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Evaluation of some halogen biocides using a microbial biofilm system
ARTICLE IN PRESS
Water Research 39 (2005) 4126–4132 www.elsevier.com/locate/watres
Evaluation of some halogen biocides using a microbial biofilm system Mariko Tachikawaa,, Masakatsu Tezukaa, Masahiro Moritab, Katsuhisa Isogaib, Shoji Okadac a
College of Pharmacy, Nihon University, 7-7-1 Narashinodai, Funabashi 274-8555, Japan K I Chemical Industry Co., Ltd., 328 Shioshindenhamano, Fukudecho 437-1213, Japan c University of Shizuoka, 2-12-7 Mariko, Shizuoka 421-0103, Japan
b
Received 19 January 2005; received in revised form 31 May 2005; accepted 20 July 2005 Available online 16 September 2005
Abstract A simple method for the formation of microbial biofilms of three species, Pseudomonas fluorescens, Pseudomonas aeruginosa, and Klebsiella pneumoniae, on a small glass slide was established, and its suitability for evaluation of disinfectant efficacy was examined. The biofilms formed were observed in situ by confocal laser scanning microscopy (CLSM). Using the biofilms established, biocidal efficacy of several halogen biocides, such as hypochlorite (HOCl), bromochlorodimethylhydantoin (Br, Cl-DMH), ammonia monochloramine (NH2Cl), a stabilized hypobromite biocide named STABREXs, and a mixed solution of NH4Br and HOCl, was evaluated. The formation of NHBrCl in the mixed solution was indicated by UV spectra analysis. Biofilm cells were more resistant to these biocides than planktonic cells and the extent of resistance varied with the biocide tested. Among the biocides tested, the biocidal potency of HOCl was the most susceptible to the change brought about by biofilm formation. By CLSM observation, differences in biofilm conformation were revealed between the microbial species. The efficacy of the biocide tested varied with the structure of biofilms formed. The assay method developed in the present study would be useful for further investigation on biofilm disinfection. r 2005 Elsevier Ltd. All rights reserved. Keywords: Biofilms; Disinfection efficacy; Halogen biocides; HOCL; Chloramines; Bromamines; Bromochloramine; Biofilm images by CLSM
1. Introduction In an aqueous system, some bacterial cells are planktonic and flow from one ecosystem to another, but a majority of bacterial cells are sessile and attach themselves to surfaces to form colonies and biofilms. In Corresponding author. Tel.: +81474655574;
fax: +81474655637. E-mail address: [email protected] (M. Tachikawa).
industrial water systems, the formation of biofilms causes heat transfer resistance and blockages in pipes, screens, and nozzles (Characklis, 1990), leads to microbiologically induced corrosion (Hamilton, 1995), and brings about proliferation of some pathogenic organisms such as Legionella pneumophila (Walker et al., 1995). Hence, the removal and disinfection of biofilms thus formed have become an important subject in the maintenance of water fields such as public spas, swimming pools, food processing lines, and industrial water systems. Since the biofilms formed may have
0043-1354/$ - see front matter r 2005 Elsevier Ltd. All rights reserved. doi:10.1016/j.watres.2005.07.039
ARTICLE IN PRESS M. Tachikawa et al. / Water Research 39 (2005) 4126–4132
altered sensitivity toward antimicrobial agents, the importance of using a biofilm system has been pointed out for evaluation of disinfectant efficacy of biocides (Anand et al., 1983; Kuchta et al., 1983; Wright et al., 1991; Camper et al., 1985; LeChevallier et al., 1988a). Several trials have thus been made to develop test methods using biofilms (Korber et al., 1994; Ceri et al., 1999; Luppens et al., 2002; LeChevallier et al., 1988b). Halogen biocides, such as hypochlorites, inorganic and organic chloramines, and bromamines are widely used for biological control of water systems (Wojtowicz, 1993). Nevertheless, the mechanism of biofilm disinfection by halogen biocides remains obscure and its clarification awaits further studies. Therefore, we attempted to establish a simple and reproducible method for the preparation of microbial biofilms and evaluation of the biocidal effects of disinfectants on them. Since the formation of biofilms by Pseudomonas fluorescens, Pseudomonas aeruginosa and Klebsiella pnuemoniae was reported (Davies et al., 1993; Korber et al., 1994; LeChevallier et al., 1988a), these three biofilms were formed and their suitability for disinfection assay was compared. The structures of the biofilms formed were observed in situ by confocal laser scanning microscopy (CLSM). Using the biofilms established, biocidal potencies of some chlorine and bromine compounds with different reactivities were evaluated. The compounds used were hypochlorite (HOCl)1, bromochlorodimethylhydantoin (Br, Cl-DMH), ammonia monochloramine (NH2Cl), a stabilized hypobromite named STABREXs, and a mixed solution of NH4Br and HOCl. Since bromamines have high disinfecting activity in a wide pH range compared to chloramines (Wojtowicz, 1993; Nalepa, 2004), mixed solutions of bromamine and chloramine were prepared under the coexistence of bromide ions (Br). The actual species of residual halogens in the mixed solutions of NH4Br and HOCl were studied by UV spectral analysis. From the results obtained, the factors influencing biofilm disinfection by halogen biocides were discussed.
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28 1C. The biofilms of P. fluorescens and K. pneumoniae were grown on clean and sterile microscope slides (14 26 mm) placed in a glass culture dish (i.d. 145 mm) containing 150 ml of EPS (extracellular polysaccharide) growth medium (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 1C 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. 2.2. Water Milli Qs water was used for preparation and dilution of halogen compound solutions and preparation of reagent solutions for the determination of available chlorine and bromine. 2.3. Chemicals and halogen biocides
2. Materials and methods
Sodium hypochlorite solution (NaOCl) was obtained from Wako Pure Chemical Industry, Osaka, Japan. 1Bromo-3-chloro-5, 5-dimethylhydantoin (Br, Cl-DMH) was from K I Chemical Industry, Shizuoka, Japan. STABREXs was from Nalco, Naperville, IL. All other chemicals were of the reagent grade and used without further purification. NH2Cl was prepared by mixing an 11 mM NH4Cl solution and 10 mM HOCl as available chlorine in a 13 mM phosphate buffer solution at pH 7.4. A mixed solution of NH4Br and HOCl was prepared by mixing 2 mM NH4Br and 1 mM HOCl, in a 13 mM phosphate buffer solution at pH 8.5. The solutions were used 0.5–1 h after preparation. Test solutions of biocides were prepared by dilution with 0.5 mM phosphate buffer solution at pH 7.4 or pH 8.5. The concentrations of residual chlorine and bromine in the test solution were determined by the DPD (N, Ndiethyl-p-phenylenediamine) method (APHA, AWWA, WEF, 1992b; Palin, 1961).
2.1. Bacterial strains and biofilm formation
2.4. Killing experiments
P. fluorescens (JCM no. 2779), K. pneumoniae (JCM no. 1662) and P. aeruginosa (JCM no. 2776) were obtained from Japan Collection of Microorganisms (JCM), Wako, Japan. Their stock cultures were kept at 80 1C with 25% (wt/vol) glycerol. Luria–Bertani’s (LB) medium was used for pre-cultivation of these bacteria, overnight at
Biofilms formed on the slide glass were passed through sterile water twice to remove planktonic cells and growth medium, and then placed in a flask containing 40 ml of test solutions of halogen biocides (2.0–3.0 105 M) for 1–60 min at room temperature. After the exposure, the residual halogens in the test solution were neutralized by addition of thiosulfate. Colony forming units (cfu) of the biofilms in the test solution were determined after sonication and dilution as described above.
1 The actual compound used was a mixture of HOCl and OCl, which is designated as HOCl in the present paper.
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2.5. CLSM observations For fluorescence staining of cells in the biofilm, LIVE/ DEADsBacLightTM, Molecular Probes Inc., OR, was used. Biofilms on the glass slide were soaked and incubated in the dye solution for 15 min in the dark. On a cover glass (25 50 mm) was placed a drop (0.1 ml) of water, and then the slide of stained biofilms upside down. Photomicrographs were taken at a magnification of 100 with an oil immersion lens under a CLSM (LSM510 Carl Zeiss). Computer image micrographs of the vertical section of biofilms were obtained by using the Z stack function of the LSM510.
was observed repeatedly in the biofilm formation of K. pneumoniae. 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. The cell density and cell distribution in the biofilms of P. fluorescens and P. aeruginosa were clearly defined by the computer image analysis by CLSM after fluorescent staining (Figs. 2 and 3). The bacteria having intact cell membranes were stained in fluorescent green, whereas those having damaged membranes stained in fluorescent red. In the biofilms of P. fluorescens, the cells formed dense aggregates (Fig. 2a), whereas P. aeruginosa cells were scattered (Fig. 3a). Since P. aeruginosa attached on
2.6. UV spectra of available halogens For the determination of residual halogen species, spectra of test solutions at the concentration of 1–4 mM as available halogen were recorded with a UV-visible spectrophotometer of Shimadzu UV-160. A 1.0-cm cell was used in all experiments.
3. Results and discussion The number of viable cells in the biofilms of P. fluorescens, K. pneumoniae, and P. aeruginosa formed on the glass slides were determined after incubation for 1–15 days (Fig. 1). After incubation for 6 days, the mean cell density of the biofilms of P. fluorescens reached 2.570.5 108 cfu/slide. P. aeruginosa biofilms reached a plateau of 1.970.2 107 cfu/slide after 2 days of incubation. K. pneumoniae reached a peak of 1 106 cfu/slide at 1 day of incubation, and decreased to 1 104 cfu/slide at 7 days, and then gradually increased again to 2 106 cfu/slide at 15 days. This rise and fall P. fluorescens
1010 109
cfu / glass slide
108 107
P. aeruginosa
106 105 104
K. pneumoniae
103 102 0
2
4
6 8 10 12 Incubation time (day)
14
16
Fig. 1. Growth of biofilms on incubation at 28 1C. Each point, P. fluorescens (), P. aeruginosa (J) and K. pneumoniae (’), and bar represents mean7S.D. of cfu on a glass slide (n ¼ 3–6).
Fig. 2. CLSM images of the vertical section of P. fluorescens biofilms exposed to HOCl solution (1.5 mg/l as available chlorine at pH 7.4). Biofilms of P. fluorescens formed by 24-h incubation were exposed to the HOCl solution for indicated times. Staining and observation under the CLSM were carried out as described in the text. The bar represents 20 mm. (a) Control. A biofilm exposed to the medium without chlorine for 1 min. (b) Exposed to HOCl for 1 min. (c) Exposed to HOCl for 3 min.
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a surface produces alginate-rich mucous (Davies et al., 1993), each of the cells in the biofilm might be covered with a relatively thick layer of the mucous. These images of biofilms revealed clearly the differences in cell density and structure between the biofilms. In the biofilms of K. pneumoniae, the cell density was sparser than that of P. aeruginosa (data not shown).
Fig. 3. CLSM images of the vertical section of P. areruginosa biofilm exposed to HOCl (0.8 mg/l as available chlorine at pH 7.4). Biofilms of P. aeruginosa formed by 120-h incubation were exposed to the HOCl solution for 1 min. Staining and observation under the CLSM were carried out as described in the text. The bar represents 20 mm. (a) Control. A biofilm exposed to the medium without chlorine for 1 min. (b) Exposed to HOCl for 1 min.
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The changes of P. fluorescens and P. aeruginosa biofilms brought about by the treatment with HOCl are shown in Fig. 2b, c and Fig. 3b, respectively. In the biofilms of P. fluorescens, HOCl (1.5 mg/l) caused cell injury and exfoliation with increasing exposure time (Fig. 2b and c). In the biofilms of P. aeruginosa, all of the cells were injured by HOCl at a low concentration (0.8 mg/l) and stained red, but did not exfoliate (Fig. 3b). The biofilm and suspended cells of P. fluorescens and P. aeruginosa were treated with HOCl and NH2Cl, respectively. Cell suspensions were prepared by ultrasonic dispersion of the biofilms. The survival fractions, listed in Table 1, indicated that the biofilm cells were more resistant to the biocides than the suspended cells and that the extent of resistibility varied with the biocide tested. In general, NH2Cl was less effective on the suspended cells than HOCl, but was as effective as HOCl on biofilms. The results from P. fluorescens indicated that the resistibility of biofilms to the biocides brought about an increase in the percentage of viable cells in the biofilms. The penetration of biocides into biofilms seems to be an important factor for efficacy. The potencies of several halogen biocides on the biofilms of P. fluorescens and K. pneumoniae were examined and are shown in Figs. 4 and Fig. 5, respectively. The efficacy varied with each biocide and biofilm. NH2Cl and the mixed solution of NH4Br and HOCl showed continuous biocidal effect on P. fluorescens biofilms, whereas HOCl and Br, Cl-DMH showed biocidal effect only in the early time of exposure. Although it is known that the disinfection potential of NH2Cl for planktonic cells is significantly lower than HOCl, the lipophilic nature of ammonia chloramine and bromamine may facilitate the penetration into the biofilms of P. fluorescens. STABREX showed weak disinfection efficacy on both biofilms of P. fluorescens
Table 1 Comparison of the biocidal efficacy of HOCl and NH2Cl on suspended and biofilm cells of P. fluorescens and P. aeruginosa Microorganisms
Chlorine
cfu/plate ( 106) Control
P. fluorescens
P. aeruginosa
HOCl HOCl NH2Cl HOCl NH2Cl
224 704 742 20.3 13.1
Biofilm cells
Suspended cells a
Treated
% (std.)
Treated
%a (std.)
0.88 44.7 51.2 0.088 0.079
0.39 6.35 6.90 0.43 0.60
0.35 5.29 25.0 0.0015 0.0104
0.16 0.75 3.37 0.01 0.08
(0.39) (0.17)*b (5.60) (0.08)**b (0.41)
(0.14) (0.65)*b (0.32) (0.005)**b (0.13)
Since the suspended cells were prepared by ultrasonic dispersion of the biofilm formed in an aliquot volume of 0.5 mM phosphate buffer solution at pH 7.4, the number of control cells was common between the biofilm and suspended cells in the same line. The cells were exposed to the chlorine solutions of 1.7–2.4 105 M as available chlorine at pH 7.4 for 5 min. Significant difference was examined by Student’s t-test. (n ¼ 3). a cfu treated/cfu control 100. b Significant difference (Po0.05) between the same numbers of asterisks.
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Survival fraction (%)
100
HOCl NH2Cl Br,Cl-DMH STABREX NH4Br + HOCl
P. fluorescens
Biocide solution
10
Fractions determined (%) Free
Combined as NH2Cl
Combined as NHCl2
100 63 0 11 33
0 28 100 89 49
0 9 0 0 18
1 HOCl Br, Cl-DMH NH2Cl NH4Br+HOCl STABREX
0.1
0.01
1E-3 0
10
20
30 40 Time (min)
50
60
Fig. 4. Efficacy of several biocides on the biofilms of P. fluorescens. The biofilms were exposed to the biocide solutions of 2.1–2.9 105 M as available halogen at 20 1C for indicated times. The pH of the test solutions was 7.4 and that of the mixed solution of NH4Br and HOCl was 8.5. Each point represents the mean value of 3 plates.
K. pneumoniae
100
HOCl NH2Cl Br,Cl-DMH STABREX NH4Br + HOCl
10 Survival fraction (%)
Table 2 Differentiation of available halogen species in each biocide test solution (1.8–2.4 105 M as total available halogen) by the DPD method (APHA, AWWA, WEF, 1992b; Palin, 1961)
1 0.1 0.01
Separated fractions were defined according to the reactivity of free chlorine and combined chlorine as NH2Cl and NHCl2. Each fraction was given as percentage to the total halogen.
free chlorine fractions of HOCl and Br, Cl-DMH might work effectively. To analyze the products in the mixed solution of NH4Br and HOCl, a photometric study was undertaken. The UV spectra of the mixed solution of NH4Br and HOCl (Fig. 6a) indicated the formation of absorption peaks at 243 and 330 nm corresponding to NH2Cl and hypobromite ions (OBr), respectively (Gazda et al., 1993), both of which decreased gradually with time. The ether extracts of the mixed solutions showed absorption peaks at 220, 245, and 330 nm (Fig. 6b), which agreed to the UV spectrum of the ether extract of the mixture of NHBrCl and NH2Cl (Gazda et al., 1993). Most of the residual halogen in the solutions was differentiated as a monochloramine fraction together with a small amount of free chlorine as revealed by the DPD method (Table 2). The formation of NHBrCl might have an effect on the yield of the free chlorine fraction. For evaluation of the disinfection efficacy of NHBrCl, further study will be required.
0 0
10
20 Time (min)
30
Fig. 5. Efficacy of several biocides on the biofilms of K. pneumoniae. The biofilms were exposed to the biocide solutions of 2.0–3.0 105 M as available halogen at 20 1C for indicated times. The pH of the test solutions was 7.4 and that of the mixed solution of NH4Br and HOCl was 8.5. Each point represents the mean value of 3 plates.
and K. pneumoniae. Since the DPD analysis showed that about two-thirds of residual halogen of STABREX is differentiated as NH2Cl and NHCl2 fractions (Table 2), its oxidation potential is considered to be lower than any other biocide tested. In the biofilms of K. pneumoniae having a sparse cell population, large amounts of the
4. Conclusions 1. For the evaluation of biocides in an aqueous environment, a simple laboratory system to form biofilms from some ubiquitous bacteria was established. 2. By CLSM observation, differences in cell density and architecture among biofilms were revealed. The difference of architecture in biofilms may affect the efficacy of biocides. The biofilms formed by K. pneumoniae were too sparse to be used for the CLSM observation. 3. Using the biofilms, the efficacy of several chlorine and bromine biocides was evaluated. Biofilm forma-
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References 2.0
1 min 10 min 30 min 60 min
Absorbance
1.5
1.0
0.5
0.0 200
250
(a)
300 Wavelength (nm)
350
400
2.0
1 min 10 min 30 min 60 min
Absorbance
1.5
1.0
0.5
0.0 200
250
(b)
300 Wavelength (nm)
350
400
Fig. 6. (a) The changes of UV spectra of a mixture solution of 6 mM NH4Br and 3 mM HOCl at pH 8.5 at indicated times. (b) The UV spectra of an ether extract (1:1) of the mixed solution (a) at indicated times.
tion lowered the biocidal efficacy. The potency of HOCl was most markedly affected by biofilm formation among the biocides tested. 4. The efficacy of the halogen biocides tested varied with their chemical properties, such as oxidation potential and lipophilicity. 5. The assay methods developed in the present study would be useful for further investigation of biofilms.
Acknowledgements This study was supported by a grant from the Ministry of Education, Culture, Sports, Science and Technology to promote multi-disciplinary research projects and a Joint Research Grant from Nihon University, College of Pharmacy.
Anand, C.M., Skinner, A.R., Malic, A., Kurtz, J.B., 1983. Interaction of L. pneumophila and a free-living amoeba (Acanthamoeba palestinensis). J. Hyg. 91, 167–178. APHA, AWWA, WEF, 1992a. In: Greenberg, A.H., Clesceri, L.S., Eaton, A.D. (Eds.), Standard Method for the Examination of Water and Wastewater, 18th ed. American Public Health Association, Washington, DC, pp. 9-32–34. APHA, AWWA, WEF, 1992b. In: Greenberg, A.H., Clesceri, L.S., Eaton, A.D. (Eds.), Standard Method for the Examination of Water and Wastewater, 18th ed. American Public Health Association, Washington, DC, pp. 4-45–46. Camper, A.K., LeChevallier, M.W., Broadway, S.C., McFeters, G.A., 1985. Evaluation of procedures to desorb bacteria from granular activated carbon. J. Microbiol. Meth. 3, 187–198. Ceri, H., Olson, M.E., Stremick, C., Read, R.R., Morck, D., Buret, A., 1999. The Calgary biofilm device: new technology for rapid determination of antibiotic susceptibilities of bacterial biofilms. J. Clin. Microbiol. 37, 1771–1776. Characklis, W.G., 1990. Microbial Fouling. In: Characklis, W.G., Marshal, K.C. (Eds.), Biofilm. Wiley, New York, pp. 528–529. Davies, D.G., Chakrabarty, A.M., Geesey, G.G., 1993. Expolysaccharide production in biofilms: substratum activation of alginate gene expression by Pseudomonas aeruginosa. Appl. Environ. Microbiol. 59, 1181–1186. Gazda, M., Dejarme, L.E., Choudhury, T.K., Graham Cooks, R., Margerum, D.W., 1993. Mass spectrometric evidence for the formation of bromochloramine and N-bromo-Nchloromethylamine in aqueous solution. Environ. Sci. Technol. 27, 551–557. Hamilton, W.A., 1995. Biofilms and microbially influenced corrosion. In: Lappin-Scott, H.M., Costerton, J.W. (Eds.), Microbial Biofilms. Cambridge University Press, Cambridge, UK, pp. 171–182. Korber, D.R., James, G.A., Costerton, J.W., 1994. Evaluation of fleroxacin activity against established Pseudomonas fluorescens biofilms. Appl. Environ. Microbiol. 60, 1663–1669. Kuchta, J.M., States, S.J., McNamara, A.M., Wadowsky, R.M., Yee, R.B., 1983. Susceptibility of Legionella pneumophila to chlorine in tap water. Appl. Environ. Microbiol. 46, 1134–1139. LeChevallier, M.W., Cawthon, C.D., Lee, R.G., 1988a. Factors promoting survival of bacteria in chlorinated water supplies. Appl. Environ. Microbiol. 54, 649–654. LeChevallier, M.W., Cawthon, C.D., Lee, R.G., 1988b. Inactivation of biofilm bacteria. Appl. Environ. Microbiol. 54, 2492–2499. Luppens, S.B.I., Reij, M.W., Van der Heijden, R.W.L., Rombouts, F.M., Abee, T., 2002. Development of a standard test to assess the resistance of Staphylococcus aureus biofilm cells to disinfectants. Appl. Environ. Microbiol. 68, 4194–4200. Nalepa, C.J., 2004. 25 years of bromine chemistry in industrial water systems: a review. In: Conference Papers of Corrosion NACExpo 2004 in New Orleans, No. 04087, pp. 9–10.
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Palin, A.T., 1961. The determination of free residual bromine in water. Water Sewage Works 461–462. Walker, J.T., Mackerness, C.W., Rogers, J., Keevil, C.W., 1995. Heterogeneous mosiac biofilm—a haven for waterborne pathogens. In: Lappin-Scott, H.M., Costerton, J.W. (Eds.), Microbial Biofilms. Cambridge University Press, Cambridge, UK, pp. 196–204.
Wojtowicz, J.A., 1993. Chloramines and bromamines. In: Kroschwitz, J.I., Howe-Grant, M. (Eds.), Kirk-Othmer Encyclopedia of Chemical Technology, 4th ed. Wiley, New York, pp. 911–932. Wright, J.B., Rusesca, I., Costerton, J.W., 1991. Decreased biocide susceptibility of adherent Legionella pneumophila. J. Appl. Bacteriol. 71, 531–538.
Water Research 39 (2005) 4126–4132 www.elsevier.com/locate/watres
Evaluation of some halogen biocides using a microbial biofilm system Mariko Tachikawaa,, Masakatsu Tezukaa, Masahiro Moritab, Katsuhisa Isogaib, Shoji Okadac a
College of Pharmacy, Nihon University, 7-7-1 Narashinodai, Funabashi 274-8555, Japan K I Chemical Industry Co., Ltd., 328 Shioshindenhamano, Fukudecho 437-1213, Japan c University of Shizuoka, 2-12-7 Mariko, Shizuoka 421-0103, Japan
b
Received 19 January 2005; received in revised form 31 May 2005; accepted 20 July 2005 Available online 16 September 2005
Abstract A simple method for the formation of microbial biofilms of three species, Pseudomonas fluorescens, Pseudomonas aeruginosa, and Klebsiella pneumoniae, on a small glass slide was established, and its suitability for evaluation of disinfectant efficacy was examined. The biofilms formed were observed in situ by confocal laser scanning microscopy (CLSM). Using the biofilms established, biocidal efficacy of several halogen biocides, such as hypochlorite (HOCl), bromochlorodimethylhydantoin (Br, Cl-DMH), ammonia monochloramine (NH2Cl), a stabilized hypobromite biocide named STABREXs, and a mixed solution of NH4Br and HOCl, was evaluated. The formation of NHBrCl in the mixed solution was indicated by UV spectra analysis. Biofilm cells were more resistant to these biocides than planktonic cells and the extent of resistance varied with the biocide tested. Among the biocides tested, the biocidal potency of HOCl was the most susceptible to the change brought about by biofilm formation. By CLSM observation, differences in biofilm conformation were revealed between the microbial species. The efficacy of the biocide tested varied with the structure of biofilms formed. The assay method developed in the present study would be useful for further investigation on biofilm disinfection. r 2005 Elsevier Ltd. All rights reserved. Keywords: Biofilms; Disinfection efficacy; Halogen biocides; HOCL; Chloramines; Bromamines; Bromochloramine; Biofilm images by CLSM
1. Introduction In an aqueous system, some bacterial cells are planktonic and flow from one ecosystem to another, but a majority of bacterial cells are sessile and attach themselves to surfaces to form colonies and biofilms. In Corresponding author. Tel.: +81474655574;
fax: +81474655637. E-mail address: [email protected] (M. Tachikawa).
industrial water systems, the formation of biofilms causes heat transfer resistance and blockages in pipes, screens, and nozzles (Characklis, 1990), leads to microbiologically induced corrosion (Hamilton, 1995), and brings about proliferation of some pathogenic organisms such as Legionella pneumophila (Walker et al., 1995). Hence, the removal and disinfection of biofilms thus formed have become an important subject in the maintenance of water fields such as public spas, swimming pools, food processing lines, and industrial water systems. Since the biofilms formed may have
0043-1354/$ - see front matter r 2005 Elsevier Ltd. All rights reserved. doi:10.1016/j.watres.2005.07.039
ARTICLE IN PRESS M. Tachikawa et al. / Water Research 39 (2005) 4126–4132
altered sensitivity toward antimicrobial agents, the importance of using a biofilm system has been pointed out for evaluation of disinfectant efficacy of biocides (Anand et al., 1983; Kuchta et al., 1983; Wright et al., 1991; Camper et al., 1985; LeChevallier et al., 1988a). Several trials have thus been made to develop test methods using biofilms (Korber et al., 1994; Ceri et al., 1999; Luppens et al., 2002; LeChevallier et al., 1988b). Halogen biocides, such as hypochlorites, inorganic and organic chloramines, and bromamines are widely used for biological control of water systems (Wojtowicz, 1993). Nevertheless, the mechanism of biofilm disinfection by halogen biocides remains obscure and its clarification awaits further studies. Therefore, we attempted to establish a simple and reproducible method for the preparation of microbial biofilms and evaluation of the biocidal effects of disinfectants on them. Since the formation of biofilms by Pseudomonas fluorescens, Pseudomonas aeruginosa and Klebsiella pnuemoniae was reported (Davies et al., 1993; Korber et al., 1994; LeChevallier et al., 1988a), these three biofilms were formed and their suitability for disinfection assay was compared. The structures of the biofilms formed were observed in situ by confocal laser scanning microscopy (CLSM). Using the biofilms established, biocidal potencies of some chlorine and bromine compounds with different reactivities were evaluated. The compounds used were hypochlorite (HOCl)1, bromochlorodimethylhydantoin (Br, Cl-DMH), ammonia monochloramine (NH2Cl), a stabilized hypobromite named STABREXs, and a mixed solution of NH4Br and HOCl. Since bromamines have high disinfecting activity in a wide pH range compared to chloramines (Wojtowicz, 1993; Nalepa, 2004), mixed solutions of bromamine and chloramine were prepared under the coexistence of bromide ions (Br). The actual species of residual halogens in the mixed solutions of NH4Br and HOCl were studied by UV spectral analysis. From the results obtained, the factors influencing biofilm disinfection by halogen biocides were discussed.
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28 1C. The biofilms of P. fluorescens and K. pneumoniae were grown on clean and sterile microscope slides (14 26 mm) placed in a glass culture dish (i.d. 145 mm) containing 150 ml of EPS (extracellular polysaccharide) growth medium (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 1C 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. 2.2. Water Milli Qs water was used for preparation and dilution of halogen compound solutions and preparation of reagent solutions for the determination of available chlorine and bromine. 2.3. Chemicals and halogen biocides
2. Materials and methods
Sodium hypochlorite solution (NaOCl) was obtained from Wako Pure Chemical Industry, Osaka, Japan. 1Bromo-3-chloro-5, 5-dimethylhydantoin (Br, Cl-DMH) was from K I Chemical Industry, Shizuoka, Japan. STABREXs was from Nalco, Naperville, IL. All other chemicals were of the reagent grade and used without further purification. NH2Cl was prepared by mixing an 11 mM NH4Cl solution and 10 mM HOCl as available chlorine in a 13 mM phosphate buffer solution at pH 7.4. A mixed solution of NH4Br and HOCl was prepared by mixing 2 mM NH4Br and 1 mM HOCl, in a 13 mM phosphate buffer solution at pH 8.5. The solutions were used 0.5–1 h after preparation. Test solutions of biocides were prepared by dilution with 0.5 mM phosphate buffer solution at pH 7.4 or pH 8.5. The concentrations of residual chlorine and bromine in the test solution were determined by the DPD (N, Ndiethyl-p-phenylenediamine) method (APHA, AWWA, WEF, 1992b; Palin, 1961).
2.1. Bacterial strains and biofilm formation
2.4. Killing experiments
P. fluorescens (JCM no. 2779), K. pneumoniae (JCM no. 1662) and P. aeruginosa (JCM no. 2776) were obtained from Japan Collection of Microorganisms (JCM), Wako, Japan. Their stock cultures were kept at 80 1C with 25% (wt/vol) glycerol. Luria–Bertani’s (LB) medium was used for pre-cultivation of these bacteria, overnight at
Biofilms formed on the slide glass were passed through sterile water twice to remove planktonic cells and growth medium, and then placed in a flask containing 40 ml of test solutions of halogen biocides (2.0–3.0 105 M) for 1–60 min at room temperature. After the exposure, the residual halogens in the test solution were neutralized by addition of thiosulfate. Colony forming units (cfu) of the biofilms in the test solution were determined after sonication and dilution as described above.
1 The actual compound used was a mixture of HOCl and OCl, which is designated as HOCl in the present paper.
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2.5. CLSM observations For fluorescence staining of cells in the biofilm, LIVE/ DEADsBacLightTM, Molecular Probes Inc., OR, was used. Biofilms on the glass slide were soaked and incubated in the dye solution for 15 min in the dark. On a cover glass (25 50 mm) was placed a drop (0.1 ml) of water, and then the slide of stained biofilms upside down. Photomicrographs were taken at a magnification of 100 with an oil immersion lens under a CLSM (LSM510 Carl Zeiss). Computer image micrographs of the vertical section of biofilms were obtained by using the Z stack function of the LSM510.
was observed repeatedly in the biofilm formation of K. pneumoniae. 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. The cell density and cell distribution in the biofilms of P. fluorescens and P. aeruginosa were clearly defined by the computer image analysis by CLSM after fluorescent staining (Figs. 2 and 3). The bacteria having intact cell membranes were stained in fluorescent green, whereas those having damaged membranes stained in fluorescent red. In the biofilms of P. fluorescens, the cells formed dense aggregates (Fig. 2a), whereas P. aeruginosa cells were scattered (Fig. 3a). Since P. aeruginosa attached on
2.6. UV spectra of available halogens For the determination of residual halogen species, spectra of test solutions at the concentration of 1–4 mM as available halogen were recorded with a UV-visible spectrophotometer of Shimadzu UV-160. A 1.0-cm cell was used in all experiments.
3. Results and discussion The number of viable cells in the biofilms of P. fluorescens, K. pneumoniae, and P. aeruginosa formed on the glass slides were determined after incubation for 1–15 days (Fig. 1). After incubation for 6 days, the mean cell density of the biofilms of P. fluorescens reached 2.570.5 108 cfu/slide. P. aeruginosa biofilms reached a plateau of 1.970.2 107 cfu/slide after 2 days of incubation. K. pneumoniae reached a peak of 1 106 cfu/slide at 1 day of incubation, and decreased to 1 104 cfu/slide at 7 days, and then gradually increased again to 2 106 cfu/slide at 15 days. This rise and fall P. fluorescens
1010 109
cfu / glass slide
108 107
P. aeruginosa
106 105 104
K. pneumoniae
103 102 0
2
4
6 8 10 12 Incubation time (day)
14
16
Fig. 1. Growth of biofilms on incubation at 28 1C. Each point, P. fluorescens (), P. aeruginosa (J) and K. pneumoniae (’), and bar represents mean7S.D. of cfu on a glass slide (n ¼ 3–6).
Fig. 2. CLSM images of the vertical section of P. fluorescens biofilms exposed to HOCl solution (1.5 mg/l as available chlorine at pH 7.4). Biofilms of P. fluorescens formed by 24-h incubation were exposed to the HOCl solution for indicated times. Staining and observation under the CLSM were carried out as described in the text. The bar represents 20 mm. (a) Control. A biofilm exposed to the medium without chlorine for 1 min. (b) Exposed to HOCl for 1 min. (c) Exposed to HOCl for 3 min.
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a surface produces alginate-rich mucous (Davies et al., 1993), each of the cells in the biofilm might be covered with a relatively thick layer of the mucous. These images of biofilms revealed clearly the differences in cell density and structure between the biofilms. In the biofilms of K. pneumoniae, the cell density was sparser than that of P. aeruginosa (data not shown).
Fig. 3. CLSM images of the vertical section of P. areruginosa biofilm exposed to HOCl (0.8 mg/l as available chlorine at pH 7.4). Biofilms of P. aeruginosa formed by 120-h incubation were exposed to the HOCl solution for 1 min. Staining and observation under the CLSM were carried out as described in the text. The bar represents 20 mm. (a) Control. A biofilm exposed to the medium without chlorine for 1 min. (b) Exposed to HOCl for 1 min.
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The changes of P. fluorescens and P. aeruginosa biofilms brought about by the treatment with HOCl are shown in Fig. 2b, c and Fig. 3b, respectively. In the biofilms of P. fluorescens, HOCl (1.5 mg/l) caused cell injury and exfoliation with increasing exposure time (Fig. 2b and c). In the biofilms of P. aeruginosa, all of the cells were injured by HOCl at a low concentration (0.8 mg/l) and stained red, but did not exfoliate (Fig. 3b). The biofilm and suspended cells of P. fluorescens and P. aeruginosa were treated with HOCl and NH2Cl, respectively. Cell suspensions were prepared by ultrasonic dispersion of the biofilms. The survival fractions, listed in Table 1, indicated that the biofilm cells were more resistant to the biocides than the suspended cells and that the extent of resistibility varied with the biocide tested. In general, NH2Cl was less effective on the suspended cells than HOCl, but was as effective as HOCl on biofilms. The results from P. fluorescens indicated that the resistibility of biofilms to the biocides brought about an increase in the percentage of viable cells in the biofilms. The penetration of biocides into biofilms seems to be an important factor for efficacy. The potencies of several halogen biocides on the biofilms of P. fluorescens and K. pneumoniae were examined and are shown in Figs. 4 and Fig. 5, respectively. The efficacy varied with each biocide and biofilm. NH2Cl and the mixed solution of NH4Br and HOCl showed continuous biocidal effect on P. fluorescens biofilms, whereas HOCl and Br, Cl-DMH showed biocidal effect only in the early time of exposure. Although it is known that the disinfection potential of NH2Cl for planktonic cells is significantly lower than HOCl, the lipophilic nature of ammonia chloramine and bromamine may facilitate the penetration into the biofilms of P. fluorescens. STABREX showed weak disinfection efficacy on both biofilms of P. fluorescens
Table 1 Comparison of the biocidal efficacy of HOCl and NH2Cl on suspended and biofilm cells of P. fluorescens and P. aeruginosa Microorganisms
Chlorine
cfu/plate ( 106) Control
P. fluorescens
P. aeruginosa
HOCl HOCl NH2Cl HOCl NH2Cl
224 704 742 20.3 13.1
Biofilm cells
Suspended cells a
Treated
% (std.)
Treated
%a (std.)
0.88 44.7 51.2 0.088 0.079
0.39 6.35 6.90 0.43 0.60
0.35 5.29 25.0 0.0015 0.0104
0.16 0.75 3.37 0.01 0.08
(0.39) (0.17)*b (5.60) (0.08)**b (0.41)
(0.14) (0.65)*b (0.32) (0.005)**b (0.13)
Since the suspended cells were prepared by ultrasonic dispersion of the biofilm formed in an aliquot volume of 0.5 mM phosphate buffer solution at pH 7.4, the number of control cells was common between the biofilm and suspended cells in the same line. The cells were exposed to the chlorine solutions of 1.7–2.4 105 M as available chlorine at pH 7.4 for 5 min. Significant difference was examined by Student’s t-test. (n ¼ 3). a cfu treated/cfu control 100. b Significant difference (Po0.05) between the same numbers of asterisks.
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Survival fraction (%)
100
HOCl NH2Cl Br,Cl-DMH STABREX NH4Br + HOCl
P. fluorescens
Biocide solution
10
Fractions determined (%) Free
Combined as NH2Cl
Combined as NHCl2
100 63 0 11 33
0 28 100 89 49
0 9 0 0 18
1 HOCl Br, Cl-DMH NH2Cl NH4Br+HOCl STABREX
0.1
0.01
1E-3 0
10
20
30 40 Time (min)
50
60
Fig. 4. Efficacy of several biocides on the biofilms of P. fluorescens. The biofilms were exposed to the biocide solutions of 2.1–2.9 105 M as available halogen at 20 1C for indicated times. The pH of the test solutions was 7.4 and that of the mixed solution of NH4Br and HOCl was 8.5. Each point represents the mean value of 3 plates.
K. pneumoniae
100
HOCl NH2Cl Br,Cl-DMH STABREX NH4Br + HOCl
10 Survival fraction (%)
Table 2 Differentiation of available halogen species in each biocide test solution (1.8–2.4 105 M as total available halogen) by the DPD method (APHA, AWWA, WEF, 1992b; Palin, 1961)
1 0.1 0.01
Separated fractions were defined according to the reactivity of free chlorine and combined chlorine as NH2Cl and NHCl2. Each fraction was given as percentage to the total halogen.
free chlorine fractions of HOCl and Br, Cl-DMH might work effectively. To analyze the products in the mixed solution of NH4Br and HOCl, a photometric study was undertaken. The UV spectra of the mixed solution of NH4Br and HOCl (Fig. 6a) indicated the formation of absorption peaks at 243 and 330 nm corresponding to NH2Cl and hypobromite ions (OBr), respectively (Gazda et al., 1993), both of which decreased gradually with time. The ether extracts of the mixed solutions showed absorption peaks at 220, 245, and 330 nm (Fig. 6b), which agreed to the UV spectrum of the ether extract of the mixture of NHBrCl and NH2Cl (Gazda et al., 1993). Most of the residual halogen in the solutions was differentiated as a monochloramine fraction together with a small amount of free chlorine as revealed by the DPD method (Table 2). The formation of NHBrCl might have an effect on the yield of the free chlorine fraction. For evaluation of the disinfection efficacy of NHBrCl, further study will be required.
0 0
10
20 Time (min)
30
Fig. 5. Efficacy of several biocides on the biofilms of K. pneumoniae. The biofilms were exposed to the biocide solutions of 2.0–3.0 105 M as available halogen at 20 1C for indicated times. The pH of the test solutions was 7.4 and that of the mixed solution of NH4Br and HOCl was 8.5. Each point represents the mean value of 3 plates.
and K. pneumoniae. Since the DPD analysis showed that about two-thirds of residual halogen of STABREX is differentiated as NH2Cl and NHCl2 fractions (Table 2), its oxidation potential is considered to be lower than any other biocide tested. In the biofilms of K. pneumoniae having a sparse cell population, large amounts of the
4. Conclusions 1. For the evaluation of biocides in an aqueous environment, a simple laboratory system to form biofilms from some ubiquitous bacteria was established. 2. By CLSM observation, differences in cell density and architecture among biofilms were revealed. The difference of architecture in biofilms may affect the efficacy of biocides. The biofilms formed by K. pneumoniae were too sparse to be used for the CLSM observation. 3. Using the biofilms, the efficacy of several chlorine and bromine biocides was evaluated. Biofilm forma-
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References 2.0
1 min 10 min 30 min 60 min
Absorbance
1.5
1.0
0.5
0.0 200
250
(a)
300 Wavelength (nm)
350
400
2.0
1 min 10 min 30 min 60 min
Absorbance
1.5
1.0
0.5
0.0 200
250
(b)
300 Wavelength (nm)
350
400
Fig. 6. (a) The changes of UV spectra of a mixture solution of 6 mM NH4Br and 3 mM HOCl at pH 8.5 at indicated times. (b) The UV spectra of an ether extract (1:1) of the mixed solution (a) at indicated times.
tion lowered the biocidal efficacy. The potency of HOCl was most markedly affected by biofilm formation among the biocides tested. 4. The efficacy of the halogen biocides tested varied with their chemical properties, such as oxidation potential and lipophilicity. 5. The assay methods developed in the present study would be useful for further investigation of biofilms.
Acknowledgements This study was supported by a grant from the Ministry of Education, Culture, Sports, Science and Technology to promote multi-disciplinary research projects and a Joint Research Grant from Nihon University, College of Pharmacy.
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