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

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.

The effect of chlorination and hydrodynamic shear stress on the persistence of bacteriophages associated with drinking water biofilms.

AIMS: This work aimed to assess at pilot scale the effect of chlorination and water flushing on 2-month-old drinking water biofilms and, above all, on biofilm-associated F-specific RNA bacteriophages MS2, GA and Qß. METHODS AND RESULTS: Chlorination (4 mg l(-1) ) was applied first with a hydrodynamic shear stress of 1 Pa and second with an increase in hydrodynamic shear stress to 10 Pa. Despite a rapid decrease in the number of biofilm bacteria and associated phages, infectious phages were still detected on surfaces after completion of the 150 min cleaning procedure. The resulting sequence of phage removal was: GA > Qߠ≫ MS2. CONCLUSIONS: The effect of chlorine on biofilm bacteria and biofilm-associated phages was limited to the upper layers of the biofilm and was not enhanced by an increase in hydrodynamic shear stress. A smaller decrease was observed for MS2 than for GA or Qß after completion of the cleaning procedure. SIGNIFICANCE AND IMPACT OF THE STUDY: The differences observed between the three phages suggest that the location of the viral particles in the biofilm, which is related to their surface properties, affects the efficiency of chlorine disinfection.

Drinking water biofilm cohesiveness changes under chlorination or hydrodynamic stress.

Attempts at removal of drinking water biofilms rely on various preventive and curative strategies such as nutrient reduction in drinking water, disinfection or water flushing, which have demonstrated limited efficiency. The main reason for these failures is the cohesiveness of the biofilm driven by the physico-chemical properties of its exopolymeric matrix (EPS). Effective cleaning procedures should break up the matrix and/or change the elastic properties of bacterial biofilms. The aim of this study was to evaluate the change in the cohesive strength of two-month-old drinking water biofilms under increasing hydrodynamic shear stress τw (from ∼0.2 to ∼10 Pa) and shock chlorination (applied concentration at T0: 10 mg Cl2/L; 60 min contact time). Biofilm erosion (cell loss per unit surface area) and cohesiveness (changes in the detachment shear stress and cluster volumes measured by atomic force microscopy (AFM)) were studied. When rapidly increasing the hydrodynamic constraint, biofilm removal was found to be dependent on a dual process of erosion and coalescence of the biofilm clusters. Indeed, 56% of the biofilm cells were removed with, concomitantly, a decrease in the number of the 50-300 µm(3) clusters and an increase in the number of the smaller (i.e., <50 µm(3)) and larger (i.e., >600 µm(3)) ones. Moreover, AFM evidenced the strengthening of the biofilm structure along with the doubling of the number of contact points, NC, per cluster volume unit following the hydrodynamic disturbance. This suggests that the compactness of the biofilm exopolymers increases with hydrodynamic stress. Shock chlorination removed cells (-75%) from the biofilm while reducing the volume of biofilm clusters. Oxidation stress resulted in a decrease in the cohesive strength profile of the remaining drinking water biofilms linked to a reduction in the number of contact points within the biofilm network structure in particular for the largest biofilm cluster volumes (>200 µm(3)). Changes in the cohesive strength of drinking water biofilms subsequent to cleaning/disinfection operations call into question the effectiveness of cleaning-in-place procedures. The combined alternating use of oxidation and shear stress sequences needs to be investigated as it could be an important adjunct to improving biofilm removal/reduction procedures.

Drinking water and biofilm disinfection by Fenton-like reaction.

A Fenton-like disinfection process was conducted with Fenton's reagent (H2O2) at pH 3 or 5 on autochthonous drinking water biofilms grown on corroded or non-corroded pipe material. The biofilm disinfection by Fenton-like oxidation was limited by the low content of iron and copper in the biomass grown on non-corroded plumbing. It was slightly improved by spiking the distribution system with some additional iron source (soluble iron II or ferrihydrite particles appeared as interesting candidates). However successful in situ disinfection of biofilms was only achieved in fully corroded cast iron pipes using H2O2 and adjusting the pH to 5. These new results provide additional support for the use of Fenton's processes for cleaning drinking water distribution systems contaminated with biological agents or organics.

Assessment of Damage to Nucleic Acids and Repair Machinery in Salmonella typhimurium Exposed to Chlorine.

Water disinfection is usually evaluated using mandatory methods based on cell culturability. However, such methods do not consider the potential of cells to recover, which should also be kept as low as possible. In this paper, we hypothesized that a successful disinfection is achieved only when the applied chlorine leads to both intracellular nucleic acid damage and strong alterations of the DNA repair machinery. Monitoring the SOS system responsiveness with a umuC'-'lacZ reporter fusion, we found that the expression of this important cellular machinery was altered after the beginning of membrane permeabilization but prior to the total decline of both the cell culturability and the nucleic acid integrity as revealed by Sybr-II staining. Rapid measurement of such nucleic acid alterations by fluorochrome-based staining could be used as an alternative method for assessing the effectiveness of disinfection with chlorine.

Reversible shift in the alpha-, beta- and gamma-proteobacteria populations of drinking water biofilms during discontinuous chlorination.

As disinfection strategies could support a shift of some bacterial populations, the biodiversity of drinking water biofilms depending on the disinfectant concentrations was explored. The effect of different chlorine sequences applied for several weeks (0.1-0.4-0.1 mg Cl(2)L(-1) or vice versa) was tested on the abundance of the alpha-, beta- and gamma-proteobacteria populations, used as indicators of changes in bacterial populations within drinking water biofilms. Using dynamic (industrial pilot) and batch (bench scale) conditions, our work demonstrated the ability of the 3 proteobacteria subclasses to re-organize following discontinuous chlorinations. The beta- and gamma-proteobacteria subclasses were favoured by high free residual chlorine concentrations (0.4 mg Cl(2)L(-1)) while alpha-proteobacteria population was sensitive to this oxidant level. The proteobacteria population shifts within the biofilm exposed to discontinuous chlorination were reversible. The resilience of the biofilm proteobacteria populations exposed to oxidant stress questioned the emergence of bacterial population less sensitive to chlorine.

Survival of Mycobacterium avium attached to polyethylene terephtalate (PET) water bottles.

AIMS: The main objective of our study was to assess the persistence of Mycobacterium avium in an oligotrophic environment such as bottled groundwater. METHODS AND RESULTS: Filtered groundwater samples were spiked with washed Myco. avium suspension and stored in dark and under static conditions, at 20 degrees C, for 3 months in 500 ml PET bottles. The loss of Myco. avium cultivability was slow in water. On the contrary, after a 3-month storage at 20 degrees C, growth of attached cells was observed and cell adhesiveness to the PET wall increased with time. It could probably be because of the presence of an extracellular matrix. CONCLUSIONS: This study has shown the great stability of Myco. avium in bulk water as well as their adhesiveness and their growth on a PET bottle wall in an oligotrophic environment. SIGNIFICANCE AND IMPACT OF THE STUDY: Slowly growing mycobacteria are well adapted to oligotrophic environments such as groundwater. As they stick very well to surfaces, they could be used for determining the efficiency of the cleaning of contaminated surfaces.

Highly chlorinated Escherichia coli cannot be stained by propidium iodide.

Several studies have shown that the staining by fluorochromes (DAPI, SYBR Green II, and TOTO-1) of bacteria is altered by chlorination. To evaluate the effect of chlorine (bleach solution) on propidium iodide (PI) staining, we studied Escherichia coli in suspension and biomolecules in solution (DNA, RNA, BSA, palmitic acid, and dextran) first subjected to chlorine and then neutralized by sodium thiosulphate. The suspensions and solutions were subsequently stained with PI. The fluorescence intensity of the PI-stained DNA and RNA in solution dramatically decreased with an increase in the chlorine concentration applied. These results explain the fact that for chlorine concentrations higher than 3 micromol/L Cl2, the E. coli cells were too damaged to be properly stained by PI. In the case of highly chlorinated bacteria, it was impossible to distinguish healthy cells (with a PI-impermeable membrane and undamaged nucleic acids), which were nonfluorescent after PI staining, from cells severely injured by chlorine (with a PI-permeable membrane and damaged nucleic acids) that were also nonfluorescent, as PI penetrated but did not stain chlorinated nucleic acids. Our results suggest that it would be prudent to be cautious in interpreting the results of PI staining, as PI false-negative cells (cells with compromised membranes but not stained by PI because of nucleic acid damage caused by chlorine) are obtained as a result of nucleic acid damage, leading to an underestimation of truly dead bacteria.

Behaviour of Campylobacter jejuni in experimentally contaminated bottled natural mineral water.

AIMS: The main objective of the present study was to estimate the survival of microaerophilic Campylobacter jejuni in filtered natural mineral water at 4 degrees C and 25 degrees C. The influence of the presence of biodegradable organic matter was tested, assuming that the bacterial contamination of a bottled natural mineral water could be associated with contamination by organic matter. METHODS AND RESULTS: Washed Campylobacter cultures were inoculated in natural mineral water and sterile natural mineral water, and incubated in the dark at 4 degrees C and 25 degrees C. The effect of temperature, the biodegradable organic matter added, incubation atmosphere and autochthonous microflora were tested on the cultivability of Camp. jejuni. CONCLUSIONS: The survival of Camp. jejuni in natural mineral water was better at 4 degrees C than at 25 degrees C, and the presence of organic matter led to a deceleration in the loss of cultivability and to the multiplication of Camp. jejuni in natural mineral water. SIGNIFICANCE AND IMPACT OF THE STUDY: This study highlighted the fact that, in the event of dual contamination of a bottled natural mineral water (Campylobacter and biodegradable organic matter), the pathogen could survive (and even grow) for a relatively long time, especially at low temperature and in spite of the presence of oxygen.

Coliforms and other microbial indicators occurrence in water and biofilm in full-scale distribution systems.

Biofilm and microbial water quality were studied in four middle size full-scale distribution systems (DS) in France serving 5,000-30,000 inhabitants (maximum residence time 23-160h) through three sampling campaigns over 1 year. Three of these DSs were chosen because of a quite high occurrence of bacterial indicators (i.e. total coliforms), the last DS was considered as a reference. Biofilm was studied on cast iron coupons incubated for more than 1 month in devices continuously fed with water from the DS in conditions imitating those met in DS. The devices were located at different points (4-6) along each DS. The abundance of bacteria in biofilm was estimated by heterotrophic plate counts (HPC) after detachment of the biofilm from the support by sonication. Microbiological water quality was estimated in parallel; analysis of total coliforms, E. coli, enterococci and anaerobic sulphide-reducing bacteria spores (ASRB spores) was carried out in biofilm and water. Over the period of the study, 171 water samples and 57 biofilm samples were collected. Over these 171 waters, 19 (11%) were positive for at least one of the measured indicators while two biofilm samples were positive (3.5%). Significant differences were observed in the levels of contamination between the DSs. High residence time in the DS, low disinfectant residual and high temperature increased the risk of indicator occurrence in the water phase. Due to the low number of biofilm samples positive for bacterial indicators, the data collected in the present study did not allow observation of a direct association between biofilm and water contaminations, even if the occurrence of indicators in water appeared on DSs with the highest density of biofilm (HPC).

Iron uptake is essential for Escherichia coli survival in drinking water.

AIMS: The aim of this study was to elucidate if the need for iron for Escherichia coli to remain cultivable in a poorly nutritive medium such as the drinking water uses the iron transport system via the siderophores. METHODS AND RESULTS: Environmental strains of E. coli (isolated from a drinking water network), referenced strains of E. coli and mutants deficient in TonB, an essential protein for iron(III) acquisition, were incubated for 3 weeks at 25 degrees C, in sterile drinking water with and without lepidocrocite (gamma-FeOOH), an insoluble iron corrosion product. Only cells with a functional iron transport system were able to survive throughout the weeks. CONCLUSIONS: The iron transport system via protein TonB plays an essential role on the survival of E. coli in a weakly nutritive medium like drinking water. SIGNIFICANCE AND IMPACTS OF THE STUDY: Iron is a key parameter involved in coliform persistence in drinking water distribution systems.

Adsorption/desorption of linear alkylbenzenesulfonate (LAS) and azoproteins by/from activated sludge flocs.

Our study investigated the adsorption/desorption by/from activated sludge flocs, dispersed in river water or in diluted wastewater, of organic compounds (C(11)-LAS, azoalbumin and azocasein) at concentrations relevant to environmental conditions. Activated sludge flocs, used as a model of biological aggregates, are characterized by a very heterogeneous matrix able to sorb the three organic compounds tested at 4 degrees C. The adsorbed amount of C(11)-LAS by activated sludge flocs was higher than that of azocasein or azoalbumin, as shown by the Freundlich parameters (K(ads)=8.6+/-1.7, 1.6+/-0.3 and 0.3+/-0.1 micromol(1-1/n)g(-1)l(1/n) for C(11)-LAS, azocasein and azoalbumin, respectively; n=3 sludges). C(11)-LAS sorption from activated sludge appeared to be partially reversible in river water, while a marked hysteresis phenomenon was observed for azocasein and azoalbumin, implying a low degree of reversibility in their exchange between activated sludge and river water. It has also been displayed that the conductivity variation of bulk water (comprised between 214 and 838 microS cm(-1)) exerted no dramatic effect on the C(11)-LAS desorption from activated sludge flocs, while a little effect of it on azocasein desorption was observed. Thus, biological aggregates as activated sludge flocs can serve as an intermediate carrier for C(11)-LAS, while it represents a sink for proteins.

Variations of respiratory activity and glutathione in activated sludges exposed to low ozone doses.

Ozonation is one of the most effective treatments for reducing the production of activated sludges in wastewater treatment plants. However, because microorganisms are present in the form of microcolonies, some bacteria may be exposed to sub-lethal ozone doses that could lead to adaptation and resistance to further exposition to oxidative treatment. This represents a major question as it may limit the effect of the treatment, especially when low ozone doses are applied. The critical ozone dosage, defined as the lowest specific transferred ozone concentration leading to a decrease in the maximum oxygen uptake rate was estimated to range between 0.9 and 13.6mg O(3)g(-1) COD(sludges), according to the sludges tested. The lowest ozone dosage leading to the decrease of GSH and GSHt concentrations could be estimated to be lower than 10mg O(3)g(-1) COD(sludges) for GSH, and close to 10mg O(3)g(-1) COD(sludges) for GSHt. After sludge exposure to low ozone doses, no higher amounts of glutathione were synthesized, suggesting that no development of resistance to ozonation occurred after sludge treatment with low ozone doses.

Coliform culturability in over- versus undersaturated drinking waters.

The culturability of Escherichia coli in undersaturated drinking water with respect to CaCO3 (corrosive water) or in oversaturated water (non-corrosive water) was tested in different reactors: glass flasks (batch, "non-reactive" wall); glass reactors (chemostat, "non-reactive" wall) versus a corroded cast iron Propella reactor (chemostat, "reactive" wall) and a 15-year-old distribution system pilot (chemostat, "reactive" wall with 1% corroded cast iron and 99% cement-lined cast iron). The E. coli in E. coli-spiked drinking water was not able to maintain its culturability and colonize the experimental systems. It appears from our results that the optimal pH for maintaining E. coli culturability was around 8.2 or higher. However, in reactors with a reactive wall (corroded cast iron), the decline in E. coli culturability was slower when the pH was adjusted to 7.9 or 7.7 (i.e. a reactor fed with corrosive water; pHpHs). We tentatively deduce that corrosion products coming from chemical reactions driven by corrosive waters on the pipe wall improve E. coli culturability.

Influence of lepidocrocite (gamma-FeOOH) on Escherichia coli cultivability in drinking water.

A washed suspension of the bacteria Escherichia coli, pre-grown on a complex culture medium, was stored in sterilized drinking water for 21 days at 25 degrees C in glass flasks in order to assess the effect of iron corrosion products on the persistenceof the bacteria in drinking water. Four conditions were tested: aerobic with 50 mM lepidocrocite (gamma-FeOOH, an insoluble iron corrosion product), anaerobic with 50 mM lepidocrocite, aerobic without lepidocrocite and anaerobic without lepidocrocite. The survival of E. coli was monitored by their cultivability and their membrane integrity (propidium iodide staining). When the samples were not supplemented with the iron oxide, the cultivability and cell integrity of the bacteria were dramatically altered: from the 10(7) initially added, only 10 CFU ml(-1) remained after 21 days; 90% of the cells exhibited membrane alteration after 2 weeks of storage. In contrast, bacteria with lepidocrocite preserved their cultivability and integrity over the 21 days of storage. In the presence of di-oxygen and without iron oxide, the alteration of cell cultivability was more pronounced than that in anaerobic conditions, suggesting that oxidative stress was part of the phenomenon. When the cells were pre-grown in a growth medium supplemented by a large excess of an easily available form of iron (ferric-citrate), the cells stored a higher amount of iron and persisted one week longer in the iron-free drinking water than cells pre-grown in the standard growth medium. Therefore, in an oligotrophic environment like drinking water, E. coli cells can find the ability to survive a long time through the presence of iron corrosion products. The necessity of controlling the corrosiveness of drinking water for sanitary reasons is therefore emphasized by this study.

Chlorination effect on the fluorescence of nucleic acid staining dyes.

An alternative to culture methods for the control of drinking water disinfection would use fluorescent dyes that could evidence the nucleic acid damages provoked by sodium hypochlorite treatment. The two dyes selected in this study, SYBR Green II RNA gel stain and TOTO-1 iodide, efficiently stain nucleic acids (DNA and RNA) and quite poorly the other biomolecules considered (Bovine serum albumin, palmitic acid and dextrane). After treatment of nucleic acid solutions with increasing amounts of sodium hypochlorite, a decrease of fluorescence intensity is observed for both DNA and RNA stained with either SYBR-II or TOTO-1. However, the two fluorochromes do not lead to the same results, which shows that the two dyes are not bound to nucleic acids in the same way. Contrary to TOTO-1, SYBR-II reveals to be sufficiently sensitive to indicate both DNA or RNA damages as soon as the latter are in contact with hypochlorite even at concentrations of HClO lower than 10 micromol/L. Moreover, SYBR-II offers the opportunity to make quantitative titration of chlorine treated DNA and therefore seems to be the appropriate candidate to control the efficiency of the hypochlorite disinfection process of drinking water samples.

Measurement of glutathione in activated sludges.

Thermal, electric, mechanical or oxidative stress seem a promising way to reduce the production of excess activated sludge during biological wastewater treatment. However, the adaptation and the resistance of the sludge microbial ecosystem to stress conditions is a major question as it may definitively limit the effect of some treatments. Defence mechanisms developed by aerobic organisms, in particular, in response to oxidative stress involve various antioxidant activities and compounds such as glutathione. An HPLC method was developed for measuring reduced and total glutathione (GSH and GSHt) in perchloric acid sludge extracts. The method was sensitive, highly specific and validated for linearity, precision and recovery. Considering the extraction yield and the oxidation of GSH during extract storage, the measured GSH concentration was estimated to represent 60% of the GSH content from activated sludges. GSHt ranged from 0.32 to 3.34micromolg(-1) volatile solids and the GSH/GSHt ratio ranged from 32% to 91%. Measurements performed on sludges stressed in precise conditions selected to reach a reduction of sludge production showed a decrease of GSH and GSHt concentrations with thermal, mechanical, electric and ozone stress.

Release of organic matter in a discontinuously chlorinated drinking water network.

The effects of discontinuous chlorination on the characteristics of the water in a pilot drinking water distribution network were investigated. The release or consumption of organic matter (as dissolved organic carbon, DOC) following chlorination and non-chlorination periods were estimated, as were changes in bacterial cell production. In each unchlorinated network 0.3 mg DOCl(-1) was consumed and the average cell production was approximately 1.3 x 10(5) cells ml(-1). In discontinously chlorinated networks (chlorine treatment: 3.3 mg Cl2l(-1), chlorine residual: 0.1 mg Cl2l(-1)) the DOC release (DOCout-DOCin) was between 0.1 and 0.2 mg Cl(-1). Biomass production (cells(out)-cells(in)) during this chlorination period was lower (approximately 2 x 10(4) cells ml(-1)). The delay before DOC was released in chlorinated networks appeared to be less than 24 h, which corresponds to one hydraulic residence time. Likewise, when chlorination was stopped, 24 h or less were required before an efficient DOC removal was resumed. When chlorination was prolonged the observed release of DOC was progressively reduced from 0.2 mg l(-1) to zero, thus after 6 weeks of continuous chlorination the DOCin was equivalent to the DOCout.

Characterization of extracellular polymeric substances in rotating biological contractors and activated sludge flocs.

Extracellular polymeric substances (EPS) from biofilms of a rotating biological contactor (RBC) system and from activated sludge flocs were extracted using ultrasound and a cation exchange resin. In both wastewater treatment systems, the EPS matrix was mainly constituted of proteins, humic-like substances and polysaccharides, although other components--DNA and uronic-like substances--were also present. The composition of the biofilm EPS was different in the succesive RBC wastewater treatment stages: protein had its highest concentration in the first RBC unit, while humic substances and polysaccharides in the third RBC. Significant differences between biofilm and sludge floc EPS were also reported. The amount of proteins was 3.5 times higher for RBC biofilms than for sludge flocs, and quantities of humic substances and polysaccharides extracted from biofilms were twice as high than in sludges. Moreover, biofilm exopolymers were two times more hydrophobic than those of sludge flocs. Implications of wastewater chemistry and microbial growth strategies into EPS composition and properties are suggested.

Hydrolysis of wastewater colloidal organic matter by extracellular enzymes extracted from activated sludge flocs.

Enzymatic activities associated with the exopolymeric substances (EPSs) extracted from activated sludges were tested for their ability to hydrolyse the organic colloidal fraction of wastewater. Bacteria extracted with EPS and concentrated by wastewater microfiltration were inhibited with NaN3 or KCN. The protein hydrolysis mainly resulted from the enzymatic activity of EPS, whereas the glycolytic activity was mainly present in the organic colloidal fraction of the wastewater.