influence of phosphate and disinfection on the composition of biofilms produced from drinking water,...

Influence of phosphate and disinfection on thecomposition of biofilms produced from drinkingwater, as measured by fluorescence in situhybridization

M. Batté, L. Mathieu, P. Laurent, and M. Prévost

Abstract: Biofilms were grown in annular reactors supplied with drinking water enriched with 235 µg C/L. Changes inthe biofilms with ageing, disinfection, and phosphate treatment were monitored using fluorescence in situ hybridization.EUB338, BET42a, GAM42a, and ALF1b probes were used to target most bacteria and the alpha (α), beta (β), andgamma (γ) subclasses of Proteobacteria, respectively. The stability of biofilm composition was checked after the onsetof colonization between T = 42 days and T = 113 days. From 56.0% to 75.9% of the cells detected through total directcounts with DAPI (4′-6-diamidino-2-phenylindole) were also detected with the EUB338 probe, which targets the 16S rRNAof most bacteria. Among these cells, 16.9%–24.7% were targeted with the BET42a probe, 1.8%–18.3% with the ALF1bprobe, and <2.5% with the GAM42a probe. Phosphate treatment induced a significant enhancement to the proportion ofγ-Proteobacteria (detected with the GAM42a probe), a group that contains many health-related bacteria. Disinfectionwith monochloramine for 1 month or chlorine for 3 days induced a reduction in the percentage of DAPI-stained cellsthat hybridized with the EUB338 probe (as expressed by percentages of EUB338 counts/DAPI) and with any of theALF1b, BET42a, and GAM42a probes. The percentage of cells detected by any of the three probes(ALF1b+BET42a+GAM42a) tended to decrease, and reached in total less than 30% of the EUB338-hybridized cells.Disinfection with chlorine for 7 days induced a reverse shift; an increase in the percentage of EUB338 counts targetedby any of these three probes was noted, which reached up to 87%. However, it should be noted that the global bacterialdensities (heterotrophic plate counts and total direct counts) tended to decrease over the duration of the experiment.Therefore, those bacteria that could be considered to resist 7 days of chlorination constituted a small part of the initialbiofilm community, up to the point at which the other bacterial groups were destroyed by chlorination. The results sug-gest that there were variations in the kinetics of inactivation by disinfectant, depending on the bacterial populations in-volved.

Key words: biofilm, phosphate, chlorine, monochloramine, FISH, drinking water.

753Résumé : Des biofilms ont été développés dans des réacteurs annulaires à partir d’eau potable enrichie en carbone àraison de 235 µg C/L. Un suivi par hybridation in situ avec des sondes oligonucléotidiques fluorescentes a été réaliséafin d’évaluer les changements dans ces biofilms en fonction de leur âge et pendant la désinfection ou un traitement àbase de phosphates. Les sondes utilisées ont été EUB338, BET42a, GAM42a et ALF1b, ciblant, respectivement, la plupartdes bactéries et les sous-classes alpha (α), beta (β) et gamma (γ) des Proteobactéries. Dans un premier temps, la stabi-lité de la composition des biofilms a été vérifiée entre les 42ème et 113ème jours à partir du début de la phase de co-lonisation. De 56,0 % à 75,9 % des bactéries dénombrées par un marquage au DAPI (4′-6-diamidino-2-phenylindole)ont également été détectées par la sonde EUB338 ciblant l’ARNr 16S de la plupart des bactéries. Parmi ces cellules,de 16,9 % – 24,7 % ont été ciblées par la sonde BET42a, de 1,8 % – 18,3 % par la sonde ALF1b et <2,5 % par lasonde GAM42a. Un traitement aux phosphates a induit une augmentation significative de la proportion des bactéries ap-partenant aux γ-Protéobactéries (détectées par la sonde GAM42a), un groupe contenant de nombreuses bactéries d’intérêtsanitaire. Un traitement de désinfection avec du chlore pendant 3 jours ou avec de la monochloramine pendant un mois aengendré une diminution de la proportion de cellules s’hybridant avec EUB338 et une diminution des cellules détectées

Can. J. Microbiol. 49: 741–753 (2003) doi: 10.1139/W03-094 © 2003 NRC Canada

741

Received 7 April 2003. Revision received 14 October 2003. Accepted 15 October 2003. Published on the NRC Research Press Website at http://cjm.nrc.ca on 22 January 2004.

M. Batté. NSERC Industrial Chair on Drinking Water, École Polytechnique de Montréal, C.P. 6079, Succ. Centre Ville, Montréal,QC H3C 3A7, Canada; and Service de Recherche en Environnement et Santé, Faculté de médecine, 9 avenue de la Forêt de Haye,B.P. 184, 54 505 Vandoeuvre-lès-Nancy, France.L. Mathieu. Service de Recherche en Environnement et Santé, Faculté de médecine, 9 avenue de la Forêt de Haye, B.P. 184,54 505 Vandoeuvre-lès-Nancy, France.P. Laurent1 and M. Prévost. NSERC Industrial Chair on Drinking Water, École Polytechnique de Montréal, C.P. 6079, Succ.Centre Ville, Montréal, QC H3C 3A7, Canada.

1Corresponding author (e-mail: [email protected]).

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avec une des sondes ALF1b, BET42a ou GAM42a. Au total, l’ensemble des bactéries détectées par l’une de ces 3 son-des représentaient moins de 30 % des cellules hybridées par EUB338. Au contraire, la chloration pendant 7 jours aconduit à une communauté bactérienne dont 87 % des bactéries détectées par EUB338 ont été également détectées parl’une des sondes (ALF1b+BET42a+GAM42a). Cependant, il faut remarquer que les densités globales de bactéries(comptes sur géloses et comptes directs totaux) ont eu tendance à diminuer fortement pendant l’expérience. Ceci per-met de considérer que les bactéries qui ont résisté à 7 jours de chloration constituaient une petite partie de la commu-nauté initiale du biofilm et qu’elles ont été détectées lorsque les autres groupes bactériens ont été éliminés par lachloration. Les résultats indiquent qu’il existe des variations dans les cinétiques d’inactivation par les désinfectants enfonction des différentes sous-populations étudiées.

Mots clés : biofilms, phosphate, chlore, monochloramine, FISH, eau potable.

[Traduit par la Rédaction] Batté et al.

Introduction

Maintaining the microbial quality of drinking water fromthe treatment plant to the consumer’s tap is, in many cases, amajor challenge. The control of bacterial growth in distribu-tion systems is usually achieved by limiting dissolved nutri-ents or by adding disinfectants, such as chlorine ormonochloramine, to the water (LeChevallier et al. 1988,1990, 1991; Mathieu et al. 1992; Prévost et al. 1998; van derKooij 1992; Camper et al. 1997). The chemical and aestheticquality of drinking water may also be controlled by limitingcorrosion through the addition of phosphate (Abernathy andCamper 1997, 1998; Jones 1996; Lyons et al. 1995; Lytle etal. 1996; Rompré et al. 2000; Vik et al. 1996). However,phosphate addition can influence the growth of bacteria,given (i) that inorganic phosphate is an essential nutrient forbacteria and (ii) the low concentration of phosphate (gener-ally less than 20 µg P/L) in some drinking water sources(Miettinen et al. 1997; Sathasivan et al. 1997; Lehtola et al.2002).

Chlorine and monochloramine are the most frequentlyused disinfectants. Chlorine reactivity is greater than that ofmonochloramine, leading to correspondingly greater effi-ciency (Jacangelo and Olivieri 1985). However,monochloramine is sometimes preferred because it is fre-quently, but not systematically, reported as a more effectiveagent for biofilm disinfection (Berman et al. 1988; Mathieuet al. 1992; Chen et al. 1993; Griebe et al. 1994; Kirmeyer etal. 1993; Camper et al. 1997). Most studies have comparedchlorine and monochloramine efficiency in terms of globalbacterial density decrease (Koudjonou et al. 1998). A fewstudies (Ridgway and Olson 1982; Mir et al. 1997; Nortonand LeChevallier 2000) have attempted to measure the quali-tative effect of these disinfectants on bacterial communitypopulations, even though it is important to identify the partsof a population that resist disinfection.

Indeed, most of the studies on bacterial growth in drink-ing water distribution systems (DWDS) that have been car-ried out on either fixed or suspended bacterial populationshave involved the use of global methods, such asculturability or total direct counts (Appenzeller et al. 2001;Batté et al. 2003; Crespi and Ferra 1997; Mathieu et al.1992; Miettinen et al. 1997; Norton and LeChevallier 2000).However, the bacteriological quality of drinking water is de-pendent on the species encountered. For example, it may bepreferable to try to limit opportunistic pathogens, such as

Salmonella spp., rather than environmental bacteria that arenot related to public health issues. Only a few studies basedon cultivation techniques have looked at the composition ofsuspended bacterial communities in DWDS, and even fewerhave studied the composition of DWDS biofilm, i.e., thebacterial community fixed on the surfaces of pipes in aDWDS (Briganti and Wacker 1995; Rompré et al. 1998;Norton and LeChevallier 2000). Nevertheless, since mostbacteria are not culturable in drinking water (Kalmbach1998), only about 1% of the total bacterial population maybe detected in this way. Furthermore, agar culturing intro-duces a selection bias in favour of the α- and γ-Proteobacteria phylogenetic groups to the detriment of β-Proteobacteria (Wagner et al. 1993), even though these lat-ter bacteria form the major part of the bacterial population indrinking water (Kalmbach et al. 1997).

The main objective of this study was to monitor the ef-fects of various drinking water treatments on the composi-tion of the biofilm community, using a culture-independentmethod. The underlying hypothesis was that depending onthe drinking water treatment applied, small but significantvariations in biofilm community composition may occur andthat this may have a significant impact on water quality anddegree of compliance with regulations. Biofilms were grownin the laboratory from drinking water, and the impacts onbiofilm composition of three of the most common chemicaltreatments used in DWDS were then assessed: phosphate ad-dition, chlorination, and chloramination.

The fluorescence in situ hybridization (FISH) method wasused to detect shifts in the composition of the bacterialbiofilm community of the drinking water. rRNA bio-molecules are numerous in bacteria (Fegatella et al. 1998),and their nucleotidic sequence provides information on theidentity of the bacteria present, in terms of class, family, andspecies. The FISH method uses probes complementary to re-gions of rRNA specific to the subpopulation under study(Amann et al. 1990). The method does not involve a cultiva-tion step, thus avoiding the selection bias (Wagner et al.1993).

Materials and methods

Experimental setupAnnular reactor experimental systems (Characklis and

Marshall 1990) were used to allow the colonization of sur-faces with biofilm bacteria. The reactors were supplied with

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carbon-enriched dechlorinated drinking water from the Cityof Montréal DWDS (Quebec, Canada). During one experi-ment, at least two reactors (and up to four) were put into op-eration to maintain at least one control reactor. The reactorswere run in parallel and supplied with the same water. Thetreatments (such as chlorine, monochloramine, or phos-phate), when applied, were directly plugged into the reactorsat the end of a colonization step (Fig. 1).

During the colonization period, sampling was performedevery 2 weeks to monitor changes in biofilm composition.The bacterial densities of the biofilm were evaluated usingFISH, cultivation (heterotrophic plate count, HPC), and totaldirect count (TDC) methods.

To assess the impact of treatments on biofilm composi-tion, five experiments were performed (Table 1) and threetreatments were tested on the biofilms: phosphate addition(experiment A), chlorination (experiments B and C), andchloramination (experiments D and E). Additionally, the ef-fect of the age of the biofilm during disinfection was evalu-ated for the chlorine treatment (experiments B and C), andthe impact of disinfectant dosage on biofilm compositionwas evaluated for monochloramine treatment (experiments Dand E). Experiments were performed during the autumn pe-riod (experiment B), spring period (experiment A), andspring to summer period (experiments C, D, and E).

Annular reactors are bench-scale systems, which to alarge degree, reproduce conditions observed in a drinkingwater system and facilitate adjustment of operational param-eters (pH, temperature, disinfectant, etc.) (Characklis andMarshall 1990). The annular reactors used in this study werecomposed of an internal drum and an external cylinder madeof polycarbonate, as described in Batté et al. (2003). The in-ternal surface of the external cylinder was equipped with 12polycarbonate slides for biofilm sampling. During the exper-iments, the mean hydraulic residence time was set to 2 h bycontrolling the inlet flow rate. The rotational speed was setto 40 r/min to induce a shear stress similar to that of waterflowing at 0.6 m/s (ref.) through a pipe 6 in. (15.24 cm) indiameter (Davies 1994).

Water supplyThe drinking water used contained a very low concentra-

tion of biodegradable organic carbon (200 µg BDOC/L).Such a low value is mentioned by several authors (Laurent etal. 1999) as a threshold defining a biologically stable water.Therefore, it seemed necessary to ensure an additionalsource of carbon for the microorganisms to obtain a popula-tion response to the parameters tested. Thus, the annular re-actors were slightly supplied with carbon-enriched tap water.The tap water taken from the City of Montréal DWDS isproduced from St. Lawrence River water. The average char-acteristics of the City of Montréal tap water are presented inTable 2. This water was stored for 20 h in a 200-L tank toallow the chlorine residual to decay to less than the measure-ment detection limit (0.01 mg Cl2/L) and to achieve a con-stant temperature (22 ± 2 °C) ahead of the annular reactorinlet.

To enhance bacterial colonization in the reactors, we en-riched the dechlorinated tap water with a sterile solutioncontaining a mixture of bioavailable organic componentsthat are not reactive to chlorine (Camper 1995). This stock

solution contained a total carbon concentration of 20 mg C/L,obtained with ethyl alcohol at 94%, propionaldehyde,parahydroxybenzoic acid, benzoic acid, and acetate, addedfollowing an equimolar carbon concentration ratio. The solu-tion was prepared with ultrapure water (MilliQ-UV plus,Millipore), autoclaved at 121 °C for 20 min, and renewedweekly. The reactor was supplied at 6.5 mL/min through amixing bottle containing a continuous-flow dilution suppliedwith 32.3 mL/min of dechlorinated tap water and0.38 mL/min of carbon stock solution. Thus, thedechlorinated tap water sent to the inlet of the reactors con-tained an additional 235 µg C/L of bioavailable carbon.

Phosphate solution and measurementsThe phosphate solution was prepared in autoclaved

ultrapure water by diluting a filter-sterilized commercialproduct (AquaMag (Na56H3P57O147), Kjell Inc., Canada)containing two-thirds polyphosphate and one-thirdorthophosphate (w/w of phosphorus). The solution contain-ing 11 mg P/L was stored in a brown glass bottle and re-newed weekly. Neither storage nor dilution affected the two-thirds polyphosphate ratio in this solution, which was testedwith a phosphate solution through orthophosphate andpolyphosphate analysis over a period of more than 2 weeks(data not shown). A continuous-flow dilution was made updirectly in the annular reactor, fed with a 6.5 mL/min ofdechlorinated, carbon-enriched water and 0.3 mL/min ofphosphate stock solution corresponding to an additional con-centration of approximately 0.5 mg P/L.

The concentration of phosphate in the liquid phase wasobtained by colorimetry, and measurements were performedon the day of sampling. Polyphosphates were hydrolysed toorthophosphate by the persulphate digestion method (Ameri-

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Fig. 1. Schematic diagram of one annular reactor and the set upperformed during the experiments to supply it.



© 2003 NRC Canada


can Public Health Association 1998) prior to analysis.Orthophosphate concentrations were determined by theascorbic acid method, with a detection limit of approxi-mately 50 µg P/L with a light path of 1.9 cm (AmericanPublic Health Association 1998). Absorbency was measuredat 880 nm on a spectrophotometer (Milton Roy, Spectronic1001plus, New York, U.S.A.). All results were expressed inmilligrams of P-PO4 per litre.

Monochloramine and chlorine solutions andmeasurements

To test monochloramine disinfection at two concentra-tions, we prepared two monochloramine stock solutions on-line by mixing together one chlorine solution and one am-monium chloride solution (45 mg Cl2/L with 33.75 mgNH4Cl/L, and 150 mg Cl2/L with 113.75 mg NH4Cl/L re-spectively). The mass ratio was 5:1 and the contact time be-fore injection into the reactors was 30 min.

The chlorine solutions were prepared in ultrapure water,from a commercial solution of bleach. Monochloramine for-mation was regularly controlled through chlorine residualmeasurements, and the mixture was injected at 0.3 mL/mindirectly into the reactors corresponding to an injection of 0.6(Experiment D) or 1.9 mg NH2Cl/L (Experiment E). Chlo-rine and monochloramine residuals were measured at the re-actor outlets by using the DPD colorimetric method(American Public Health Association 1998). The detectionlimits of chlorine and monochloramine are approximately0.01 mg Cl2/L. Absorbance was measured at 515 nm on aspectrophotometer.

Biofilm sampling and conditioning prior to analysisThe polycarbonate sampling slides colonized by biofilm

were removed from the reactors and placed in themultivessel slide holder, described in Batté et al. (2003). Foreach slide, ten identical vessels delimiting identical biofilmsurfaces (1.2 cm2) allowed simultaneous measurements to beperformed.

Three methods were used to characterize the biofilm bac-teria: cultivation (HPCs), TDCs, and FISH. The biofilm wassuspended (in 3 mL of 0.2-µm-filtered tap water) bysonication (2 W, 2 min) (ultrasonic processor equipped witha probe 3-mm in diameter, Cole Palmer, Anjou, Que., Can-ada). Sonication parameters (power and time of exposure)had previously been optimized to reach the maximal TDCsand HPCs (data not shown).

Heterotrophic plate countsFor enumeration by HPCs (CFU/cm2), bacteria were col-

lected by filtration on 0.45-µm porosity, 45-mm diameter,acetated cellulose filters. The filters were placed on R2Aagar plates (ref. 1826-17-1, Difco Laboratories, Detroit,Mich., U.S.A.) for cultivation and incubated for 7 days at 22 ±2 °C. Duplicate counts were performed through single-platedilution for each measurement.

Total direct countsThe total numbers of bacteria or TDCs (cells/cm2) were

determined using DAPI (4′,6-diamidino-2-phenyl-indol)staining, according to the protocol of Saby et al. (1997), ex-cept that the reagent Triton X-100 was not used. Duplicatesamples were taken from two different vessels of themultivessel system, and counts were performed with anepifluorescence microscope (BX60; Olympus) equippedwith a × 100 objective lens and a set of DAPI-specific filters(ref. U-MWU, Olympus: Dichroic Mirror 400 nm, BandPass

ExperimentAge atT = 0* (days) Treatment agent Dosage (mg/L)

Treatmentduration (weeks)

A 63 Phosphate 0.5 P-PO4 6B 49 Chlorine 0.75 Cl2 1C 113 Chlorine 1 Cl2 1D 104 Monochloramine 0.6 NH2Cl 4E 104 Monochloramine 1.9 NH2Cl 4

*Biofilm age at the beginning of treatment.

Table 1. Treatments applied on biofilms.

Parameter (unit) N* Mean (min.–max.)

Turbidity (NTU) 334 0.23 (0.08–1.26)Alkalinity (mg CaCO3-eq/L) 53 83 (76–87)Total hardness (mg CaCO3-eq/L) 53 117 (109–125)Oxygen demand (mg KMnO4/L) 47 1.0 (0.6–1.6)Dissolved organic carbon (mg C/L) 232 2.42 (2.00–3.51)pH 331 7.84 (7.58–8.21)Total iron (mg Fe/L) 24 0.018 (0.005–0.046)Chloride (mg/L) 53 21 (18–22)Conductivity (µS/cm) 365 290 (270–310)

Note: NTU, nephelometric turbidity unit.*Samples taken throughout the year 2000.

Table 2. Physicochemical characteristics of the tap water (before enrichment) used to supplythe annular reactors (Montréal, Que., Canada).



330–385 nm, and Barrier 420 nm). Ten to sixty microscopicfields were counted, depending on cell concentration, to ar-rive at a count of at least 100 cells for one sample.

FISH procedureBy targeting rRNAs with oligonucleotidic probes labelled

with a fluorochrome, bacteria can be detected even if theyare not culturable. Usually, cells are fixed to limit the bio-chemical reactions occurring in them that lead to cellularmultiplication. In many cases, the fixed cells are permeableto short oligonucleotidic probes (Amann et al. 1990;Giovannoni et al. 1988). However, preventive permeabilisationcan be carried out, generally with ethanol, methanol, and en-zymes (DeLong 1993; Bidnenko et al. 1998). It is importantthat the conditions be well defined to permit good hybridiza-tion with the target, while at the same time avoiding match-ing errors. These conditions are optimized by addingproducts, such as formamide, NaCl, SDS, and Tris–HCl, tothe hybridization solution. Formamide permits thelinearization of the RNA strand and the formation of doublestrands between the probe and the target region of the rRNA(Moter and Göbel 2000). NaCl reduces the electrostatic re-pulsions between target and probe (Swiger and Tucker1996), SDS limits the nonspecific fixation of the probes andthus limits the background, and Tris–HCl is added to controlthe pH of the solution.

Here, the universal bacterial probe EUB338 (5′-GCT-GCCTCCCGTAGGAGT-3′) was used to estimate the densityof cells which can be detected by FISH (Amann et al. 1990).To assess the composition of biofilm populations, we usedthree other FISH probes (Manz et al. 1992): ALF1b (5′-CGTTCGYTCTGAGCCAG-3′) specific to α-Proteobacteria,BET42a (5′-GCCTTCCCACTTCGTTT-3′) specific to β-Proteobacteria, and GAM42a (5′-GCCTTCCCACATCG-TTT-3′) specific to γ-Proteobacteria.

All probes were labelled with CY3 dye (Interactiva, Ulm,Germany). Since probes BET42a and GAM42a differed onlyin one base pair, hybridization was performed with an unla-belled competitor (i.e., unlabelled GAM42a acts as a com-petitor for BET42a labelled with CY3, and unlabelledBET42a acts as a competitor for CY3-labelled GAM42a), asdescribed by Manz et al. (1992); an unlabelled BET42aprobe (5 ng/µL) was added for hybridization with theGAM42a probe, and an unlabelled GAM42a probe(5 ng/µL) was added for hybridization with the BET42aprobe.

The FISH protocol used was the one described byKalmbach (1998). Biofilm cells detached from the slides bysonication were collected by filtration onto a 25-mm diame-ter, 0.2-µm porosity, white polycarbonate membrane(ref. GTTP0025, Millipore). To fix the sample, 3 mL ofparaformaldehyde at 4% (w/w) prepared from PBS (pH 7.2)were placed on the filter for 30 min at 4 °C. The sample wasrinsed with PBS, air-dried, and stored at –20 °C, up to thestart of the hybridization procedure. Prior to hybridization,the filter was dehydrated with 200 µL of ethanol (50%, 80%,and 94% for 3 min each) to permeabilise the cellular mem-brane. Hybridization was performed for 2 h at 46 °C in a hu-mid chamber placed in an oven, as suggested by Kalmbach(1998). Fifty microlitres of hybridization solution were ap-plied to the filters. The hybridization solution was composed

of 5 ng/µL of probe (and also 5 ng/µL of unlabelled probeused as a competitor when necessary) suspended in 0.9 mol/LNaCl, 20 mmol/L Tris–HCl (pH 7.2), 0.01% SDS, and 35%formamide (for EUB338 probe hybridization) or 40% for theother probes hybridizations. The washing was performed for15 min at 46 °C with 50 mL of solution. This solution con-tained 20 mmol/L Tris–HCl (pH 7.2) and 0.01% SDS, and88 mmol/L or 62.4 mmol/L NaCl (corresponding to the hy-bridization performed with 35% or 40% formamide, respec-tively). The filter was then stained with DAPI (1 mL of a0.05 µg/mL DAPI solution) for 10 min, rinsed withautoclaved ultrapure water (ref. MilliQ-UV plus, Millipore),air-dried, and then mounted on a slide with an antifading so-lution (Citifluor AF1, Citifluor Ltd., London, U.K.). Cellswere visualized by epifluorescence microscopy (BX60,Olympus) with green light for CY3 staining (ref. U-MWG,Olympus: Dichroic Mirror 570 nm, BandPass 510–550 nmand Barrier 590 nm) or UV light for DAPI staining, as de-scribed above. Comparison of the DAPI counts performedon slides for TDCs (DAPI staining alone) and for FISH(DAPI staining and oligonucleotidic probe hybridization onthe same slide) allowed us to establish that less than 20% ofcells were lost during the hybridization procedure.

Systematic checks of the quality of the hybridization pro-cedures were performed through double counting (FISH andDAPI labelling were observed for each type of bacteria).Moreover, the probes (already validated in Manz et al. 1992)were tested using known species that should (or should not)be hybridized with the probe tested (data not shown).

Statistical estimationThe probability, p, associated with a student test (t test)

was calculated with the software “Excel” with a bilateral ttest, which consists of a comparison of two sets of data bypairs and with equal variance. The p value gave the probabil-ity that the difference was due only to random chance. Thetwo sets of data are statistically different when p < 0.05.

Results

Composition of tested biofilmsThe environmental conditions under which the experi-

ments were performed were similar during the colonizationsteps for all the experiments. To evaluate the similarities inbiofilm composition during Experiments B, C, D, and E, weevaluated the proportions of cells belonging to the α, β, or γsubclasses of Proteobacteria during the colonization step(from 42 to 56 days old for Experiment B, 61 to 113 daysold for Experiment C, and 34 to 104 days old for Experi-ments D and E) (Table 3). The stability of the compositionof the suspended population was evaluated through FISHmeasurements during Experiments A, D, and E to be surethat the evolution of the biofilm studied by FISH was a con-sequence of the applied treatments and not of a change ofthe community in the inlet water (data not shown).

The age of the biofilms did not induce significant changesin biofilm composition. On average (all experiments), lessthan 3% of TDCs (2.4% ± 1.3%) were culturable on R2Aagar, whereas 58%–76% of TDCs were detected with theEUB338 probe, meaning that the FISH method allowed de-tection of at least 24 times more cells than cultivation. Few

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of the biofilm bacteria detected with the EUB338 probe(<2.5%) in any of the experiments were γ-Proteobacteria(detected with the GAM42a probe), and 16.9%–24.7% ofthem were β-Proteobacteria (detected with the BET42aprobe). Even if there are no clearly identified causes for thisphenomenon, it should be noted that the populations of bac-teria belonging to the α-Proteobacteria group represented1.8% of the global population group during Experiment B,but reached 12.6%–18.3% in Experiments C, D, and E.

Effect of phosphate addition on biofilm populationcomposition

Duplicate reactors containing 63-day-old biofilms weretreated with 0.5 mg P/L over a 6-week period and sampledafter 2, 4, and 6 weeks of treatment (Experiment A, Ta-ble 1). Two other reactors with biofilms of the same agewere monitored as controls over the same period (Fig. 2).Throughout the 6 weeks of phosphate treatment, the TDCsapproached 2.5 ± 107 cells/cm2, and no differences werenoted between the controls and the phosphate-treatedbiofilms following the addition of the phosphate. Culturablebacteria, evaluated as HPCs, accounted for less than 3% of

the bacteria in the biofilms, irrespective of phosphate treat-ment.

The addition of phosphate neither enhanced nor reducedglobal population detection by FISH with the EUB338probe; after 6 weeks of treatment (t test, p = 0.81), 60% ofTDC cells were detected by the EUB338 probe in both reactors(Fig. 2). The monitored subpopulations were similar prior toand following the start of phosphate addition (p > 0.05). Theexception was the proportion of the γ-Proteobacteriasubpopulation, which was significantly enhanced after6 weeks of phosphate treatment (t test, p = 0.0006). How-ever, the biofilm was still dominated by other bacteria (β-, α-Proteobacteria, and unidentified bacteria), which representmore than 95% of the bacteria targeted by the EUB338probe.

Effect of chlorine on biofilm population compositionThe experiments to determine chlorination effect were

performed on biofilms of various ages. Chlorination wasperformed continuously by dosing 0.75 mg Cl2/L and1 mg Cl2/L on a 49-day-old and a 113-day-old biofilm, re-spectively. Because of the small proportion of the population

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Experiment

B C D E

Period of sampling (age of biofilm) (days) 42–56 61–113 34–104 34–104N samples 5 5 6 6TDC (cells/cm2) 5.3×106 (±1.8×106) 2.3×107 (±1.2×107) 1.7×107 (±1.1×107) 1.5×107 (±1.0×107)HPC (CFU/cm2) ND 3.6×105 (±1.3×105) 4.4×105 (±4.0×105) 3.2×105 (±4.0×105)In situ hybridization*

%EUB338/TDC 75.9 (±4.2) 64.8 (±6.1) 56.0 (±6.9) 59.6 (±5.8)%ALF1b/TDC 1.8 (± 1.2) 12.6 (± 4.5) 18.3 (±6.1) 13.7 (± 8.3)%BET42a/TDC 21.6 (±1.7) 24.7 (±3.2) 16.9 (±6.5) 20.5 (±4.9)%GAM42a/TDC 0.4 (±0.1) 1.1 (±0.2) 2.4 (±1.2) 1.2 (±0.6)

Note: Values are the means of all samples measurements (± standard deviation). TDC, total direct count; HPC, heterotrophic plate count; ND, no data.*Expressed as percentages of TDC.

Table 3. Cellular abundance in biofilms measured with TDC and HPC and biofilm composition estimated by in situ hybridizationmeasurements during colonization).

Fig. 2. Effect of phosphate treatment on composition and densities of biofilms grown on polycarbonate slides. Biofilms exposed tophosphate (n = 2) (black). Controls (n = 2) (white). Means of three sampling campaigns over the 6 weeks of phosphate treatment.Bars indicate standard deviation. (A) Effect of phosphate measured by fluorescence in situ hybridization, expressed as percentage ofprobe hybridized cells/TDC. For GAM42a, the difference between control and phosphate-treated biofilms is statistically significant(p < 0.01). (B) Effect of phosphate measured by total direct count (TDC) and heterotrophic plate count (HPC).



labelled by the various probes in terms of TDC, the percent-age contributions are expressed relative to the number ofcells hybridized with the EUB338 probe by considering thisnumber as the maximum that could be achieved by a sub-bacterial group (Table 4).

During Experiment B (0.75 mg Cl2/L applied to a 49-day-oldbiofilm), TDC and EUB338 counts were dramatically re-duced by chlorination in the biofilm (Table 4). After 7 daysof disinfection, TDCs had dropped by more than 2 log.Since only 0.9% of these TDC cells could be detected withthe EUB338 probe, it was not possible to determine thecomposition of the biofilm after 7 days of chlorination (toofew bacteria to count). The very small number of bacteriadetected by FISH may be indicative of the viable population,in a similar way to the very low numbers of bacteria de-tected by HPCs.

In Experiment C (1 mg Cl2/L applied to a 113-day-oldbiofilm), chlorination for 7 days led to a significant decreasein the percentage of DAPI-stained cells detected by FISH(% EUB338/DAPI). However, this process was not as fast asdescribed previously in Experiment B, whereas TDC countshad been decreasing by the same rate (around 2 log lost per7 days in Experiments B and C), the percentage forEUB338/DAPI decreased by a factor of 85 during Experi-ment B versus 4.5 during Experiment C. It is unclear why aslightly higher dosage of chlorine would favourably affectthe ability of the probe to label bacterial rRNA, and is rathersuggestive of an ageing effect (the more developed matrixsurrounding bacteria in an older biofilm may induce a stron-ger limitation on chlorine efficiency).

The composition of older and younger biofilm populationscan be compared after 3 days of chlorination. After 3 daysof chlorination of the younger biofilm (Experiment B), itwas only possible to hybridize 4.6% of the TDC cells withthe EUB338 probe, and among the bacteria labelled with theEUB338 probe, 15.6% were identified as belonging to the α-,β-, or γ-Proteobacteria groups (versus 38.2% before chlori-nation), whereas 84.4% of the EUB338-detected bacteriacould not be identified successfully with those probes. After3 days of chlorination, even with a higher dosage, the olderbiofilm in Experiment C contained a higher proportion ofEUB338-detected cells (23.3%).

Interestingly, although the age of the biofilms and the pro-portions of cells detected by the EUB338 probe were differ-ent in the two experiments (B and C), there were similaritiesin the compositions of the biofilms after 3 days of chlorina-tion (Table 4). The 3-day chlorinated biofilms presented adecrease in TDC counts, EUB338 counts, and EUB338 cellstargeted by the ALF1b, BET42a, or GAM42a probes. Amongthe bacteria detected with the EUB338 probe, 15.6%–21.1%were labelled with the ALF1b, BET42a, or GAM42 probeafter 3 days of chlorination, whereas these proportions were38.2% and 53.4%, respectively, before chlorination. Thismeans that the proportion of cells identified as belonging tothe (α + β + γ)-Proteobacteria group decreased by a factorof 2.5 as a result of 3 days of chlorination, whatever the ageof the biofilm.

A different population pattern was observed after 1 weekof disinfection applied to the older biofilm (Experiment C).In this case, the cells labelled with ALF1b, BET42a, orGAM42a probes represented 87% of the cells labelled with

© 2003 NRC Canada

Batté et al. 747

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the EUB338 probe. It should also be noted that the propor-tion of γ-Proteobacteria in the resisting community seems tohave increased during Experiment C. This result needs to beconfirmed by specific additional experiments before any dis-cussion on its implications for health and water quality canbe considered.

Effect of monochloramine on biofilm compositionChloramination (Experiments D and E), applied continuously

on 104-day-old biofilms at rates of 0.6 and 1.9 mg NH2Cl/Lrespectively, generated gradual decreases in both the TDCand EUB338 counts (Tables 5A and 5B), but at a muchslower rate than chlorination. Regardless of the chloraminedosage applied, the TDC decreased progressively by a factorof 3 (Experiment D) to 5 (Experiment E) over a 1-month pe-riod, whereas EUB338 counts progressively declined from58.0% to 64.7% to 11.9% (Experiment D) and to 7.2% (Ex-periment E) of TDCs. The culturable cells (HPCs) weremuch more sensitive to chloramine dosage, as shown by thedrastic decrease in HPC of 2–5 log, 1 week after the onset ofchloramination at 0.6 or 1.9 mg NH2Cl/L, respectively.

Among the bacteria detected with the FISH probes, theBET42a-labelled proportion dropped drastically followingthe onset of chloramination (Tables 5A and 5B). This dropwas six times greater when 1.9 mg NH2Cl/L was applied.The proportion of BET42a-detected cells reached 3.3% after7 days of 1.9 mg NH2Cl/L chloramination, and 18.4% ofEUB338-detected cells when 0.6 mg NH2Cl/L was applied.After 14–28 days of treatment, the proportion of β-Proteobacteria still decreased with 0.6 mg NH2Cl/L applied,reaching 2.9% after 28 days, while it increased to reach

20.9% in biofilms treated for 28 days with 1.9 mg NH2Cl/L.Unlike the BET42a-detected cells, the proportion of ALF1b-detected cells increased during the first week after the onsetof disinfection, but then also diminished to proportions <5–6%.The proportion of γ-Proteobacteria increased from 1.2–2.6% to 6.1–7.9% after 2 weeks of chloramination, but alsodiminished after 4 weeks.

The impact of chloramination on the biofilm included(i) gradual decreases in TDCs and in EUB338-detected cells,(ii) drastic dosage-sensitive HPC decreases, and (iii) majordecreases in labelling by FISH probes when general sites(EUB338) or specific sites (ALF1b, BET42a, and GAM42a)were targeted. As a consequence of the changes in the vari-ous bacterial groups, most of the diminution in EUB338counts was noted after 15 days of monochloramination.

Discussion

Evolution of biofilm composition with ageingThe experiments were performed to evaluate the effect of

treatments (phosphate, chlorine, monochloramine) onbiofilm population. Batté et al. (2003) have noted thatbiofilm age may influence the matrix composition ofbiofilm, i.e., the exopolymer components synthesized bybacteria to surround them in the process leading to biofilmformation. Therefore, our experiments regarding the effectsof phosphate treatment, chlorination, or chloramination wereperformed on biofilms of various ages to arrive, as much aspossible, at an exhaustive study. Biofilms were allowed tosettle for 49–113 days before any treatment was applied.The suspended population of the carbon-enriched drinking

© 2003 NRC Canada


Before treatment

N days of NH2Cl treatment

7 14 28

(A) Application of 0.6 mg NH2Cl/LTDC (×107cells/cm2) 2.7 (±0.01) 2.3 (±0.4) 1.8 (±0.04) 1.0 (±0.03)HPC (CFU/cm2) 1.2×106 (±0.5×106) 1.1×104 (±0.9×104) 1.1×102 (±0.1×102) 6.1 (±0.00001)FISH

%EUB338/TDC* 58.0 (±7.0) 48.9 (±11.2) 22.7 (±1.9) 11.9 (±6.6)%ALF1b/EUB338 18.3 (±2.1) 22.3 (±11.8) 15.6 (±0.2) 5.9 (±0.4)%BET42a/EUB338 38.4 (±4.4) 18.4 (±4.3) 9.3 (±12) 2.9 (±0.2)%GAM42a/EUB338 2.6 (±1.0) 1.7 (±1.8) 6.1 (±1.9) 0.3 (±0.2)

Total/EUB338 59.3 42.4 31 9.1

(B) Application of 1.9 mg NH2Cl/LTDC (×107cells/cm2) 3.0 (±0.1) 1.4 (±0.1) 1.5 (±0.3) 0.63 (±0.13)HPC (CFU/cm2) 1.1×106 (±0.01×106) 4.9×101 (±0.9×101) 2.4 (±1.7) 5.5 (±0.9)FISH

%EUB338/TDC* 64.7 (±8.9) 42.9 (±14.3) 9.7 (±1.0) 7.2 (±0.03)%ALF1b/EUB338 14.1 (±2.2) 28.2 (±1.4) 9.3 (±7.4) 5.0 (±3.8)%BET42a/EUB338 30.9 (±2.5) 3.3 (±0.2) 16.5 (±15) 20.9 (±7.7)%GAM42a/EUB338 1.2 (±0.7) 1.9 (±1.42) 7.9 (±0.9) 2.3 (±1.5)

Total/EUB338 46.2 33.4 33.7 28.2

Note: Data are the means of all samples measurements (± standard deviation). Age of biofilms at T = 0: 104 days. TDC, total direct count; HPC,heterotrophic plate count; FISH, fluorescence in situ hybridization.

*Expressed as percentages of TDC.

Table 5. Evolution of biofilm abundances (TDC and HPC) and composition (FISH) during monochloramination.



water biofilm was allowed to colonize reactor surfaces toform biofilm for at least 35 days prior to treatment. Phos-phate (0.5 mg P/L), chlorine (0.75 and 1 mg Cl2/L), andmonochloramine (0.6 and 1.9 mg NH2Cl/L) were then ap-plied for 1–6 weeks, depending on the experiment.

During Experiments C (chlorine treatment at 1 mg/L), D(monochloramine treatment at 0.6 mg/L), and E(monochloramine treatment at 1.9 mg/L), prior to the appli-cation of any treatment, it was noted that the composition ofbiofilm populations developed in untreated drinking waterdid not change much, at least not within the time frame ofour experiments (<113 days). The cells that were not de-tected by FISH might be Planctomycetes (Daims et al. 1999)or cells that do not contain enough rRNA to be observed bymicroscopy (Rompré et al. 2002). During these experiments,the β-Proteobacteria constituted the largest labelled fractionof TDCs, whereas γ-Proteobacteria were in a minority, afinding that is in good agreement with results reported byother authors (Kalmbach 1998; Kalmbach et al. 1997) forother types of drinking water. However, some differenceswere noted in the proportions of bacterial groups, as illus-trated by the contribution of α-Proteobacteria, which varieddepending on the experiment, even though the same experi-mental protocol and the same tap water were used for all theexperiments. Seasonal variations in this treated water mighthave resulted in the observed shifts in bacterial community,since Experiment B was the only one performed in the au-tumn; the others were performed from winter to summer.Kalmbach et al. (1997) noted that α-Proteobacteria may ormay not form a major part of communities, ranging from<5% of TDCs in biofilm grown in groundwater to 40% ofTDCs in biofilm grown in drinking water from a DWDSsupplied with treated groundwater (aerated, subjected tosand filtration, and not chlorinated).

Effect of phosphates on biofilm compositionPhosphate addition is a common treatment applied to con-

trol corrosion in DWDS (Rompré et al. 2000; Appenzeller etal. 2001; McNeill and Edwards 2002). Since phosphatesconstitute nutrients for the survival and growth of bacteria,as do carbon and nitrogen, it has been suggested that theiruse may amplify the regrowth of bacteria in a distributionsystem. Some studies performed through batch experimentsusing very particular drinking water have shown that the ad-dition of phosphates may influence drinking water quality,leading to increased bacterial culturability (Miettinen et al.1997; Sathasivan and Ohgaki 1999) and sometimes to bacte-rial growth (Sathasivan et al. 1997; Lehtola et al. 2002).However, in most types of drinking water in which carbon isthe limiting nutrient for bacterial growth, the addition ofphosphates does not induce relevant regrowth phenomena(Lyons et al. 1995; Cohen et al. 1999; Appenzeller et al.2001; Batté et al. 2003). Data presented here are in agree-ment with these latter results, as the bacterial concentrationsmeasured either through TDC or HPC did not increase sig-nificantly following phosphate addition (Fig. 2).

Through the use of FISH probes, the study presented herewas aimed at pointing out the effects of phosphate on thebacterial composition of biofilm. During phosphate treat-ment, the proportion of γ-Proteobacteria is significantly en-hanced. Since this group constitutes a minor part of the total

community, the detection of this increase in bacterial counts maynot be possible when global measurement techniques (HPC orTDC) are used. The γ-Proteobacteria subclass contains severalfrank or opportunistic pathogens, such as Salmonella spp.,Escherichia spp., and Legionnella spp., and also indicatorbacteria, such as total or fecal coliforms.

It should be remembered that some bacteria are not capa-ble of growing when the usual plating methods are used, andtherefore may only be detected through methods such asFISH. These microorganisms are described as viable butnonculturable (VBNC) (Colwell and Grimes 2000). In thecase of which pathogenic bacteria would still be pathogenswhen VBNC (Baudart et al. 2002), our results might point toa real health-related effect of phosphate treatment. The out-break of Legionellosis, described by Crespi and Ferra(1997), in a building following a phosphate treatment mayhave resulted from the same shift in the bacterial communityas that reported here.

Effects of chlorine on biofilm composition (ExperimentsC and D)

In all cases studied here, chlorination of the biofilm in-duced significant decreases in TDCs, HPCs, and counts withFISH probes. The proportion of cells hybridized with theEUB338 probe decreased 3- to 85-fold because of the chlori-nation. This sharp decrease might be linked to (i) the reac-tivity of nucleotides, which are the most reactive cellulartargets of chlorine (Dennis et al. 1979; Jacangelo andOlivieri 1985; Doré 1989) and also to (ii) intracellular mate-rial leakage following chlorination (Haas and Englebrecht1980). Such bacteria, depleted in rRNA to the point wherehybridization is not sufficient to be detected, might be mori-bund or at best have limited regrowth potential. However,the dosage of chlorine applied in our experiments should notinduce a cellular lysis (Jacangelo et al. 1991).

The various rates of decrease in cells hybridized with theEUB338 probe following chlorination (3- to 85-fold) de-pended on biofilm ageing and chlorination duration. Thegreater resistance to disinfection by older biofilms in com-parison with younger ones (Experiment C versus ExperimentB) can be noted here through the proportion of bacteria hy-bridizing with the EUB338 probe. This is in agreement withresults obtained by LeChevallier et al. (1988). The enhance-ment of exopolymer content within biofilms through ageing(Dott and Schoenen 1985; Batté et al. 2003), which maylimit the diffusion of chlorine and enhance the reduction ofchlorine (Morin et al. 1999), could partly explain the higherresistance of older biofilms noted here. The age of biofilmmay slow down the deep penetration of disinfectant into thebiofilm because of changes in biofilm composition andwidth with ageing. With the duration of disinfection, phe-nomena known as “bacterial resistance to oxidative treat-ment”, are likely to be induced. This resistance is linked tomechanisms, such as the up-regulation of resistance pumps(Arc), the induction of the SOS system or the mar operon(Chesney et al. 1996; Lisle et al. 1998; Saby 1999; Maira-Litran et al. 2000).

Moreover, the FISH results presented here make it possi-ble to observe that the decreases in numbers of the variousbacterial groups follow different kinetics within the biofilmcommunity. Chlorination continuously reduced the number

© 2003 NRC Canada

Batté et al. 749



of bacteria belonging to the (α + β + γ)-Proteobacteriagroup. However, the reduction in their densities was slowerthan that noted for the overall biofilm community. Therefore,there was a shift in the biofilm, with an enhancement of theproportion of bacteria belonging to the (α + β + γ)-Proteobacteria group (from <25% after 3 days of chlorina-tion to >85% after 1 week of chlorination). Norton andLeChevallier (2000) noted that the biofilm in a chlorinatedenvironment is mainly composed of Gram-negative bacteria.Indeed, an analysis of the species encountered in biofilmperformed by Rompré et al. (1998), revealed that most ofthem were Gram-negative bacteria, belonging to theProteobacteria family. Therefore, the results presented inthis paper are in agreement with observations made byNorton and LeChevallier (2000).

Effect of monochloramine on biofilm compositionThe mechanisms of bacterial inactivation by monochloramine

are not as well documented as those by chlorine. The weakdecline in TDCs (<1 log) compared with the noticeable de-cline in HPCs (>5 log) observed during Experiments D andE (after 28 days with 1.9 and 0.6 mg/L applied, respectively)were in accordance with observations previously reported(Stewart et al. 1994; Morin and Camper 1997).

Since monochloramine has been observed to break downgenetic macromolecules (Jacangelo and Olivieri 1985;Suzuki et al. 1998; Shibata et al. 1999), rather than to chem-ically react with nucleotides or nucleosides, it can be arguedthat the chloraminated community labelled by FISH probesis likely to contain nonviable bacteria. However, it is sup-posed that detection by FISH of cells killed decreases overtime, since rRNA degradation starts within 3 days of cellulardeath (Van der Vliet et al. 1994; McKillip et al. 1998). In theexperiments reported here, exposure to monochloraminelasted 28 days, hence it is likely that the labelled communityis the fraction of the bacterial biofilm that resists disinfec-tion.

The bacterial community evolved over time when exposedto chloramines (Tables 5A and 5B). We cannot exclude thepossibility that some variations in counts could reflect somedifficulties in enumerating so few cells. But these variationsmay also be due to differences in bacterial ability to resist todisinfection (Ridgway and Olson 1982; Mir et al. 1997).Such differences in resistance to disinfection (chlorine andmonochloramine) have been observed among the sporulatingGram-positive, nonsporulating Gram-positive, and Gram-negative bacteria, and have been attributed to differences incell-wall composition (Ridgway and Olson 1982). While allsubgroups undergo a reduction in density, the decrease insome is faster than in others. The bacterial group labelled bythe EUB338 probe showed a clear reduction in the propor-tion of bacteria belonging to the α-, β-, and γ-Proteobacteriasubclasses, to the benefit of other nontargeted bacteria. It isnotable that the proportion of Proteobacteria decreased overtime. Given that this group predominated in untreated biofilm,monochloramine disinfection seemed to provide an adverseecological environment for these bacteria.

Our results regarding the chlorine and monochloraminedisinfection of drinking water biofilms suggest that over thecourse of a long period of exposure, the resistance ofProteobacteria to disinfection is enhanced when chlorine is

used and hindered when monochloramine is used. These re-sults are in agreement with the literature, which reports abacterial resistance of biofilm exposed to chlorine(LeChevallier et al. 1988; Mathieu et al. 1992; Camper et al.1997).

Conclusion

The impacts of different water treatments on the bacterialcommunity of a biofilm were investigated by using differentmicrobiological techniques, including FISH. If no treatmentwas applied, the composition of the biofilm in annular reac-tors was observed to be fairly stable for more than 100 days.The drinking water biofilms studied were mainly composedof β- and α-Proteobacteria, with a small subgroup composedof γ-Proteobacteria.

The application of a phosphate blend commonly used forcorrosion control did not influence total bacterial densities,no matter what bacterial enumeration method was used. Thecomposition of the community was slightly modified, how-ever, since the γ-Proteobacteria were detected in a higherproportion after a phosphate addition. This result raisesquestions about the global consequences of phosphate treat-ment on microbial populations in water systems, since thisphylogenetic group contains many of the health-related mi-croorganisms and indicator bacteria that are usually moni-tored in drinking water.

Chlorination was the second treatment evaluated. Forbiofilms less than 2 months old, the monitoring of bacterialcomposition was only possible for a short treatment period,by which time too few bacteria were detected by the FISHmethod. For biofilms over 100 days old, the bacterial com-munity of the biofilm detected just after the application ofchlorine was composed of bacteria that were neither α-, β-,or γ-Proteobacteria. By contrast, after a longer period oftreatment, the biofilm population was composed mainly ofthese subclasses of bacteria.

The last treatment evaluated was monochloramination.After treatment, it was noted that the population was mainlycomposed of bacteria that do not belong to the α-, β-, or γ-subclasses, with some differences in the kinetics of decreaseof those three subgroups.

As reported in the Discussion section, Proteobacteria areGram-negative bacteria. Therefore, our results are in accor-dance with the observations of Ridgway and Olson (1982),Mir et al. (1997), and Norton and LeChevallier (2000): chlo-rination has been noted to lead to a predominance of Gram-negative bacteria in the bacterial community, whereasmonochloramination has been noted to lead to a predomi-nance of Gram-positive bacteria in the community.

The FISH technique was a very useful tool for evaluatingchanges in the composition of biofilms produced from drink-ing water. The FISH method makes it possible to study evenVBNC part of the populations in drinking water. Furtherwork is needed (i) to confirm the results obtained during Ex-periment C, concerning the γ-Proteobacteria and the impli-cations for public health, and (ii) to understand the reasonsfor the observed changes in bacterial populations as a resultof treatment.

© 2003 NRC Canada




Acknowledgements

This research was supported by the partners of the NSERC(Natural Sciences and Engineering Research Council of Canada)Industrial Chair on Drinking Water; namely, the City ofMontréal, the City of Laval, Triax, Vivendi Water-JohnMeunier-U.S. Filter. The authors would like to thank Dr. C.Greer of the NRC-BRI, Dr. B.K. Koudjonou, Dr. J. Coallier,and M. Rivard of the NSERC Industrial Chair on DrinkingWater, and J. Douki, Master’s student, for their advice andvaluable assistance.

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