Biofilms in Drinking Water Distribution

Biofilms in drinking water
distribution systems.
M. Batté, B.M.R. Appenzeller, D. Grandjean, S.
Fass, V. Gauthier, F. Jorand, L. Mathieu, M.
Boualam, S. Saby, and J.C. Block
1. INTRODUCTION
Potable drinking water carries with it both: (i) extremely diverse but poorly
identified microbial flora; and (ii) complex organic matter (Block et al. 1997;
Croué et al. 2000; Doggett 2000; Percival et al. 2000). Most of the organic
matter in distribution sytems is present in dissolved form, usually described as
DOC (dissolved organic carbon), a large fraction of which is biodegradable and
monitored as biodegradable dissolved organic matter (BDOC) (Block et al.
1992; Servais et al. 1992a).
[1]
2 Authors
Some environments, such as ground waters or waters treated by

nanofiltration, are described as oligotrophic (less than 0.5 mg DOC/l and 0.2
mg DOC/l respectively). However, even in such a poor environment, the
transport of water through a distribution system permits microorganisms to
multiply (Crissot-Laruade et al. 1999; Devender 1995; Lévi et al. 1992; Sibille
et al. 1997). There are three important reasons why this situation is difficult to
manage:
In most drinking water networks, the interface between water and pipe wall is
a prime site for the accumulation of cells and organic matter, and for bacterial
multiplication (Block et al. 1993; Servais et al. 1992b and c, 1995; van der
Wende et al. 1989). This proliferation is followed by: (i) the detachment of
bacteria (Ascon-Cabrera et al. 1995) and/or the removal of bacteria from the
network pipewalls through shearing/erosion (Rittman 1989); and (ii) transport
of these bacteria into the circulating water (Figure 1).
Water flow
Release
Corrosion products,
Chlorine
Organic Bacterial carbonates...
Oxygen
growth
BDOC matter
Biofilm
thickness Bacteria
FeIII
FeII
0.1 to 10 µg organic
106 to 10 8 bacterial
carbon/cm2
cells /cm2
Figure 1. Reaction mechanisms involved in the accumulation of biofilms on a

surface in contact with drinking water.
This bacterial population is adapted to the oligotrophic environment of

drinking water (less than 2 mg of organic matter per litre of drinking water in
many cases). There is evidence that these microbial ecosystems may be very
difficult to eradicate. Even when the nutrient concentration is low (less than 200
Short chapter title 3
µg of biodegradable organic matter per litre of drinking water), the bacterial

biomass is only slightly reduced (Sibille et al. 1997).
In a network where the system is biologically unstable, the water-borne
bacterial biomass and the bacterial biomass that multiplies on the pipes are the
starting points of a complex trophic chain, which includes free-living protozoan
and other bugs (Sibille et al. 1998).
The entry of biomass into the distribution network and its multiplication
generate at least two complementary problems:
(1) The system is continuously inoculated with unknown micro-organisms

which are both saprophytes and opportunistic pathogens. Non-culturable
bacteria are very likely to be present and are estimated to account for less
than 0.1 to 1% of the biomass (Roszak and Colwell 1987; Colwell and
Grimes 2000). In some instances, network conditions may be suitable for
the survival and growth of these bacteria.
(2) A fraction of the micro-organisms introduced into the network (and which
multiply there principally within the biofilm) pose a potential danger to the
consumer, leading to an increase in gastroenteric symptom frequency, as
reported in various epidemiological studies:
- Payment et al. (1991a and b, 1997) demonstrated that, in the
general population, 0.1 incident case of gastrointestinal tropism
per person per year results from the consumption of water
meeting the accepted norms for drinking water.
- Gofti et al. (1999) reported even higher values in a different
context. Their study of groups of children who are clearly more
susceptible showed close to four digestive problems per person
per year and one episode of diarrhoea per person per year.
- Zmirou et al. (1995) reported similar results in an entirely
different situation (a rural environment, simple chlorination of
water resources, water conforming to norms and two thousand
children from 7 to 11 years old), which revealed a relative risk of
1.4.
Most of these low-impact epidemiological results (i.e. not epidemic in nature
or associated with an accident and large-scale contamination event) are identified
by the classical medical system, and are only detected in specific studies of
exposed populations.
Assuming that 10% of infected adults are absent from work for one day due
to their illness, the economic consequences are non-negligible (Payment 1997;
4 Authors
Garthright et al. 1988). Then all the strategies developed for the 20 last years
combine actions as control of the water resource quality, and control and
maintenance of the distribution system. Drinking water of low quality (high
nutrient content, high particle content, unpleasant taste and odours, inefficient
disinfection, presence of opportunistic pathogens) will always be unstable and
associated with the systematic occurrence of indicators which do not meet
microbiological regulations following transport through the network. Such a
situation will be exacerbated by other factors, such as corroded materials,
stagnation of the water in dead-ends, etc.
Some of the classic problems currently encountered in drinking water
distribution systems are presented in this chapter:
(i) the presence of biofilms and loose deposits, which represent
active zones of biomass accumulation and development;
(ii) the behaviour of health-related micro-organisms introduced in
distribution systems;
(iii) the limited effect of chlorine addition to networks (a long-
standing practice still in use today which does not prohibit
biofilm growth);
(iv) the effect of the pipe-wall material (plastic and iron-based
materials) on the biofilm and on water quality; and
(v) the microbial growth risk associated with phosphate-based
anti-corrosion treatments.
2. BIOFILMS AND LOOSE DEPOSITS

2.1 Biofilms
The microbial activity in distribution systems occurs in water, on pipe
surfaces and in all kinds of deposits which have accumulated in pipes and
storage tanks. Such typical surface pipe colonisation is usually described as
biofouling (Flemming 2002). The structure of the biofilm found in real
drinking water networks is unclear, and has not yet been described in detail due
to difficulties involved in investigating such a very small amount of biomass
without disturbing it. This process is rendered even more complicated by the
presence of debris, corrosion products and mineral deposits, and by the
formation of corrosion tubercles which provide new niches or surfaces to
colonise (Allen et al. 1980; Tuovinen et al. 1980; Ridgway and Olson 1981;
LeChevallier et al. 1987; Sly et al. 1988; Stolzenbach 1989; Flemming and
Geesey 1990).
Most of the descriptive variables (thickness, porosity, density, fractal
dimensions) have generally been measured on thicker biofilms generated in the
laboratory, and are therefore not directly relevant to the issue of drinking water.
However, by analogy, description of the biofilms found in drinking water

networks can be approached in several ways:
(1) A large number of microbial cells are generally counted on surfaces in
contact with drinking water (in the order of 106 to 108 cells per cm2)
(Donlan and Pipes 1988; Pedersen 1990; Lévi et al. 1992; Mathieu et
al. 1992). This biomass represents only 0.1 to 20 micrograms of
organic carbon per cm2 (Niquette et al. 2000; Fass et al. 2003).
(2) Biofilms have a heterogeneous and discontinuous structure, displaying
a non-uniform distribution over the surface of the materials in contact
with the water (van der Kooij et al. 1995). The aggregates in these
biofilms are surrounded by channels, accounting for up to 50% of the
volume of the biofilm, in which water, particles and protozoa circulate
(Stewart et al. 1993; de Beer et al. 1994a and b; Gjaltema et al. 1994;
Stoodley et al. 1994; Devender 1995; Massol-Deyá et al. 1995).
(3) The biofilm consists of a mixture of micro-organisms which differ in
activity level according to their position in the aggregate (Bouwer 1989;
Rittman and Manem 1992). Even micro-colonies (around 50 cells) may
constitute an association of several species (Manz et al. 1993), and
plasmid exchanges may occur within these biofilms (Lisle and Rose
1995).
(4) Although new material exposed to drinking water colonises rapidly
(Figure 2), a stationary state is never reached in a real network due to
frequent discontinuities: variations in the hydraulic regime, changes in
the nature and concentration of nutrients and disinfectants and the
introduction of new micro-organisms. Zacheus et al. (2000) showed a
slow but continuous increase in biomass after five months of exposure
to drinking water. Percival et al. (1998) observed a change in dominant
bacterial populations between the "pioneers", which colonise steel
material within a month, and the species present after five months of
immersion in water. Because a biofilm never reaches a real equilibrium
in a drinking water network, its composition and density vary
depending on its age, even for young biofilms (5 to 15 weeks). (Table
1).
(5) Finally, by analogy with current concepts concerning the differentiation
of biofilms as they age (Davies et al. 1998; Allison et al. 2000), the
biofilm system is almost certainly continuously re-organised during its
development (formation of micro-aggregates and channels, etc.).
6 Authors
Bacterial density Cells marked with DAPI

(Cells or CFU) cm-2
Culturable bacteria on R2A agar
1E+8
1E+7
1E+6
1E+5
1E+4
1E+3
0 2 4 6 8 10 12 14 16
Time of colonisation (weeks)
Figure 2. Colonisation of polycarbonate coupons exposed to drinking water for 1 6

weeks, at 20°C (adapted from Batté et al. 2003).
Table 1 Concentration of total carbohydrates, total amino acids and phosphate i n

9-week-old and 15-week-old biofilms per unit surface area (polycarbonate) (adapted
from Batté et al. 2003).
Bacterial density Carbohydrates Amino Acids

(Cells cm-2) (ng-C cm-2) (ng-C cm-2)
9 weeks-old biofilm 9.25 106 242 ± 9 49 ± 2
15 weeks-old biofilm 2.84 107 1069 ± 211 54 ± 1
Biofilms accumulate on the surface of materials in zones where water

circulation is restricted by friction against pipe walls, creating a viscous layer
which may, for example, reach 70 µm in thickness, in a cast-iron tube lined
with cement mortar, for a flow rate of 1 m/s, at 20°C. The transfer of various
molecules (oxygen, disinfectant, nutrients) across this layer is limited by the
diffusion rate of these molecules. If the biofilm (or micro-colony) is thin (<40
µm), then the transfer of nutrients and oxygen is probably not limited, and the
indicators of activity of the whole system (µ, Ks, Y) are similar to those for
bacteria in suspension (Bakke et al. 1984). If the biofilm (or colony) is thick
(>80 µm), then the level of respiratory activity is lower in the layers closest to
the support material (de Beer et al. 1994b). This accounts for the low level of
activity of a fraction of the fixed bacteria (Zhang and Bishop 1994; Kalmbach et
al. 2000).
It has not been definitively demonstrated that the biodegradable organic
matter content of distributed water directly controls the density of cells in the
biofilm, although it has been shown to affect activity and bacterial production
(Prévost et al. 1998; Volk and LeChevallier 1999). Biofilm appears to be a
meta-stable system supplied by the multiplication of its inhabitants and by the
arrival of cells from the aqueous phase (the rate of deposition/adhesion of cells
on a material surface is correlated with the density of the cells in the circulating
water) The biofilm is also subject to erosion due to continual shearing off,
leading to a leakage of biofilm bacteria into the aqueous phase.
2.1 Loose deposits

The other kind of biomass accumulation in drinking water is in the form of a
deposit. Accumulated material may adhere to pipe walls (hard deposits or
incrustations mainly consisting of scale or corrosion products) or may just be
deposited on the surface (soft or loose deposits)(Smith et al. 1997). Loose
deposits may be readily transferred into tap water if re-suspended during
hydraulic changes in the distribution system. Such a re-suspension may affect
routine coliform monitoring results (Gauthier et al. 1999a and b) or even public
health (Kent et al. 1988).
Deposit accumulation in distribution systems is generated by many
mechanisms, among them the precipitation, flocculation and settling of material
during water treatment or the corrosion of pipe surfaces (Gauthier et al. 2001).
A wide range of cultivable bacteria densities have been found, most of the
recorded measurements being in the 2x106 to 2x109 CFU/g range. Total cell
counts (epifluorescence microscopy after acridine orange staining) were found to
be in the range of 1010 to 1011 cells/g of deposit (Barbeau et al. 1999; Zacheus
et al 2001). Zacheus et al. (2001) also measured bacterial activity in loose
deposits using the 3H-thymidine incorporation technique. They found that this
activity was 80x greater in recent deposits than in old ones, and 220x higher in
old deposits than in distributed water.
The variations in HPC densities in deposits are probably caused by a
combination of factors. Ainsworth (1978) and Gauthier et al. (1996 and 1999a)
pointed out a relationship between the HPC density and the concentration of
organic matter in deposits (expressed as volatile solids or organic carbon). The
fraction of TOC may be as high as 11% in storage tank deposits (Schreiber and
Schoenen 1994; Gauthier et al. 1999a and b). Ainsworth (1978) also observed
8 Authors
that bacteria are found in greater numbers in sediments and precipitated layers
than in corrosion products. In the UK and Germany, more cultivable bacteria
were measured in deposits from distribution systems supplied with treated
surface water (as compared to groundwater) (De Rosa 1993; Schreiber and
Schoenen 1994).
Carrière et al. (2002) measured deposit densities varying from 0.01 to
40 g/m2 in three Canadian distribution systems, and found that it was not
unusual for the results obtained from two pipe sections in the same distribution
system to differ by a factor of 10. When computing the total number of deposit-
associated cultivable bacteria from such quantities and comparing this number
to the total number of biofilm bacteria (Gauthier 1998), it appears that the
majority of cultivable bacteria may be located in the deposits rather than in the
biofilm for deposit quantities >1 g/m 2. When more than 10 g/m2 of deposits
have accumulated, the bacteria in them represent >80% of the total bacteria in
the pipe (Figure 3). Such deposit concentrations are not uncommon, especially
in distribution systems which are rarely cleaned by flushing or mechanical
means, or which are prone to deposit accumulation (Zacheus et al. 2001;
Carrière 2002).
100%
Fraction of bacteria in the

80%
biofilm (%)
60%
40%
20%
0%
0,2 200
1 150
Loo r
se
5 100 ete
dep 10 80 Diam
e
(g/m osits Pip (mm)
)
Figure 3. Biofilm fraction of cultivable bacteria in a 1-m pipe section – compared t o

the total number (biofilm+deposits) – as a function of deposit amount and pipe
diameter (hypotheses: average concentration of cultivable bacteria in biofilms: 105

CFU/cm2 (Mathieu et al. 1992); average concentration of cultivable bacteria i n
deposits: 2.5 108 CFU/g (Gauthier et al. 1996).
Therefore, it would be worth managing distribution systems to limit

the accumulation of deposits (through preventive actions and/or regular
cleaning), and to provide a better understanding of the factors that favour the
survival of pathogens (including Legionella) and bacterial indicators in these
deposits.
To summarise, drinking water systems are biologically unstable, with most
of the bacterial activity occurring in deposits and biofilm (Boe-Hansen et al.
2002). Limiting biofilm requires a combination of actions, such as limitation of
biodegradable organic matter, bacterial control of finished water, disinfectants,
etc.
3. THE FATE OF UNDESIRABLE MICROFLORA

Various undesirable water-borne microorganisms are more or less
systematically present in drinking water distribution systems and may cause
epidemic events (Payment et al. 1994; Leclerc et al. 2002; Sharma et al. 2003).
They enter the network in different ways, either directly, through the water
treatment plant, or indirectly, through intrusion due to cross-connection or pipe
breakdown (Besner et al. 2002). It is quite impossible to prevent them from
penetrating the system through the treatment plant. Even with the best available
technologies for water treatment (excluding full treatment with membrane
filtration) which remove a large fraction of the colloids, at least 107 cells (dead
or alive) remain per litre of finished water. From a statistician’s point of view,
pathogens may also escape treatment entirely and flow into the distribution
system, some of them in an injured state. These undesirable flora implant
biofilm or deposits, which may then be regarded as reservoirs of opportunistic
pathogens.
This implantation is controlled initially by the kinetics of attachment
(transport to the surface, followed by the action of an efficient sticking
mechanism governed by van der Waals, electrostatic, and acid-base
(hydrophobic) interactions) (Azeredo et al. 1999). Whether the colonisation of
the biofilm is stable or transitory can be explained by the competition between
the species within the biofilm, the availability of nutrients or the presence of
disinfectants. The changes in environmental conditions (temperature, nutrients,
toxics, etc.) may, therefore, lead to drastic shifts in bacterial population
dominance (Kalmbach 1998; Rompré et al. 1998; Sibille et al. 1998; Percical
10 Authors
et al. 2000; Norton and LeChevallier 2000). The fate of undesirable microflora
cannot be attributed to any one behaviour (Figure 4). A washout from the
drinking water distribution system is observed for strains not adapted to such an
oligotrophic environment, i.e. those unable to grow in the biofilm
(Cryptosporidium, Giardia, viruses, etc.), while transitory contamination (from
a few days to a few months) is possible for micro-organisms such as
Pseudomonas, Helicobacter, Campylobacter and some strains of coliforms,
(Fass et al. 1996; Batté 2001; Boualam et al. 2003). Finally, the most
problematic class is that of undesirable micro-flora which have colonised
biofilm, leading to the true formation of a reservoir. These microorganisms
(Mycobacterium spp., Legionella, etc.) can adapt and persist in biofilms which
provide a mosaic of electrochemical and nutritive micro-environments, a chance
for cooperative strategies to succeed and a protection against disinfectant
diffusion (cf. section 6; this chapter): for example Flavobacterium release
cysteine which is favourable to Legionella (Wadowski and Yee 1983); and
micro-colonies (around 50 cells) may correspond to an association of several
species (Manz et al. 1993).
c/c 0
2
3
Time
1 Adapted strains
2 Partially adapted strains
3 Non adapted strains
Figure 4. Possible behaviours of microorganisms in biofilm spiked at To by a single

dose of a microbial contaminant: Q The strain adapts and grows continuously,
leading to a constant leakage into the drinking water; R The strain is stressed b y
the water environment, but adapts with a transitory colonisation. z The strain i s
stressed by the water environment and does not adapt or has no growth potential.
Dashed line : Theoretical dilution rate curve.
However, the behaviour of these pathogens may be misunderstood as the

consequence of choosing an inaccurate detection method. Boualam et al. (2003),
for example, noted that a coliform species may grow (here, evaluated through
the number of cells counted by epifluorescence microscopy) while its
culturability (CFU) is in continuous decline (Figure 5).
In other words, looking for pathogens by using cultures will underestimate
the real number of viable microorganisms, while using RNA probes will
overestimate their number
Cells mL -1 CFU mL-1

1E+5
1E+4
1E+6
1E+3
1E+2
1E+1
1E+5 1E+0
0 50 100 150 200 250 0 50 100 150 200 250
Time (h) Time (h)
Figure 5. Changes of coliform densities in sterile drinking water (0.2 µm filtration)

with different DOC concentrations (: 0.5 mg L-1; : 1.2 mg L-1) evaluated with two
different methods: Total number of coliform bacteria (cells mL-1); culturable
coliform bacteria (CFU mL-1) (adapted from Boualam et al. 2003).
3.1. Coliforms
Fass et al. (1996) demonstrated that a strain of E. coli takes a few minutes to
contaminate the biofilm when it has been introduced rapidly in a single
experimental injection into a drinking water distribution pilot. Following rapid
experimental contamination, 40% of the injected coliforms were found to stick
to the biofilm, whereas one week later 95% of the few coliforms still present in
the network were located in the biofilm. Sibille et al. (1998) also observed that
E. coli introduced experimentally into a network fed with nanofiltred drinking
water survive better because of the low biodiversity of such an oligotrophic
environment and the limitation of protozoan grazing.
12 Authors
3.2. Cryptosporidium, Giardia, virus…

Some pathogens, such as Cryptosporidium, Giardia and enteroviruses, do
not behave in the way coliforms do, since their ecology does not allow them to
settle in the biofilm. However, these micro-organisms, which need hosts in
order to multiply, are able to stick to the biofilm (e.g. Quignon et al. 1997).
Because they are able to survive in water, live through the treatment protocol
and reach the consumer, drinking water may be a route for contamination by
these parasites. In this case, the distribution system constitutes more a route of
contamination than a reservoir problem.
3.3. Helicobacter pylori

Helicobacter pylori demonstrates well the difficulties encountered in trying
to determine whether or not a pathogen is transported in drinking water. This
pathogen is implicated in chronic gastritis, gastric cancer and peptic ulcers
(Ernst and Gold 2000), and several studies have confirmed the presence of the
genetic material of H. pylori (DNA and 16Sr RNA) in drinking water (Hulten et
al. 1996; Park et al. 2001) and drinking water supply (Hulten et al. 1996,
1998; McKeown et al. 1999). The survival of H. pylori in water in a non-
cultivable coccoid form may be as long as 20 to 30 days (Hegarty et al. 1999;
Mizoguchi et al. 1999; Sasaki et al. 1999).
3.4. Non-tuberculosis mycobacteria

Non-tuberculosis mycobacterial species constitute quite a good example of
undesirable water-borne micro-flora. The presence of some of them, namely
Mycobacterium avium, M. intracellulare and M. fortuitum, has been found to
lead to wound infections in AIDS patients through gastro-intestinal tract
contamination, and has also been identified in drinking water systems (Morrisey
et al. 1992; Vess et al. 1993; Von Reyn et al. 1994; Dailloux et al. 1999;
Livanainen et al. 1999; Bodmer et al. 2000; Chang et al. 2002). As well, their
presence in drinking water biofilms has been clearly demonstrated. Schulze-
Robbecke et al. (1992) studied 50 biofilm samples collected from water
treatment plants and domestic water supply systems, and found that 90% tested
positive for Mycobacteria spp. Falkinham et al. (2001) reported the presence of
cultivable M. intracellulare in the biofilms of six drinking water systems
among the eight studied. Mycobacterial species are affected by their
environment, and those found in water leaving a treatment plant differ from
those isolated in a distribution system (Le Dantec et al. 2002). Their presence
may also be explained by their ability to resist disinfection (Bardouniatis et al.
2003). Pelletier et al. (1988) reported that chlorination with 0.15 mg/L of free
chlorine had no bactericidal effect on these species. Several experimental data

suggest a role for protozoa in water environments as a host for mycobacteria
such as M. avium (Cirillo et al. 1997; Steinert et al. 1998).
4. THE LIMITED EFFECT OF CHLORINE
4.1. Relative efficiency of chlorine

Chlorine, chloramine, chlorine dioxide, and chlorine compounds generally, have
been used successfully for many years to disinfect drinking water. Chlorine is
able to limit bacterial regrowth both by injuring bacteria, thereby preventing
their growth, and by limiting the production of bacteria in the system (Figure
6), either in the planktonic phase or in the biofilm (Mathieu et al. 1992).
5
2.8 10
2.4 10 5
∆Biomass per mL (cells out - cells in)
2 105
5
1.6 10
1.2 10 5
8 104
4
4 10
0
Time (days)
70 80 90 100 110 120
Figure 6. Variations in biomass produced in a unchlorinated drinking water

distribution pilot (expressed as the difference between cellsout and cellsin) during a
new chlorination period from day 68 to day 120 (adapted from Fass et al. 2003).
However, the chlorine residual resulting from such a treatment, which

is usually less than 1 mg Cl2/L, is insufficient to kill and remove all the
attached biomass. Even large doses and drastic treatments are quite unable to
eradicate biofilms (Figure 7). Several mechanisms may explain such a resistance
(see paragraph 4.3), but the end result is that most of the biofilm remains alive
Cells or CFU/cm2 Total counts (DAPI staining)

1E+7 Colony Forming Units (CFU)
1E+6
1E+5
1E+4
1E+3
1E+2
1E+1
1E+0
J0 J1 J2 J3 J10 J17 Days
Sodium Chlorine
Hydroxyde
14 Authors
Figure 7. Densities of bacterial cells and culturable bacteria (expressed as colony

forming units on R2A agar plates after 14 days at 20°C) after alkaline treatment (pH
11.8 for 12 hours) followed by chlorination (24 hours; with an initial concentration
of 1,225 mg Cl2/L) (Davrainville et al. 2002).
Another limitation is that chlorination of unchlorinated network

determines a release of adsorbed organic matter (10 µg DOC cm-2) to which the
bacterial cells contribute about 1%. It takes one to two months of continuous
chlorination to release all the desorbable biofilm-entrapped organic matter (with
a chlorination dosage of approximately 3.5 mg Cl 2 l-1 and a resulting chlorine
residual of approximately 0.1 mg Cl2 l-1) (Fass et al. 2003).
4.2 Bacterial injuries generated by chlorine

Very early work by the McFeters group has shown that many bacteria which
are only injured by chlorine are unable to grow on stringent media and need
soft-culture conditions (no inhibitors, low nutrient concentration, longer
incubation period, etc.). Chlorine is able to oxidize superficial structures (i.e.
envelopes), as well as diffuse inside the cells and react with key polymers such
as nucleic acids. This has been demonstrated clearly by staining the bacteria
with fluorochromes, such as in DAPI staining (Saby et al. 1997), and by the
presence of weakly fluorescent bacteria, observed following chlorination with
bleach.
Respiration, cultivability, and even nucleic acids, are affected by
chlorination, as demonstrated by a decrease in (i) CTC+ bacterial densities, (ii)
culturable bacterial densities, and (iii) the fluorescence intensity of DAPI-stained
bacteria (Figure 8). With 1.5 mg Cl2/L applied (residual of up to 0.5 mgCl2/L
after 4 hours), there are still 10 slow-growing bacteria/mL and 200 CTC+
bacteria/mL. At least 90% of the total population is poorly stained by the
fluorochrome DAPI-staining process, which means that the nucleic acids have
been altered in a significant way.
Cells mL-1
1E+6
Total DAPI-stained bacteria
1E+5
Poorly fluorescent
1E+4
DAPI-stained bacteria
1E+3
Limit of detection CTC CTC + bacteria
1E+2
1E+1 C.F.U. (15 days)
1E+0
0 C.F.U. (3 days)
0 1 2 3 4
Time (hours)
Figure 8. Total DAPI-stained bacteria, low-fluorescence bacteria, bacteria CTC+, and

culturable bacteria (CFU 3 days or 15 days) as a function of time and concentration
of NaClO (1.5 mg chlorine L-1) added to drinking water (Saby et al. 2000).
4.3 Resistance to oxidative stress

Most of the disinfectants used in the field of drinking water, e.g. NaClO,
ClO2, chloramines, peracetic acid and their by-products (reactive oxygen and
nitrogen species: OH, O -2, H2O2, ONOO-, NO2, NO)(Nathan and Shiloh 2000),
efficiently alter the micro-organisms. At the same time, however, a large
fraction of the bacterial population escapes disinfection (Morin et al. 1999).
This is because of both the microbial aggregation state (Gauthier et al. 1999c),
which limits diffusion of the oxidant (De Beer et al. 1994b; Stewart and
Raquepas 1995), and the sophisticated antioxidant strategies developed by many
micro-organisms (Storz and Zheng 2000). This resistance might, in principle,
be acquired through selection or adaptation (i.e. genetic induction of resistance
to oxidative stress).
Adaptation to non-chlorinated bactericides has been demonstrated for a

number of micro-organisms (Brözel and Cloete 1993; Dukan and Touati 1996),
which adapt rapidly to hydrogen peroxide (H2O2) via the so-called oxidative
stress response, leading to increased resistance after exposure to low levels of
H2O2. Antioxidant defense systems have also been characterized in E. coli,
where the OxyR and SoxR transcription factors activate antioxidant genes
(Leyval et al. 1984; Christman et al. 1985; Brözel and Cloete 1993).
16 Authors
Bacterial protection against O 2, H2O2, and HOCl has been correlated to

reduced intracellular glutathione: γ-glutamylcysteinylglycine (GSH), the major
non-protein thiol in many gram-negative bacteria, which controls the redox
status of the cells (Meister 1983; Katsuvon and Anderson 1989; Hartford and
Dowds 1992; Chesney et al. 1996; Saby et al. 1999). It is oxidized to
glutathione disulfide (GSSG) and recycled by glutathione reductase.
In this context, Saby (1999) assesses whether or not the resistance of a
bacterial strain to chlorine is an induced response. E. coli (AB1157)
suspensions were subjected to two successive chlorinations at various chlorine
doses, without an intermediate sub-culture. Colony-forming ability was used to
quantify viability. Thiol concentrations (GSH and its precursor, the γ-
glutamylcysteine Glucys) in E. coli AB1157 after the first chlorination were
also measured in order to test their ability to explain bacterial resistance to the
second chlorine exposure. The number of surviving bacteria after the second
chlorination was dependent on the conditions (i.e. dose, time) of the first
chlorination (Figure 9). Generally, the shorter the first chlorination time, the
higher the number of CFU recovered after the second chlorination. Only the low
initial chlorinations that affected cell culturability led to a slight increase in
resistance to a second oxidative exposure, confirming that sub-lethal stress
induces resistance (Christman et al. 1985; Demple and Halbrook, 1983).
B
Surviving bacteria after second chlorination (CFU mL-1)

7
10000000
10
6
1000000
10
5
100000
10
4
10000
10
3 Short chapter title 17
1000
10
2
10100
60 min
1
10 10 120 min
0 240 min
10 1
0 2.8 7 14 28 42
First chlorination dose (µM)
Figure 9. Survival of prechlorinated Escherichia coli to a second chlorination.
Saby (1999) assumed that the first chlorination also induced intracellular
accumulation of scavengers as reduced glutathione (GSH: γ-
glutamylcysteinylglycine). In our study, low and intermediate chlorine doses
induced time-dependent responses in E. coli (Fig. 10). For example, in
comparison to the control without chlorine, exposure of bacteria to a dose of
2.8 µM showed GSH and Glucys increases of 17% and 72% respectively. These
results confirm the hypothesis that E. coli AB1157 reacts against oxidative
stress via a shift of glutathione homeostasis towards synthesis and the uptake of
higher GSH concentrations.
gl
lut
ut
at
a
hi
m
chlorination
Figure 10. Thiol concentrations in E. coli AB1157 after prechlorination for 30 min
(A), 60 min (B), 120 min (C) and 240 min (D). Symbols : reduced glutathione
(GSH); oxidized glutathione forms (GSSG); γ-glutamylcysteine (Glucys).
18 Authors
Values represent data averages from three independent experiments with standard
deviation.
The over-production of GSH (calculated as the difference between the GSH

content of each assay and its own non-pre-chlorinated control) appeared to be
correlated to bacterial survival during the second chlorination. Therefore, the
protection afforded by GSH was not only due to a rapid reaction of this
antioxidant with chlorine oxidants, but also to its role in the signal
transcription of its genetic defense system (oxyr, SoxRS, SOS) (Ding and
Demple, 1996; Demple, 1996; Dukan et al. 1996; Dukan and Touati, 1996;
Zheng et al. 1998; Müller and Janz, 1993).
Because biofilms provide an intrinsic resistance to biocide transfer and

deep zones where bacteria are exposed to sub-lethal traces of oxidants,
disinfection efficiency may be significantly limited by induced resistance.
5. EFFECT OF MATERIALS USED IN DRINKING WATER

DISTRIBUTION SYSTEMS
Most of the pipes used in drinking water distribution systems are made of
plastic (PVC, PE, etc.) or metal (copper, cast iron) which can become highly
corroded (Figure 11). A recent survey of public distribution system pipes in
France showed that a large proportion of them are PVC (40%), while the rest are
grey iron (22%) or ductile iron (20%) (Cador 2002).
A B
Figure 11. Highly corroded cast iron pipes (A) and new PVC pipe (B).
Whatever they are made of, pipes become extensively colonised by micro-
organisms (Niquette et al. 2000; Zacheus et al. 2000). However, the nature of
the pipe material plays a major role in the selection of biomass and its
organisation. Through their roughness, wettability, adhesive properties, etc.,
materials affect the adhesion efficiency of pioneers and may act as a source of
nutrients or growth factors (Pedersen 1990; Rogers et al. 1994; van der Kooij et
al. 1995; Kerr et al. 1999; Bryers 2000; Kielemoes et al. 2000). For example,
analysis based on the detection of cells by the oligonucleotide probe ALF1b has
shown that biofilm can be made up of 4 or 26% alpha proteobacteria, depending
on whether it was formed on glass or on a polyethylene material (Kalmbach et
al. 2000). In another study, polyethylene, PVC, steel and copper materials were
found to be covered with almost equal amounts of biomass, but there was much
less bacterial activity on copper due to the toxicity of the released ions
(Schwartz et al. 2000).
5.1. Plastic materials

The effects of the organic nutrients released by plastic pipes on bacterial
growth in drinking water have long been questioned. Organic additives which
leach out of plastic piping (Brocca et al. 2000; Forslund et al. 1991) have a
measurable impact on biofilm accumulation potential (Mathieu et al. 1998; Kerr
et al. 1999; Van der Kooij 1999; Niquette et al. 2000; Zacheus et al. 2000;
Hallam et al. 2001), and are known to promote the multiplication of
opportunistic, pathogenic bacteria in laboratory tests. However, no field studies
have looked at these events.
Several tests have been carried out to estimate the amounts of
biodegradable compounds released from plastic pipes and the amounts of
biomass growth on these materials (British Standard 1988; DVGW 1990; Van
der Kooij and Veenendaal 1993). The KIWA test (Van der Kooij and
Veenendaal 1993) mainly looks at the biofilm formation potential of various
pipe materials. It also addresses the question of how the compounds released
affect the growth of selected micro-organisms. The static test designed by Van
der Kooij and Veenendaal allows materials to be screened rapidly; it is simple
and permits estimation of the amount of biomass (by directly measuring the
amount of ATP released) in the water and in the biofilm (see the chapter written
by D. van der Kooij; this book). However, static tests cannot predict the effects
of plastic material on the quality or safety of drinking water in distribution
systems (i.e. in dynamic conditions). Indeed, in real systems, biomass
production leading to contamination of the bulk water is governed not only by
the organic material released from plastic pipes, but also by the flux of
biodegradable organic matter carried by the drinking water. Furthermore, the
release of bacterial biofilms into the bulk water is independent of the mass and
20 Authors
thickness of the biofilm. The material does not always affect water quality in
real conditions, as flowing drinking water can be highly nutritive (Zacheus et
al. 2000) or contain disinfectants (Hallam et al. 2001). In other words, it is
only through dynamic testing (e.g. continuous flow through a reactor, see
section 5.3, this chapter) that an accurate way of testing materials and their
effect on the bacteriological quality of drinking water may be found.
5.2. Iron-based corroded materials

Corrosion of metals in contact with drinking water is an electrochemical
process with anodic oxidation of the metal and cathodic reduction of species
such as H+, O2 or H2O (see the chapter written by W. Sand, this book). It leads,
for example, to the formation of a mixture of iron oxide-bearing and iron(II)-
bearing minerals (Figure 12).
The isoelectric point of such corrosion products (for example goethite) is
between 8 and 9, which facilitates the adhesion of negatively charged bacteria
(isoelectric point lower than 3) (Appenzeller et al. 2002; van der Wal et al.
2003). Therefore, the microbial colonisation of pipe walls is probably promoted
when they are highly corroded. As shown in Figure 13, biomass production is
very considerable in highly corroded pipes compared to lightly corroded ones.
Figure 12. Some examples of corrosion products scraped from a corrosion tubercle:
A, magnetite (Fe3O4); B, goethite (α-FeOOH); and C, carbonated green rust
(Fe6(OH)12CO3) (from Appenzeller 2002).
3.50E+06
3.00E+06
2.50E+06
2.00E+06
cells/mL
cells/mL
1.50E+06
1.00E+06
5.00E+05
0.00E+00
Stainless steel Corroded cast iron
Figure 13 : Bacterial production in number of cells per millilitre of water (difference

between entry into and exit from the reactor) in a Propella™ reactor fed
continuously with drinking water (Hydraulic residence time of 24 hours,
temperature of 25°C) (adapted from Appenzeller et al. 2001).
How iron oxy-hydroxides favour bacterial growth is not well known. It has
been shown that they increase the growth of aerobic gram-negative bacteria like
Pseudomonas sp., probably because they were used as a nutrient (Hersman et al.
2000; Hersman et al. 2001). In addition, the use of iron as a terminal electron
acceptor in a respiratory chain (anaerobic respiration) is also well-known in
several bacterial strains (Lovley 1995). Iron corrosion products have also been
reported to support the growth of E. coli (Victoreen 1984; LeChevallier et al.
1993). Complementary assays showed that the culturability of E. coli (CFU) in
drinking water can be maintained for 550 h in the presence of iron oxy-
hydroxide, compared to the results of an assay conducted in the absence of iron
oxy-hydroxide, in aerobic as well as anaerobic conditions (Figure 14).
22 Authors
1,00E+07 CFU mL-1

cells mL-1
1,00E+06
1,00E+05
aerobic cond. aerobic anaerobic anaerobic
cond./lepido cond. cond./lepido
Figure 13. Total and cultivable E. coli after starvation for 23 days in sterilised
drinking water (T = 25°C; pH = 8; initial total cells and CFU concentration were
around 2 106 mL-1) in aerobic (moderate aeration by gentle shaking) and anaerobic
conditions, with and without 50 mM lepidocrocite (γ-FeOOH) added to the media
(Block et al. 2002).
To conclude, it is clear that the control of corrosion by decreasing water

aggressivity or by anti-corrosion treatments (see section 6 this chapter) is needed
to limit the excessive growth of biomass in the network and to alleviate the
coliform nightmare.
5.3. Lab scale testing the effect of material on biofilm and

drinking water
The lack of information on biofilm dynamics is a limiting factor in
managing the quality of water in distribution system and conducting drinking
water surveys. In spite of the difficulty of gaining access to the inner surfaces of
distribution pipes, biofilm measurement on pipe walls is indispensable if more
information on the water contamination risks is to be obtained. New methods
need to be developed, adapted, evaluated and optimised. Such methods will
create important advantages: continuous, non-destructive, simple, in situ, on-
line information on biofilm location and development (see the chapter written
by L. Melo, this book). However, biofilm monitoring requires laboratory-scale
tools adapted to biofilm sampling, so that the hydraulics of real distribution
systems can be reproduced as closely as possible.
PropellaTM and annular reactors such as RototorqueTM may be considered to

be the best laboratoty-scale systems currently available, since they permit the
simultaneous control of temperature, flow velocity, hydraulic residence time and
Reynolds number, as well as the nature of the material being tested (Figure 15).
motor Axis to motorise the

rotation of the internal
cylindre
water inlet water outlet Inlet
Internal rotating
cylindre
drilled of two
coupon Outlet tunnels
propeller
Cork to allow slide

inner sampling
cylinder
blade Slide on the

internal surface
stainless steel Coupons

PVC, PP or PE
External cylindre
equipped with coupons
control of and slides to sample
temperature biofilm
Figure 14. Schematic representation of a Propella™ and a RototorqueTM reactor

(adapted from Appenzeller et al. 2001 and Batté 2001).
The PropellaTM reactor is a perfectly mixed reactor, in which the drinking

water is pushed by a propulsive propeller through an internal tube. It is easy to
impose a defined hydraulic regime (Reynolds number) by fixing the water
circulation rate in the pipe. Using this reactor, the biostability of the drinking
water can be measured, and biofilm analysis on coupons and adaptation of on-
line sensors can be easily performed.
The RotoTorqueTM consists of two concentric cylinders, a stationary outer
cylinder and a rotating inner cylinder. Twelve removable slides form an integral
part of the inner wall of outer cylinder and permit the sampling of any biofilm
growing on them. The bulk liquid is mixed by cylinder rotation and by draft
tubes bored through the solid inner cylinder. The rotation of the inner cylinder
causes fluid to rise in the draft tubes, resulting in internal recirculation. Sensor
heads can be implemented both in the outer cylinder and on the slides, in order
to test on-line and in-situ monitoring devices.
24 Authors
6. PHOSPHATE BASED ANTICORROSION TREATMENT
6.1. Who is afraid of phosphate?

Phosphate addition is a recent practice in drinking water treatment which
capitalises on the ability of phosphate to react with cations such as FeII, FeIII
and PbII (Hiemstra and Van Riemsdijk 1996; Persson et al. 1996; Appenzeller
et al. 2001). Specifically, it leads to passivation of the metal surface, which
slows down metal oxidation by forming a stable complex at this surface.
Phosphate treatment is aimed at improving drinking water by limiting iron pipe
corrosion in distribution systems, limiting copper and lead dissolution (Besner
et al. 2002) and exercising better control of red waters (Lytle and Snoeyink
2002). Several phosphate-based products may be used, from simple ortho-
phosphoric acid to various phosphate blends of ortho-phosphate and poly-
phosphate molecules. In all cases, phosphate addition is limited by regulations
which defining a maximum concentration of 5 mg P205 /L in drinking water
(i.e. 1.1 mg P/L).
The practice of adding phosphate in this way has raised questions about the
possible effects of phosphate on bacterial growth in drinking water distribution
systems (Miettinen et al. 1997; Sathasivan et al. 1997). The main point is that
phosphate is one of the major nutrients required by bacteria, since it is their
usual source of phosphorus which is vital to their survival, as it is for any
living cell. Phosphates are important because: (i) they are a constituent of bio-
molecules such as DNA, RNA, ATP, polyphosphate granules, etc.; and (ii) they
participate in various vital functions (through energy delivery or regulation of
cellular physiology) (Kornberg 1994; Shiba et al. 1997, Kornberg and Fraley
2000; Rashid et al. 2000a, b). Finally, since bacterial cells without phosphate
uptake contain around 1% (w/w) phosphorus, bacteria need to incorporate
phosphates continuously in order to survive and multiply.
No phosphate treatment can be performed without considering the
induced microbiological response. Most drinking waters usually contain less
than 10 µg P /L. The main microbiological risk of adding phosphate, therefore,
would be a change in microbial physiology and bacterial dominance, as well as
increased health concerns and the emergence of opportunistic pathogens in
drinking water distribution systems.
6.2. The response of biomass to phosphate

There is no consensus in the literature about the effect of phosphate on
bacterial populations in drinking water distributions systems. There are at least
three reasons for this:
(1) tested waters may not be phosphate-limited, in which case the addition
of phosphate will not change the “life” of micro-organisms (Appenzeller
et al. 2001);
(2) laboratory tests carried out in clean glass flasks to measure the
phosphorus available to bacteria (Lethola et al. 1999; Sathasivan and
Ohgaski, 1999) do not take into account the reactivity of corroded
surfaces and the potential effect of such treatments on reduced bacterial
adhesion and on the limitation of bacterial activity (Appenzeller et al.
2001 and 2002);
(3) total biomass may not be affected by phosphate treatment (Batté et al.
2003), in particular when there is room for improvement in the
culturability of some heterotrophic bacteria and some bacterial species
(Batté et al. submitted).
A review of several publications reveals that any addition of phosphate to

drinking water distribution systems leads either to a very limited change in
biomass (Appenzeller et al. 2001; Frias et al. 2001; Batté et al. 2003) or to a
significant increase in biomass (Lethola et al 2002). For example, Fass
(personal communication) has measured an increase in biofilm density by a
factor two in a pipe loop pilot system following a phosphate treatment of
several weeks’ duration. In the case of phosphorus-limited waters, Lethola et al.
(2002) noted that the addition of 1 µg /L of phosphorus doubled the total
number of bacteria in the biofilm. Also, Keinänen et al. (2002) have reported
phospholipid fatty acid changes in biofilm following phosphate treatment.
Batté et al. (submitted) have noted that phosphate treatment can enhance the
proportion of gamma-Proteobacteria measured by FISH, although the total cell
counts were not affected by P-treatment (Figure 16). Appenzeller (personal
communication) also noted that the number of selected species, such as
Legionella sp., increased following phosphate treatment of a corroded propella
reactor. Finally, Abernathy and Camper (1998) indirectly demonstrated that
phosphate treatment influences the efficiency of biofilm disinfection.
Bacterial density
(Cells or CFU) cm-2
1E+8
1E+7
1E+6
1E+5
Total cells PEPA CFU
26 Authors
Figure 15. Impact of phosphate addition on Total Direct Counts (total cells (cells
cm-2)), Potential Exoproteolytic Activity (PEPA (cells cm-2)) and Heterotrophic Plate
Counts (CFU cm-2) of 15-week-old biofilms exposed to drinking water with (grey
columns) or without (white columns) phosphate addition. Phosphate treatment was
applied after the 9th week of colonisation. Each point is a mean value derived from
duplicate reactor and duplicate sample measurements. Error bars represent standard
deviations (adapted from Batté et al. 2003).
6.3 Corrosion and biomass limitation through phosphate

treatment
The effect of phosphate addition on biofilm is clearly dependent on the
environmental conditions occurring in the drinking water system. In a corroded
environment, phosphate treatment has a combined effect on the corrosion
reaction and on the bacterial population.
Corrosion limitation induced by phosphate treatment leads to a decrease of
the bacterial number in the biofilm (LeChevallier et al. 1993; Abernathy and
Camper 1998; Rompré et al. 2000; Appenzeller et al. 2001) as in, for example,
the drastic decrease in the bacterial production of phosphate-treated cast iron
reactors (Figure 17). Such an unexpected change may be related either to the fact
that there is less bacterial adhesion on the negatively charged phosphated iron
oxides (Appenzeller et al. 2002), or to the reduced bioavailability of FeII by
stabilisation of some iron oxide-phosphate complexes.
3.5 E+6
Without phosphate
3.0 E+6
With phosphate
Produced bacteria (cells/mL)
2.5 E+6
2.0 E+6
1.5 E+6
1.0 E+6
0.5 E+6
0 E+6
(0.5 E+6)
Stainless steel Cast iron
Figure 16. Produced bacteria (expressed as the difference between the outlet and the
inlet of the reactor) in a stainless steel reactor and in a corroded cast iron reactor,
with and without 1 mg P-PO4/L addition (n = 4 to 10 according to the assay)

(Appenzeller et al. 2001).
Phosphate treatment in cases of corroded iron has some significant

limitations. First, it is not able to limit the corrosion reaction completely.
Second, since the phosphate changes the nature of iron oxides (from well
crystallised to a soft and more amorphous material), the red water problem is
not always solved.
7. CONCLUSIONS
The microbial activity in distribution systems occurs in water, on pipe
surfaces and in all kinds of deposits which have accumulated in pipes and
storage tanks. A large number of microbial cells are generally counted on
surfaces in contact with drinking water (in the order of 106 to 108 cells per cm2).
Through their roughness, wettability, adhesive properties, etc., materials affect
the adhesion efficiency of pioneers and may act as a source of nutrients or
growth factors
Although new material exposed to drinking water colonises rapidly, a
stationary state is never reached in a real network due to frequent discontinuities
Deposit accumulation in distribution systems is generated by many

mechanisms, among them the precipitation, flocculation and settling of material
during water treatment or the corrosion of pipe surfaces. Deposit densities
varying from 0.01 to 40 g/m2 were measured in several netwoks. Total cell
counts (epifluorescence microscopy after acridine orange staining) were found to
be in the range of 1010 to 1011 cells/g of deposit
Various undesirable water-borne microorganisms are more or less

systematically present in drinking water distribution systems and may cause
epidemic events. These undesirable flora implant biofilm or deposits, which
may then be regarded as reservoirs of opportunistic pathogens
Biomass production is very considerable in highly corroded pipes compared

to lightly corroded ones. The control of corrosion by decreasing water
aggressivity or by anti-corrosion treatments is needed to limit the excessive
growth of biomass in the network
Prediction of the material impact on the biofilm is impossible and need to be

tested in careffully controled dynamic conditions. Biofilm monitoring requires
28 Authors
laboratory-scale tools adapted to biofilm sampling. PropellaTM and annular

reactors such as RototorqueTM may be considered to be the best systems
currently available, since they permit the simultaneous control of temperature,
flow velocity, hydraulic residence time and Reynolds number, as well as the
nature of the material being tested.
Phosphate treatment is aimed at improving drinking water by limiting iron

pipe corrosion in distribution systems, limiting copper and lead dissolution and
exercising better control of red waters. The main microbiological risk of adding
phosphate, therefore, would be a change in microbial physiology and bacterial
dominance, as well as increased health concerns and the emergence of
opportunistic pathogens in drinking water distribution systems.
In practice any addition of phosphate to drinking water distribution systems
leads either to a very limited change in biomass or to a significant increase in
biomass.
Biofilm response to phosphate is highly dependent to the environment
characteristics. The corrosion products clearly affect the biofilm behaviour
during a phosphate treatment of corroded cast iron pipes.
The complex response of biofilm to phosphate treatment suggests that
simple flask test is not sufficient to evaluate the risk of growth in distribution
system. Indeed the risk of phosphate-based growth can be strongly balanced by
the reduction of corrosion and bacterial activity. The use of bench-scale test
including corrosion products is highly recommended.
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Biofilms in drinking water

Some environments, such as ground waters or waters treated by

Figure 1. Reaction mechanisms involved in the accumulation of biofilms on a

This bacterial population is adapted to the oligotrophic environment of

µg of biodegradable organic matter per litre of drinking water), the bacterial

(1) The system is continuously inoculated with unknown micro-organisms

2. BIOFILMS AND LOOSE DEPOSITS

However, by analogy, description of the biofilms found in drinking water

Bacterial density Cells marked with DAPI

Figure 2. Colonisation of polycarbonate coupons exposed to drinking water for 1 6

Table 1 Concentration of total carbohydrates, total amino acids and phosphate i n

Bacterial density Carbohydrates Amino Acids

Biofilms accumulate on the surface of materials in zones where water

2.1 Loose deposits

Fraction of bacteria in the

Figure 3. Biofilm fraction of cultivable bacteria in a 1-m pipe section – compared t o

diameter (hypotheses: average concentration of cultivable bacteria in biofilms: 105

Therefore, it would be worth managing distribution systems to limit

3. THE FATE OF UNDESIRABLE MICROFLORA

2 Partially adapted strains

3 Non adapted strains

Figure 4. Possible behaviours of microorganisms in biofilm spiked at To by a single

However, the behaviour of these pathogens may be misunderstood as the

Cells mL -1 CFU mL-1

Figure 5. Changes of coliform densities in sterile drinking water (0.2 µm filtration)

3.2. Cryptosporidium, Giardia, virus…

3.3. Helicobacter pylori

3.4. Non-tuberculosis mycobacteria

chlorine had no bactericidal effect on these species. Several experimental data

4. THE LIMITED EFFECT OF CHLORINE

4.1. Relative efficiency of chlorine

Figure 6. Variations in biomass produced in a unchlorinated drinking water

However, the chlorine residual resulting from such a treatment, which

Cells or CFU/cm2 Total counts (DAPI staining)

Figure 7. Densities of bacterial cells and culturable bacteria (expressed as colony

Another limitation is that chlorination of unchlorinated network

4.2 Bacterial injuries generated by chlorine

Figure 8. Total DAPI-stained bacteria, low-fluorescence bacteria, bacteria CTC+, and

4.3 Resistance to oxidative stress

Adaptation to non-chlorinated bactericides has been demonstrated for a

Bacterial protection against O 2, H2O2, and HOCl has been correlated to

Surviving bacteria after second chlorination (CFU mL-1)

Figure 9. Survival of prechlorinated Escherichia coli to a second chlorination.

The over-production of GSH (calculated as the difference between the GSH

Because biofilms provide an intrinsic resistance to biocide transfer and

5. EFFECT OF MATERIALS USED IN DRINKING WATER

5.1. Plastic materials

5.2. Iron-based corroded materials

Figure 13 : Bacterial production in number of cells per millilitre of water (difference

1,00E+07 CFU mL-1

To conclude, it is clear that the control of corrosion by decreasing water

5.3. Lab scale testing the effect of material on biofilm and

PropellaTM and annular reactors such as RototorqueTM may be considered to

motor Axis to motorise the

Cork to allow slide

blade Slide on the

stainless steel Coupons

Figure 14. Schematic representation of a Propella™ and a RototorqueTM reactor

The PropellaTM reactor is a perfectly mixed reactor, in which the drinking

6. PHOSPHATE BASED ANTICORROSION TREATMENT