Selective Reactivity of Monochloramine with Extracellular Matrix

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Selective Reactivity of Monochloramine with Extracellular Matrix Components Affects the Disinfection of Biofilm and Detached Clusters Zheng Xue,† Woo Hyoung Lee,‡ Kimberly M. Coburn,† and Youngwoo Seo*,†,§,∥ †

Department of Civil Engineering, University of Toledo, Toledo, Ohio 43606, United States Department of Civil, Environmental, and Construction Engineering, University of Central Florida, Orlando, Florida 32816, United States § Department of Chemical and Environmental Engineering, University of Toledo, Toledo, Ohio 43606, United States ∥ School of Green Chemistry and Engineering, University of Toledo, Toledo, Ohio, United States ‡

S Supporting Information *

ABSTRACT: The efficiency of monochloramine disinfection was dependent on the quantity and composition of extracellular polymeric substances (EPS) in biofilms, as monochloramine has a selective reactivity with proteins over polysaccharides. Biofilms with proteinbased (Pseudomonas putida) and polysaccharide based EPS (Pseudomonas aeruginosa), as well as biofilms with varied amount of polysaccharide EPS (wild-type and mutant P. aeruginosa), were compared. The different reactivity of EPS components with monochloramine influenced disinfectant penetration, biofilm inactivation, as well as the viability of detached clusters. Monochloramine transport profiling measured by a chloramine-sensitive microelectrode revealed a broader diffusion boundary layer between bulk and biofilm surface in the P. putida biofilm compared to those of P. aeruginosa biofilms. The reaction with proteins in P. putida EPS multiplied both the time and the monochloramine mass required to achieve a full biofilm penetration. Cell viability in biofilms was also spatially influenced by monochloramine diffusion and reaction within biofilms, showing a lower survival in the surface section and a higher persistence in the middle section of the P. putida biofilm compared to the P. aeruginosa biofilms. While polysaccharide EPS promoted biofilm cell viability by obstructing monochloramine reactive sites on bacterial cells, protein EPS hindered monochloramine penetration by reacting with monochloramine and reduced its concentration within biofilms. Furthermore, the persistence of bacterial cells detached from biofilm (over 70% for P. putida and ∼40% for polysaccharide producing P. aeruginosa) suggested that currently recommended monochloramine residual levels may underestimate the risk of water quality deterioration caused by biofilm detachment.

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INTRODUCTION It is becoming evident that the presence of biofilm in water distribution systems can greatly impact water quality and safety.1,2 Biofilm development involves initial colonization of planktonic cells, biofilm maturation with cell proliferation and extracellular polymeric substances (EPS) secretion, as well as cell dispersion. However, the cell detachment from a parent biofilm has not been well studied, although this life-cycle process greatly contributes to biomass redistribution and cell recolonization downstream.3 These detached cells are continuously released from biofilms and may possess the potential to form new biofilms with relatively high disinfectant resistance.4 Hence, inactivating preformed biofilm and controlling viable detached biofilm are both challenging issues for water authorities to deliver high-quality water. The increased attention on drinking water safety requires a more carefully administered and tightly controlled disinfectant application in the distribution systems. © 2014 American Chemical Society

Recently, many water utilities have been adapting monochloramine disinfection for water distribution systems in order to meet the more stringent Stage 2 Disinfectants and Disinfection Byproducts Rule.5 Accordingly, a growing number of studies have been conducted to investigate the disinfection efficacy of monochloramine6−8 in laboratory settings9−11 and in actual distribution systems.12,13 Among them, studies with both artificial biofilm comprised of alginate and bacterial biofilm reported that monochloramine maintained more stable residuals and achieved better biofilm penetration than chlorine due to its low reactivity with biofilm components.14 However, these studies mainly focused on the penetration efficacy of monochloramine without considering biofilm structure, EPS Received: Revised: Accepted: Published: 3832

November 30, 2013 February 24, 2014 February 27, 2014 February 27, 2014 dx.doi.org/10.1021/es405353h | Environ. Sci. Technol. 2014, 48, 3832−3839

Environmental Science & Technology

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chlorine-to-ammonia-nitrogen mass ratio, where monochloramine is the dominant species at this pH. The monochloramine concentration as free chlorine was determined with the indophenols method using a DR/2700 spectrophotometer (Hach Co., Loveland, CO).24 2.3. Biofilm Cultivation and Disinfection Tests. Biofilms were cultivated in two channel flowcells (BioSurface Technologies Corp., Bozeman, MT) fitted with a glass microscope slide opposing a glass coverslip at room temperature (22 ± 2 °C) (see a schematic diagram of the flowcell system in SI Figure S1).25 Detail information on flowcell preparation, operation and biofilm cultivation was stated elsewhere.26 Biofilms were grown six days to reach structural maturity prior to the application of monochloramine.27 For each flowcell, one channel was used as a control, while disinfectant was applied to the other. The monochloramine concentrations were maintained at 2 mg/L at each flow cell inlet throughout the disinfection process by premixing the media and disinfectant solutions in a bubble trap (HRT = 25 min) before the solutions entering the flow cells. Flowcell effluent was collected every 30 min for 2 h during the disinfection process14 and quenched with 0.1 M sodium thiosulfate before further analysis.28 The selected monochloramine concentration and exposure time yielded a Ct value of 240 mg·min/L as recommended for the distribution system.29 For each bacterial strain and each disinfectant, the experiment was repeated more than three times. 2.4. Analysis of Biofilm Structure and Cell Viability. Immediately after 2 h disinfection, biofilms were sampled and prepared for image analysis. The biofilm content on glass slides was labeled with BacLight LIVE/DEAD staining kit (Molecular Probes Inc., 1.5 μL/mL of SYTO 9 and propidium iodide (PI) each) to differentiate live and dead cells. Polysaccharide contents in biofilm EPS were visualized by applying Alexa 633 conjugated concanavalin A (ConA-Alexa 633, 20 μg/mL), which specifically targets the polysaccharides (D-glucose and Dmannose residues).30 Proteins in EPS were labeled with SYPRO tangerine (10 μg/mL).31 The biofilm samples were stained with a mixture of the LIVE/DAED stains for 15 min, rinsed with CDF buffer, exposed to the EPS stain for 15 min,31,32 and then rinsed again. The stained biofilms were visualized with a Leica SP5 confocal laser scanning microscope (CLSM) (See SI Text S2 for detailed CLSM settings). For each biofilm sample slide, at least six positions were randomly selected for image acquisition and further image analysis. CLSM images were further analyzed using the COMSTAT program to determine total biomass, EPS content, and biofilm structural parameters, as defined in detail elsewhere.33 Important structural parameters discussed in this study include (i) the roughness coefficient, a measure of how the thickness of biofilm varies; (ii) surface area to volume ratio, representing the spatial complexity of biofilm structure; and (iii) diffusion distance, defined as the shortest distance from a pixel containing biomass to a pixel not containing biomass. Average diffusion distance is the average of diffusion distances for all pixels containing biomass. This parameter indicates the extent of void spaces in the biofilm structure. Stained EPS content per unit area was generated from image analysis to evaluate the role of specific EPS components on monochloramine penetration and disinfection efficacy. Cell viability in biofilms was obtained from both CLSM imaging and heterotrophic plate count (HPC). From the CLSM images, cell viability was presented as viable ratio and

reactivity, or the spatial distribution of cell viability within biofilms.15 The influence of biofilm EPS composition and its reactivity on monochloramine efficacy is not yet completely understood. With the continuous discoveries of biofilm EPS in the past decades, besides the originally identified polysaccharides, proteins have been accepted as another major components of biofilm EPS. Our previous study revealed that monochloramine reacted rapidly with proteins while it had minimal reactivity with polysaccharides; this selective reactivity of monochloramine affected the inactivation efficacy on planktonic cells with different quantity of capsular EPS.16 It was also reported that monochloramine selectively attacks amino acids on cell membrane to achieve bacterial inactivation.17 Therefore, the presence of monochloramine-reactive or monochloraminenonreactive components in biofilms, specifically in EPS, which comprise over 90% of biofilm, may have a great influence on monochloramine transport as well as disinfection efficacy. Furthermore, even though detachment and redistribution of biofilm have been recognized as essential stages in biofilm life cycle, relatively little is known about the disinfection efficacy of monochloramine on these clusters released from biofilms in a dynamic flow system. Detached cell viability with free chlorine residuals was recently reported to be affected by the quantity of polysaccharide EPS and the presence of chlorine demanding substrate;4 however, the response of these detached clusters to monochloramine has not been explored yet. This study aimed to investigate the influence of monochloramine reactivity with different EPS components on the viability of biofilm and detached clusters in a model distribution system. Protein-based EPS producing Pseudomonas putida and polysaccharide-based EPS producing Pseudomonas aeruginosa strains were selected to construct model biofilms, in order to understand how EPS reactivity with monochloramine affects monochloramine transport, cell viability distribution within biofilms, and detached cell viability.

2. MATERIALS AND METHODS 2.1. Bacteria Culture. P. aeruginosa and P. putida are microorganisms identified in water distribution systems, which are known to produce polysaccharide-based and protein-based EPS, respectively.18,19 Three P. aeruginosa strains and the wildtype P. putida were selected to construct confluent biofilms for monochloramine disinfection. The three P. aeruginosa strains, a wild-type (PAO1) and two mutant strains (alginate deficient algT(U) and alginate overproducing mucA22) have varied capacity in alginate EPS production (see Supporting Information (SI) Text S1 for details of mutant strain construction and Table S1 for EPS quantification of the tested four strains). The three P. aeruginosa strains were selected to investigate the effect of EPS quantity on disinfection, as alginate is the predominant component in the polysaccharide EPS in established biofilms and it is involved in the defense response of biofilms.20 Wild-type P. putida strain and wild-type P. aeruginosa strain were compared to determine the influence of EPS composition on biofilm disinfection. 2.2. Solution Preparation. Biofilms were cultivated in 0.02 strength LB broth (0.5 g/L) to create a nutrient limited growth condition.21,22 A chlorine demand free buffer (CDF) buffer (0.54 g/L Na2HPO4, 0.88 g/L KH2PO4, pH 6.98) was used as the buffer solution for culture suspension and all other assay preparation.23 Monochloramine solution was prepared by combining solutions of sodium hypochlorite (6%, pH 8.3) and ammonium chloride (0.2 mmol/L, pH 8.3) in a 4:1 3833

dx.doi.org/10.1021/es405353h | Environ. Sci. Technol. 2014, 48, 3832−3839


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Table 1. Biofilm Structural Parametersa, Biomass Contenta and Viable Ratiob algT(U) roughness coefficient surface area to volume ratio (μm2/μm3) average diffusion distance (μm) average thickness (μm) stained EPS3 (μm3/μm2) total biomass (μm3/μm2) viable rate (%), control viable rate (%), disinfected

0.97 1.57 0.43 37.49 0.19 23.14 65.54 6.5

± ± ± ± ± ± ± ±

PAO1

0.34def 0.66a 0.37a 5.13a 0.02a 12.92ac 14.09 3.27a

1.05 1.39 0.29 46.91 1.46 28.86 63.39 9.68

± ± ± ± ± ± ± ±

0.29ad 0.39a 0.15a 9.17b 0.48b 13.06a 18.50 9.39a

mucA22 0.69 1.17 0.25 45.85 2.24 22.33 65.65 17.25

± ± ± ± ± ± ± ±

0.24be 0.28a 0.19a 7.19b 0.40c 4.23ac 19.42 9.47b

putida 1.08 8.22 0.93 48.42 1.98 15.10 64.04 25.41

± ± ± ± ± ± ± ±

0.42acf 1.88b 1.04b 19.61b 1.75bc 5.54bc 11.63 9.44c

a

Biofilm structural parameters and biomass content shown in Table 1 were quantified based on control biofilms. No statistical differences were found between control and disinfected conditions for all tested strains. bViable rate was calculated as the biomass of live cells (stained with SYTO 9) divided by the biomass of total cells ×100%. cThe stained EPS were alginate EPS for the three P. aeruginosa strains and protein EPS for P. putida. Values represent average ± standard deviation (N ≥ 15). For each parameter, the uncommon letters denote statistical difference (p < 0.05).

monochloramine microelectrode was positioned at the center of a well-shaped biofilm structure and a flow media was switched to a monochloramine solution (5 mM borate buffer solution, pH 8.0, 2.1 mg Cl2/L (4:1 Cl2:N), 8.3 mg DO/L, and 23 °C). Then, monochloramine concentration profiles were measured with a high spatial resolution of 20 μm using a 3D micromanipulator and a microsensor multimeter until monochloramine fully penetrates biofilms. Monochloramine concentrations in bulk solutions were maintained constant during the experiments. All experiments for monochloramine biofilm penetration microprofile measurements were conducted under the same conditions inside a faraday cage. Biofilm thicknesses were measured using a microelectrode under direct microscopic observation. 2.7. Statistical Analysis. Data were presented as mean ± standard deviation. Differences were analyzed using unpaired t test or one-way ANOVA test. P < 0.05 was accepted as statistically significant. All calculations were performed using SigmaPlot (Jandel Scientific, Sausalito, CA).

determined as the biomass ratio of live cell (SYTO 9 labeled) to total cell. Using the HPC method, both control and disinfected biofilms were scraped from the flowcells, resuspended in CDF buffer, and plated on R2A agar plates (Difco Laboratories, Detroit, MI) in duplicate. As the HPC method only yields culturable cell counts, the cell viability was defined as viability ratio and calculated as the ratio of culturable cell counts in disinfected biofilms to those in control biofilms. 2.5. Viability Analysis of the Detached Clusters. The collected flowcell effluent was examined for detached cell viability by HPC and flow cytometry. Enumerations of the culturable cells by HPC followed the same procedure described above and the viability ratio was derived from the ratio of cell counts in the disinfected effluent samples to those in the control samples. In addition to the HPC method, flow cytometry was applied to differentiate and quantify dead, injured and live cells as a function of fluorescence intensity based on the extent of cell membrane damage. Data were acquired in “log” mode using a FACScalibur flow cytometer (BD Biosciences, San Jose, CA). The flow cytometer was equipped with an argon laser set at 15 mV and an excitation wavelength of 488 nm. PI and SYTO 9 were used in combination to determine membrane compromised cells and intact cells, respectively. Stains were simultaneously added at concentrations of 0.15 μL PI and 0.1 μL of SYTO 9 to 1 mL of sample and incubated as described above.26 Cell concentration was determined by comparing cell events to events from a microsphere standard of known concentration (InVitrogen, Carlsbad, CA). On the basis of negative and positive controls, flow cytometry analysis was performed on two fluorescent channels (PI and SYTO 9) to evaluate the cell viability. Each acquired data plot was analyzed using WinMDI (J. Trotter 1993−1998) in four quadrants:34 (A) PI positively stained dead cells with a permeabilized cell membrane; (B) both PI and SYTO 9 positively stained membrane compromised cells; (C) SYTO 9 positively stained live cells with intact cell membrane; and (D) negative signals.26 2.6. Monochloramine Microprofile Measurements in Biofilms. Monochloramine penetration in biofilm was directly measured using a monochloramine sensitive microelectrode as previously described.15 The 6-day old biofilm samples were fully saturated in a phosphate buffer saline (PBS) (10 g/L NaCl, 0.25 g/L KCl, 1.8 g/L Na2HPO4, and 0.3 g/L KH2PO4) for 1 h. It was then moved to a flow chamber and acclimated in a 5 mM borate buffer solution (pH 8.0 and 23 °C) for 1 h to reach steady-state under the flow condition of 15 mL/min. A

3. RESULTS AND DISCUSSION 3.1. Effect of EPS Content on Biofilm Structure. Structural characteristics of 6-day old biofilm were quantitatively analyzed using CLSM images (Table 1). No significant changes in biofilm structural parameters, cell biomass and major EPS content were observed when comparing control and disinfected biofilms for all tested strains (data not shown for disinfected biofilms). Image analysis revealed that both the quantity and type of the major EPS component affected biofilm structure. For the three P. aeruginosa strains, the production of polysaccharide EPS (alginate) resulted in a higher average biofilm thickness. P. putida biofilm with protein-based EPS had a significantly higher surface area to volume ratio and average diffusion distance compared to the polysaccharide EPS producing P. aeruginosa biofilms. These higher values suggested more void spaces within a biofilm, which benefits mass transport in biofilms.35 3.2. Monochloramine Reaction and Penetration in Biofilms. Monochloramine concentration microprofiles in mucA22 biofilm and P. putida biofilm were compared in Figure 1 (monochloramine microprofiles for algT(U) and PAO1 biofilms are shown in SI Figure S2). Each monochloramine penetration profile represents the average value of concentrations measured at each depth from the bulk to the biofilm substratum at a specific exposure time (0−2 h). Biofilm sloughing or detachment was negligible during the monochloramine profiling with microelectrodes.15 Monochloramine was 3834



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mucA22 biofilm for only 20 min exposure. Deer et al.37 and Jang et al.17 reported significant chlorine and chlorine dioxide decline in the biofilm diffusion boundary layer by microelectrode profiling. Both chlorine and chlorine dioxide are highly reactive disinfectants and their penetration is predominantly affected by the reactive substrate in the bulk or the disinfectant consuming material of the substratum. Although monochloramine is previously known to be less reactive and able to better penetration biofilm, the observed profiling data suggests a strong influence of EPS composition on monochloramine diffusion and reaction in biofilm. Based on the monochloramine concentration microprofiles, the mass transport was determined to further investigate monochloramine penetration into biofilm. Two mass values, Cz and Czmax, were calculated using the time based concentration profile.15 Cz (mg Cl2) is the total mass delivered to a biofilm unit area (cm2) throughout the biofilm depth (z, cm) at a given time and Czmax (mg Cl2) is the maximum Cz when full penetration is achieved. The ratio of Cz and Czmax (Cz/Czmax) indicates the extent of monochloramine penetration, with 0% and 100% representing no penetration and complete penetration, respectively. Previous studies reported that monochloramine penetrate biofilm quicker and further than free chlorine;15 however, in this study, monochloramine penetration rates were significantly influenced by the quantities or the component of biofilm EPS. The mass transfer profile in Figure 2 showed a similar trend for PAO1 and mucA22, while Figure 1. Monochloramine concentration microprofiles measured during 2 h exposure time: (a) P. aeruginosa mucA22 biofilm, (b) P. putida biofilm. The vertical line at distance value of 0 indicates biofilm surface. The left side of this line is the bulk; and right side is the biofilm.

transported from the bulk through the diffusion boundary layer in a biofilm. For the mucA22 biofilm (Figure 1(a)), the diffusion boundary layer was approximately 200 μm. The monochloramine concentration at the biofilm surface was 70% of the bulk concentration at time 0 and nearly bulk concentration after 1 h exposure. The diffusion boundary layer was much larger in P. putida biofilms than in mucA22 biofilms (Figure 1(b)), even with the same flow rate and experimental conditions. The monochloramine concentration started to decrease approximately 600 μm away from the biofilm surface and it was continuously reduced when approaching the biofilm surface. The monochloramine concentration was only 50% of the bulk concentration at the biofilm surface at time 0 and 90% bulk concentration at 2 h. The significant differences in monochloramine decline at the biofilm-bulk fluid interface of these two biofilms could be linked to the presence of soluble EPS, released bacterial cells with cell bound EPS from biofilm, as well as biomass floating or loosely bound near the biofilm surface.36 This finding may further indicate that different EPS composition affects monochloramine penetration rate. From biofilm surface to the substratum, monochloramine penetration was modulated by both reaction and diffusion mechanisms. Comparing the monochloramine concentration at the same depth inside the two biofilms, monochloramine penetrated nearly three times faster in mucA22 biofilm than in P. putida biofilm as presented in Figure 1. For example, at the same distance from biofilm surface, it took 1 h for monochloramine to reach the same concentration (∼1.7 m/L at 100 μm thickness) in the P. putida biofilm as that in the

Figure 2. Monochloramine penetration in biofilms as a function of disinfection time. The Cz/Czmax value indicates the extent of monochloramine mass transport into biofilms.

monochloramine transport was impeded in algT(U) and P. putida biofilms. The time for a full biofilm penetration (>90%) was 40 min for PAO1 and mucA22; while it took approximately 2 h to approach a full penetration in algT(U) and P. putida biofilms. Within the first 30 min of monochloramine exposure, monochloramine transported more rapidly in polysaccharide producing P. aeruginosa biofilms than in P. putida biofilm. This could be due to the reactivity between polysaccharide EPS and monochloramine is minimal, while protein EPS react rapidly with monochloramine.16 The high reaction rate between protein EPS and monochloramine immediately reduced monochloramine concentration at the surface layer of biofilm, resulting in a lower amount of total monochloramine delivered into the deeper layer of biofilms. After the first 30 min, monochloramine transport was delayed in algT(U) biofilm compared to the other two polysaccharide EPS producing P. 3835



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aeruginosa biofilms. The presence of polysaccharide EPS may shield the reactive sites on bacterial cells from monochloramine inactivation according to the multiple-hit concept.17 Thus, the total reaction between polysaccharide producing biofilm was lower than polysaccharide deficient biofilm, which also led to a higher resistance of cells in the polysaccharide producing biofilms (results further discussed in the following section). 3.3. Cell Viability in Biofilms by Monochloramine Disinfection. The cell viability in biofilms was investigated using both heterotrophic plate count (HPC) and CLSM image analysis to better understand the influence of EPS components on monochloramine disinfection. The HPC results showed that P. putida biofilm had the highest viability ratio (37.62 ± 6.67%) followed by mucA22 (32.70 ± 8.70%), PAO1 (13.04 ± 8.87%), and the least viable algT(U) (1.14 ± 1.33%) after 2-h monochloramine disinfection. The same trend in biofilm overall cell viability with monochloramine disinfection was observed using CLSM analysis (Table 1). The overall cell viability was similar for all four strains under the control condition (approximately 64 ± 1% on average). Upon disinfectant exposure, the cell viability was influenced differently among strains, showing the highest viability in the P. putida biofilm with protein-based EPS and an enhanced resistance observed with increased polysaccharide EPS content in the three P. aeruginosa biofilms. Both the HPC results and CLSM analysis suggested that biofilm resistance to monochloramine was not only promoted by EPS quantity but also influenced by EPS composition (P < 0.05). Beyond the overall cell viability in biofilms, a spatial distribution of cell viability in biofilms was also profiled by CLSM image analysis to investigate disinfectant efficacy during monochloramine penetration. To evaluate the viability spatial distribution, data were acquired by imaging at 1 μm depth increments throughout the image acquisition process and calculating a viable ratio of live over total cells in each optical slice. Data were plotted as the average viable ratio determined in each depth within biofilm versus the normalized distance from the substratum to biofilm surface where each curve represents an individual strain under control or disinfected condition (Figure 3). A similar pattern in the viability distribution was observed for all control biofilms, ranging from 70 to 100% viable ratio. For

the monochloramine disinfected biofilms, cell viability was low in the surface and bottom sections and high in the middle section along the biofilm thickness. The maximum viable ratio observed for algT(U), PAO1, and mucA22 biofilms was less than 10%, about 17% and up to 25%, respectively, whereas the maximum viable ratio was close to 40% for P. putida. In the surface section of biofilms (80−100% distance away from the substratum), the cell viability was slightly higher in the polysaccharide EPS producing strains (PAO1 and mucA22), whereas close to zero for algT(U) and P. putida. The difference of viability in biofilm surface sections could be related to the different EPS-monochloramine reactivity and the structural characteristics of P. putida biofilm (higher values of the surface area to volume ratio and average diffusion distance than P. aeruginosa biofilms), which led to a faster monochloramine penetration in the top layer of biofilm. However, monochloramine concentration was also reduced rapidly by the protein components in EPS during the penetration, resulting in higher cell viability in the middle section (40−80% distance away from the substratum) of P. putida biofilm compared to the P. aeruginosa biofilms. On the other hand, among the three P. aeruginosa biofilms, due to the minimal reactivity between polysaccharide and monochloramine, the presence of polysaccharide EPS may act as a protective shield for cells in PAO1 and mucA22 biofilms. Thus, a higher cell viability and a lower monochloramine consumption rate was observed in the polysaccharide producing P. aeruginosa biofilms than the polysaccharide (alginate) deficient biofilm. In the bottom section (0−30%), the viability of P. putida and algT(U) was again the lowest among the tested strains, which could be due to the structural characteristics of biofilms. Specifically, algT(U) biofilm was slightly thinner than the other tested biofilms and P. putida had more void spaces within the biofilm, facilitating monochloramine transport to the bottom of a biofilm. The disinfectant impact depth, defined as the distance from the top surface of biofilm to the point where maximum viable ratio was observed, appeared in the middle for all three P. aeruginosa biofilms (50% thickness from the biofilm surface). However, for P. putida, the disinfectant impact depth was reduced to approximately 25% thickness from the biofilm surface. This finding again suggested that high reactivity of protein-based EPS with monochloramine led to a fast monochloramine decrease during biofilm penetration thus resulting in a higher cell viability in the middle section of biofilms. In addition to the effects of biofilm/EPS structural properties on the distribution of cell viability, the high resistance of cells within the middle section and the almost complete inactivation of cells near the substratum may also be attributed to the physiology of cell located at different depth of biofilms. Due to the nutrient and oxygen gradient in a biofilm, the cells enclosed in the interior of a biofilm may be in a dormant state, which may lead to an enhanced disinfectant resistance.38 Although monochloramine reacted very differently with protein-based EPS and polysaccharide-based EPS, it does not significantly affect its transport into biofilm under sufficient exposure time. In this study, monochloramine achieved 90% penetration in all tested biofilms after 2 h disinfection. However, based on the flux calculation, monochloramine consumption for P. putida was twice than that of mucA22 in terms of a full penetration in biofilms. The additional monochloramine consumption by protein EPS in P. putida biofilm reduced available monochloramine for bacterial

Figure 3. Spatial distribution of the viable ratio in control and monochloramine disinfected biofilms. The vertical axis represents the normalized distance from the substratum increasing to the biofilm surface, where the viability ratio (horizontal axis) was determined at each cross-section. 3836



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inactivation, thus a higher cell viability in the middle section of biofilm was observed. 3.4. Detached Cluster Viability during Biofilm Disinfection. As an element in biofilm lifecycle, the viability of detached clusters from biofilms was investigated using HPC and flow cytometry. HPC results indicated a higher viability ratio of P. putida over P. aeruginosa and an increased viability ratio with elevated polysaccharide production among the three P. aeruginosa strains (Figure 4). The highly susceptible algT(U)

Figure 4. Viability ratio of detached biofilm clusters determined by HPC. Data points represent mean ±1 standard deviation (N ≥ 12).

strain showed a below 10% viability ratio in their detached cells. Cells detached from PAO1 and mucA22 biofilms had higher viability ratios, which were approximately 40%. Meanwhile, the putida detached cells were the most viable showing an over 60% viability ratio. These results suggested that monochloramine was less efficient in controlling viability of cell detached from biofilms with protein-based EPS than polysaccharide-based EPS. In addition to HPC, flow cytometry provided a measure of both cell concentration and cell viability. The detached cell concentration was found consistent for all tested strains and conditions. To analyze cell viability, the flow cytometry acquired a data plot of PI versus SYTO 9 fluorescence intensity and analyzed in four quadrants (Figure 5(a)). Reports from previous studies have shown that the staining of bacterial cells with SYTO 9 and PI did not always produce distinct “live” and “dead” populations.39,40 The appearance of yellow or orange stained cells observed in previous studies indicated an intermediate state of membrane compromised cells. In this study, the intermediate state cells were observed in quadrant B (upper right), indicating both SYTO 9 and PI positive signals. No significant difference in the dead cell percentage was found for the control samples when comparing cells detached from all tested biofilms. The percentage of live, dead and membrane compromised cells were plotted in a bar graph (Figure 5(b)), suggesting an influence of EPS composition on detached cell viability with a similar trend as observed from the HPC results. Although monochloramine was not completely depleted after passing through the biofilm reactor (approximately 1.2 mg/L monochloramine concentration in the effluent throughout the 2-h disinfection period), detached cells were still largely viable except the most susceptible algT(U) strain. The detached cell viability could be directly related to the monochloramine concentration profiles in biofilms. The composition of EPS significantly affected monochloramine concentration at the biofilm diffusion boundary layer (Figure

Figure 5. Detached biofilm viability quantified using flow cytometry. (a) Flow cytometry data in dot plot for control and disinfected PAO1. Quadrant A: PI positive signal (dead cells); Quadrant B: PI and SYTO 9 positive signal (injured cells); Quadrant C: SYTO 9 positive signal (live cells); and Quadrant D: negative signal. (b) Percentages of live, dead, and injured cells in total detached cells. Notations in x-axis: C stands for control; D stands for disinfected. Data points represent mean ±1 standard deviation (N ≥ 24).

1), where detached cells immediately entered upon their release from biofilms. Overall, the monochloramine concentration decline was the most pronounced in the P. putida biofilm, which could be one important cause for the high viability of detached P. putida cells. In addition, we observed with planktonic cells that the presence of either protein-based or polysaccharide-based EPS resulted in a longer lag time and a higher Ct value to achieve a 2-log reduction level in the inactivation kinetics of P. aeruginosa and P. putida cells. The lag time of monochloramine inactivation for planktonic cells was 10−30 min, depending on the quantity of EPS.16 Considering the presence of cell clusters in the detached biofilm, lag time could be even longer than that of their planktonic cells.4 The combined time of effluent sample collection interval (30 min) and flowcell retention time (5 min) did not exceed 35 min, which reflects the end of lag phase or early exponential reduction phase in the inactivation kinetics. Due to the different length of lag phase, differences in viable cell numbers among strains were the most significant during the exponential reduction phase of cell viability and then the viability tended to reach a steady state after extended exposure time (over 60 min). In this study, the sample residence time reflected the differences in the susceptibility of detached clusters among the tested strains, showing a significant survival of cells detached from P. putida, PAO1, and mucA22 biofilms. 3837



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This study demonstrated that EPS quantity and composition significantly affected monochloramine penetration, biofilm inactivation, and detached cell viability due to the selective reactivity of monochloramine. The monochloramine concentration recommended by current regulations may be adequate to fully penetrate biofilms; however, cell inactivation may not necessarily be achieved within biofilms. Furthermore, monochloramine could be inefficient to control detached cell viability, especially for the EPS producing strains. When applying monochloramine as a residual disinfectant in drinking water distribution systems, the Ct value should be properly adjusted considering the monochloramine consumption by biofilm EPS in order to disinfect both biofilm and detached clusters efficiently.

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ASSOCIATED CONTENT

* Supporting Information S

Tables S1, Figures S1−S2, and Text S1−S2 are included. This material is available free of charge via the Internet at http:// pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*Phone: (419) 530-8131; fax: (419) 530-8116; e-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This project was supported by National Science Foundation (Award Number: CBET0933288) and UT faculty summer grant. We also acknowledge the UT Imaging Center for assisting CLSM analyses and Dr. Daniel Hassett for providing the Pseudomonas aeruginosa mutant strains. Dr. Woo Hyoung Lee at the U.S EPA was supported by the postdoctoral fellowship of the Oak Ridge Institute for Science and Education.

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REFERENCES

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Selective Reactivity of Monochloramine with Extracellular Matrix

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