilms

Abstract

Low intensity and very low-frequency electromagnetic fields (EMF) used for preventing scaling in water distribution systems were tested for the first time for their potential impact on drinking water biofilms. The assays were carried out in laboratory-scale flow-through reactors that mimic water distribution systems. The drinking water biofilms were not directly exposed to the core of the EMF generator and only subjected to waterborne electromagnetic waves. The density and chlorine susceptibility of nascent or mature biofilms grown under exposure to EMF were evaluated in soft and hard water. This EMF treatment was able to modify CaCO₃ crystallization but it did not significantly affect biofilms. Indeed, over all the tested conditions, there was no significant change in cell number, or in the integrity of the cells (membrane, culturability), and no measurable effect of chlorine on the biofilm.     

Chlorine inactivation of human norovirus,murine norovirus and poliovirus in drinking water

Aims: To evaluate the reduction of human norovirus (HuNoV) by chlorine disinfection under typical drinking water treatment conditions. Methods and Results: HuNoV, murine norovirus (MNV) and poliovirus type 1 (PV1) were inoculated into treated water before chlorination, collected from a drinking water treatment plant, and bench‐scale free chlorine disinfection experiments were performed for two initial free chlorine concentrations, 0·1 and 0·5 mg l^?1. Inactivation of MNV reached more than 4 log₁₀ after 120 and 0·5 min contact time to chlorine at the initial free chlorine concentrations of 0·1 and 0·5 mg l^?1, respectively. Conclusions: MNV was inactivated faster than PV1, and there was no significant difference in the viral RNA reduction rate between HuNoV and MNV. The results suggest that appropriate water treatment process with chlorination can manage the risk of HuNoV infection via drinking water supply systems. Significance and Impact of the Study: The data obtained in this study would be useful for assessing or managing the risk of HuNoV infections from drinking water exposure.     

Identification of Bacteria in Biofilm and Bulk Water Samples from a Nonchlorinated Model Drinking Water Distribution System: Detection of a Large Nitrite-Oxidizing Population Associated with Nitrospira spp.

In a model drinking water distribution system characterized by a low assimilable organic carbon content (<10 μg/liter) and no disinfection, the bacterial community was identified by a phylogenetic analysis of rRNA genes amplified from directly extracted DNA and colonies formed on R2A plates. Biofilms of defined periods of age (14 days to 3 years) and bulk water samples were investigated. Culturable bacteria were associated with Proteobacteria and Bacteriodetes, whereas independently of cultivation, bacteria from 12 phyla were detected in this system. These included Acidobacteria, Nitrospirae, Planctomycetes, and Verrucomicrobia, some of which have never been identified in drinking water previously. A cluster analysis of the population profiles from the individual samples divided biofilms and bulk water samples into separate clusters (P = 0.027). Bacteria associated with Nitrospira moscoviensis were found in all samples and encompassed 39% of the sequenced clones in the bulk water and 25% of the biofilm community. The close association with Nitrospira suggested that a large part of the population had an autotrophic metabolism using nitrite as an electron donor. To test this hypothesis, nitrite was added to biofilm and bulk water samples, and the utilization was monitored during 15 days. A first-order decrease in nitrite concentration was observed for all samples with a rate corresponding to 0.5 × 10⁵ to 2 × 10⁵ nitrifying cells/ml in the bulk water and 3 × 10⁵ cells/cm² on the pipe surface. The finding of an abundant nitrite-oxidizing microbial population suggests that nitrite is an important substrate in this system, potentially as a result of the low assimilable organic carbon concentration. This finding implies that microbial communities in water distribution systems may control against elevated nitrite concentrations but also contain large indigenous populations that are capable of assisting the depletion of disinfection agents like chloramines.     

Inactivation of Pseudomonas aeruginosa biofilm by dense phase carbon dioxide

Dense phase carbon dioxide (DPCD) is one of the most promising techniques available to control microorganisms as a non-thermal disinfection method. However, no study on the efficiency of biofilm disinfection using DPCD has been reported. The efficiency of DPCD in inactivating Pseudomonas aeruginosa biofilm, which is known to have high antimicrobial resistance, was thus investigated. P. aeruginosa biofilm, which was not immersed in water but was completely wet, was found to be more effectively inactivated by DPCD treatment, achieving a 6-log reduction within 7 min. The inactivation efficiency increased modestly with increasing pressure and temperature. This study also reports that the water-unimmersed condition is one of the most important operating parameters in achieving efficient biofilm control by DPCD treatment. In addition, observations by confocal laser scanning microscopy revealed that DPCD treatment not only inactivated biofilm cells on the glass coupons but also caused detachment of the biofilm following weakening of its structure as a result of the DPCD treatment; this is an added benefit of DPCD treatment.     

Interaction between phosphorus and biodegradable organic carbon on drinking water biofilm subject to chlorination

Aims: To examine whether phosphorus and biodegradable organic carbon interact to impact biofilm density and physiological function of biofilm‐forming bacteria under conditions relevant to chlorinated drinking water distribution systems. Materials and Results: The 2 × 2 factorial experiments with low and high levels of phosphorus and biodegradable organic carbon were performed on 4 ‐week‐old drinking water biofilms in four separate pipe systems in the presence of chlorine. Experimental results revealed that biofilm heterotrophic plate count levels increased with the increase in biodegradable organic carbon concentration, showed no response to increases in levels of phosphorus and was not affected by interaction between phosphorus and biodegradable organic carbon. However, a significant positive interaction between phosphorus and biodegradable organic carbon was found to exist on biofilm mass and physiological function and/or metabolic potentials of biofilm communities; the effects of biodegradable organic carbon on biofilm mass and physiological function of biofilm‐forming bacteria were accelerated in going from low to high level of phosphorus. Conclusions: Biodegradable organic carbon was found to be the primary nutrient in regulating biofilm formation in drinking water regardless of the presence of chlorine. It can be therefore concluded that the removal of an easily biodegradable organic carbon is necessary to minimize the biofilm growth potential induced by the intrusion of phosphorus. Significance and Impact of the Study: Phosphorus introduced to drinking water may interact with biodegradable organic carbon, thus leading to measurable impact on the biofilm formation.