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Jul 7, 2006 - National Homeland Security Research Center, Water. Infrastructure Protection Division, United States. Environmental Protection Agency ...
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Environ. Sci. Technol. 2006, 40, 4996-5002

Persistence of Klebsiella pneumoniae on Simulated Biofilm in a Model Drinking Water System J E F F R E Y G . S Z A B O , * ,† EUGENE W. RICE,‡ AND PAUL L. BISHOP† Department of Civil and Environmental Engineering, University of Cincinnati, Cincinnati, Ohio 45221, and National Homeland Security Research Center, Water Infrastructure Protection Division, United States Environmental Protection Agency, Cincinnati, Ohio 45268

Persistence of Klebsiella pneumoniae on corroded iron surfaces in drinking water was studied using biofilm annular reactors operated under oligotrophic conditions. Reactors were inoculated with K. pneumoniae, and persistence was monitored in the bulk and biofilm phases. Initial cell concentration of 106 MPN/mL in the bulk water phase resulted in significantly longer adhesion than initial concentrations 1 and 2 orders of magnitude lower. K. pneumoniae cultured in low nutrient growth medium persisted longer in dechlorinated tap water than those cultured in full strength medium. Cell surface charge was more negative under low nutrient conditions, and this influenced electrostatic attraction between the cells and the oxidized iron surface. Cells grown in full strength media persisted longer in water with both low (0.5 mg/L) free chlorine residuals. Growth media injected with the cells dechlorinated the water allowing adhesion without inactivation. Microelectrode measurements showed a 4070% drop in free chlorine from the bulk to the coupon surface, which decreased disinfectant potency against adhered cells. Growth and injection conditions clearly influenced cell adhesion and persistence, but permanent colonization of the corroded iron surface by K. pneumoniae was not observed.

Introduction Concerns about microbial persistence in a water distribution system following contamination have shifted from accidental events such as wastewater cross connections and treatment plant failures to intentional contamination. How long allochthonous pathogens survive on pipe surfaces and whether successful colonization can occur under oligotrophic drinking water conditions are topics of interest. Research examining microbial persistence in water distribution systems following contamination is limited in the open literature. Past research has focused on coliform regrowth (1-3) and wastewater cross connections (4, 5). Other researchers examined how long specific pathogens such as Helicobacter pylori (6), Legionella pneumophila (7), and viruses (8) persist on simulated biofilm. * Corresponding author phone: (513)487-2823, fax: (513)569-7052; e-mail: [email protected]. Current address: United States Environmental Protection Agency, National Homeland Security Research Center, Water Infrastructure Protection Division (MS 163), Cincinnati, OH 45268. † University of Cincinnati. ‡ United States Environmental Protection Agency. 4996

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Most research in this area has focused on drinking water biofilm grown on materials such as PVC or stainless steel. Both materials are readily available and easily sampled, but they are not representative of the pipe material that constitutes much of the nation’s drinking water infrastructure. The American Water Works Association’s WATER: [\]STATS 2002 survey of U.S. drinking water utilities concluded that unlined cast and ductile iron pipes make up 18% of the total pipe miles (9). A “corroding biofilm” is formed in these pipes where the cells and extracellular polymeric substances (EPS) are mixed with the corrosion products. Corroded iron supports faster biofilm growth and more biomass compared to PVC or stainless steel. It has been theorized that this is due to a combination of nutrient accumulation, surface roughness, and disinfectant demand (10). Corroded iron may represent a worst case scenario in terms of biofilm density and microbial adhesion (1, 10). Cell surface properties such as hydrophobicity and surface charge also play an important role in adhesion and persistence of cells to surfaces (11). Simple actions such as changing how a microorganism is cultured can have a dramatic impact on cell surface properties and, possibly, adhesion to surfaces (12-14). The presence of disinfectant residual will also impact how long pathogens not accustomed to a drinking water environment will survive. However, the dechlorinated environment is important when evaluating persistence after intentional contamination. Persistence of a laboratory strain of Klebsiella pneumoniae on a corroded iron surface representative of distribution system pipe material is the focus of this research. K. pneumoniae was chosen since it is easily enumerable, and it contains a distinct cell surface capsule, which can be manipulated to change cell surface properties. Biofilm annular reactors containing iron coupons corroded in drinking water were contaminated, and the presence of K. pneumoaniae in the bulk water and biofilm phases was monitored. The effect of initial concentration on K. pnuemoniae persistence in the reactor was evaluated, as was the effect of altering growth conditions. Survival with and without the presence of a free chlorine residual was also assessed.

Materials and Methods Experimental Reactor System. Temperature, substrate, and shear stress conditions of a drinking water pipe were reproduced in biofilm annular reactors (BioSurface Technologies Corporation, Bozeman, MT), described previously (1, 3). The reactors consist of a glass outer cylinder and a rotating polycarbonate inner cylinder with 20 flush mounted rectangular polycarbonate slides. Shear stress was applied to the slide surface by setting the inner cylinder rotation to 100 rpm, which produces shear similar to 30.5 cm/s (1 ft/s) flow in a 10.2 cm (4 in.) pipe. It should be noted that this shear is valid for a smooth surface on the inner drum of the reactor, and the rough surface of the corroded iron coupons protruded from the slide surface. As exact quantification of the shear on such a tortuous surface is impossible, the reported shear should be considered approximate. A mixed population “corroding biofilm” developed on 1 cm2 × 0.5 mm thick 99.5% pure iron coupons that were attached to the polycarbonate slides. Fouling biofilm is defined as the mixture of corrosion products and cells that form on the iron surface. Coupons were mounted on the polycarbonate slides with acrylic cement (TAP Plastics, Oakland, CA). Coupon surfaces in contact with the reactor bulk phase were abraded with medium grit emery paper 10.1021/es060857h CCC: $33.50

 2006 American Chemical Society Published on Web 07/07/2006

FIGURE 2. Persistence of K. pneumoniae cultured in full strength TSB on iron coupons when injected at different initial concentrations in the biofilm annular reactor. Tap water is dechlorinated. Initial concentration is listed in the legend. Curves represent the average between experiments in two independent reactors. Error bars represent the range between duplicate experiments.

FIGURE 1. Development of HPC on iron coupons. Figure 1a (top) shows colonization in two independent BARs with dechlorinated tap water. Figure 1b (bottom) has chlorinated water with the free chlorine concentrations of 0.1 and 0.5 mg/L. The curves in both plots represent two separate reactors. Error bars represent standard deviation from replicate measurements carried out in one reactor. before attachment to the slides to allow for corrosion over the entire coupon surface. A corroding biofilm was cultivated using Cincinnati tap water (chlorinated and dechlorinated) with a heterotrophic plate (HPC) count of approximately 102 CFU/mL as determined by spread plates (R2A, 20° C, 7 day incubation) (15). Dechlorination was performed with a 10% (w/v) sterile sodium thiosulfate solution. Thiosulfate solution was added in slight excess for dechlorination, but this did not effect pH or dissolved oxygen. HPC analysis was also performed on bulk water in the biofilm annular reactor and the corrosion material scraped from the coupons. Tap water pH (sample daily) and TOC (sampled monthly) were between 8.5 and 8.7 and 0.8-1.2 mg/L, respectively. An online TOC monitor at a nearby EPA facility confirmed the TOC readings. Reactor temperature, which was measured daily, was maintained at 21-23 °C. Free chlorine, also measured daily, ranged from 0.6 mg/L during low demand days (weekends, holidays) to 1.0 mg/L on high demand days. Tap water was fed at 0.5 L/h, which resulted in a 2 h residence time in the 1 L annular reactors. Reactors were disassembled, cleaned, reassembled, plumbed, and sterilized in an autoclave (121 °C, 19 psig, 25 min) before new uncorroded coupons were attached. Coupons were not autoclaved due to excessive corrosion and weakening of the adhesive during sterilization. Instead, the polycarbonate slides with adhered coupons were submerged in 100 mg/L sodium hypochlorite for 30 s, rinsed with sterile water, and aseptically inserted into the reactor. The disinfected coupons were scraped with a sterile scalpel into 10 mL of sterile 0.05 M KH2PO4 buffer (pH 7.2) and plated on R2A (20 °C, 7 days). Two coupons were analyzed each time

the reactors were started, and no colonies were detected on the plates after incubation. Sampling of Coupon Corrosion Material. Polycarbonate slides containing the coupons were aseptically removed from the annular reactor using a sterile slide removal tool (BioSurface Technologies Corporation, Bozeman, MT). Coupons were removed from the slide using a flame sterilized scalpel and tweezers. Corrosion material was scraped with the flame sterilized scalpel from the coupon into 10 mL of 0.05 M sterile KH2PO4 buffer (pH 7.2). When chlorine residual was maintained in the reactor, 0.1 mL of sterile 10% (w/v) sodium thiosulfate was added to the buffer. Corrosion particles were broken up with a tissue homogenizer (Tekmar, Cincinnati, OH) for 30 s. The homogenized samples were then serially diluted and analyzed. Cell Culturing, Analysis, and Inoculation. Annular reactors were contaminated with a laboratory strain of Klebsiella pneumoniae (ATCC 13883). K. pneumoniae injected into the biofilm annular reactors was cultured in Trypticase Soy Broth (TSB) (Becton Dickenson, Sparks, MD) at full strength or in 1:1000 TSB diluted in 0.05 M KH2PO4 (pH 7.2). K. pneumoniae was inoculated into either full strength TSB and incubated for 24 h or 1:1000 TSB and incubated for 72 h at 23 °C. Biofilm annular reactors were seeded with these suspensions by adding volumes of the cell suspensions necessary to achieve 104-106 MPN/mL inside the reactors. Cell densities were estimated using McFarland standards (bioMerieux, Marcy l’Etoile, France) so this volume could be approximately determined. However, the concentration of K. pneumoniae in the culture flask was directly measured, so the initial concentration of K. pneumoniae in the reactor could be accurately reported by accounting for dilution of the injected volume by the volume of liquid in the reactor. K. pneumoniae was enumerated using Colilert and QuantiTray/2000 (Idexx Corporation, Westbrook, ME) (15). Homogenized corrosion material suspended in 10 mL of sterile 0.05 M KH2PO4 buffer (pH 7.2) and 10 mL of bulk water from the biofilm annular reactor were used for enumeration of the biofilm and bulk water phases, respectively. Serial dilutions were made using sterile Nanopure water. Positive wells were counted after incubation, and the most probable number (MPN) was determined using the Quanti-Tray/2000 MPN Table. All analyses were performed in duplicate, and blanks containing dilution water were run with each sample. Tap water and fouling biofilm were monitored before experimentation, and no coliforms or false positives were VOL. 40, NO. 16, 2006 / ENVIRONMENTAL SCIENCE & TECHNOLOGY

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FIGURE 3. Persistence of K. pneumoniae cultured in 1:1000 TSB on iron coupons when injected at different initial concentrations in the biofilm annular reactor. Tap water is dechlorinated. Initial concentration is listed in the legend. Curves represent the average between experiments in two independent reactors. Error bars represent the range between duplicate experiments. detected. Further detail on K. pneumoniae enumeration with Colilert is included in the Supporting Information. Hydrophobicity. The bacterial adhesion to hydrocarbons (BATH) test, also known as the microbial adhesion to hydrocarbons (MATH) test, was performed according to the protocol used by Matz and Jurgens (16), which is based on the original method published by Rosenberg et al. (17) but incorporates the recommendations of Pembrey et al. (18). Cell suspensions were washed and suspended in dechlorinated tap water, and the optical density (OD) at 550 nm was measured. Four milliliters of the suspension (ODinitial) were mixed with 1 mL of n-hexadecane. The suspension was vortexed for 1 min and settled for 15 min, and the OD of the resulting aqueous layer (ODfinal) was measured. The hydrophobicity index, reported as a percentage, was calculated using the following equation: [(ODinitial-ODfinal)/ODinitial] × 100. Detailed discussion of the assay is in the Supporting Information. Electrophoretic Mobility. Electrophoretic mobility (EPM) was measured with a Zetasizer 3000 HSA (Malvern Instruments Ltd., Malvern, England) following the procedure used by Lytle et al. (19). Cell suspensions of approximately 107 MPN/mL were used for all measurements. EPM measurements were conducted in 0.05 M KH2PO4 buffer at pH 7.2 and in dechlorinated tap water. Six replicates were performed for each suspension. A detailed procedure is included in the Supporting Information. Microelectrode Fabrication and Measurements. Microelectrode assembly and use followed the original design of De Beer et al. (20). Calibration and profile measurements were conducted at an applied potential of 200 mV and with an Ag/AgCl reference electrode in a specially designed flow cell inside a Faraday cage, which minimized electrical interferences. Microprofile measurements were carried out in 0.05 M KH2PO4 buffer at pH 7.2 supplemented with free chlorine at concentrations of 0.25, 0.5, 1.4 and 4.5 mg/L in the flow cell. Control measurements with no chlorine showed no response from the microelectrode. Coupons taken from the biofilm annular reactors had been corroding under shear as described above for at least 1 month in tap water with a stable chlorine residual of 0.6-1.0 mg/L. Chlorine profiles were measured after at least 10 min of contact time. Further description of microelectrode fabrication and use is included in the Supporting Information. Quality Control. Experiments were repeated independently in two biofilm annular reactors. HPC was performed 4998

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FIGURE 4. Comparison of K. pneumoniae persistence when cultured in full strength and 1:1000 TSB. Initial concentration in the reactor is in the legend in parentheses. Tap water is dechlorinated. Curves represent the average between experiments in two independent reactors and are the same curves presented in Figures 2 and 3 injected at the 106 MPN/mL level. Error bars represent the range between duplicate experiments. in triplicate, and Colilert was performed in duplicate. Replicate analyses allowed for quantification of random errors associated with sampling, dilution, and enumeration. Relative standard deviation for HPC and Klebsiella pneumoniae enumeration using Colilert were generally between 10 and 15% and 10-20%, respectively. The only exception to this was at very low cell concentrations (less than 10 CFU or MPN per mL) when relative standard deviation was sometimes over 50%. K. pneumoniae adhesion/detachment curves (Figures 2-5) are reported as the average of two independent experiments, with the range reported as error bars. This allows visualization of experimental reproducibility when performed independently under identical conditions. K. pneumoniae was considered extinct from the reactor when three consecutive coupon or bulk samples showed none present. The point that the curves reach zero in Figures 2-5 is the first of these consecutive points. Free chlorine concentrations were determined by using the N,N-diethyl-p-phenylenediamine (DPD) colorimetric method (15). Analyses were performed in duplicate with relative standard deviation usually less than 10%. Free chlorine profiles measured with a microelectrode were performed in duplicate. Reproducibility of the measurements is reported by plotting the duplicate curves together (Figure 6). The signal generated from the microelectrode during measurement in a flowing environment is not completely stable. Error bars on profiles indicate the range in which the microelectrode signal fluctuated and the individual points represent the average of this range.

Results and Discussion Formation of a Corroding Biofilm. Figure 1a,b shows the colonization of the iron coupons over time by microorganisms indigenous to Cincinnati tap water. Figure 1a shows that HPC levels on the coupons stabilized between 5 and 7 days when dechlorinated tap water was fed to the reactors. HPC in the biofilm annular reactor bulk liquid was approximately 105 CFU/mL, which was higher than tap water fed to the reactor. This is likely due to biofilm sloughing inside the reactor, which has been observed in other studies using biofilm annular reactors (1, 3). Figure 1b shows coupon HPC with chlorine residual present in the bulk water. An order of magnitude drop in HPC is seen between days 15 and 28. Chlorinated tap water was fed to the reactors for the first 22 days of operation, at

FIGURE 5. Comparison of K. pneumoniae persistence on corroded iron coupons in chlorinated water. Dechlorinated water (shown previously) is compared with low and high chlorine residual experiments. Experiments with low chlorine residual fluctuated between 0.07 and 0.17 mg/L, while high residual experiments ranged from 0.59 to 0.64 mg/L. Error bars represent the range between duplicate experiments. which time it was discovered that the iron coupons consumed all of the free chlorine and the bulk liquid chlorine residual was zero. From that time on, chlorine residual was maintained by adding a concentrated sodium hypochlorite solution with syringe pumps. HPC stabilized after 28 days at two chlorine residuals, with some variation at day 38. Bulk liquid HPC levels at 0.1 and 0.5 mg/L free chlorine were approximately 104 and 103 CFU/mL, respectively, compared to 105 CFU/mL in dechlorinated water. HPC levels similar to those in Figure 1b were found on cast iron coupons installed in chloraminated distribution mains (21). Biofilm formation was influenced most by temperature and disinfectant concentration under oligotrophic conditions found in a distribution system. For that reason, both parameters were kept constant in the biofilm annular reactors during biofilm formation in this study. Reactor temperature was maintained at the laboratory level of 21-23 °C. Free chlorine residual was kept within 0.1 mg/L of the reported values. Furthermore, in both chlorinated and dechlorinated water, 30 days were allowed for corrosion and biofilm formation on the iron coupons before experiments began. In addition to stable HPC, visual inspection of the coupons showed heavy corrosion and tuberculation after 1 month. The corrosion surface ranged from thin layers of rust barely covering the unoxidized metal surface to tubercles ranging from 0.5 to 2 mm deep. Tubercle depth was measured with a microelectrode, and the procedure is described in the Supporting Information. Reactor Contamination with Klebsiella pneumoniae. Effect of Initial Concentration. Unwashed K. pneumoniae suspensions were pulsed injected into the biofilm annular reactors in less than 1 min. Figure 2 shows the persistence of K. pneumoniae on the biofilm when cultured in full strength TSB and injected into dechlorinated water. Initial bulk water concentrations were 4.8 × 104, 1.5 × 105, and 9.6 × 105 MPN/ mL. The suspensions with initial concentrations of 4.8 × 104 and 1.5 × 105 MPN/mL show similar initial trends, although the lower concentration was still detected at low levels 2 days after the higher concentration had disappeared. When the initial concentration in the reactor was increased to just under 106 MPN/mL, K. pneumoniae disappeared from the coupon 9 days after injection. K. pneumoniae was still present at 102 MPN/cm2 5 days after injection and did not drop below 10 MPN/cm2 until day 7. From day 1 on, less than 10 MPN/ mL were detected in the bulk water, and none were detected after day 5. The K. pneumoniae suspensions injected at the

FIGURE 6. Free chlorine microprofiles leading from the bulk to the coupon surface. Zero represents the biofilm/corrosion layer that covered the entire coupon surface and the values on the x-axis represent distance from the surface. Duplicate profiles are plotted together. Error bars represent the range in which the microelectrode signal fluctuated at each point during measurement, with each point representing the average of that range. two lower concentrations both disappeared from the bulk at day 2. Exponential decay in an ideal CSTR at initial bulk concentrations of 106, 105, and 104 MPN/mL leads to 1 MPN/ mL or less at 1.2, 1.0, and 0.8 days, respectively. Figure 3 shows the persistence of the 1:1000 TSB culture inoculated at initial bulk concentrations of 5.5 × 104 and 1.2 × 106 MPN/mL. The higher concentration was detected on the coupons up to 16 days after inoculation, while the low concentration was last found at day 4. K. pneumoniae in the bulk phase was found up to 6 days after inoculation at the high initial concentration but had disappeared at day 1 at the low concentration. The presence of K. pneumoniae in the bulk at the high concentration for 6 days after inoculation indicates that some were detaching from the coupon surface since the Klebsiella initially suspended in the bulk phase should have washed out of the reactor. These results indicate that the concentration of K. pneumoniae in the bulk liquid will impact how long the microorganisms persist on the coupon surface. Fass et al. (2) injected Escherichia coli into a recirculating pipe loop with a biofilm grown from dechlorinated tap water on PVC coupons. Persistence results were similar to those found in this study at an initial bulk concentration of 106 CFU/mL. However, the authors did not determine the effect of initial concentration on persistence. Warren et al. (22) found that concentration indeed impacted persistence on a complex, multispecies biofilm. When challenged with Pseudomonas fluorescens at 103 and 106 CFU/mL, the 106 CFU/mL culture colonized a complex biofilm grown from lake water. The fouled iron coupons represent a complex biofilm system with many microbial species colonizing the surface. Significant persistence or long term colonization may only be seen at high initial concentrations of allochthonous cells since high concentrations contain more cells able to compete with the established bioiflm organisms or able to adhere strongly and resist being sheared from the surface. Effect of K. pneumoniae Growth Conditions. Figure 4 compares the persistence of K. pneumoniae on the coupons when cultured in full strength and 1:1000 TSB and injected at initial concentrations of 9.6 × 105 and 1.2 × 106 MPN/mL, respectively. K. pneumoniae cultured in low strength media persisted longer on the coupons in dechlorinated water than when cultured in full strength media. The low strength culture was found at 16 days after inoculation, while the full strength culture was detected only up to day 7. The initial concentraVOL. 40, NO. 16, 2006 / ENVIRONMENTAL SCIENCE & TECHNOLOGY

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tion of K. pneumoniae on the coupon was higher when cultured in 1:1000 TSB, even though cultures grown in both medias were injected at approximately the same concentration. As with all of the injections, a sharp decrease in K. pneumoniae on the coupon was observed 1 day after injection. Approximately 8% of the K. pneumoniae grown in full strength media initially adhered to the coupon remained at day 1, while 0.1% remained of the 1:1000 culture. However, the culture grown in full strength TSB continuously decreased over time, while the low strength culture persisted at roughly 103 MPN/cm2 for 5 days (days 1-6) before beginning a gradual decline. The hydrophobicity index of 1:1000 TSB K. pneumonaie was 8.5 ( 6.0%, which represents a slight increase from the full strength TSB index of 1.2 ( 0.9%. Still, both cultures remained relatively hydrophilic. Electrophoretic mobility was significantly more negative in cells cultured in 1:1000 TSB. Electrophoretic mobility of the full strength culture was -1.96 ( 0.26 µm cm V-1 s-1 (zeta potential: -24.7 ( 3.3 mV), while the dilute culture was -2.85 ( 0.17 µm cm V-1 s-1 (zeta potential: -35.9 ( 2.2 mV). The increased persistence of K. pneumoniae grown in 1:1000 TSB may be related to the change in cell surface properties that sometimes occur when microorganisms are cultured under low nutrient or starvation conditions. Numerous researchers have noted that bacteria cultured under these conditions have become more hydrophobic as determined by BATH or other hydrophobicity assays (12-14, 2325). The K. pneumoniae used in this study showed a very small increase in hydrophobicity index, and both cultures remained relatively hydrophilic, which indicates that the cultures had the same affinity for the hydrophilic portions of the oxidized iron surface when cultured in either media. Electrophoretic mobility of the 1:1000 TSB K. pneumoniae culture showed a significant increase in surface negativity compared to the full strength culture. Fox (26) found that Cryptosporidium oocysts suspended in deionized water gradually changed from a zeta potential of -6 mV to -14 mV over 8 days. The iron oxides such as goethite, magnetite, and lepidocrocite, among others, are common corrosion products on the iron surface in water pipes (27). These iron oxides generally have an isoelectric point at pH of 6-10 (28), which means the net surface charge will be predominantly negative at the pH of 8.5 used in this study. As the cell surfaces of both cultures were negatively charged, the conditions were not favorable for attachment and persistence from an electrostatic perspective. However, since electrophoretic mobility of the 1:1000 TSB culture was significantly more negative than those of the full strength TSB culture, the resistance to adhesion was greater and may explain why only 0.1% of the initially adhered 1:1000 K. pneumoniae cells were detected on the coupon surface 1 day after inoculation (Figure 4), compared to 8% of the full strength culture. Still, when considering the rough surface of the corroded iron coupons, it is also quite possible that cells were simply lodged in a crevice or ravine that prevented detachment due to shear. If shear forces did not detach the organisms, competition for nutrients with established biofilm organisms caused detachment of the K. pneumoniae or left them in a stressed and noncultureable state. After the initial steep decrease of the 1:1000 TSB culture occurs in Figure 4, what remained of the culture at day 1 persisted longer than the full strength TSB culture. When grown in low nutrient conditions, it has been shown that bacteria alter cell surface properties and metabolic pathways, and cell surfaces become more sticky and amenable to adhesion (1, 12, 29). The low nutrient culture was more acclimated to the low nutrient conditions that a drinking water environment presents and survived longer, although permanent colonization did not occur. Past research has 5000

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shown that long-term colonization is possible when coliforms are adhered to a sterile surface and grow with the rest of a heterotrophic biofilm (1). However, when adhered to an established biofilm/corrosion layer, K. pneumoniae did not colonize the surface, although the trend of cells cultured in a low nutrient environment persisting longer than those in a rich medium was maintained. Effect of Chlorine Residual. K. pneumoniae cultured in full strength TSB was injected into reactors containing free chlorine in the bulk water. Initial K. pneumoniae concentrations were approximately 106 MPN/mL. Figure 5 compares the experiments conducted in dechlorinated water with water containing high (0.59-0.64 mg/L) and low chlorine residual (0.07-0.17 mg/L). Chlorine residual fluctuated between the ranges reported during the duplicate experiments. At both the high and low chlorine levels, the number of K. pneumoniae on the coupons at day 1 was reduced significantly. However, the injected cells still persisted in the presence of chlorine for as long as the culture in dechlorinated water at concentrations between 10 and 102 MPN/cm2. Few K. pneumoniae cells were detected in the bulk at any chlorine level. None were detected at day 1 or beyond at the high range, and less than 10 MPN/mL were found at the lower levels, with none detected at 3 days after injection. The same experiments with similar chlorine levels were conducted with a 1:1000 TSB culture. Low chlorine ranged from 0.06 to 0.13 mg/L, and high chlorine levels were 0.68-0.84 mg/L. When the reactors were challenged with K. pneumoniae grown in a 1:1000 TSB, none of these organisms were recovered from coupons or the chlorinated water. K. pneumoniae cultured in full strength TSB persisted on the corroded coupon surface, while those grown in 1:1000 TSB were quickly inactivated when introduced into chlorinated water. Past research has shown that bacteria cultured in low nutrient growth media are more resistant to disinfectants than the same cells grown in rich media (30-33). However, inactivation studies carried out by Virto et al. (34) compared the survival of various bacteria introduced into chlorinated water with and without a chlorine demanding substrate (growth media). When the growth media was introduced with the microorganisms, they survived longer than when they were introduced without. Any disinfectant resistance K. pneumoniae developed from cultivation in 1:1000 TSB did not help them survive beyond 30 min, which was the first sample taken after injection, at the chlorine levels used in this study. When K. pneumoniae suspensions cultured in full strength TSB were injected (10 mL), all of the free chlorine in the reactor was consumed by the growth media. This allowed transport and adhesion to the coupon surface without substantial cell inactivation. Separate inactivation experiments in chlorinated water show that washed K. pneumoniae cultured in full strength TSB were not detectable 30 s after inoculation in water with 0.35 mg/L (CT < 0.18 mg‚min/L) when no other substrate demands the free chlorine (Supporting Information, Figure S-2). Even though chlorine residual returned to the high and low level ranges at day 1, the cells survived at low concentration for as long as when injected into dechlorinated water. Therefore, increased persistence of K. pneumoniae cells cultured in full strength TSB in chlorinated water was due to dechlorination of the bulk water with growth media. K. pneumoniae cultured in 1:1000 TSB did not have this advantage but will persist longer if the water is dechlorinated by other means. Persistence in the Presence of Free Chlorine. Although the experiments were not as reproducible, Figure 5 shows that low levels of K. pneumoniae definitely persisted at low levels on the coupons in chlorinated water as long as in dechlorinated water. Free chlorine concentration at the surface of the coupon was investigated as a reason for survival.

Chlorine microprofiles from the bulk water to the corroded iron surface were recorded using a chlorine microelectrode (20) and are displayed in Figure 6 (calibration curve is included in the Supporting Information, Figure S-1). Disinfectant profiles were only measured to the surface of the coupon since the microelectrode tip would have broken against the corrosion surface. All of the profiles show similar trends, with stable residual chlorine in the bulk liquid until 100-200 µm from the coupons surface where the diffusion boundary layer begins. Although the chlorine profile inside the corroding biofilm layer could not be determined, the profiles are characteristic for disinfectant diffusing into a matrix where it is being consumed (20, 35-36). Free chlorine concentrations were 40-70% lower at the surface that the microelectrode could reach compared to the bulk liquid. Past studies have recorded pH profiles leading up to the surface of a corroded cast iron drinking water pipe with a pH microelectrode. Dechlorinated Cincinnati tap water pH increased from 7.2 in the bulk water to between 8 and 9 at the corrosion surface (37). This is likely due to the release of hydroxyl ions from iron hydroxide formed during corrosion. Past research has also shown that the chlorine microelectrode signal decreases with increasing pH (20, 36). This could lead to underestimation of the true chlorine concentration close to the corrosion surface when using this microelectrode. A pH increase close to the surface would decrease the potency of free chlorine reaching the surface since hypochlorous acid speciates to the less biocidal hypochlorite ion at a pH above its pKa of 7.5 (27, 38). K. pneumoniae persisted on the corroded iron coupons in the presence of free chlorine due to ineffective transport of disinfectant to the coupon surface. Changes in pH that favor hypochlorite ion over hypochlorous acid could also be a factor. It has been established that microorganisms associated with biofilm are more persistent than their planktonic counterparts (3, 39). This is primarily due to the biofilm matrix consuming disinfectant and diffusion resistance though the hydrodynamic boundary layer (35, 40, 41). These factors allow K. pneumoniae persistence for multiple days even in the presence of free chlorine. These results provide important knowledge from a homeland security perspective. Although electrostatic effects and protection from free chlorine may have played a part in the initial persistence on the corroding biofilm surface, K. pneumoniae did not successfully colonize the corroded iron surface or persist for longer than 2 weeks in chlorinated or dechlorinated conditions. This could have been due to shear forces, competition with established biofilm organisms, or inactivation by chlorine. The primary conclusion is that, given enough time, vegetative K. pneumoniae will not be detectable on, and presumably be extinct from, the corrosion surface. Free chlorine drastically decreased the density of K. pneumoniae on the coupon surfaces, but inoculated cells still persisted for as long as in dechlorinated water at low concentrations. This is interesting in that adding high concentrations of free chlorine may not decontaminate pipe material faster than normal disinfection residual. Based on the microelectrode measurements presented earlier, there may be places on the tortuous iron surface that free chlorine cannot reach, and any adhered cells must detach by other means. As this study only dealt with vegetative K. pneumoniae, future work will focus on bacterial spores that are more resistant to free chlorine.

Acknowledgments The authors thank Noreen Adcock for preparing the frozen K. pneumoniae and Darren Lytle for allowing use of the Zetasizer. This research was supported by EPA contract 68C-00-159.

Supporting Information Available Detailed description of K. pneumoniae enumeration, hydrophobicity assay, use of the Zetasizer for electrophoretic mobility measurements, microelectrode fabrication, and a calibration curve for the microelectrode used in this study and the fate of washed K. pneumoniae cells in chlorinated water and a method for measuring biofilm/tubercle depth with a microelectrode. This material is available free of charge via the Internet at http://pubs.acs.org.

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Received for review April 10, 2006. Revised manuscript received June 14, 2006. Accepted June 15, 2006. ES060857H