Ozone: Science & Engineering, 31: 39

Copyright # 2009 International Ozone Association

ISSN: 0191-9512 print / 1547-6545 online
DOI: 10.1080/01919510802586566

Studies on the Disinfection and Removal of Biofilms by

Ozone Water Using an Artificial Microbial Biofilm System
Mariko Tachikawa,1 Kenzo Yamanaka,1 and Katsuhiko Nakamuro2
1
2

College of Pharmacy, Nihon University, 7-7-1 Narashinodai, Funabashi, Japan

Faculty of Pharmaceutical Sciences, Setsunan University, 45-1 Nagaotougemachi, Hirakata, Japan

Inactivation rates of the biofilms of P. fluorescence and

P. aeruginosa established on a small slide glass in ozone
water (0.93.2 mg/L, 120 min) were determined in a
batch or flow-through system. The effects of ozone water
on the biofilm matrices were defined clearly in situ by
confocal laser scanning microscopy. These results indicate
that ozone is an effective biocide against biofilms and it can
remove exopolysaccharides in the biofilm matrices. However, the effective concentration of ozone for disinfection of
biofilms varied with the biofilms formed, mainly due to
reactions of ozone with constituents of the biofilms.
Keywords

Ozone, Disinfection Efficacy, Biofilms, P. fluorescens,

P. aeruginosa, Removal, Extracellular Polysaccharides

INTRODUCTION
The disinfection and removal of biofilms has become
an important subject in maintaining water quality management in the fields of swimming pools, food processing
lines, industrial water systems, etc. Microorganisms, by
attaching to surfaces to form biofilms which are protected
by matrices of excreted exopolysaccharides (EPS), are
usually highly resistant to antimicrobial agents. The
importance of tests using a biofilm system has been
pointed out in order to evaluate disinfection efficacy of
biocides (LeChevallier et al., 1988a and Wright et al.,
1991). We have attempted to establish a simple method
of producing microbial biofilm from ubiquitous bacteria,
Pseudomonas fluorescens, Pseudomonas aeruginosa and
Klebsiella pnuemoniae in water environments and in
biofilms, and compared the efficacy of several halogen
biocides using the biofilms established. By using confocal
Received 4/7/2008; Accepted 8/20/2008
Address correspondence to Mariko Tachikawa, College of
Pharmacy, Nihon University, 7-7-1 Narashinodai, Funabashi,
Japan 274-8555. E-mail: tachikaw@pha.nihon-u.ac.jp

laser scanning microscopy (CLSM), differences in cell

density and structure among the biofilms established were
visualized clearly, and the changes of biofilm structures
caused by halogen biocides were described (Tachikawa
et al., 2005).
Although ozone water is widely used as a potent
oxidant in water treatments (White, 1999), the efficacy
of ozone water for biofilm disinfection remains to be
established. Thomas et al. (2004) studied the resistance
of the amoebae, Legionella pneumophila, and biofilms of
b-proteobacteria to disinfection treatments with ozone,
chlorine dioxide, chlorine, etc. in pilot-scale domestic
water systems. Planktonic and biofilm populations in
the systems were reduced markedly, but they were still
detectable after ozone treatment at 0.5 mg/L, which
decreased to the undetectable limit (<0.1 mg/L) at the
end of the contact column. These results may indicate
the importance of choosing a good exposure system and
an optimal ozone concentration for disinfection of the
biofilms.
The purpose of the present study is to evaluate the
changes of resistibility of bacterial cells to ozone after
forming biofilms and to determine inactivation rates of
biofilms by ozone using a biofilm model established on a
glass slide. The survival fractions of biofilm-retained cells
and suspended cells, which were prepared by ultrasonic
dispersion of the biofilm, were compared after the treatment with ozone at 1.51.7 mg/L in a batch system. Since
it is necessary to keep a constant ozone concentration and
minimize the water stream force during exposure for
evaluation of the effectiveness of ozone against biofilms,
a glass apparatus for continuous flow- through exposure
was devised. Using this glass apparatus, P. fluorescens
and P. aeruginosa biofilms were treated with ozone
water for an extended duration ( 20 min.) at a constant
ozone concentration (0.9  3.2 mg/L). Inactivation rates
of the biofilms were obtained and compared. The changes

Disinfection and Removal of Biofilms by Ozone

JanuaryFebruary 2009

of biofilm structures by ozone exposure were observed in

situ by CLSM after staining with a fluorescent dye,
LIVE/DEADBacLightTM. The effects of ozone water
on the matrix EPS of P. fluorescens biofilms were visualized in situ by confocal laser scanning microscopy
(CLSM) after staining with a fluorescent conjugate of
concanavaline A, a lectin binding to oligosaccharide
selectively. Through these experiments, the implications
of EPS and cell density in the disinfection treatments with
ozone against biofilms would be clarified.

EXPERIMENTAL
Bacterial Strains and Preparation of Biofilm
Pseudomonas fluorescens (JCM no. 2779) and
Pseudomonas aeruginosa (JCM no. 2776) were obtained
from the Japan Collection of Microorganisms (JCM),
Wako, Japan. They were Gram-negative, rod-shaped ubiquitous microorganisms, found in our daily environments
such as biofilms, water, soil, humans, sewage, hospitals,
etc. P. aeruginosa is an opportunistic human pathogen
(Kapatral et al., 2000). Their stock cultures were kept at
80 C with 25% (wt/vol) glycerol. Luria-Bertanis (LB)
medium was used for pre-cultivation of these bacteria,
overnight at 28 C. The biofilms of P. fluorescens were
grown on clean and sterile microscope slides (14 26 mm)
placed in a glass culture dish (i.d. 145 mm) with 150 ml of
EPS growth medium, containing 1% of glucose and phosphate and small amounts of minerals (LeChevallier et al.,
1988b), which was inoculated with each overnight culture.
For the formation of biofilms of P. aeruginosa, 50 mL of
LB medium was added to 100 mL of EPS growth medium.
The dishes were incubated at 28  C with continuous slow
stirring with magnetic stirrers. The number of viable cells
in the biofilms formed on the slide was determined by
colony counting on tryptone glucose yeast agar (APHA,
AWWA, WEF, 1992a) following ultrasonic dispersion and
serial dilution.
Water
Milli Q water was used for preparation and dilution
of reagent solutions for the determination of available
chlorine and ozone. For the preparation of ozone water,
tap water distributed by Funabashi municipal water supply was dechlorinated by passing through an activated
carbon column and then led to an ozone water generator
(AOD-TH, Ai Electronic Ind. Co. Ltd, Japan). The tap
water containing 0.6 0.7 mg/L of free residual chlorine
was used as a comparative control for ozone water. The
temperature of the test water ranged between 14 and
18  C and its pH was 6.56.7. Concentrations of residual
chlorine and ozone in test water were determined by the
DPD (N,N-diethyl-p-phenylenediamine, APHA, AWWA,
WEF, 1992b) and indigo colorimetric (APHA, AWWA,
WEF, 1992c) methods, respectively.
4

Inactivation Experiments
Biofilms formed on the slide glass were passed through
sterile water twice to remove planktonic cells and growth
medium. For the batch treatment, the biofilm formed on
a slide glass was placed in a sterile flask containing 40 mL
of test water for 5 to 10 min at room temperature. A
suspended cell fraction was prepared by placing a glass
slide of biofilms established in a sterile flask containing
5 mL of sterile water and sonicating for 90 sec. 40 mL of
ozone water was added into the dispersed cell suspension,
and the cells were treated for 5 min. For the continuous
flow-through treatment, a glass apparatus shown in Figure 1
was used. By using this apparatus, the ozone concentration was kept constant, and mechanical removal of
biofilms by water stream could be minimized. The volume
of the apparatus was ca. 500 mL and test water was
circulated at a rate of 25 mL/sec. Biofilms on the slide
glass were placed onto the wire-net stool placed in the
apparatus. After the treatment, the slide was placed immediately into a flask containing a sterile solution of thiosulfate for neutralization of residual ozone and chlorine.
Colony forming units (CFU) of the biofilms in the flask
solution were determined after ultrasonic dispersion and
serial dilution as described above.
CLSM Observations
For fluorescent staining of cells in the biofilms, LIVE/
DEADBacLightTM, a mixture of SYTO 9 and propidium iodide, Molecular Probes Inc. OR, was used. For
fluorescent staining of EPS in a biofilm matrix, Alexa
Fluor 633 conjugate of concanavalin A (ConA-Fluor),
Molecular Probes Inc. OR, was used. Biofilms on the

Water flow :
FIGURE 1. Schematic diagram of the flow-through exposure
apparatus.

M. Tachikawa, K. Yamanaka, and K. Nakamuro

JanuaryFebruary 2009

glass slide were soaked and incubated in the dye solution

for 15 min in the dark at room temperature (Tachikawa
et al., 2005). A 0.1 mL of water was dropped on a cover
glass (25 50 mm), and then the slide of stained biofilms
was placed upside down. Photomicrographs were taken at
a magnification of x100 with an oil immersion lens under
a CLSM (LSM 510, Carl Zeiss). Computer image micrographs of the vertical section of biofilms were obtained by
using the Z-stack function of the LSM 510.
RESULTS AND DISCUSSION
Formation of Biofilms
The number of viable cells in the biofilms of P. fluorescens and P. aeruginosa formed on the glass slide was
determined after incubation for 1 to 9 days (Figure 2).
After incubation for 6 days, the mean cell density of the
biofilms of P. fluorescens reached 2.5 0.5 108 cfu / slide.
That of the P. aeruginosa biofilms reached a plateau of
1.9 0.2 x 107 cfu / slide after 2 days of incubation. The
variation of the numbers of cfu between the biofilms formed
on the slide was small enough for evaluation of the disinfection efficacy of biocides.
Effects of Biofilm Formation on Cellular Resistance
to Ozone Water
For investigation of changes of disinfection resistibility
of cells against ozone by forming biofilms, the biofilms
and suspended cells of P. fluorescens and P. aeruginosa
were treated with ozone water at 1.7 and 1.6 mg/L for,
respectively, 5 min in the batch-type system. In both
bacteria, the biofilm cells were more resistant to ozone
water than the suspended cells (Table 1). Although the
survival fractions of suspended P. fluorescens and
P. aeruginosa were similar, i.e., 0.006 % and 0.008 %,

respectively, those in their biofilms differed greatly, i.e.,

22.7 % and 0.13 %, respectively. By forming biofilms,
P. fluorescens and P. aeruginosa increased their resistibility
against ozone more than 3000 and 10 times, respectively. It
is known that, by forming biofilms, sessile cells may have
altered their sensitivity toward antimicrobial agents not
only by physical changes of their environment but also
by changes of their gene expression (Costerton and
Lewandowski, 1997). Therefore, the difference in sensitivity
between biofilm-retained cells and suspended cells in the
present experiment may represent physical hindrance to
ozone penetration by biofilm formation. In a previous
study (Tachikawa et al., 2005), we determined survival
fractions of biofilm and suspended cells of P. fluorescence
and P. aeruginosa after treatments with hypochlorite (HOCl)
and ammonia monochloramine (NH2Cl) at 1.21.7 mg/L
for 5min. By forming biofilms, P. fluorescence increased its
resistibility to HOCl and NH2Cl 8 and 2 times, respectively,
and P. aeruginosa increased its resistibility to HOCl and
NH2Cl, 40 and 7 times, respectively. Compared to these
results, P. fluorescens greatly increased its disinfection
resistibility against ozone by forming biofilms.
Biofilms of P. fluorescens were treated with ozone at
different concentrations for different time intervals in the
batch-type system. The survival fractions of biofilm cells
treated by ozone at 0.6 and 1.7 mg/L for 5 min were 42 %
and 18 %, respectively, and those treated for 10 min were
31 % and 14 %, respectively (Table 2). Thus increasing
the exposure time did not cause further decrease in the
survival fraction at both ozone concentrations. Residual
ozone in the test water of the batch system was determined under similar conditions (Table 3). It indicated
that more than 50% of initial ozone concentrations
were remained in 10 min at both concentrations, and
that the ozone consumption by biofilm was very slight.

1010

Cfu / Glass slide

109
108

P. aeruginosa
P. fluorescens

107
106
105
104
103

5
6
Time (days)

10

), with a bar represents a mean S.D.

FIGURE 2. Growth of biofilms on incubation at 28  C. Each point, P. aeruginosa (~) and P. fluorescens (
of cfu on the glass slide (n = 36).

Disinfection and Removal of Biofilms by Ozone

JanuaryFebruary 2009

TABLE 1. Disinfection Efficacy of Ozone Water on Biofilm-Retained and Suspended Cells of P. fluorescens and P. aeruginosa in the Batch
Treatment for 5 min. Mean S.D.(n = 3)

Biofilm cells cfu / plate ( 106)

Microorganisms

O3 (mg/L)

(%)b)

O3 (mg/L)

Treated

(%)b)

22.7 15.5

1.5

0.009 0.012

0.006 0.008

0.13 0.04

1.4

0.002 0.001

0.008 0.004

Treated

P. fluorescens

control

159 26

P. aeruginosa

1.7
control
1.6

36.1 24.7
23.8 9.7
0.03 0.01

Suspended cellsa) cfu / suspension ( 106)

a)

Suspended cells were obtained by ultrasonic dispersion of the biofilms established on glass plates. Therefore, the control biofilm-retained and
suspended cells should contain almost the same number of cfu.
b)
(cfu treated / cfu control) 100.

(Figure 3a and 3b). As shown in Figure 3a, the survival

fractions of the biofilms of P. fluorescens after the treatment with ozone water at 0.9 and 1.4 mg/L for 5 min
exposure were ca. 1%, and the inactivation rates became
slow with increasing exposure time. With a higher concentration of ozone at 3.2 mg/L, the survival fractions of
the biofilms reached 0.01% in 5 min and further
decreased to 0.00002% in 20 min. The survival fraction
of the biofilm of P. aeruginosa after the treatment with
ozone water at 1.0 and 3.0 mg/L for 5 min were ca. 0.2
and 0.003 %, respectively, and then their inactivation rates
declined gradually as shown in Figure 3b. At the lower
concentrations of ozone, the inactivation rates were retarded
in both bacteria and the retardation was lessened at the
higher concentrations. The changes of inactivation rates
during the treatment may support the occurrence of diffusional resistance to ozone within the biofilm as suggested by
Viera et al. (1999). The inactivation rates in chlorinated tap
water also became slow with increasing exposure time in
both biofilms. This indicates that chlorine as well as ozone
encounters diffusional resistance within the biofilms. The
different inactivation rates and their changes during the
treatment between P. fluorescens and P. aeruginosa may
indicate that the diffusional impediment may vary with
ozone concentrations and constituents of the biofilms,
such as biomass, growth medium, EPS excreted etc.
Effective concentrations of ozone for disinfection of

TABLE 2. Effects of Concentration and Time on the Disinfection

Efficacy of Ozone Water on the Biofilms of P. fluorescens in Batch
Treatments. Mean S.D. (n = 3)

O3 mg/L
0 (control)
0.6
0.6
1.7
1.7
a)

Exposure
Biofilm cells
Survival
time min. cfu / plate ( 106) fractiona) %
380
161
118
69
54

5
10
5
10

43
82
24
4
8

100
42
31
18
14

21
6
1
2

(cfu treated / cfu control) 100.

The decrease of ozone concentration may be mainly due

to the decomposition of ozone molecules in water
(Staehelin and Hoigne, 1982; Buhler et al., 1984;
Staehelin et al., 1984). Therefore the slowdown of inactivation rate with increasing exposure time could not be
explained only by the decrease in ozone concentration.
Viera et al. (1999) suggested occurrence of diffusional
resistance to ozone penetration into biofilms by reactions
of ozone with constituents of the biofilms.
For evaluation of inactivation rates of biofilms
in ozone water, biofilms of P. fluorescens and P. aeruginosa were treated with ozone water and chlorinated tap
water in a flow-through system for 1 to 20 min

TABLE 3. Changes of Ozone Concentrations with Increasing Exposure Time in the Batch Treatments of
Biofilms of P. fluorescens. Mean S.D. (n  3) or Ranges (n = 2)

Residual O3 (mg/L) in test water at exposure time (min)

0
a)

Low O3 water

0.48

0.51

High O3 water

1.57 0.03a)

10

0.43 0.04

0.35 0.01

1.09

1.27

0.26 0.01
(0.29 0.30)b)
0.96 1.04
(0.99 1.07)b)

1.51

1.30

a)

Concentration of ozone water determined just before a glass plate of P. fluorescens biofilm was placed in the flask.
Concentration of ozone water in the flask without biofilms.

b)

M. Tachikawa, K. Yamanaka, and K. Nakamuro

JanuaryFebruary 2009

(a) P. fluorescens

Ozone 3.2 mg/L

Ozone 1.4 mg/L
Ozone 0.9 mg/L
Chlorine* 0.6 mg/L

100

(a) P. fluorescens biofilm (control).

Cover glass

Survival fraction (%)

10
1
0.1
Slide glass
0.01

(b) P. fluorescens biofilm treated with ozone water (1.2 mg/L) for 1min.

0.001
Cover glass

0.0001
0.00001

10
15
Time (min)

25
Slide glass

(b) P. aeruginosa

Ozone 3.0 mg/L

Ozone 1.3 mg/L
Ozone 1.0 mg/L
Chlorine* 0.7 mg/L

100
10
Survival fraction (%)

20

(c) P. aeruginosa biofilm (control).

Cover glass

1
0.1
0.01

Slide glass
(d) P. aeruginosa biofilm treated with ozone water (1.1 mg/L) for 1 min.
Cover glass

0.001
0.0001
0.00001
0

10

15

20

25

Time (min)
Slide glass

FIGURE 3. Efficacy of ozone water (ozone,

, ~ and &)
and chlorinated tap water (free chlorine*,*) on the biofilms of
P. fluorescens (a) and P. aeruginosa (b) treated in the flowthrough system at indicated ozone concentrations. Each point
with a bar represents the mean cfu value and S.D. of 3 slides.

biofilms may vary with the biofilm matrix formed.

Therefore, the concept of CT (concentration time),
which is used for the evaluation of disinfection efficacy of
biocides for planktonic cells, could not be applied to the
evaluation of disinfection efficacy of ozone for biofilms.

Observation of Biofilm Matrixes by CLSM and

Effects of Ozone Water
Since the results obtained above strongly suggest the
participation of biofilm matrices in the biocidal efficacy
of ozone, changes of biofilm matrix by the treatment with
ozone were observed in situ by the computer image
analysis by CLSM after staining with the fluorescent
dye, LIVE/DEADBacLightTM. The differences in cell
density and cell distribution in the biofilm matrices of

FIGURE 4. Changes of the biofilms of P. fluorescens (a and b)

and P. aeruginosa (c and d) by treatment with ozone water. CLSM
images of the vertical section of biofilms were obtained after
staining with LIVE/DEADBacLightTM . (a) and (c), Controls; (b)
and (d), biofilms treated with ozone water in the flow-through
system. The bars represent 20 mm.

P. fluorescens and P. aeruginosa were clearly defined

(Figure 4). By using LIVE/DEADBacLightTM, bacteria
having intact cell membranes were stained in fluorescent
green, whereas those having damaged membranes were
stained in fluorescent red. In the biofilms of P. fluorescens,
the cells formed dense aggregates (Figure 4a), whereas in
P. aeruginosa, the cells were scattered in the biofilm matrix
(Figure 4c). The biofilms of P. fluorescens and P. aeruginosa
after the treatment with ozone at 1.11.2 mg/L for 1 min are
shown in Figure 4b and 4d, respectively. In the biofilms
of P. fluorescens, ozone caused cell exfoliation mainly
(Figure 4b). In the biofilms of P. aeruginosa, ozone
lessened the thickness of the biofilm and increased the

Disinfection and Removal of Biofilms by Ozone

JanuaryFebruary 2009

proportion of injured cells, which were stained red, in the

biofilm matrix, indicating ozone penetration into the biofilm matrix (Figure 4d). It was reported that P. fluorescens
excretes glucuronic acid- and gulcuronic acid-rich EPS
(Kives et al., 2006), while P. aeruginosa excretes alginic
acid (a polymer of mannuron acid)-rich EPS in the biofilms (Davis et al., 1993). Korber et al. (1995) indicated
that the biofilm structure might be dependent on the constituents of EPS. The difference of the biofilm structures,
consisting of biomass and EPS, may influence the local
reactions of ozone within the biofilms.
Since we found that EPS excreted in the P. fluorescens
biofilm was stained with the lectine-conjugated fluorescent (ConA-Fluor), we attempted to visualize the matrix
of EPS by CLSM for observation of removal of EPS
matrix by ozone. Computer images of a vertical section
of the P. fluorescens biofilm, stained with SYTO 9
(component A of LIVE/DEADBacLightTM) and ConAFluor in turn, are shown in Figure 5. The bacteria cells were
observed as green areas (Figure 5a), the EPS matrix as red
areas (Figure 5b) and these two images were merged as seen
in Figure 5c. These images indicate that EPS is abundant
in the bottom of the biofilm matrix and it helps keep the
bacterial cells aggregated. After the treatment with
ozone at 1.0 and 2.0 mg/L for 10 min, EPS matrices in
the biofilm were stained with ConA-Fluor and observed
by CLSM (Figure 6). Compared with the intact EPS
matrix (Figure 5, (b)), the EPS matrix decreased apparently by the treatment with ozone and the extent of

(a)

(b)

(a)

Ozone water at 1.0 mg/L

for 10 min.

(b)

Ozone water at 2.0 mg/L

for 10 min.

FIGURE 6. Decrease of EPS in the P. fluorescens biofilm matrix

by treatment with ozone water. EPS attached on the glass slide
was stained with concanavalin A, Alexa Fluor 633 conjugate
after the treatment. The bars represent 10 mm in (a), and 5 mm
in (b).

removal was dependent on the ozone concentration. At

the higher ozone concentration of 2 mg/L, the EPS
matrix was removed more effectively than at 1.0 mg/L
(Figure 6a and 6b). It was reported that one of the reactions of ozone with polysaccharides is a direct glycosidic
bond cleavage reaction by the insertion of ozone into the
anomeric C-H bond. Fragmentation of the hydrotrioxide
yields aldonic acid-lactones. The conversion to the lactone
leads to a shortening of the chain length (Pan et al., 1981).
The other is oxidation of hydroxyl groups at C2, C3, or C6
positions in polysaccharides to produce carbonyl groups
by ozone itself (Katai and Schuerch 1966). When the pH of
ozone water is >7, ozone also can react nonselectively or
indirectly through HO radicals formed by the hydroxyl
ion-catalyzed decomposition of ozone (Wojtowicz, 1998).
Taking together the above results of EPS removal shown in
Figure 6 with the inactivation rates in Figure 3a, the slow
inactivation rates in ozone water at 0.9 and 1.4 mg/L for
10 min may be explained by the lesser decrease of EPS in
the treatment with ozone at 1.0 mg/ L, and the more
effective removal of EPS at 2.0 mg/L could contribute to
the greater inactivation rate at the higher ozone concentration of 3.2 mg/L. The amounts of EPS excreted into the
biofilms in the present experiment are too small to be
analyzed by chemical assay. Hence, for further studies on
the removal of EPS in the biofilms, this way of EPS observation by CLSM will be helpful.
CONCLUSIONS

(c)

FIGURE 5. CLSM images of P. fluorescens biofilm matrix

stained with SYTO 9 and concanavalin A, Alexa Fluor 633 conjugate. Biofilm matrix stained was scanned by multi track with
argon laser at 488 nm and helium/neon laser at 633 nm. (a), a
computer image obtained by scanning at 488 nm, bacterial cells
stained with SYTO; (b), a computer image by scanning at 633 nm,
EPS stained with ConA-Fluor; (c), merged images of (a) and (b).
The bars represent 20 mm.

The cells in the biofilms of P. fluorescens and P. aeruginasa were more resistant to ozone than their suspended
cells. However, the survival cells in both biofilms were
decreased to less than 1 % by exposure to ozone at ca. 1
mg/L for 5 min in the flow-through system. Each biofilm
established showed different inactivation rates in ozone
water at different ozone concentrations after various
exposure times. The decrease of inactivation rates with
increasing exposure time may suggest occurrence of diffusional impediment a reactions of ozone with constituents
of the biofilms. These results indicate that, though
ozone is an effective biocide against biofilms, effective

M. Tachikawa, K. Yamanaka, and K. Nakamuro

JanuaryFebruary 2009

concentrations for biofilm disinfection may vary with

the biofilms formed and cannot be estimated from CT
values obtained by counting planktonic cells. The
observations of biofilms in situ by CLSM showed the
differences in cell density and structure of the biofilms
between P. fluorescens and P. aeruginosa, and suggested
that these differences might have influenced the efficacy of
ozone water. An apparent decrease of EPS in the matrix
of the P. fluorescens biofilm by ozone was visualized by
CLSM observation with a lectine-fluorescent conjugate
dye. The observation indicated a relationship between
removal of EPS and inactivation rates and suggested the
importance of EPS removal for effective disinfection of
biofilms.
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