Biofilm Removal With Ozone

7/25/2019 Biofilm Removal With Ozone

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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 byOzone Water Using an Artificial Microbial Biofilm System

Mariko Tachikawa,1 Kenzo Yamanaka,1 and Katsuhiko Nakamuro2

1College of Pharmacy, Nihon University, 7-7-1 Narashinodai, Funabashi, Japan2Faculty of Pharmaceutical Sciences, Setsunan University, 45-1 Nagaotougemachi, Hirakata, Japan

Inactivation rates of the biofilms ofP. fluorescence andP. aeruginosa established on a small slide glass in ozonewater (0.93.2 mg/L, 120 min) were determined in a

batch or flow-through system. The effects of ozone wateron the biofilm matrices were defined clearly in situ byconfocal laser scanning microscopy. These results indicatethat ozone is an effective biocide against biofilms and it canremove exopolysaccharides in the biofilm matrices. How-ever, the effective concentration of ozone for disinfection ofbiofilms varied with the biofilms formed, mainly due toreactions 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 man-

agement 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

laser scanning microscopy (CLSM), differences in cell

density and structure among the biofilms established were

visualized clearly, and the changes of biofilm structurescaused 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 (


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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 ofP. fluorescens biofilms were visua-

lized 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 ubi-

quitous 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 at80C with 25% (wt/vol) glycerol. Luria-Bertanis (LB)

medium was used for pre-cultivation of these bacteria,

overnight at 28C. 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 phos-

phate and small amounts of minerals (LeChevallier et al.,

1988b), which was inoculated with each overnight culture.

For the formation of biofilms ofP. 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 sup-

ply 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.

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 concentra-

tion 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 imme-

diately into a flask containing a sterile solution of thiosul-

fate for neutralization of residual ozone and chlorine.

Colony forming units (CFU) of the biofilms in the flasksolution 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 propi-

dium 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.

4 M. Tachikawa, K. Yamanaka, and K. Nakamuro JanuaryFebruary 2009


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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 micro-

graphs 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 ofP. fluor-

escens 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 ofP. fluorescensreached 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 disin-fection 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 ofP. 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 thepresent 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

andP. aeruginosaafter 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 ofP. 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 deter-

mined 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.

103

104

105

106

107

108

109

1010

0 1 2 3 4 5 6 7 8 9 10

Time (days)

Cfu/Glassslide P. aeruginosa

P. fluorescens

FIGURE 2. Growth of biofilms on incubation at 28 C. Each point, P. aeruginosa(~) andP. fluorescens(), with a bar represents a mean S.D.of cfu on the glass slide (n = 36).

Disinfection and Removal of Biofilms by Ozone JanuaryFebruary 2009 5


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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 inacti-

vation 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 ofP. fluorescens and P. aerugi-

nosa were treated with ozone water and chlorinated tap

water in a flow-through system for 1 to 20 min

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

fractions of the biofilms ofP. fluorescensafter the treat-

ment 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 con-

centration 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 diffu-

sional 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 ozoneencounters 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)

O3mg/L

Exposure

time min.

Biofilm cells

cfu / plate ( 106)

Survival

fractiona) %

0 (control) 380 43 100

0.6 5 161 82 42 21

0.6 10 118 24 31 6

1.7 5 69 4 18 1

1.7 10 54 8 14 2

a)(cfu treated / cfu control) 100.

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) Suspended cellsa) cfu / suspension ( 106)

Microorganisms O3(mg/L) Treated (%)b) O3(mg/L) Treated (%)

b)

P. fluorescens control 159 26

1.7 36.1 24.7 22.7 15.5 1.5 0.009 0.012 0.006 0.008

P. aeruginosa control 23.8 9.7

1.6 0.03 0.01 0.13 0.04 1.4 0.002 0.001 0.008 0.004

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.

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 1 5 10

Low O3water 0.48 0.51a) 0.43 0.04 0.35 0.01 0.26 0.01

(0.29 0.30)b)

High O3water 1.57 0.03a) 1.09 1.51 1.27 1.30 0.96 1.04

(0.99 1.07)b)

a)Concentration of ozone water determined just before a glass plate ofP. fluorescensbiofilm was placed in the flask.b)Concentration of ozone water in the flask without biofilms.



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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 andEffects 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

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 ofP. fluorescens,the cells formed dense aggregates (Figure 4a), whereas in

P. aeruginosa, the cells were scattered in the biofilm matrix

(Figure 4c). The biofilms ofP. fluorescensandP. 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

ofP. fluorescens, ozone caused cell exfoliation mainly

(Figure 4b). In the biofilms of P. aeruginosa, ozone

lessened the thickness of the biofilm and increased the

0.00001

0.0001

0.001

0.01

0.1

1

10

100

0 5 10 15 20 25

Time (min)

Survival

fraction(%)

0.00001

0.0001

0.001

0.01

0.1

1

10

100

0 5 10 15 20 25

Time (min)

Survivalfraction(%)

Ozone 3.0mg/L

Ozone 1.3mg/L

Ozone 1.0mg/L

Chlorine* 0.7mg/L

Ozone 3.2mg/L

Ozone 1.4mg/L

Ozone 0.9mg/L

Chlorine* 0.6mg/L

(a) P. fluorescens

(b) P. aeruginosa

FIGURE 3. Efficacy of ozone water (ozone, , ~ and &)and chlorinated tap water (free chlorine*,*) on the biofilms ofP. fluorescens (a) and P. aeruginosa (b) treated in the flow-

through system at indicated ozone concentrations. Each point

with a bar represents the mean cfu value and S.D. of 3 slides.

(a) P. fluorescensbiofilm (control).

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

(c) P. aeruginosa biofilm (control).

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

Cover glass

Cover glass

Slide glass

Slide glass

Cover glass

Slide glass

Cover glass

Slide glass

FIGURE 4. Changes of the biofilms ofP. fluorescens(aand b)

andP. 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.



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

biofilm matrix, indicating ozone penetration into the bio-

film 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 bio-

films (Davis et al., 1993). Korber et al. (1995) indicated

that the biofilm structure might be dependent on the con-

stituents of EPS. The difference of the biofilm structures,

consisting of biomass and EPS, may influence the localreactions of ozone within the biofilms.

Since we found that EPS excreted in the P. fluorescens

biofilm was stained with the lectine-conjugated fluores-

cent (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 ConA-

Fluor 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 appar-

ently by the treatment with ozone and the extent of

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 reac-

tions 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 concentra-

tion 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 obser-

vation by CLSM will be helpful.

CONCLUSIONS

The cells in the biofilms ofP. fluorescens and P. aeru-

ginasawere 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 diffu-

sional impediment a reactions of ozone with constituents

of the biofilms. These results indicate that, though

ozone is an effective biocide against biofilms, effective

(a)

(b)

(c)

FIGURE 5. CLSM images of P. fluorescens biofilm matrix

stained with SYTO 9 and concanavalin A, Alexa Fluor 633 con-

jugate. 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.

(a)

(b)

Ozone water at 1.0 mg/L

for 10 min.

Ozone water at 2.0 mg/L

for 10 min.

FIGURE 6. Decrease of EPS in theP. fluorescensbiofilm matrix

by treatment with ozone water. EPS attached on the glass slide

was stained with concanavalin A, Alexa Fluor

633 conjugateafter the treatment. The bars represent 10 mm in (a), and 5 mm

in (b).



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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 conjugatedye. 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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Biofilm Removal With Ozone