Microbial community structure and biomass in developing drinking water biofilms

Traditional techniques to study microbes, such as culturable counts, microbial biomass, or microbial activity, do not give information on the microbial ecology of drinking water systems. The aim of this study was to analyze whether the microbial community structure and biomass differed in biofilms collected from two Finnish drinking water distribution systems (A and B) receiving conventionally treated (coagulation, filtration, disinfection) surface water. Phospholipid fatty acid methyl esters (PLFAs) and lipopolysaccharide 3-hydroxy fatty acid methyl esters (LPS 3-OH-FAs) were analyzed from biofilms as a function of water residence time and development time. The microbial communities were rather stabile through the distribution systems, as water residence time had minor effects on PLFA profiles. In distribution system A, the microbial community structure in biofilms, which had developed in 6 weeks, was more complex than those grown for 23 or 40 weeks. The microbial communities between the studied distribution systems differed, possibly reflecting the differences in raw water, water purification processes, and distribution systems. The viable microbial biomass, estimated on the basis of PLFAs, increased with increasing water residence time in both distribution systems. The quantitative amount of LPS 3-OH-FAs increased with increasing development time of biofilms of distribution system B. In distribution system A, LPS 3-OH-FAs were below the detection limit.     

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.     

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.     

Biofilm formation in drinking water affected by low concentrations of phosphorus

Abstract: There are geographical regions where microbial growth in drinking waters is limited by phosphorus instead of organic carbon. In these drinking waters even a low amount of phosphorus can strongly enhance microbial growth. The formation of biofilm can be limited by low availability of phosphorus in drinking waters with low content of phosphorus. The formation of biofilms on polyvinyl chloride plates was studied in laboratory experiments with water containing 48 microg/L assimilable organic carbon and 0.19 microg/L microbially available phosphorus. We found that low additions of phosphate (1-5 microg/L PO4(3-)-P) to water increased microbial growth in the water and in the biofilm. The effect of phosphorus on microbial growth could be detected by determining either the microbial cell production or the content of ATP in biofilms. Also, in steady-state biofilms, microbial concentrations were higher with phosphorus addition as enumerated by heterotrophic plate counts on R2A-agar and acridine orange direct counting. This work confirms the earlier findings of the importance of phosphorus for microbial growth in humic-rich drinking waters.     

A review of drinking-water-associated endotoxin,including potential routes of human exposure

In the past decade efforts have been made to reduce the formation of harmful disinfection byproducts during the treatment and distribution of drinking water. This has been accomplished in part by the introduction of processes that involve the deliberate encouragement of indigenous biofilm growth in filters. In a controlled environment, such as a filter, these biofilms remove compounds that would otherwise be available as disinfection byproduct precursors or support uncontrolled biological activity in distribution systems. In the absence of exposure to chlorinated water, most biofilm bacteria are gram negative and have an outer layer that contains endotoxin. To date, outbreaks of waterborne endotoxin-related illness attributable to contamination of water used in hemodialysis procedures have been only infrequently documented, and occurrences linked to ingestion or through dermal abrasions could not be located. However, a less obvious conduit, that of inhalation, has been described in association with aerosolized water droplets. This review summarizes documented drinking-water-associated incidents of endotoxin exposure attributable to hemodialysis and inhalation. Typical endotoxin levels in water and conditions under which substantial quantities can enter drinking water distribution systems are identified. It would appear that endotoxin originating in tap water can be inhaled but at present there is insufficient information available to quantify potential health risks.     

An ignored and potential source of taste and odor (T&O) issues—biofilms in drinking water distribution system (DWDS)

It is important for water utilities to provide esthetically acceptable drinking water to the public, because our consumers always initially judge the quality of the tap water by its color, taste, and odor (T&O). Microorganisms in drinking water contribute largely to T&O production and drinking water distribution systems (DWDS) are known to harbor biofilms and microorganisms in bulk water, even in the presence of a disinfectant. These microbes include T&O-causing bacteria, fungi, and algae, which may lead to unwanted effects on the organoleptic quality of distributed water. Importantly, the understanding of types of these microbes and their T&O compound-producing mechanisms is needed to prevent T&O formation during drinking water distribution. Additionally, new disinfection strategies and operation methods of DWDS are also needed for better control of T&O problems in drinking water. This review covers: (1) the microbial species which can produce T&O compounds in DWDS; (2) typical T&O compounds in DWDS and their formation mechanisms by microorganisms; (3) several common factors in DWDS which can influence the growth and T&O generation of microbes; and (4) several strategies to control biofilm and T&O compound formation in DWDS. At the end of this review, recommendations were given based on the conclusion of this review.

     

Survival of Mycobacterium avium in drinking water biofilms as affected by water flow velocity, availability of phosphorus, and temperature

Mycobacterium avium is a potential pathogen occurring in drinking water systems. It is a slowly growing bacterium producing a thick cell wall containing mycolic acids, and it is known to resist chlorine better than many other microbes. Several studies have shown that pathogenic bacteria survive better in biofilms than in water. By using Propella biofilm reactors, we studied how factors generally influencing the growth of biofilms (flow rate, phosphorus concentration, and temperature) influence the survival of M. avium in drinking water biofilms. The growth of biofilms was followed by culture and DAPI (4',6'-diamidino-2-phenylindole) staining, and concentrations of M. avium were determined by culture and fluorescence in situ hybridization methods. The spiked M. avium survived in biofilms for the 4-week study period without a dramatic decline in concentration. The addition of phosphorus (10 microg/liter) increased the number of heterotrophic bacteria in biofilms but decreased the culturability of M. avium. The reason for this result is probably that phosphorus increased competition with other microbes. An increase in flow velocity had no effect on the survival of M. avium, although it increased the growth of biofilms. A higher temperature (20 degrees C versus 7 degrees C) increased both the number of heterotrophic bacteria and the survival of M. avium in biofilms. In conclusion, the results show that in terms of affecting the survival of slowly growing M. avium in biofilms, temperature is a more important factor than the availability of nutrients like phosphorus.     

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.     

Phosphorus and bacterial growth in drinking water.

The availability of organic carbon is considered the key factor to regulate microbial regrowth in drinking water network. However, boreal regions (northern Europe, Russia, and North America) contain a large amount of organic carbon in forests and peatlands. Therefore, natural waters (lakes, rivers, and groundwater) in the northern hemisphere generally have a high content of organic carbon. We found that microbial growth in drinking water in Finland is highly regulated not only by organic carbon but also by the availability of phosphorus. Microbial growth increased up to a phosphate concentration of 10 micrograms of PO4-P liter-1. Inorganic elements other than phosphorus did not affect microbial growth in drinking water. This observation offers novel possibilities to restrict microbial growth in water distribution systems by developing technologies to remove phosphorus efficiently from drinking water.     

Microbial biogeography of drinking water: patterns in phylogenetic diversity across space and time

In this study, we collected water from different locations in 32 drinking water distribution networks in the Netherlands and analysed the spatial and temporal variation in microbial community composition by high‐throughput sequencing of 16S rRNA gene amplicons. We observed that microbial community compositions of raw source and processed water were very different for each distribution network sampled. In each network, major differences in community compositions were observed between raw and processed water, although community structures of processed water did not differ substantially from end‐point tap water. End‐point water samples within the same distribution network revealed very similar community structures. Network‐specific communities were shown to be surprisingly stable in time. Biofilm communities sampled from domestic water metres varied distinctly between households and showed no resemblance to planktonic communities within the same distribution networks. Our findings demonstrate that high‐throughput sequencing provides a powerful and sensitive tool to probe microbial community composition in drinking water distribution systems. Furthermore, this approach can be used to quantitatively compare the microbial communities to match end‐point water samples to specific distribution networks. Insight in the ecology of drinking water distribution systems will facilitate the development of effective control strategies that will ensure safe and high‐quality drinking water.     

Assessment of bismuth thiols and conventional disinfectants on drinking water biofilms

AIMS: Biofilms in water distribution systems represent a far more significant reservoir of micro-organisms than the water phase. Biofilms are (i) resistant to disinfectants, (ii) nuclei for microbial regrowth, (iii) a refuge for pathogens, (iv) accompanied by taste and odour problems, and (v) corrode surfaces. The effects of the current strategies for disinfection of drinking water systems in large buildings (chlorination, copper and silver ionization, and hyper-heating) were compared with a new generation of bismuth thiol (BT) biocides. METHODS AND RESULTS: Multispecies biofilms were treated with 0.8 mg l(-1) of free chlorine, 400 and 40 microg l(-1) of copper and silver ions, respectively, at 55 and 70 degrees C, and bismuth-2,3-dimercaptopropanol (BisBAL). Furthermore, the effect of combined heat and BisBAL on planktonic cell viability was examined in monoculture using Escherichia coli suspensions. Inactivation rates for BisBAL were similar to copper-silver ions, where the effects were slower than for free chlorine or temperature. The BisBAL effect on E. coli monocultures was augmented greatly by increasing temperatures. CONCLUSIONS: Like copper-silver ions, BTs show more persistent residual effects than chlorine and hyper-heating in water systems. BT efficiency increased with temperature. Like copper-silver ions, BT action is relatively slow. SIGNIFICANCE AND IMPACT OF THE STUDY: BT presents a new approach to containing water biofilms. BT action is not as rapid, but is more thorough than chlorine, and less caustic. BTs may also be more efficacious in hot water systems. At sub-minimum inhibition concentration levels, BTs uniquely inhibit bacterial exopolysaccharide, thereby retarding biofilm formation. Thus, the combination of bactericidal and residual effects may prevent slime build-up in hot water systems.     

Survival of Mycobacterium avium in Drinking Water Biofilms as Affected by Water Flow Velocity, Availability of Phosphorus, and Temperature

Mycobacterium avium is a potential pathogen occurring in drinking water systems. It is a slowly growing bacterium producing a thick cell wall containing mycolic acids, and it is known to resist chlorine better than many other microbes. Several studies have shown that pathogenic bacteria survive better in biofilms than in water. By using Propella biofilm reactors, we studied how factors generally influencing the growth of biofilms (flow rate, phosphorus concentration, and temperature) influence the survival of M. avium in drinking water biofilms. The growth of biofilms was followed by culture and DAPI (4′,6′-diamidino-2-phenylindole) staining, and concentrations of M. avium were determined by culture and fluorescence in situ hybridization methods. The spiked M. avium survived in biofilms for the 4-week study period without a dramatic decline in concentration. The addition of phosphorus (10 μg/liter) increased the number of heterotrophic bacteria in biofilms but decreased the culturability of M. avium. The reason for this result is probably that phosphorus increased competition with other microbes. An increase in flow velocity had no effect on the survival of M. avium, although it increased the growth of biofilms. A higher temperature (20°C versus 7°C) increased both the number of heterotrophic bacteria and the survival of M. avium in biofilms. In conclusion, the results show that in terms of affecting the survival of slowly growing M. avium in biofilms, temperature is a more important factor than the availability of nutrients like phosphorus.     

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.     

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.     

Biophysical controls on cluster dynamics and architectural differentiation of microbial biofilms in contrasting flow environments

Ecology, with a traditional focus on plants and animals, seeks to understand the mechanisms underlying structure and dynamics of communities. In microbial ecology, the focus is changing from planktonic communities to attached biofilms that dominate microbial life in numerous systems. Therefore, interest in the structure and function of biofilms is on the rise. Biofilms can form reproducible physical structures (i.e. architecture) at the millimetre‐scale, which are central to their functioning. However, the spatial dynamics of the clusters conferring physical structure to biofilms remains often elusive. By experimenting with complex microbial communities forming biofilms in contrasting hydrodynamic microenvironments in stream mesocosms, we show that morphogenesis results in ‘ripple‐like’ and ‘star‐like’ architectures – as they have also been reported from monospecies bacterial biofilms, for instance. To explore the potential contribution of demographic processes to these architectures, we propose a size‐structured population model to simulate the dynamics of biofilm growth and cluster size distribution. Our findings establish that basic physical and demographic processes are key forces that shape apparently universal biofilm architectures as they occur in diverse microbial but also in single‐species bacterial biofilms.     

Microbial activity in drinking water-associated biofilms

Microbial biofilms from surfaces in contact with water may play a beneficial role in drinking water treatment as biological filters. However, detrimental effects such as biofouling (i.e., biocorrosion and water quality deterioration) may also occur. In this study microbiological processes and factors influencing the activity of bacteria in biofilms were investigated by conventional cultivation methods. The presence of bacteria belonging to different ecophysiological groups was assessed during drinking water treatment, in biofilms developed on concrete, steel and sand surfaces. Influences of the treatment process, type of immersed material and physico-chemical characteristics of raw/bulk water and biofilms upon the dynamics of bacterial communities were evaluated. Results revealed intense microbial activity in biofilms occurring in the drinking water treatment plant of Cluj. Ammonification, iron reduction and manganese oxidation were found to be the predominant processes. Multiple significant correlations were established between the evolution of biofilm bacteria and the physico-chemical parameters of raw/ bulk water. The type of immersed material proved to have no significant influence upon the evolution of microbial communities, but the treatment stage, suggesting that the processes applied restrict microbial growth not only in bulk fluid but in biofilms, too.     

Nitrite production by ammonia-oxidizing bacteria mediates chloramine decay and resistance in a mixed-species community

As water distribution centres increasingly switch to using chloramine to disinfect drinking water, it is of paramount importance to determine the interactions of chloramine with potential biological contaminants, such as bacterial biofilms, that are found in these systems. For example, ammonia-oxidizing bacteria (AOB) are known to accelerate the decay of chloramine in drinking water systems, but it is also known that organic compounds can increase the chloramine demand. This study expanded upon our previously published model to compare the decay of chloramine in response to alginate, Pseudomonas aeruginosa, Nitrosomonas europaea and a mixed-species nitrifying culture, exploring the contributions of microbial by-products, heterotrophic bacteria and AOBs to chloramine decay. Furthermore, the contribution of AOBs to biofilm stability during chloramination was investigated. The results demonstrate that the biofilm matrix or extracellular polymeric substances (EPS), represented by alginate in these experiments, as well as high concentrations of dead or inactive cells, can drive chloramine decay rather than any specific biochemical activity of P. aeruginosa cells. Alginate was shown to reduce chloramine concentrations in a dose-dependent manner at an average rate of 0.003 mg l⁻¹ h⁻¹ per mg l⁻¹ of alginate. Additionally, metabolically active AOBs mediated the decay of chloramine, which protected members of mixed-species biofilms from chloramine-mediated disinfection. Under these conditions, nitrite produced by AOBs directly reacted with chloramine to drive its decay. In contrast, biofilms of mixed-species communities that were dominated by heterotrophic bacteria due to either the absence of ammonia, or the addition of nitrification inhibitors and glucose, were highly sensitive to chloramine. These results suggest that mixed-species biofilms are protected by a combination of biofilm matrix-mediated inactivation of chloramine as well as the conversion of ammonia to nitrite through the activity of AOBs present in the community.     

Probabilistic Models to Describe the Dynamics of Migrating Microbial Communities

In all but the most sterile environments bacteria will reside in fluid being transported through conduits and some of these will attach and grow as biofilms on the conduit walls. The concentration and diversity of bacteria in the fluid at the point of delivery will be a mix of those when it entered the conduit and those that have become entrained into the flow due to seeding from biofilms. Examples include fluids through conduits such as drinking water pipe networks, endotracheal tubes, catheters and ventilation systems. Here we present two probabilistic models to describe changes in the composition of bulk fluid microbial communities as they are transported through a conduit whilst exposed to biofilm communities. The first (discrete) model simulates absolute numbers of individual cells, whereas the other (continuous) model simulates the relative abundance of taxa in the bulk fluid. The discrete model is founded on a birth-death process whereby the community changes one individual at a time and the numbers of cells in the system can vary. The continuous model is a stochastic differential equation derived from the discrete model and can also accommodate changes in the carrying capacity of the bulk fluid. These models provide a novel Lagrangian framework to investigate and predict the dynamics of migrating microbial communities. In this paper we compare the two models, discuss their merits, possible applications and present simulation results in the context of drinking water distribution systems. Our results provide novel insight into the effects of stochastic dynamics on the composition of non-stationary microbial communities that are exposed to biofilms and provides a new avenue for modelling microbial dynamics in systems where fluids are being transported.