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Biofouling: The Journal of Bioadhesion and Biofilm

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Biofilm accumulation in drinking water distribution

systems
a b a a a a
J C Block , K Haudidier , J L Paquin , J Miazga & Y Levi
a
Biofilm Research Group , (GIP Stelor, CGE, Pont‐à‐Mousson SA, District Urbain de Nancy,
Syndicat des Eaux d'Ile de France, Nancie), France
b
Faculté de Pharmacie , 5 rue Albert Lebrun, Nancy, 54000, France
Published online: 10 Jan 2009.

To cite this article: J C Block , K Haudidier , J L Paquin , J Miazga & Y Levi (1993) Biofilm accumulation in drinking
water distribution systems, Biofouling: The Journal of Bioadhesion and Biofilm Research, 6:4, 333-343, DOI:
10.1080/08927019309386235

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BIOFILM ACCUMULATION IN DRINKING WATER

DISTRIBUTION SYSTEMS
J C BLOCK†, K HAUDIDIER, J L PAQUIN, J MIAZGA and Y LEVI
Biofilm Research Group (GIP Stelor, CGE, Pont-à-Mousson SA, District Urbain de
Nancy, Syndicat des Eaux d'Ile de France, NANCIE), France
(Received 12 January 1992; infinalform 22 April 1992)
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In order to classify the relative importance of the parameters governing the accumulation of biofilm in
drinking water distribution systems, a study has been carried out, using an industrial pilot plant fed with
finished waters, with three main objectives, viz. (i) determination of biofilm density on pipe surfaces (PVC or
cement lined cast iron) as a function of the distance from the water treatment plant, (ii) evaluation of the
growth rate of attached bacteria along the distribution system, and (iii) measurement of the behavior of the
dissolved organic matter (DOC) in the water stream for system residence times of 40 to 240 h.
Biofilms were found to comprise a complex mixture of microorganisms, viz. fungi, yeasts, protozoa
(Bodo sp.), free amoebae (Hartmannela, Vannella, Cochliopodium and Naegleria), and bacteria (up to
106 cells.cm-2), 19% of which were actively respiring as determined by the INT method.
The densities of attached bacteria and the proportions of active bacteria decreased slowly along the
distribution system and fitted a non-linear polynomial regression model with six significant terms.
The highest growth rate of attached bacteria was observed at the beginning of the experimental network
(μ = 0.0017 h-1 or g = 17 d with no residual chlorine at 25°C), i.e. where the system receives the highestfluxof
biodegradable organic matter.
The apparent doubling time of attached cells in drinking water networks might be expected to be generally
relatively low (more than 100 d) because of the rapid consumption of biodegradable organic matter (within
40 to 80 h at 25°C when there is no chlorine in the water).
In large water distribution systems, far from the water treatment plant, the analysis of drinking water
quality gives more circumstantial information on the ecology of attached cells (biofilm) than on water
treatment efficiency.

KEY WORDS: biofilms, drinking water distribution systems, bacteria, water treatment.

INTRODUCTION

Formation of microbial biofilms on pipe walls has serious implications in water distri-
bution systems (bacterial blooms, biocorrosion, food resource for macroinvertebrate
e.g. Asellus) (Donlan & Pipes, 1986; Levy et al, 1986; Haudidier et al., 1988; Van der
Wende et al., 1989; Reasoner et al., 1989). A biofilm represents a very stable ecosystem
comprising a complex mixture of microbial cells embedded in a matrix of exopolymers.
Attached cells represent the major fraction of biomass in a distribution system
(Paquin et al., 1992) and generally contribute to the continuous contamination of the
water phase by bacteria that grow on and are then sheared from the surfaces of the
pipes. Thus, control of the biofilm becomes one of the main objectives of good drinking
water distribution practice. As chlorination does not prevent accumulation of biofilms
(Characklis, 1990; LeChevallier et al., 1990; Paquin et al, 1992; Mathieu et al., 1992),
other preventive measures have to be taken.

†Present address: Faculté de Pharmacie, 5 rue Albert Lebrun, 54000 NANCY, France

333
 334 J C BLOCK ET AL.

Several parameters govern biofilm accumulation (Bryers, 1987), including hydraulic

regime, flux of cells and nutrients, characteristics of the substratum. Moreover, in order
to classify the relative importance of the parameters governing the biofilm formation in
such systems, an experimental study has been carried out using an industrial pilot scale
system that simulates a drinking water distribution system (Colin et al, 1987).
The main objectives in this study were (i) to determine the level of bacterial
contamination of pipe surfaces (PVC or cement lined cast iron) and of the water phase;
(ii) to evaluate bacterial growth rate in the biofilm along the distribution system; (iii) to
measure the concentration of organic matter in the water stream for residence times of
40 to 240 h.

MATERIAL AND METHODS

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Distribution System Pilot Plant

The industrial pilot plant used in this study comprises six loops serially disposed
(Fig. 1). For each loop (31 metres in length, 100 mm in diameter, cement lined cast
iron), the water flow velocity was around 1 m-s~' and the theoretical residence time
was 40hloop~' (240 h or 10 d for the whole pilot plant). Table 1 gives most of the
parameters characterizing the pilot plant.
From a previous simulation study, it could be assumed that each loop behaves like a
perfectly mixed reactor where the whole pilot plant is equivalent to an infinite tubular
reactor with high axial dispersion coefficient.
Removable test coupons were made of PVC or cement; the circular coupons of 2 cm2
wetted surface area were put inside the water pipes of each loop using special sampling
devices which positioned the exposed coupon surface flush with the inner surface of the
pipe wall. Consequently, each coupon surface was submitted to the true hydraulic
conditions occurring inside the distribution system.
During the experiment, the pilot plant was fed with finished drinking water from the
surface water treatment plant of the city of Nancy (Imbeaux water treatment plant).
The source of supply was the Moselle river coagulated with aluminium chloride, fil-
tered through sand and GAC filters, post ozonated and chlorinated. The characteristics
of the finished water are given in Table 2.

Bacteria Counting Techniques

Bacteria were enumerated according to three different techniques applied directly to
water samples and to biofilm attached to PVC or cement coupons. In the latter,
attached bacteria were removed from the coupons by 2 min sonication (Vibra Sonic
Cells VC 250) in 25 ml of bacteria-free distilled water (pH 7).
Total number of cells (epifluorescence counts) A 9 ml quantity of each sample (water or
sonicated biofilm) was poured into sterilized glass tubes. 1 ml of acridine orange (0-2%,
aqueous solution) (Gurr Cl 40005) and lml of Triton XI00 (Prolabo) ( 0 1 % in
saccharose 0-1 M Merck, EDTA 001 M Prolabo, citric acid 002 M Prolabo, K2 HPO4
002M Prolabo) (Prolabo) were added in each tube. (All reactants were sterilized by
filtration through cellulose nitrate filters, pore size 0-22 um.)
After mixing for 30 s and a contact time of 2 min, aliquotes were filtered through
black polycarbonate filters (Nuclepore SN111156, pore size 0-22 um). Filters were also
rinsed with distilled water (5 ml), hot air dried, then hydrated with a drop of buffered
 BIOFILM ACCUMULATION 335

-3OJ-

dj A liJ - - Lf

HOI * 5 *
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-3CJ-

Fig. 1 Schematic representation of the drinking water system.

Table 1 Physical and hydraulic characteristics of

the experimental distribution system

Loop tube length (cm) 3,100

Loop volume (cm3) 240,000
Wetted surface area (cm2) 92,840
Water flow velocity (m-s"1) 1
Water flow rate (lh"') 6
Residence time-loop"1 (h) 40
Dilution rate (h" 1 ) 0025
Reynolds number 10s
Velocity gradient (-s~') 438
Peclet number 10
Viscosity (stockes) 001
 336 J C BLOCK ETAL.

Table 2 Characteristics of the finished water entering the experimental

distribution system

Parameters Average value

(n=3)

Total chlorine (mgl-1) 0-6

Free chlorine (rng-1"1) 0-4
Heterotrophic plate count
(15 d incubation) (CFUml-1) 4x10*
Active bacteria (INT+) (cells-ml"1) 2-4 xlO3
Epifluorescence count (cells-ml*') 2-6 xlO3
Organic carbon (TOC mg-1"') 204
Organic nitrogen (N mgl"1) 01
Nitrates (N-NOj_ mgl-1) 511
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Phosphates (P-PO 3 " 4 mg-1"1) 006

glycerin for immunofluorescence (Diagnostic Pasteur 74 921) and covered with a cover
slip. Filters were then examined using an epifluorescence microscope (Zeiss Universal
microscope; blue light excitation: 450-490 nm; reading: 520 nm) with an oil immersion
(Merck 4699) objective (100X). The enumeration was done on 15 fields of 10~4cm2
each (Zeiss 474068) and the results expressed in number of cells-mi"1 of water or -cm"2
of coupons.
INT actively respiring bacteria (active bacteria) Each 10 ml of sample (water or soni-
cated biofilm) was incubated for 3 h at room temperature with 0-1 ml yeast extract
(Merck in 30 g-1"1 aqueous solution), 01 ml nalidixic acid (SIGMA in 2 gl~' aqueous
solution) and lml iodo-nitrophenyl tetrazolium (INT) (SIGMA 18377 in 2 g - r '
aqueous solution). After incubation, the reaction was stopped by the addition of 1 ml
of formaldehyde (37%) (Prolabo). Then, 1 ml of 1% (aqueous solution) malachite
green (Prolabo) and 1 ml of 0-1% (see before the solution) Triton XI00, were added.
All reactants were sterilized by filtration through cellulose nitrate filters (pore size,
0-22 nm).
After 30 min contact time, each aliquot was filtered through white polycarbonate
filters (Nuclepore SN UN 06, pore size, 0-22 um). The filter was dried and examined by
optical microscopy (Zeiss) with an oil immersion objective (100X). The enumeration of
bacteria with red spots of formazan inside the cells was made on 15 fields (10~4cm2
each) and the result expressed as active cells-ml"1 of water or -cm"2 of coupons.
Heterotrophic plate count bacteria 1 ml of each sample (water or sonicated biofilm) was
incorporated in standard nutrient agar without glucose (AFNOR NF T90-1402)
(eventually dilutions were made in physiological solution NaCl 0-9%). After 15 d
incubation at 20°C±2°C, the colonies were enumerated and expressed as number of
colony forming units (CFU)ml~' of water or -cm"2 of coupons.

Analysis of Total Organic Carbon

Organic carbon was determined using a Dohrman TOC analyser, according to the
manufacturer's instructions.
After elimination of inorganic carbon by HC1 acidification and oxygen bubbling,
organic carbon was oxidized with UV in persulfate medium. The carbon dioxide
produced was measured by infra-red and the results expressed as g TOCml"'.
 BIOFILM ACCUMULATION 337

Polynomial Regression
The relative importance of the controlled experimental factors (loop number, method
of bacterial enumeration, material tested) in explaining the variations in the number
of bacteria was assessed by multiple regression analysis. The factorial design of the
experiment eliminated correlations between the regression factors and allowed for
their univoqual ranking. Ranking was made on the basis of the contribution of each
polynomial term to the regression (a saturated 36 terms polynomial with power and
interaction terms was computed). The contribution of each term corresponded to the
residual variance of an hypothetical 35 terms polynomial obtained by the removal of
this term from the saturated model.
The significance of the individual terms cannot be obtained by classical variance
analysis, as variance homogeneity across the experimental groups is not respected
(as tested with Bartlett's test). Therefore, the following significance criterion was
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used. An ordered model (on the basis of contributions) whose residual variance is just
superior to experimental intra-group variance was retained; its terms were considered
significantly important for explaining the variation of the observed variable (Bois et al.,
1986).

Protocol
One secondary aim of the study was to evaluate the quasi steady state biofilm accumu-
lated on different materials used in water distribution systems. For that purpose, 3
coupons of each tested material (PVC and cement) were introduced in each of the six
loops of the distribution system which were operated continuously for one month at
24-5±0-5°C.
Experience indicated that a quasi steady state (d Cell att/dt = 0) was reached after 3
weeks immersion. The coupons and water were then sampled for biomass and organic
carbon analysis.

RESULTS
Data which are presented in this paper are related to the proliferation of bacteria in an
industrial pilot-scale drinking water system operating with treated surface waters, at
relatively high temperatures (24°C) in which the chlorine disinfectant residual was
immediately and totally consumed.
The introduction of new coupons into the system lead to the accumulation of a quasi
steady state biofilm within a few weeks, as seen in a previous study (Colin et al., 1987).

Biofilm Cellular Density

As shown in Figure 2, the total number of attached bacterial cells on material coupons
in the no 1 loop or "first loop" is about 7-8 x 106-cm~2. The INT actively respiring
bacteria (1-1 x 106cm~2) and CFU counts (4-5x 105-cm~2) represent only 14-1%
and 5-8% respectively of the total attached cell population. Bacteria represent not the
only component of the biofilm. Fungi, yeasts, protozoa (Bodo sp.) and free amoebae
(Hartmannela vermiformis, Vannella mira, Cochliopodium minutum and Naegleria sp.)
were also identified but not enumerated.
Throughout the entire pilot study the density of attached cells was not constant with
distance {i.e. between loops) and a slight decrease is observed from loop no 1 (7-8 x 106
 338 J C BLOCK ETAL.

1.00E+07 T T

Number of cells
1.00E+06 •-
per cm2
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1.00E+05
E, (I o e cence INT
Active bacteria C.F.U.
count

Fig. 2 Total number and numbers of active and heterotrophic bacteria attached inside loop no 1. (Average
values obtained from PVC and cement coupons; n=5.)

cells-cm 2) to the loop no 6 (4-4 x 106 cells-cm 2). The same trend is shown for the
active and the viable bacteria (Fig. 3).
The values of attached cells fit a polynomial regression model with only 6 significant
terms from among the 36 used for the calculation, which gives the calculated value y of
attached cells:
y = 5-96 - 0-796 X2+0-089(3 Xf- 2) -(0077+0046 X 2 )X,- 0009 X]+0086 X3
in which X, is the number of the loop (nol to no 6, codes —5, —3, —1, + 1 , + 3 , +5
respectively), X2 is the method of enumeration (epifluorescence count, code — 1; INT
actively respiring bacterial count, code 0; CFU, code 4-1) and X3 the kind of material
tested (PVC, code - 1 , code +1).
Because of the square terms, the graphic representation of the polynomial draws a
parabola which expresses a multiplicity of interactions for the biofilm accumulation in
each loop. The different terms of the polynomial are considered significantly important
for explaining the variation in the observed variables. However, the contribution of
each term differs greatly and the enumeration method is more important than the
number of the loop and far more important than the type of material tested.
The indexes given by the proportion of active cells (or CFU) in the biofilm decreases
from loop no 1 to no 6 by a factor of 4 and 16 respectively. This suggests a significant
change in the ecology of the loops (especially the nutritional environment) even if the
total attached cell population looks relatively stable in terms of cell numbers from the
beginning to the end of the distribution system.
Growth Rate of Biofilm Cells
Accumulation of biofilm is related to at least five phenomena, viz. deposition of plank-
tonic cells, and the growth, mortality, shear loss, and the extent of protozoan grazing of
 BIOFILM ACCUMULATION 339

7 -r
• Epifluo. PVC

6.5 -- a Epifluo. Cement

A I.N.T. PVC
6 --
A I.N.T. Cement

Number of 5.5 -- A • C.F.U. PVC

• • O
bacteria
(Iog/cm2) o C.F.U. Cement
A
5
— Epifluorescence

- •
I.N.T.
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4.5 --
- -
C.F.U.

r-
Loop 1 Loop 2 Loop 3 Loop 4 Loop 5 Loop 6

log(EPI) = 6.8-0.031 X,-O.009X?+O.086X3

log (INT) =5.7-0.077X,-O.009X?+0.086X3
log(UFC) = 5.2-0.12X,-0.009X?+0.086X3
Fig. 3 Total number and numbers of active and heterotrophic attached bacteria (log-cm"2) in the different
loops. (Each value represents the average of 3 coupons.)

attached cells. Characterization of the specific growth of the biofilm can be obtained
from appropriate experiments, leading to the calculation of each kinetic rate (Bryers &
Characklis, 1982), or by applying the simplified model previously used by Van der
Wende et al. (1989). This latter model expresses the biofilm specific growth rate u (-h"1)
as
= D(X i -X i _,)V/XbA, (1)
in which X; and Xj_, are the suspended biomass concentrations at the outlet and inlet
(cells-ml"1); D is the dilution rate in the loop (-h"1); Xb is the biofilm cell density
(cells-cm ); V is the volume of each loop (1); and A is the wetted surface of each loop
(cm2).
Such a model assumes that both the mortality rate (km) and the planktonic growth
rate are negligible. As demonstrated from previous experiments by Haudidier et al.
(1988), in the absence of a chlorine residual, the km is relatively low (km=00018-h~1)
and the calculated planktonic growth rates are also very low or negative.
Moreover, by putting in equation (1) the values of Xb as epifluorescence counts of
the material coupons, the specific growth rate of the biofilm cells can be calculated for
each loop. As shown on Figure 4, the specific growth rate of cells attached to cement
was high (u = 00017-h -1 ) in the first loop which received the highest flux of organic
matter. This means that g, the cell generation or doubling time (g=In2-u~') of cells in
the biofilm of the loop no 1 (25°C, no chlorine) was approximately 17 d.
 340 J C BLOCK ETAL.

lib (h-1) TOC (mg 1-1)

0.003 T -r 2.5

0.0025 -•
-- 2

0.002 -•
-- 1.5

0.0015 --

-- 1
0.001 -•
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-• 0.5
0.0005 --

water ^°°P 1 '- 0 0 P 2 Loop 3 ^oop 4 ^oop 5 ^oop 6

Fig. 4 Growth rate of biofilm on cement coupons in each loop and TOC concentrations.

Such a value of u decreased substantially beyond loop 1 and reduced average values
of 0-0004-h"1 and 0-0001-h"1 in loops no 5 and no 6, and g values were then equal to
72 d and 288 d respectively.
The changes of u values can be related to high TOC consumption especially in loop 1
(around 82% of the biodegradable organic matter is consumed in 40 h). Calculated
specific growth rates for PVC material are generally higher or equivalent to cement cast
iron (Fig. 4).

Planktonic Cells
The total number of cells (epifluorescence counts) approximately doubled in the water
column of the first loop (from 2-57 x 105 to 5-37 x 105 cells-ml"1). This significant
increase is confirmed by a major change in the CFU from 0-36 x lO'CFU-ml"1 (Fig. 5),
i.e. the index expressed by the ratio CFU/epifluorescence increased by a factor of
around 10 in the first loop (its highest value in the whole system).
Beyond loop no 1 the total number of cells in the water was quite stable until the end
of the system and can be mainly explained by the transport of cells prouced in loop no 1.
The low production of cells in loops no 2 to no 6, and by way of consequence their
high age is shown on Figure 5 by the break in the slopes of the curves at the level of loop
no 3 for the CFU, and at the level of loop no 5 for the INT respiring bacteria.
Comparison of planktonic and attached cells shows on the one hand that the total
number of attached cells in a loop is from 7 to 14 times higher than in the water phase,
depending on the loop. On the other hand the proportion of CFU in the water phase is
systematically lower than 1 % whilst in the biofilm the proportion is, according to the
loops, between 0-5% and 6%. Clearly, most of the activity (especially in loop no 1) is
located in the biofilm.
 BIOFILM ACCUMULATION 341

6 T

"•" Epifluorescence

log cell number 3 - - • ^ Active Bacteria

/ ml
-•- C.F.U.
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0 40 80 120 160 200 240

hours
Fig. 5 Planktonic bacterial populations in the experimental water distribution system.

DISCUSSION AND CONCLUSIONS

Biofilms accumulated on pipe walls have a very complex structure and a diversified
microbial population. The number of attached bacteria found in the present study
(around 107 cells-cm"2) is similar to the values previously reported (Pedersen, 1990;
Olson &Nagy, 1984; Donlan & Pipes, 1986; Schoenen, 1986; LeChevallier et al., 1987;
Van der Wende et al., 1989; Paquin et al., 1992).
As several mechanisms explain biofilm accumulation (Bryers, 1987), it is not surpris-
ing to observe in the present study a non-linear relationship between the number of
attached cells and the sampling point on the distribution system. Clearly the growth of
attached cells can explain the bacterial bloom at the beginning of the network. The
highest growth rate as well as the highest proportion of viable cells was observed
in loop no 1 without chlorine (growth rate > deposition kinetic). In the latter stages
of the system, the transport of cells by the water flow mainly explains the bacterial
contamination.
As pointed out by Levi (1990), the flux of nutrient appears as one of the key
parameters in cell accumulation in the drinking water distribution system. In loops no 1
and no 2, organic matter is largely consumed (respectively 0-63 mg DOC-40 h~' in loop
no 1 and 014mg DOC-40 h" 1 in loop no 2). The resulting nutrient limitation leads
to a substantial decrease in growth rate and viable bacteria. If the generation time of
attached cells in loop no 1 is very close to those reported by Van der Wende et al.
(1989), it may be underlined that the highest value obtained in this study (g= 17 d) is
characteristic of situations in which the system receives a high flux of biodegradable
organic matter and is not submitted to toxicants like chlorine. On average, it may be
expected that the apparent doubling time of attached cells in drinking water distri-
bution systems is relatively low (more than 100 d). In these conditions, slow growing
attached bacteria will represent a very stable ecosystem which is not very susceptible to
chlorine (Paquin et al., 1992).
 342 J C BLOCK ETAL.

Because the bacterial bloom in water is linked to the growth of attached cells receiv-
ing enough nutrient, planktonic cells have two origins, viz. from the water treatment
plant and the continuously sheared biofilm. Such an observation leads to recognition
that bacterial water quality control has a different significance according to the
sampling point location on the network. Indeed, close to the water treatment plant
bacteriological analysis measures the plant efficiency whereas, away from the water
treatment plant the analysis gives a picture of the bacteria growing in and sheared
from the biofilm. Thus, the sanitary analysis of drinking water or more precisely
its interpretation should take into account transformations of water quality in the
distribution system.
Thus, the problems of people in charge of water distribution systems are very
obvious. Even with systematic post chlorination they are never sure that chlorine is
present continuously in all parts of the network. If one unchlorinated section allows cell
multiplication, this will represent a source of bacteria contaminating the rest of the
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system. Chlorine is thus not a panacea and has to be combined with substantial nutrient
removal at the level of the water treatment plant.

Acknowledgements
This work has been carried out as part of a larger research program including the
Centre International de l'Eau, GIP STELOR, Pont-a-Mousson SA, Anjou Recherche,
(Generale des Eaux Group), Agence de Bassin Seine-Normandie and the Syndicat des
Eaux de l'lle de France.
The authors would like to express their grateful thanks to C Harf (University of
Strasbourg) for the analysis of protozoa and to M Vaillant (Paris) for the polynomial
regression.

References
Bryers J D (1987) Biologically active surfaces: processes governing the formation and persistence of biofilms.
Biotechnol Prog 3: 57-68
Bryers J D, Characklis W G (1982) Processes governing primary biofilm formation. Biotechnol Bioeng 24:
2451-2476
Bois F, Vaillant M, Vasseur P (1986) Multiple regression analysis of toxic interactions: application to the
microtox test and general comments. Bull Environ Contam Toxicol 36: 707-714
Colin F, Grapin G, Cheron J, Levi Y, Pozzoli B, Miazga J, Pascal O (1987) Etude de revolution de la qualite
de l'eau potable dans les réseaux de distribution. TSN-L'Eau 12: 565-574
Characklis W G (1990) Microbial biofouling control. In: Characklis W G, Marshall K C (eds) Biofilms. John
Wiley and Sons, 589-633
Donlan R M, Pipes W O (1986) Pipewall biofilm in drinking water mains. 14e Ann AWWA Water Quality
Technol. Conf, Portland, Oregon
Haudidier K, Paquin J L, Français T, Hautermann P, Grapin G, Colin F, Jourdain M J, Block J C, Cheron J,
Pascal O, Léui Y, Miazga J (1988) Biofilm growth in a drinking water network: a preliminary industrial
pilot plant experiment. Wat Sci Technol 20: 109-115
LeChevallier M W, Babcock T M, Lee R G (1987) Examination and characterization of distribution system
biofilms. Appl Environ Microbiol53:2714-2724
LeChevallier M W, Lowry C D, Lee R G (1990) Disinfecting biofilms in a distribution system. J Am Wat Wks
Ass 82: 87-99
Levy R V, Hart F L, Cheetham R D (1986) Occurrence and public health significance of invertebrates in
drinking water systems. J Am Wat Wks Ass 78: 105-110
Levi Y (1990) Importance du contrôle du carbone organique dissous biodegradable (CODB) dans la
stratégie de maintien de la qualité de l'eau potable en cours de distribution. Compte rendu 13èmeSymp Int
Triatement des Eaux Usées et 2ème Atelier sur l'eau Potable. AQTE, Montréal, 273-282
Mathieu L, Paquin J L, Block J C, Hartemann P, Colin F (1992) Paramètres gouvernant la proliferation
bacterienne dans les réseaux de distribution. Sciences de l'Eau (in press)
 BIOFILM ACCUMULATION 343

Olson B H, Nagy L A (1984) Microbiology of potable water. In: Laksins A I (ed) Advances in Applied
Microbiology, 30. Academic Press, Incorporated, London, pp 73-132
Paquin J L, Block J C, Haudidier K, Hartemann P, Colin F, Miazga J, Lévi Y (1992) Effet du chlore sur la
colonisation bacterienne d'un réseau experimental de distribution d'eau. Sciences de l'Eau. (in press)
Pedersen K. (1990) Biofilm development on stainless and PVC surfaces in drinking water. Wat Res 24:
239-243
Reasoner D J, Blannon J C, Geldreich E E, Barnick J (1989) Nonphotosynthetic pigmented bacteria in a
potable water treatment and distribution system. Appl Environ Microbiol 55: 912-921
Schoenen D (1986) Microbial growth due to materials used in drinking water systems. In Rehm H J, Reed G
(eds) Biotechnology. VCH Publishers, Weinheim. pp 627-647
Van der Wende E, Characklis W G, Smith D B (1989) Biofilms and bacterial drinking water quality. Wat Res
23: 1313-1322
Downloaded by [New York University] at 02:13 07 January 2015