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Free chlorine demand and cell survival of microbial suspensions
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Free chlorine demand and cell survival of microbial suspensions Damian E. Helbling, Jeanne M. VanBriesen1 Department of Civil and Environmental Engineering, Carnegie Mellon University, 5000 Forbes Avenue, Pittsburgh, PA 15213, USA
ar t ic l e i n f o
abs tra ct
Article history:
The utility of chlorine residual and chlorine demand as a surrogate for microbial
Received 19 December 2006
contamination in the water distribution system was evaluated. The chlorine demanded
Received in revised form
by and cell survival of pure culture suspensions of Escherichia coli, Staphylococcus epidermidis,
21 March 2007
and Mycobacterium aurum were quantified in solutions with initial free chlorine concentra-
Accepted 4 June 2007
tions of 0.20, 0.40, and 0.80 mg/L. The chlorine demand increased with initial concentration
Available online 12 June 2007
of cells and free chlorine for all species. At equivalent initial cell concentrations, chlorine
Keywords: Chlorine demand Free chlorine Water security Water distribution system
demand was greatest for M. aurum, followed by S. epidermidis and E. coli. The chlorine contact time required for a 3-log inactivation of E. coli, S. epidermidis, and M. aurum was calculated as 0.03270.009, 0.22170.080, and 42.972.71 mg min/L, respectively. The ultimate chlorine demand and cell survival were directly proportional. No chlorine demand was observed at cell concentrations less than 105 CFU/mL for E. coli or 104 CFU/mL for S. epidermidis. M. aurum demanded chlorine at all initial cell concentrations including 103 CFU/mL, which was the detection limit of the cell quantification assay. Chlorine demand was determined to be a suitable surrogate indicator of the organisms studied and its utility may be enhanced in locations of the water distribution system that maintain a higher free chlorine residual. & 2007 Elsevier Ltd. All rights reserved.
1.
Introduction
The threat of accidental or intentional pathogenic intrusions into drinking water infrastructure, including distribution systems, is very serious. In the United States in the years 2001 and 2002, the Centers for Disease Control and Prevention reported that an estimated 1020 people were infected by water-borne diseases originating in drinking water (7 fatally) (Blackburn et al., 2004). Organisms identified in the individual outbreaks included Escherichia coli, Campylobacter jejuni, Yersinia enterocolitica, Vibrio cholerae, and several Legionella species. Many of the reported incidents were attributed to either failed treatment processes or contamination in the distribution system during anomalous periods of negative pressure
(Blackburn et al., 2004). Water-borne infectious diseases not only cause loss of life and illness but also have negative effects on the economy related to medical expenses and productivity losses (Corso et al., 2003). In addition to accidental outbreaks of water-borne illnesses, the United States Department of Homeland Security and the United States Department of Environmental Protection have made public statements that drinking water facilities in the United States may be a target for terrorist activity (Whitman, 2002). An intentional act of drinking water contamination could create catastrophic morbidity and mortality, overwhelm medical response personnel and infrastructure, and have significant negative effects on the economy and psyche of the nation. Continuous, real-time monitoring of critical
Corresponding author. Tel.: +412 268 3819; fax: +412 268 7813.
E-mail addresses: [email protected] (D.E. Helbling), [email protected] (J.M. VanBriesen). 1 Tel.: +412 268 4603; fax: +412 268 7813. 0043-1354/$ - see front matter & 2007 Elsevier Ltd. All rights reserved. doi:10.1016/j.watres.2007.06.006
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water infrastructure is imperative to provide early warning and minimize the effects of such contamination events (Luthy, 2002). The distribution system has been identified as a particularly vulnerable component of drinking water infrastructure (Khan et al., 2001) and monitoring water quality in the distribution system could provide early warning of contamination events. The term ‘sensor’ will be used in the remainder of this work to describe a device that is used to obtain continuous, realtime data of a specific water quality parameter. While there are no existing sensors that can detect all possible pathogens that might be introduced to a distribution system (Deisingh, 2004; Rodriguez-Mozaz et al., 2004), there are many commercially available sensors that monitor for water quality parameters such as pH, temperature, conductivity, turbidity, total organic carbon, total chlorine, combined chlorine, and free chlorine. The efficacy of any of these water quality parameters as a surrogate for pathogenic microorganisms has not been fully studied, but free chlorine has been suggested to be the water quality parameter that reacts with the widest range of contaminants (United States Environmental Protection Agency, 2005). Free chlorine sensors have been shown to be adequate surrogate monitors when analyzing for chemical species such as the pesticide aldicarb and the toxin sodium cyanide (Byer, 2005); however, very little research has been done on the effect of specific microorganisms on residual chlorine in drinking water distribution systems. Although the mechanisms involved in microbial inactivation by free chlorine are not fully understood, it is believed that the primary reaction involves oxidation of microbial membranes increasing cell permeability, which results in leakage of macromolecules and cell death (Venkobachar et al., 1977; Virto et al., 2004). More recent studies have shown that membrane damage is not the main mechanism of inactivation for cells exposed to oxidative stresses; more subtle effects such as an uncoupling of the electron chain or enzyme inactivation may be dominant (Virto et al., 2005b). The chlorine demand of a microbial suspension is defined as the difference between the initial chlorine concentration and the chlorine concentration after a specified contact time. As the contact time increases, the chlorine demand approaches an ultimate value that indicates that all organic material originally present and available for reaction with chlorine has been exhausted. It is hypothesized that the chlorine demand signal generated by the reaction between free chlorine and microbial material may be used as a surrogate indicator of microbial contamination in water distribution systems. The utility of free chlorine as a surrogate analyte depends on the chlorine demand of microorganisms. Studies that have investigated chlorine demand have shown that different organisms have unique chlorine demands, even at similar organic carbon content and concentration (Shang and Blatchley, 2001). Of particular concern is the relationship between chlorine demand and cell survival. If chlorine demand is only exerted during the inactivation of microorganisms, then it will not provide adequate early warning against chlorineresistant organisms. Chlorine-resistant organisms are of particular interest in water security problems because they persist and remain infective in oxidative environments. The naturally occurring microbial flora in water distribution
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systems is very diverse and can contain benign organisms and pathogens, despite the presence of the chlorine residual (Keinanen et al., 2002; Schmeisser et al., 2003; Eichler et al., 2006). Even the highly chlorine-sensitive indicator organism E. coli has been found to persist and even to grow in the presence of a chlorine residual (Fass et al., 1996; Williams and Braun-Howland, 2003). Resistance to free chlorine has been attributed to unique variations in the cellular structure and protein composition of different organisms (Cloete, 2003). In addition to physical attributes that aid in resistance to free chlorine, microorganisms defend against oxidative stresses by inherent or adapted resistance mechanisms that produce extracellular polymeric substances (EPSs). These substances react with the oxidative disinfectant, which effectively reduces the concentration of disinfectant at the cell wall or cell membrane where cell damage can occur (Lisle et al., 1998; Ryu and Beuchat, 2004). If this presumed mechanism is correct, resistant organisms will exert a strong chlorine demand while remaining viable and infective. The objective of this work is to investigate the limitations of chlorine demand as a surrogate indicator for microbial contamination by investigating the chlorine demand and survival of different organisms under different initial free chlorine concentrations. The relationship between chlorine demand and cell survival was investigated by selecting organisms that have a range of reported resistances to free chlorine as a disinfectant.
2.
Materials and methods
2.1.
Cultures and growth conditions
Organisms studied in this work included: American type culture collection (ATCC) 11775, E. coli; ATCC 35984 Staphylococcus epidermidis; and ATCC 23366 Mycobacterium aurum. E. coli was cultivated from a loop of pure culture organisms maintained on Tryptic Soy Broth (TSB, Soybean-Casein Digest Medium, Difco) agar (Fisher Scientific) plates at 4 1C. E. coli was aseptically transferred and incubated in 60 mL of TSB broth and harvested during stationary phase after 40 h of incubation at 37 1C. S. epidermidis was cultivated from a loop of pure culture organisms maintained on TSB (Difco) agar (Fisher Scientific) plates at 4 1C. S. epidermidis was aseptically transferred and incubated in 60 mL of TSB broth and harvested during stationary phase after 48 h of incubation at 37 1C. M. aurum was cultivated from a loop of pure culture organisms maintained on Middlebrook 7H10 Agar (Difco) plates amended with Middlebrook OADC Enrichment (Difco) and glycerol (Acros Organics) at 4 1C. M. aurum was aseptically transferred and incubated in 180 mL of Middlebrook 7H9 Broth (Difco) amended with Middlebrook ADC Enrichment (Difco) and glycerol (Arcos Organics) and harvested during stationary phase after 216 h (9 days) of incubation at 37 1C. Prior to each experiment, cell cultures were aseptically transferred to sterile plastic tubes and centrifuged at 6000g for 5 min. The liquid growth media was separated from the pellet and discarded. Cells were re-suspended in a volume of 0.1 M chlorine demand-free Sorensen’s phosphate buffer at a pH of 7.4 equal to the volume of the discarded supernatant.
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Sorensen’s phosphate buffer was prepared by combining a 0.2 M sodium phosphate monobasic stock solution with a 0.2 M sodium phosphate dibasic anhydrous stock solution and deionized water at a volumetric ratio of 57:243:600. Resuspended cells were centrifuged, separated, and re-suspended in phosphate buffer three additional times in order to completely separate the cells from their growth media, which could cause interferences in the chlorine demand experiments.
2.2.
Chlorine solutions
Chlorine solutions were prepared daily by diluting a stock solution of 5% sodium hypochlorite (LabChem, Inc.) in carbon-free deionized water (Barnstead NANOpure). Sodium hypochlorite stock was diluted to produce solutions of final free chlorine concentrations of 0.2070.02, 0.4070.02, and 0.8070.02 mg/L. Final free chlorine concentrations were verified in triplicate by the DPD colorimetric method.
2.3.
Free chlorine determination
Free chlorine concentrations were verified with the N,Ndiethyl-p-phenylenediamine (DPD) colorimetric method (APHA AWWA WEF, 2005). In this method, the DPD is oxidized by free chlorine, resulting in a solution with a color intensity proportional to the free chlorine concentration. The absorption of the resulting solution is measured by photometry. DPD reagents were provided by PPD-2 DPD Powder Pop Dispenser (HF Scientific). The absorption of the solution was quantified using the Free Chlorine Pocket Photometer (HF Scientific). The photometer has a reported accuracy of 72% within the concentration range used in this work.
2.4.
described for the chlorine demand assays. Free chlorine solutions of 0.2070.02, 0.4070.02, and 0.8070.02 mg/L of volume 198 mL were added to cleaned 250-mL glass Erlenmeyer flasks with a magnetic stir bar. The flasks were placed on a magnetic stir plate and set to 155 RPM to provide gentle, continuous mixing. At t ¼ 0, 2 mL of cells suspended in phosphate buffer were added to the flask. Samples of volume 1 mL were drawn from the flask at t ¼ 0.25, 0.50, 0.75, 1, 1.5, 2, 3, 4, and 5 min for E. coli and S. epidermidis and at t ¼ 5, 15, 30, 60, 90, 120, 150, and 180 min for M. aurum. Samples were immediately added to 9 mL of neutralizing buffer (Difco) to quench the remaining free chlorine in the sample. After sampling was completed, 1 mL of the 101 dilution in neutralizing buffer (Difco) was serially diluted in 9 mL of phosphate buffer to selected final dilutions. Each dilution was quantified in triplicate and the final concentration was reported as the average of the countable plates. E. coli and S. epidermidis were quantified by a spread plate on TSB (Difco) agar (Fisher Scientific) plates. M. aurum was quantified by a spread plate on Middlebrook 7H10 Agar (Difco) amended with Middlebrook OADC Enrichment (Difco) and glycerol (Acros Organics). All organisms were incubated at 37 1C. Plates were quantified after 18, 24, and 120 h for E. coli, S. epidermidis, and M. aurum, respectively. Cell survival assays were repeated at least once for each set of initial conditions in order to verify the reproducibility of the results. Because each 1 mL sample was immediately diluted in 9 mL of neutralizing buffer, the first quantified concentration was a 101 dilution. Plates were considered countable only if they contained a number of colonies between 30 and 300 (APHA AWWA WEF, 2005). Plates were spread with 100 mL of each sample dilution. Therefore, the cell quantification detection limit of the method was 3 103 CFU/mL.
Chlorine demand assay 2.6.
All glassware was washed with detergent twice and rinsed with hot water, rinsed twice with a solution of 0.1 N HCl, rinsed thrice with deionized water and allowed to air dry. Glassware was then capped with aluminum foil and baked at 550 1C for 6 h. This cleaning process was employed in order to prevent interferences from environmental organic material. Free chlorine solutions of 0.2070.02, 0.4070.02, and 0.8070.02 mg/L of volume 198 mL were added to cleaned 250-mL glass Erlenmeyer flasks with a magnetic stir bar. The flasks were placed on a magnetic stir plate and set to 155 RPM to provide gentle, continuous mixing. At t ¼ 0, 2 mL of cells suspended in phosphate buffer was added to the flask. At t ¼ 0.25, 0.50, 1, 1.5, 2, 3, 4, 5, 10, 15, 20, 30, 45, 60, 90, and 120 min, 2.5 mL of samples were drawn in duplicate from the flask and analyzed for free chlorine via the DPD method. Free chlorine was analyzed additionally at 150, 180, 210, and 240 min for M. aurum assays. The reported free chlorine concentration was the average of the samples taken. Chlorine demand assays were repeated at least once for each set of initial conditions in order to verify the reproducibility of the results.
2.5.
Cell survival assays
Cell survival assays were conducted in parallel with the chlorine demand assays. Glassware was prepared as
Organism selection and analytical conditions
The type of residual chlorine selected for this study was free chlorine as it is the most common form of chlorine used as a disinfectant in the United States (Haas et al., 1992), is used in the City of Pittsburgh (the location of the study) water distribution system, and has a well-documented efficacy as a disinfectant. Organisms were selected to provide a range of cell wall structures, EPS production, and reported resistance to free chlorine as a disinfectant. The organisms selected were: the Gram-positive S. epidermidis, which is a known EPS producer; the Gram-negative E. coli, which is a common water indicator organism, is not known to produce a significant amount of EPS, and is considered to be very sensitive to free chlorine when grown in ideal environmental conditions; and M. aurum (neither Gram positive nor Gram negative), which has been isolated in drinking water distribution systems (Le Dantec et al., 2002) and is an organism that has been observed to be very resistant to oxidative stresses. In order to meet the objectives of this work, each organism was combined with various initial concentrations of free chlorine in batch reactors. Primary experiments analyzed each organism grown to their maximum concentration under the described growth conditions and inoculated into batch reactors with initial free chlorine concentrations of 0.2070.02, 0.4070.02, and 0.8070.02 mg/L. Chlorine demand
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was measured by analyzing samples for free chlorine throughout the assay. Sensitivity to free chlorine was determined by quantification on agar plates. Subsequently, secondary experiments were completed by diluting the primary cell inoculum by an order of magnitude; this was repeated until either a cell concentration was observed that had negligible chlorine demand or the initial cell concentration fell below the limits of detection for the cell quantification assay.
3.
Results and discussion
3.1.
Escherichia coli
E. coli is an organism known to be very sensitive to free chlorine at doses consistent with those in water distribution systems. The specific strain used in this study is not known to produce significant quantities of EPS under the growth conditions employed, nor is it known to show chlorine resistance. Therefore, it was expected that E. coli would be very sensitive to free chlorine at all concentrations, and that it would exert a chlorine demand during its oxidation. Chlorine demand assays for E. coli were carried out at initial free chlorine concentrations of 0.20, 0.40, and 0.80 mg/L. The primary initial cell concentration was 1.43 107 CFU/mL and secondary experiments were completed at initial cell concentrations of 1.14 106 and 1.34 105 CFU/mL. Cells were serially diluted and quantified immediately prior to chlorine demand and cell survival assays; the difference in the initial concentrations was due to the natural variability inherent in the growth assay and in the separation of the cells from their growth media. Fig. 1 shows the results of the chlorine demand assay for E. coli as both measured free chlorine concentration and chlorine demand. Chlorine demand is defined as the difference between the initial chlorine concentration and the chlorine residual after a specified contact time, t (or as C0–Ct, where C0 is the initial free chlorine concentration and Ct is the free chlorine concentration at contact time t). For all sets of initial conditions, chlorine demand increases with increasing contact time and approaches a constant value, the ultimate chlorine demand. Fig. 1 shows that the ultimate chlorine demand is greatest at the initial cell concentration of 1.43 107 CFU/mL (panel b) and decreases with decreasing initial cell concentration. Fig. 1 also shows that the rate of chlorine demand is most rapid at the initial cell concentration of 1.43 107 CFU/mL and decreases with decreasing initial cell concentration; the initial slope is steepest in panel b. Further, the contact time required to reach the ultimate chlorine demand is shortest for the highest concentration (20 min for 107 CFU/mL) and increases with decreasing initial cell concentration (60 min for 106 CFU/mL). These results are as expected, as higher cell concentrations are expected to react with free chlorine longer and more rapidly than lower cell concentrations. A more interesting result is that higher initial free chlorine concentrations resulted in greater chlorine demands. In each of the chlorine demand curves in Fig. 1, the initial free chlorine concentration of 0.80 mg/L produced a greater ultimate chlorine demand than did the lower initial free
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chlorine concentrations at equivalent initial cell concentrations. Fig. 1a shows that, for an initial cell concentration of 1.43 107 CFU/mL, free chlorine concentrations were reduced to approximately 0.10 mg/L regardless of the initial free chlorine concentration. This results in an approximate ultimate chlorine demand of 0.70 mg/L for the 0.80 mg/L assay and only 0.10 mg/L for the 0.20 mg/L assay. At 1.14 106 CFU/mL (panel c), free chlorine concentrations were not reduced to a single value, but the ultimate chlorine demand was still the greatest at the initial free chlorine concentration of 0.80 mg/L. This counter-intuitive result is supported by re-evaluation of the work of Virto et al. (2004). Their data show that a suspension of fixed concentration of Listeria monocytogenes combined with three solutions of different free chlorine concentrations produces the same fraction of chlorine demand, which means that the higher initial free chlorine concentration systems had higher ultimate chlorine demand. Similarly, evaluation of data presented in Shang and Blatchley (2001) for E. coli with free chlorine concentrations ranging between 1.0 and 5.0 mg/L also suggests that chlorine demand is dependent on the initial free chlorine concentration. Finally, Lin and Evans (1974) observed that higher chlorine doses of sewage effluents produced greater chlorine demands at all contact times. This observation is also confirmed for S. epidermidis and M. aurum (as shown later in Fig. 4) in the present study. Trendlines applied to the observed ultimate chlorine demand data for each species show that the ultimate chlorine demand of a constant initial cell concentration increases linearly with increasing initial free chlorine concentration. The slope of the linear trendline increases with increasing initial cell concentration, indicating that this relationship is more significant at higher initial cell concentrations. It is hypothesized that this observation of increasing chlorine demand with increasing initial free chlorine concentration is the result of solutions with higher oxidation potential (higher free chlorine concentration) either reacting longer with the microbial material or continuing to react with intermediates formed by the initial oxidation reaction. Regardless of the specific mechanisms that lead to the relatively greater chlorine demand in solutions with higher free chlorine concentrations, this is an important consideration for water utility operators considering placement of free chlorine sensors in the distribution system. Sensor placement in a distribution system is a multi-objective problem that often requires consideration of several optimization criteria in order to minimize the risk of a potential contamination event. In situations where multiple sensor locations provide equivalent risk reduction, sensor placement should focus on those locations with higher free chlorine concentrations in order to capture the greater chlorine demand signals produced under that condition. For E. coli, significant chlorine demand was observed at the maximum stationary phase concentration of 1.43 107 CFU/mL. Subsequent 10-fold dilutions of the inoculum resulted in the ultimate chlorine demand decreasing and approaching zero at the initial cell concentration of 1.34 105 CFU/mL. Free chlorine sensors placed in the distribution system have sensitivities on the order of 0.01 mg/L and detect the natural
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Fig. 1 – Free chlorine concentration remaining during each assay is shown on the left and chlorine demand is shown on the right for initial E. coli suspensions of (a and b) 1.43 107 CFU/mL, (c and d) 1.14 106 CFU/mL, and (e and f) 1.34 105 CFU/mL in solutions with initial free chlorine concentrations of 0.20 mg/L (E), 0.40 mg/L (’), and 0.80 mg/L (m). Note the change in scale on the abscissa of the chlorine demand plots in (d) and (f).
variability in residual free chlorine concentration of the water. In order to successfully employ free chlorine sensors as surrogate detection devices for microorganisms, a chlorine demand signal must be produced that is statistically shown to be anomalous and outside the observed natural variability. It is concluded that E. coli grown under the conditions of this experiment would not be detected by free chlorine sensors in the distribution system at concentrations below 105 CFU/mL. Cell survival assays were carried out under the same set of initial conditions as the chlorine demand assays. Samples
were taken for 5 min and quantified by the spread plate method on TSB agar. E. coli was very sensitive to free chlorine exposure and viable quantifiable cells (after 0.25 min of exposure) were only obtained at the lowest initial free chlorine concentration, 0.20 mg/L. Even at initial free chlorine concentrations of 0.20 mg/L, 499.9% inactivation was achieved within 0.50 min for all initial E. coli concentrations (results not shown). The relationship between chlorine demand and cell inactivation is not direct. Cells are completely inactivated
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within 0.50 min, but chlorine demand is observed for as long as 20 min. Thus, the chlorine demand signal produced from E. coli is almost entirely from the oxidation of inactive cellular material including leaked intracellular macromolecules.
3.2.
Staphylococcus epidermidis
S. epidermidis is a ubiquitous organism often found on human skin. Staphylococcus has been identified to the genus level in water distribution system biofilms (Daly et al., 1998). Induced protein and enzyme synthesis has been observed for Staphylococcus spp. under free chlorine stress that has been attributed to cellular defense (Maalej et al., 2006; Abid et al., 2004). The strain of S. epidermidis in this study has been observed in our laboratory to produce significant quantities of EPS. The chlorine demand assays were carried out as described in Section 2. The primary initial cell concentration was 2.27 106 CFU/mL and secondary experiments were completed at initial cell concentrations of 2.81 105 and 2.04 104 CFU/mL. As was seen with E. coli, the ultimate chlorine demand of S. epidermidis increased with increasing initial cell concentration. The maximum stationary phase concentration of S. epidermidis (2.27 106 CFU/mL) demanded chlorine to approximately 0.05 mg/L, regardless of the initial free chlorine concentration (data not shown). This result parallels the result for E. coli, but the ultimate chlorine demand of S. epidermidis was greater even though its maximum stationary phase concentration was an order of magnitude less than that of E. coli. In addition to a fewer number of cells, the spherical S. epidermidis is significantly less voluminous than the rod-shaped E. coli, meaning that far less cellular material is producing a greater chlorine demand. A possible explanation for this observation is the oxidation of the EPS produced by S. epidermidis. The literature has shown that EPS affords additional protection from oxidizing disinfectants by reacting with the disinfectant and minimizing its concentration at the cell membrane (Ryu and Beuchat, 2004). The greater chlorine demand also agrees with previously reported results comparing the chlorine demand of Gram-positive and Gram-negative organisms (Shang and Blatchley, 2001), suggesting that cell wall structure may play an additional role in the higher chlorine demand observed for S. epidermidis. Chlorine demand was also observed to increase with increasing initial free chlorine concentration, as shown for E. coli and cited in the literature for other organisms and sewage effluents. At constant initial cell concentrations, chlorine demand increased with increasing initial free chlorine concentration, and the slope of a linear trendline applied to the data shows that the effect of increasing chlorine demand with increasing initial free chlorine concentration is magnified at greater initial cell concentrations, as shown subsequently in Fig. 4b. This adds support to the idea that higher free chlorine concentrations in the distribution system will produce a greater chlorine demand signal. Secondary chlorine demand assays continued until a cell concentration was identified for which there was negligible chlorine demand. For S. epidermidis, chlorine demand approached zero at approximately 104 CFU/mL. Free chlorine sensors as a surrogate detection method for S. epidermidis
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would therefore have a detection limit of approximately 104 CFU/mL, which is an order of magnitude less than that observed for E. coli. Cell survival assays were carried out under the same set of initial conditions as the chlorine demand assays. S. epidermidis survived longer in free chlorine solutions than E. coli, but inactivation to below the assay detection limit occurred within 1.25 and 0.75 min for initial free chlorine concentrations of 0.20 and 0.40 mg/L, respectively. No survival above the assay detection limit was observed at initial free chlorine concentrations of 0.80 mg/L. Inactivation occurred rapidly enough that it is not likely that induced production of proteins or enzymes contributed to the overall chlorine demand or cell survival. It is likely that the EPS produced by S. epidermidis provided some defense against free chlorine’s oxidative stress, allowing them to persist slightly longer than E. coli. Because the cells were inactivated rapidly by free chlorine, the observed chlorine demand is attributed to inactive cellular material and EPS produced and in solution prior to combination with the free chlorine solution.
3.3.
Mycobacterium aurum
Mycobacterium spp. are neither Gram positive nor Gram negative and are characterized by a cell wall rich in mycolic acids, which are identified as structural elements contributing to Mycobacterium resistance to chemicals and antibiotics. Mycobacterium spp. are known to be very resistant to free chlorine and M. aurum has been isolated in drinking water distribution systems (Le Dantec et al., 2002). The primary initial cell concentration was 4.43 105 CFU/mL and secondary experiments were completed at initial cell concentrations of 2.97 104 and 2.68 103 CFU/mL. Chlorine demand assays were extended to 240 min and cell survival assays were extended to 120 min. The mechanism of M. aurum resistance to oxidative stress is poorly understood. Thus, prior to the present work, it was unclear whether M. aurum would demand chlorine despite its resistance. Results of the experiments presented herein show significant chlorine demand among all initial concentrations of M. aurum. At the maximum initial cell concentration (4.43 105 CFU/mL), the higher initial free chlorine concentrations (0.40 and 0.80 mg/L) produced free chlorine curves that did not display the characteristic rapid increase in chlorine demand followed by more gradual demand increase seen for E.coli and S. epidermidis. Chlorine demand under these initial conditions produced more linear results (data not shown) and an ultimate chlorine demand was never observed, it instead continued to increase beyond 240 min. All other sets of initial conditions in the M. aurum experiments produced the chlorine demand trends seen previously for E. coli and S. epidermidis, characterized by an approach to a constant chlorine demand level after a period of rapid oxidation. Unlike E. coli and S. epidermidis experiments, M. aurum demanded chlorine at all three initial cell concentrations and the assays were concluded only when the initial cell concentration fell below the detection limits of the cell quantification assay. Therefore, no detection limit was observed for M. aurum.
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Fig. 2 – (a) Chlorine demand and (b) cell survival fraction of a 2.68 103 CFU/mL suspension of M. aurum. Chlorine demand approaches a constant value, while cells continue to be inactivated.
Cell survival assays were carried out under the same set of initial conditions as the chlorine demand assays. As expected, M. aurum survived well when exposed to free chlorine. At the initial free chlorine concentration of 0.20 mg/L, all cell concentrations remained quantifiable for the entire 120-min assay length. At 0.40 mg/L, the 2.68 103 CFU/mL initial cell concentration was inactivated to below the assay detection limit after approximately 70 min of exposure. At 0.80 mg/L, the 2.68 103 and 2.97 104 CFU/mL concentrations were inactivated to below the assay detection limit after 45 and 70 min, respectively. Comparing the chlorine demand and the cell survival of a suspension of M. aurum in Fig. 2 shows that cells continue to be inactivated after free chlorine concentration is stabilized. For E. coli and S. epidermidis, cells were inactivated rapidly and continued chlorine demand was attributed to oxidation of dead cellular material including leaked intracellular macromolecules. The converse trend was
observed for M. aurum, where chlorine demand was rapid and the inactivation of the cells continued after the free chlorine concentration stabilized. Taylor et al. (2000) report that Mycobacterium grown in media tend to form aggregates that may make quantification by spread plate difficult due to inadequate disaggregation during plate preparation. This is not likely a factor in the results shown in Fig. 2 as a clear trend was observed for all initial cell concentrations between survivability and initial free chlorine concentration.
3.4.
Organism comparison
Due to differences in the maximum stationary phase concentration of each of the organisms, only a single common cell concentration was considered for comparison (105 CFU/mL). Fig. 3 shows the chlorine demand (left) and cell
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Fig. 3 – Chlorine demand and survivability of each organism at initial free chlorine concentrations of (a and b) 0.20 mg/L, (c and d) 0.40 mg/L, and (e and f) 0.80 mg/L, and initial cell concentrations of 1.34 105 CFU/mL (E. coli, m), 2.81 105 CFU/mL (S. epidermidis, ’), and 4.43 105 CFU/mL (M. aurum, E).
survival fraction (right) for each organism at an initial cell concentration of approximately 105 CFU/mL and initial free chlorine concentrations of 0.20, 0.40, and 0.80 mg/L. Fig. 3 clearly shows that the chlorine demand of microbial suspensions is species specific. Of the organisms studied, ultimate chlorine demand was greatest in M. aurum, followed by S. epidermidis and E. coli. Further, it is shown that, at 0.40 and 0.80 mg/L, chlorine demand of M. aurum continues to increase after 120 min, while the other species have reached their ultimate chlorine demand. Cell survivability is also strongly species dependent. E. coli is most sensitive to free
chlorine, followed by S. epidermidis. M. aurum is shown to be very resistant to free chlorine. In Figs. 3b and d, cell survivability is only shown to 5 min in order to capture the survival curves for S. epidermidis, and E. coli, but M. aurum survived for the entire 120-min assay. A trend that was common among all species was that chlorine demand was linearly dependent on initial free chlorine concentration. Fig. 4 shows that all organisms had the greatest chlorine demand at 0.80 mg/L. This result is significant because it shows the importance of maintaining higher residual free chlorine concentrations in the
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Fig. 4 – Chlorine demand versus initial free chlorine concentration for (a) E. coli, (b) S. epidermidis, and (c) M. aurum at each initial cell concentration.
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distribution system. In order for free chlorine sensors to have utility in distribution systems, chlorine demand signals from contamination events must be detected within the natural variability of free chlorine concentrations. Maintenance of higher free chlorine residuals at sensor locations will magnify the chlorine demand signal of microbial contamination events and minimize the false alarm rate.
3.5.
CT comparison
Observed chlorine demand for each organism followed the biphasic demand kinetics predicted and observed previously in the literature for pure microbial cultures (Shang and Blatchley, 2001; Virto et al., 2004; Virto et al., 2005a) and sewage effluents (Lin and Evans, 1974; Haas and Karra, 1984). Chlorine demand curves were modeled with a biphasic exponential decay model: C ¼ ð1 QÞek1 t þ Qek2 t ,
(1)
where C is the free chlorine fraction at time t defined as (Ct/C0); Ct is the free chlorine concentration at time t; C0 is the initial free chlorine concentration; (1Q) is the free chlorine fraction demanded in the initial phase of chlorine demand; Q is the free chlorine fraction demanded in the second phase of chlorine demand; k1 is the chlorine decay kinetic coefficient for the initial phase of chlorine demand; and k2 is the chlorine decay kinetic coefficient for the second phase of chlorine demand. The parameters Q, k1, and k2 are dependent on initial free chlorine concentration (C0), initial cell concentration (X0), and cell species. Observed free chlorine data for each species were fitted using the biphasic exponential decay model by nonlinear regression (Microsoft Office Excel 2003, Solver) to minimize the root mean squared error between the observed chlorine fraction and the model predicted chlorine fraction (results not shown). Using both the chlorine demand and cell survival data collected for each organism, a quantification of each organism’s ability to resist free chlorine can be calculated. This quantification is the organism’s chlorine contact time (CT) and, for this analysis, was calculated as the CT required to produce a three log (3-log CT) inactivation in cell concentration. In being consistent with recent literature on calculation of CT values (Haas et al., 1996), the time-dependent residual biphasic exponential decay model (Eq. (1)) was used to improve the accuracy of the CT value. Interpolations of the cell survival curves were used to determine the amount of time required for a 3-log inactivation (t3-log inactivation) of each organism under each set of initial conditions. The fitted biphasic decay model can be integrated to yield k t e 1 ek2 t þQ , (2) C ¼ ð1 QÞ k1 k2 which can then be evaluated from t0 to t3-log inactivation to yield the 3-log CT for each organism in units of (mg min/L) interpreted as the concentration of free chlorine required to reduce the active population by 3-logs over a 1-min duration. The calculated CT values for E. coli, S. epidermidis, and M. aurum are 0.03270.009, 0.22170.080, and 42.972.71 mg min/L, respectively. These values show that there is a direct relationship between the 3-log CT values of the organisms studied
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and their ultimate chlorine demand. It is thus hypothesized that chlorine demand may be related to characteristics of the cell that aid in resistance to chlorine. This hypothesis may be tested in future work with additional organisms.
4.
Conclusions
The objective of this work was to investigate the limitations of free chlorine sensors as a surrogate monitor for microorganisms by investigating the chlorine demand of different organisms under different initial free chlorine concentrations. The persistence of each organism under oxidative stress was also investigated. It was determined that microorganisms do exert a chlorine demand in proportion to their concentration. Chlorine-resistant organisms also demand chlorine, and their resistance mechanisms are hypothesized to be attributed to extracellular material sacrificially reacting with free chlorine to reduce the concentration at the cell membrane or wall. In the organisms studied, chlorineresistant organisms demanded more chlorine than more sensitive organisms. Chlorine demand also increased with increasing free chlorine concentration. Maintenance of higher chlorine residuals in the distribution system will produce greater chlorine demand signals during contamination events and a greater chance of detecting biological contamination. For E. coli and S. epidermidis, chlorine demand approached zero at initial cell concentrations of 105 and 104 CFU/mL, respectively. Contamination at concentrations at or below these levels will not be detectable by free chlorine sensors. However, these organisms were inactivated below the assay detection limits within 30 s for all initial conditions, so it is not likely that a concentration of these organisms grown under these experimental conditions could survive without producing a detectable signal. M. aurum demanded chlorine at all concentrations studied. They also remained viable for long periods, indicating that M. aurum could persist in water distribution systems, but free chlorine sensors should detect chlorine demand signals generated by the presence of M. aurum.
Acknowledgement This work was funded by the National Science Foundation Sensors Program through the Division of Biological and Environmental Engineering under grant BES-0329549. R E F E R E N C E S
Abid, N., Maalej, S., Rouis, S., 2004. Morphological and physiological changes of staphylococcus aureus exposed to hypochlorous acid. Lett. Appl. Microbiol. 38 (3), 245–250. APHA AWWA WEF, 2005. Standard Methods for the Examination of Water and Wastewater, 21st ed. APHA AWWA WEF, Washington, DC. Blackburn, B., Craun, G., Yoder, J., Hill, V., Calderon, R., Chen, N., Lee, S., Levy, D., Beach, M., 2004. Surveillance for waterborne-disease outbreaks associated with drinking
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41 (2007) 4424– 4434
water—United States, 2001–2002. Morbid. Mortal. Wkly. Rep. 53 (SS-08), 23–45. Byer, D.C.K., 2005. Real time detection of intentional chemical contamination in the distribution system. J. Am. Water Works Assoc. 97. Cloete, T.E., 2003. Resistance mechanisms of bacteria to antimicrobial compounds. Int. Biodeterior. Biodegrad. 51 (4), 277–282. Corso, P., Kramer, M., Blair, K., Addiss, D., Davis, J., Haddix, A., 2003. Cost of illness in the 1993 waterborne cryptosporidium outbreak, Milwaukee, Wisconsin. Emerging Infect. Dis. 9 (4), 426–431. Daly, B., Betts, W.B., Brown, A.P., O’neill, J.G., 1998. Bacterial loss from biofilms exposed to free chlorine. Microbios 96 (383), 7–21. Deisingh, A.T.M., 2004. Biosensors for the detection of bacteria. Can. J. Microbiol. 50, 69–77. Eichler, S., Christen, R., Holtje, C., Westphal, P., Botel, J., Brettar, I., Mehling, A., Hofle, M.G., 2006. Composition and dynamics of bacterial communities of a drinking water supply system as assessed by RNA- and DNA-based 16s rRNA gene fingerprinting. Appl. Environ. Microbiol. 72 (3), 1858–1872. Fass, S., Dincher, L., Reasoner, D., Gatel, D., Block, J., 1996. Fate of Escherichia coli experimentally injected in a drinking water distribution pilot system. Water Res. 30 (9), 2215–2221. Haas, C., Karra, S., 1984. Kinetics of wastewater chlorine demand and exertion. J. Water Pollut. Control Fed. 56, 170–173. Haas, C.N., Jacangelo, J.G., Bishop, M.M., Cameron, C.D., Chowdhury, Z.K., Connell, G.F., Doty, G.A., Finch, G.R., Gates, D.J., Greenberg, A.E., Hoehn, R.C., Huebner, W.B., Jensen, J.N., Lange, A.L., Long, B.W., Moyer, N.P., Nagel, W.H., Noran, P.F., Palin, A.T., Regli, S.E., Routt, J.C., Symons, J.M., Thompson, C.K., Voyles, C.F., 1992. Survey of water utility disinfection practices. J. Am. Water Works Assoc. 84 (9), 121–128. Haas, C.N., Joffe, J., Anmangandla, U., Jacangelo, J., Heath, M., 1996. Water quality and disinfection kinetics. J. Am. Water Works Assoc. 88 (3), 95–103. Keinanen, M.M., Korhonen, L.K., Lehtola, M.J., Miettinen, I.T., Martikainen, P.J., Vartiainen, T., Suutari, M.H., 2002. The microbial community structure of drinking water biofilms can be affected by phosphorus availability. Appl. Environ. Microbiol. 68 (1), 434–439. Khan, A.S., Swerdlow, D.L., Juranek, D.D., 2001. Precautions against biological and chemical terrorism directed at food and water supplies. Public Health Rep. 116 (1), 3–14. Le Dantec, C., Duguet, J.P., Montiel, A., Dumoutier, N., Dubrou, S., Vincent, V., 2002. Chlorine disinfection of atypical mycobacteria isolated from a water distribution system. Appl. Environ. Microbiol. 68 (3), 1025–1032. Lin, S., Evans, R., 1974. Chlorine demand study of secondary sewage effluents. Water Sewage Works 121 (1), 35–44. Lisle, J.T., Broadaway, S.C., Prescott, A.M., Pyle, B.H., Fricker, C., Mcfeters, G.A., 1998. Effects of starvation on physiological
activity and chlorine disinfection resistance in Escherichia coli O157: H7. Appl. Environ. Microbiol. 64 (12), 4658–4662. Luthy, R.G., 2002. Bioterrorism and water security. Environ. Sci. Technol. 36 (7), 123A. Maalej, S., Dammak, I., Dukan, S., 2006. The impairment of superoxide dismutase coordinates the derepression of the perr regulon in the response of Staphylococcus aureus to Hocl stress. Microbiology 152, 855–861. Rodriguez-Mozaz, S., Marco, M., Lopez de Alda, M., Barcelo, D., 2004. Biosensors for environmental applications: future development trends. Pure Appl. Chem. 76 (4), 723–752. Ryu, J.H., Beuchat, L.R., 2004. Factors affecting production of extracellular carbohydrate complexes by Escherichia coli O157: H7. Int. J. Food Microbiol. 95 (2), 189–204. Schmeisser, C., Stockigt, C., Raasch, C., Wingender, J., Timmis, K.N., Wenderoth, D.F., Flemming, H.C., Liesegang, H., Schmitz, R.A., Jaeger, K.E., Streit, W.R., 2003. Metagenome survey of biofilms in drinking-water networks. Appl. Environ. Microbiol. 69 (12), 7298–7309. Shang, C.B.E., Blatchley, E.R., 2001. Chlorination of pure bacterial cultures in aqueous solution. Water Res. 35 (1), 244–254. Taylor, R.H., Falkinham, J.O., Norton, C.D., Lechevallier, M.W., 2000. Chlorine, chloramine, chlorine dioxide, and ozone susceptibility of Mycobacterium Avium. Appl. Environ. Microbiol. 66 (4), 1702–1705. United States Environmental Protection Agency, 2005. WaterSentinel Online Water Quality Monitoring as an Indicator of Drinking Water Contamination. December 12. /http:// www.epa.gov/safewater/watersecurity/pubs/watersentinel_ wq_monitoring.pdfS. Venkobachar, C., Iyengar, L., Rao, A., 1977. Mechanism of disinfection: effect of chlorine on cell membrane functions. Water Res. 11, 727–729. Virto, R., Alvarez, I., Condon, S., Raso, J., 2004. Relationship between inactivation kinetics of a Listeria moncytogenes suspension by chlorine and its chlorine demand. J. Appl. Microbiol. 97, 1281–1288. Virto, R., Alvarez, I., Condon, S., Raso, J., 2005a. Modeling the effect of initial concentration of Escherichia coli suspensions on their inactivation by chlorine. J. Food Saf. 25 (2), 120–129. Virto, R., Manas, P., Alvarez, I., Condon, S., Raso, J., 2005b. Membrane damage and microbial inactivation by chlorine in the absence and presence of a chlorine-demanding substrate. Appl. Environ. Microbiol. 71 (9), 5022–5028. Whitman, C.T. Strategic Plan for Homeland Security. September 2002. /http://www.epa.gov/epahome/downloads/epa_ homeland_security_strategic_plan.pdfS. Williams, M.M., Braun-Howland, E.B., 2003. Growth of Escherichia coli in model distribution system biofilms exposed to hypochlorous acid or monochloramine. Appl. Environ. Microbiol. 69 (9), 5463–5471.
41 (2007) 4424– 4434
Available at www.sciencedirect.com
journal homepage: www.elsevier.com/locate/watres
Free chlorine demand and cell survival of microbial suspensions Damian E. Helbling, Jeanne M. VanBriesen1 Department of Civil and Environmental Engineering, Carnegie Mellon University, 5000 Forbes Avenue, Pittsburgh, PA 15213, USA
ar t ic l e i n f o
abs tra ct
Article history:
The utility of chlorine residual and chlorine demand as a surrogate for microbial
Received 19 December 2006
contamination in the water distribution system was evaluated. The chlorine demanded
Received in revised form
by and cell survival of pure culture suspensions of Escherichia coli, Staphylococcus epidermidis,
21 March 2007
and Mycobacterium aurum were quantified in solutions with initial free chlorine concentra-
Accepted 4 June 2007
tions of 0.20, 0.40, and 0.80 mg/L. The chlorine demand increased with initial concentration
Available online 12 June 2007
of cells and free chlorine for all species. At equivalent initial cell concentrations, chlorine
Keywords: Chlorine demand Free chlorine Water security Water distribution system
demand was greatest for M. aurum, followed by S. epidermidis and E. coli. The chlorine contact time required for a 3-log inactivation of E. coli, S. epidermidis, and M. aurum was calculated as 0.03270.009, 0.22170.080, and 42.972.71 mg min/L, respectively. The ultimate chlorine demand and cell survival were directly proportional. No chlorine demand was observed at cell concentrations less than 105 CFU/mL for E. coli or 104 CFU/mL for S. epidermidis. M. aurum demanded chlorine at all initial cell concentrations including 103 CFU/mL, which was the detection limit of the cell quantification assay. Chlorine demand was determined to be a suitable surrogate indicator of the organisms studied and its utility may be enhanced in locations of the water distribution system that maintain a higher free chlorine residual. & 2007 Elsevier Ltd. All rights reserved.
1.
Introduction
The threat of accidental or intentional pathogenic intrusions into drinking water infrastructure, including distribution systems, is very serious. In the United States in the years 2001 and 2002, the Centers for Disease Control and Prevention reported that an estimated 1020 people were infected by water-borne diseases originating in drinking water (7 fatally) (Blackburn et al., 2004). Organisms identified in the individual outbreaks included Escherichia coli, Campylobacter jejuni, Yersinia enterocolitica, Vibrio cholerae, and several Legionella species. Many of the reported incidents were attributed to either failed treatment processes or contamination in the distribution system during anomalous periods of negative pressure
(Blackburn et al., 2004). Water-borne infectious diseases not only cause loss of life and illness but also have negative effects on the economy related to medical expenses and productivity losses (Corso et al., 2003). In addition to accidental outbreaks of water-borne illnesses, the United States Department of Homeland Security and the United States Department of Environmental Protection have made public statements that drinking water facilities in the United States may be a target for terrorist activity (Whitman, 2002). An intentional act of drinking water contamination could create catastrophic morbidity and mortality, overwhelm medical response personnel and infrastructure, and have significant negative effects on the economy and psyche of the nation. Continuous, real-time monitoring of critical
Corresponding author. Tel.: +412 268 3819; fax: +412 268 7813.
E-mail addresses: [email protected] (D.E. Helbling), [email protected] (J.M. VanBriesen). 1 Tel.: +412 268 4603; fax: +412 268 7813. 0043-1354/$ - see front matter & 2007 Elsevier Ltd. All rights reserved. doi:10.1016/j.watres.2007.06.006
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water infrastructure is imperative to provide early warning and minimize the effects of such contamination events (Luthy, 2002). The distribution system has been identified as a particularly vulnerable component of drinking water infrastructure (Khan et al., 2001) and monitoring water quality in the distribution system could provide early warning of contamination events. The term ‘sensor’ will be used in the remainder of this work to describe a device that is used to obtain continuous, realtime data of a specific water quality parameter. While there are no existing sensors that can detect all possible pathogens that might be introduced to a distribution system (Deisingh, 2004; Rodriguez-Mozaz et al., 2004), there are many commercially available sensors that monitor for water quality parameters such as pH, temperature, conductivity, turbidity, total organic carbon, total chlorine, combined chlorine, and free chlorine. The efficacy of any of these water quality parameters as a surrogate for pathogenic microorganisms has not been fully studied, but free chlorine has been suggested to be the water quality parameter that reacts with the widest range of contaminants (United States Environmental Protection Agency, 2005). Free chlorine sensors have been shown to be adequate surrogate monitors when analyzing for chemical species such as the pesticide aldicarb and the toxin sodium cyanide (Byer, 2005); however, very little research has been done on the effect of specific microorganisms on residual chlorine in drinking water distribution systems. Although the mechanisms involved in microbial inactivation by free chlorine are not fully understood, it is believed that the primary reaction involves oxidation of microbial membranes increasing cell permeability, which results in leakage of macromolecules and cell death (Venkobachar et al., 1977; Virto et al., 2004). More recent studies have shown that membrane damage is not the main mechanism of inactivation for cells exposed to oxidative stresses; more subtle effects such as an uncoupling of the electron chain or enzyme inactivation may be dominant (Virto et al., 2005b). The chlorine demand of a microbial suspension is defined as the difference between the initial chlorine concentration and the chlorine concentration after a specified contact time. As the contact time increases, the chlorine demand approaches an ultimate value that indicates that all organic material originally present and available for reaction with chlorine has been exhausted. It is hypothesized that the chlorine demand signal generated by the reaction between free chlorine and microbial material may be used as a surrogate indicator of microbial contamination in water distribution systems. The utility of free chlorine as a surrogate analyte depends on the chlorine demand of microorganisms. Studies that have investigated chlorine demand have shown that different organisms have unique chlorine demands, even at similar organic carbon content and concentration (Shang and Blatchley, 2001). Of particular concern is the relationship between chlorine demand and cell survival. If chlorine demand is only exerted during the inactivation of microorganisms, then it will not provide adequate early warning against chlorineresistant organisms. Chlorine-resistant organisms are of particular interest in water security problems because they persist and remain infective in oxidative environments. The naturally occurring microbial flora in water distribution
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systems is very diverse and can contain benign organisms and pathogens, despite the presence of the chlorine residual (Keinanen et al., 2002; Schmeisser et al., 2003; Eichler et al., 2006). Even the highly chlorine-sensitive indicator organism E. coli has been found to persist and even to grow in the presence of a chlorine residual (Fass et al., 1996; Williams and Braun-Howland, 2003). Resistance to free chlorine has been attributed to unique variations in the cellular structure and protein composition of different organisms (Cloete, 2003). In addition to physical attributes that aid in resistance to free chlorine, microorganisms defend against oxidative stresses by inherent or adapted resistance mechanisms that produce extracellular polymeric substances (EPSs). These substances react with the oxidative disinfectant, which effectively reduces the concentration of disinfectant at the cell wall or cell membrane where cell damage can occur (Lisle et al., 1998; Ryu and Beuchat, 2004). If this presumed mechanism is correct, resistant organisms will exert a strong chlorine demand while remaining viable and infective. The objective of this work is to investigate the limitations of chlorine demand as a surrogate indicator for microbial contamination by investigating the chlorine demand and survival of different organisms under different initial free chlorine concentrations. The relationship between chlorine demand and cell survival was investigated by selecting organisms that have a range of reported resistances to free chlorine as a disinfectant.
2.
Materials and methods
2.1.
Cultures and growth conditions
Organisms studied in this work included: American type culture collection (ATCC) 11775, E. coli; ATCC 35984 Staphylococcus epidermidis; and ATCC 23366 Mycobacterium aurum. E. coli was cultivated from a loop of pure culture organisms maintained on Tryptic Soy Broth (TSB, Soybean-Casein Digest Medium, Difco) agar (Fisher Scientific) plates at 4 1C. E. coli was aseptically transferred and incubated in 60 mL of TSB broth and harvested during stationary phase after 40 h of incubation at 37 1C. S. epidermidis was cultivated from a loop of pure culture organisms maintained on TSB (Difco) agar (Fisher Scientific) plates at 4 1C. S. epidermidis was aseptically transferred and incubated in 60 mL of TSB broth and harvested during stationary phase after 48 h of incubation at 37 1C. M. aurum was cultivated from a loop of pure culture organisms maintained on Middlebrook 7H10 Agar (Difco) plates amended with Middlebrook OADC Enrichment (Difco) and glycerol (Acros Organics) at 4 1C. M. aurum was aseptically transferred and incubated in 180 mL of Middlebrook 7H9 Broth (Difco) amended with Middlebrook ADC Enrichment (Difco) and glycerol (Arcos Organics) and harvested during stationary phase after 216 h (9 days) of incubation at 37 1C. Prior to each experiment, cell cultures were aseptically transferred to sterile plastic tubes and centrifuged at 6000g for 5 min. The liquid growth media was separated from the pellet and discarded. Cells were re-suspended in a volume of 0.1 M chlorine demand-free Sorensen’s phosphate buffer at a pH of 7.4 equal to the volume of the discarded supernatant.
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Sorensen’s phosphate buffer was prepared by combining a 0.2 M sodium phosphate monobasic stock solution with a 0.2 M sodium phosphate dibasic anhydrous stock solution and deionized water at a volumetric ratio of 57:243:600. Resuspended cells were centrifuged, separated, and re-suspended in phosphate buffer three additional times in order to completely separate the cells from their growth media, which could cause interferences in the chlorine demand experiments.
2.2.
Chlorine solutions
Chlorine solutions were prepared daily by diluting a stock solution of 5% sodium hypochlorite (LabChem, Inc.) in carbon-free deionized water (Barnstead NANOpure). Sodium hypochlorite stock was diluted to produce solutions of final free chlorine concentrations of 0.2070.02, 0.4070.02, and 0.8070.02 mg/L. Final free chlorine concentrations were verified in triplicate by the DPD colorimetric method.
2.3.
Free chlorine determination
Free chlorine concentrations were verified with the N,Ndiethyl-p-phenylenediamine (DPD) colorimetric method (APHA AWWA WEF, 2005). In this method, the DPD is oxidized by free chlorine, resulting in a solution with a color intensity proportional to the free chlorine concentration. The absorption of the resulting solution is measured by photometry. DPD reagents were provided by PPD-2 DPD Powder Pop Dispenser (HF Scientific). The absorption of the solution was quantified using the Free Chlorine Pocket Photometer (HF Scientific). The photometer has a reported accuracy of 72% within the concentration range used in this work.
2.4.
described for the chlorine demand assays. Free chlorine solutions of 0.2070.02, 0.4070.02, and 0.8070.02 mg/L of volume 198 mL were added to cleaned 250-mL glass Erlenmeyer flasks with a magnetic stir bar. The flasks were placed on a magnetic stir plate and set to 155 RPM to provide gentle, continuous mixing. At t ¼ 0, 2 mL of cells suspended in phosphate buffer were added to the flask. Samples of volume 1 mL were drawn from the flask at t ¼ 0.25, 0.50, 0.75, 1, 1.5, 2, 3, 4, and 5 min for E. coli and S. epidermidis and at t ¼ 5, 15, 30, 60, 90, 120, 150, and 180 min for M. aurum. Samples were immediately added to 9 mL of neutralizing buffer (Difco) to quench the remaining free chlorine in the sample. After sampling was completed, 1 mL of the 101 dilution in neutralizing buffer (Difco) was serially diluted in 9 mL of phosphate buffer to selected final dilutions. Each dilution was quantified in triplicate and the final concentration was reported as the average of the countable plates. E. coli and S. epidermidis were quantified by a spread plate on TSB (Difco) agar (Fisher Scientific) plates. M. aurum was quantified by a spread plate on Middlebrook 7H10 Agar (Difco) amended with Middlebrook OADC Enrichment (Difco) and glycerol (Acros Organics). All organisms were incubated at 37 1C. Plates were quantified after 18, 24, and 120 h for E. coli, S. epidermidis, and M. aurum, respectively. Cell survival assays were repeated at least once for each set of initial conditions in order to verify the reproducibility of the results. Because each 1 mL sample was immediately diluted in 9 mL of neutralizing buffer, the first quantified concentration was a 101 dilution. Plates were considered countable only if they contained a number of colonies between 30 and 300 (APHA AWWA WEF, 2005). Plates were spread with 100 mL of each sample dilution. Therefore, the cell quantification detection limit of the method was 3 103 CFU/mL.
Chlorine demand assay 2.6.
All glassware was washed with detergent twice and rinsed with hot water, rinsed twice with a solution of 0.1 N HCl, rinsed thrice with deionized water and allowed to air dry. Glassware was then capped with aluminum foil and baked at 550 1C for 6 h. This cleaning process was employed in order to prevent interferences from environmental organic material. Free chlorine solutions of 0.2070.02, 0.4070.02, and 0.8070.02 mg/L of volume 198 mL were added to cleaned 250-mL glass Erlenmeyer flasks with a magnetic stir bar. The flasks were placed on a magnetic stir plate and set to 155 RPM to provide gentle, continuous mixing. At t ¼ 0, 2 mL of cells suspended in phosphate buffer was added to the flask. At t ¼ 0.25, 0.50, 1, 1.5, 2, 3, 4, 5, 10, 15, 20, 30, 45, 60, 90, and 120 min, 2.5 mL of samples were drawn in duplicate from the flask and analyzed for free chlorine via the DPD method. Free chlorine was analyzed additionally at 150, 180, 210, and 240 min for M. aurum assays. The reported free chlorine concentration was the average of the samples taken. Chlorine demand assays were repeated at least once for each set of initial conditions in order to verify the reproducibility of the results.
2.5.
Cell survival assays
Cell survival assays were conducted in parallel with the chlorine demand assays. Glassware was prepared as
Organism selection and analytical conditions
The type of residual chlorine selected for this study was free chlorine as it is the most common form of chlorine used as a disinfectant in the United States (Haas et al., 1992), is used in the City of Pittsburgh (the location of the study) water distribution system, and has a well-documented efficacy as a disinfectant. Organisms were selected to provide a range of cell wall structures, EPS production, and reported resistance to free chlorine as a disinfectant. The organisms selected were: the Gram-positive S. epidermidis, which is a known EPS producer; the Gram-negative E. coli, which is a common water indicator organism, is not known to produce a significant amount of EPS, and is considered to be very sensitive to free chlorine when grown in ideal environmental conditions; and M. aurum (neither Gram positive nor Gram negative), which has been isolated in drinking water distribution systems (Le Dantec et al., 2002) and is an organism that has been observed to be very resistant to oxidative stresses. In order to meet the objectives of this work, each organism was combined with various initial concentrations of free chlorine in batch reactors. Primary experiments analyzed each organism grown to their maximum concentration under the described growth conditions and inoculated into batch reactors with initial free chlorine concentrations of 0.2070.02, 0.4070.02, and 0.8070.02 mg/L. Chlorine demand
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was measured by analyzing samples for free chlorine throughout the assay. Sensitivity to free chlorine was determined by quantification on agar plates. Subsequently, secondary experiments were completed by diluting the primary cell inoculum by an order of magnitude; this was repeated until either a cell concentration was observed that had negligible chlorine demand or the initial cell concentration fell below the limits of detection for the cell quantification assay.
3.
Results and discussion
3.1.
Escherichia coli
E. coli is an organism known to be very sensitive to free chlorine at doses consistent with those in water distribution systems. The specific strain used in this study is not known to produce significant quantities of EPS under the growth conditions employed, nor is it known to show chlorine resistance. Therefore, it was expected that E. coli would be very sensitive to free chlorine at all concentrations, and that it would exert a chlorine demand during its oxidation. Chlorine demand assays for E. coli were carried out at initial free chlorine concentrations of 0.20, 0.40, and 0.80 mg/L. The primary initial cell concentration was 1.43 107 CFU/mL and secondary experiments were completed at initial cell concentrations of 1.14 106 and 1.34 105 CFU/mL. Cells were serially diluted and quantified immediately prior to chlorine demand and cell survival assays; the difference in the initial concentrations was due to the natural variability inherent in the growth assay and in the separation of the cells from their growth media. Fig. 1 shows the results of the chlorine demand assay for E. coli as both measured free chlorine concentration and chlorine demand. Chlorine demand is defined as the difference between the initial chlorine concentration and the chlorine residual after a specified contact time, t (or as C0–Ct, where C0 is the initial free chlorine concentration and Ct is the free chlorine concentration at contact time t). For all sets of initial conditions, chlorine demand increases with increasing contact time and approaches a constant value, the ultimate chlorine demand. Fig. 1 shows that the ultimate chlorine demand is greatest at the initial cell concentration of 1.43 107 CFU/mL (panel b) and decreases with decreasing initial cell concentration. Fig. 1 also shows that the rate of chlorine demand is most rapid at the initial cell concentration of 1.43 107 CFU/mL and decreases with decreasing initial cell concentration; the initial slope is steepest in panel b. Further, the contact time required to reach the ultimate chlorine demand is shortest for the highest concentration (20 min for 107 CFU/mL) and increases with decreasing initial cell concentration (60 min for 106 CFU/mL). These results are as expected, as higher cell concentrations are expected to react with free chlorine longer and more rapidly than lower cell concentrations. A more interesting result is that higher initial free chlorine concentrations resulted in greater chlorine demands. In each of the chlorine demand curves in Fig. 1, the initial free chlorine concentration of 0.80 mg/L produced a greater ultimate chlorine demand than did the lower initial free
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chlorine concentrations at equivalent initial cell concentrations. Fig. 1a shows that, for an initial cell concentration of 1.43 107 CFU/mL, free chlorine concentrations were reduced to approximately 0.10 mg/L regardless of the initial free chlorine concentration. This results in an approximate ultimate chlorine demand of 0.70 mg/L for the 0.80 mg/L assay and only 0.10 mg/L for the 0.20 mg/L assay. At 1.14 106 CFU/mL (panel c), free chlorine concentrations were not reduced to a single value, but the ultimate chlorine demand was still the greatest at the initial free chlorine concentration of 0.80 mg/L. This counter-intuitive result is supported by re-evaluation of the work of Virto et al. (2004). Their data show that a suspension of fixed concentration of Listeria monocytogenes combined with three solutions of different free chlorine concentrations produces the same fraction of chlorine demand, which means that the higher initial free chlorine concentration systems had higher ultimate chlorine demand. Similarly, evaluation of data presented in Shang and Blatchley (2001) for E. coli with free chlorine concentrations ranging between 1.0 and 5.0 mg/L also suggests that chlorine demand is dependent on the initial free chlorine concentration. Finally, Lin and Evans (1974) observed that higher chlorine doses of sewage effluents produced greater chlorine demands at all contact times. This observation is also confirmed for S. epidermidis and M. aurum (as shown later in Fig. 4) in the present study. Trendlines applied to the observed ultimate chlorine demand data for each species show that the ultimate chlorine demand of a constant initial cell concentration increases linearly with increasing initial free chlorine concentration. The slope of the linear trendline increases with increasing initial cell concentration, indicating that this relationship is more significant at higher initial cell concentrations. It is hypothesized that this observation of increasing chlorine demand with increasing initial free chlorine concentration is the result of solutions with higher oxidation potential (higher free chlorine concentration) either reacting longer with the microbial material or continuing to react with intermediates formed by the initial oxidation reaction. Regardless of the specific mechanisms that lead to the relatively greater chlorine demand in solutions with higher free chlorine concentrations, this is an important consideration for water utility operators considering placement of free chlorine sensors in the distribution system. Sensor placement in a distribution system is a multi-objective problem that often requires consideration of several optimization criteria in order to minimize the risk of a potential contamination event. In situations where multiple sensor locations provide equivalent risk reduction, sensor placement should focus on those locations with higher free chlorine concentrations in order to capture the greater chlorine demand signals produced under that condition. For E. coli, significant chlorine demand was observed at the maximum stationary phase concentration of 1.43 107 CFU/mL. Subsequent 10-fold dilutions of the inoculum resulted in the ultimate chlorine demand decreasing and approaching zero at the initial cell concentration of 1.34 105 CFU/mL. Free chlorine sensors placed in the distribution system have sensitivities on the order of 0.01 mg/L and detect the natural
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Fig. 1 – Free chlorine concentration remaining during each assay is shown on the left and chlorine demand is shown on the right for initial E. coli suspensions of (a and b) 1.43 107 CFU/mL, (c and d) 1.14 106 CFU/mL, and (e and f) 1.34 105 CFU/mL in solutions with initial free chlorine concentrations of 0.20 mg/L (E), 0.40 mg/L (’), and 0.80 mg/L (m). Note the change in scale on the abscissa of the chlorine demand plots in (d) and (f).
variability in residual free chlorine concentration of the water. In order to successfully employ free chlorine sensors as surrogate detection devices for microorganisms, a chlorine demand signal must be produced that is statistically shown to be anomalous and outside the observed natural variability. It is concluded that E. coli grown under the conditions of this experiment would not be detected by free chlorine sensors in the distribution system at concentrations below 105 CFU/mL. Cell survival assays were carried out under the same set of initial conditions as the chlorine demand assays. Samples
were taken for 5 min and quantified by the spread plate method on TSB agar. E. coli was very sensitive to free chlorine exposure and viable quantifiable cells (after 0.25 min of exposure) were only obtained at the lowest initial free chlorine concentration, 0.20 mg/L. Even at initial free chlorine concentrations of 0.20 mg/L, 499.9% inactivation was achieved within 0.50 min for all initial E. coli concentrations (results not shown). The relationship between chlorine demand and cell inactivation is not direct. Cells are completely inactivated
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within 0.50 min, but chlorine demand is observed for as long as 20 min. Thus, the chlorine demand signal produced from E. coli is almost entirely from the oxidation of inactive cellular material including leaked intracellular macromolecules.
3.2.
Staphylococcus epidermidis
S. epidermidis is a ubiquitous organism often found on human skin. Staphylococcus has been identified to the genus level in water distribution system biofilms (Daly et al., 1998). Induced protein and enzyme synthesis has been observed for Staphylococcus spp. under free chlorine stress that has been attributed to cellular defense (Maalej et al., 2006; Abid et al., 2004). The strain of S. epidermidis in this study has been observed in our laboratory to produce significant quantities of EPS. The chlorine demand assays were carried out as described in Section 2. The primary initial cell concentration was 2.27 106 CFU/mL and secondary experiments were completed at initial cell concentrations of 2.81 105 and 2.04 104 CFU/mL. As was seen with E. coli, the ultimate chlorine demand of S. epidermidis increased with increasing initial cell concentration. The maximum stationary phase concentration of S. epidermidis (2.27 106 CFU/mL) demanded chlorine to approximately 0.05 mg/L, regardless of the initial free chlorine concentration (data not shown). This result parallels the result for E. coli, but the ultimate chlorine demand of S. epidermidis was greater even though its maximum stationary phase concentration was an order of magnitude less than that of E. coli. In addition to a fewer number of cells, the spherical S. epidermidis is significantly less voluminous than the rod-shaped E. coli, meaning that far less cellular material is producing a greater chlorine demand. A possible explanation for this observation is the oxidation of the EPS produced by S. epidermidis. The literature has shown that EPS affords additional protection from oxidizing disinfectants by reacting with the disinfectant and minimizing its concentration at the cell membrane (Ryu and Beuchat, 2004). The greater chlorine demand also agrees with previously reported results comparing the chlorine demand of Gram-positive and Gram-negative organisms (Shang and Blatchley, 2001), suggesting that cell wall structure may play an additional role in the higher chlorine demand observed for S. epidermidis. Chlorine demand was also observed to increase with increasing initial free chlorine concentration, as shown for E. coli and cited in the literature for other organisms and sewage effluents. At constant initial cell concentrations, chlorine demand increased with increasing initial free chlorine concentration, and the slope of a linear trendline applied to the data shows that the effect of increasing chlorine demand with increasing initial free chlorine concentration is magnified at greater initial cell concentrations, as shown subsequently in Fig. 4b. This adds support to the idea that higher free chlorine concentrations in the distribution system will produce a greater chlorine demand signal. Secondary chlorine demand assays continued until a cell concentration was identified for which there was negligible chlorine demand. For S. epidermidis, chlorine demand approached zero at approximately 104 CFU/mL. Free chlorine sensors as a surrogate detection method for S. epidermidis
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would therefore have a detection limit of approximately 104 CFU/mL, which is an order of magnitude less than that observed for E. coli. Cell survival assays were carried out under the same set of initial conditions as the chlorine demand assays. S. epidermidis survived longer in free chlorine solutions than E. coli, but inactivation to below the assay detection limit occurred within 1.25 and 0.75 min for initial free chlorine concentrations of 0.20 and 0.40 mg/L, respectively. No survival above the assay detection limit was observed at initial free chlorine concentrations of 0.80 mg/L. Inactivation occurred rapidly enough that it is not likely that induced production of proteins or enzymes contributed to the overall chlorine demand or cell survival. It is likely that the EPS produced by S. epidermidis provided some defense against free chlorine’s oxidative stress, allowing them to persist slightly longer than E. coli. Because the cells were inactivated rapidly by free chlorine, the observed chlorine demand is attributed to inactive cellular material and EPS produced and in solution prior to combination with the free chlorine solution.
3.3.
Mycobacterium aurum
Mycobacterium spp. are neither Gram positive nor Gram negative and are characterized by a cell wall rich in mycolic acids, which are identified as structural elements contributing to Mycobacterium resistance to chemicals and antibiotics. Mycobacterium spp. are known to be very resistant to free chlorine and M. aurum has been isolated in drinking water distribution systems (Le Dantec et al., 2002). The primary initial cell concentration was 4.43 105 CFU/mL and secondary experiments were completed at initial cell concentrations of 2.97 104 and 2.68 103 CFU/mL. Chlorine demand assays were extended to 240 min and cell survival assays were extended to 120 min. The mechanism of M. aurum resistance to oxidative stress is poorly understood. Thus, prior to the present work, it was unclear whether M. aurum would demand chlorine despite its resistance. Results of the experiments presented herein show significant chlorine demand among all initial concentrations of M. aurum. At the maximum initial cell concentration (4.43 105 CFU/mL), the higher initial free chlorine concentrations (0.40 and 0.80 mg/L) produced free chlorine curves that did not display the characteristic rapid increase in chlorine demand followed by more gradual demand increase seen for E.coli and S. epidermidis. Chlorine demand under these initial conditions produced more linear results (data not shown) and an ultimate chlorine demand was never observed, it instead continued to increase beyond 240 min. All other sets of initial conditions in the M. aurum experiments produced the chlorine demand trends seen previously for E. coli and S. epidermidis, characterized by an approach to a constant chlorine demand level after a period of rapid oxidation. Unlike E. coli and S. epidermidis experiments, M. aurum demanded chlorine at all three initial cell concentrations and the assays were concluded only when the initial cell concentration fell below the detection limits of the cell quantification assay. Therefore, no detection limit was observed for M. aurum.
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Fig. 2 – (a) Chlorine demand and (b) cell survival fraction of a 2.68 103 CFU/mL suspension of M. aurum. Chlorine demand approaches a constant value, while cells continue to be inactivated.
Cell survival assays were carried out under the same set of initial conditions as the chlorine demand assays. As expected, M. aurum survived well when exposed to free chlorine. At the initial free chlorine concentration of 0.20 mg/L, all cell concentrations remained quantifiable for the entire 120-min assay length. At 0.40 mg/L, the 2.68 103 CFU/mL initial cell concentration was inactivated to below the assay detection limit after approximately 70 min of exposure. At 0.80 mg/L, the 2.68 103 and 2.97 104 CFU/mL concentrations were inactivated to below the assay detection limit after 45 and 70 min, respectively. Comparing the chlorine demand and the cell survival of a suspension of M. aurum in Fig. 2 shows that cells continue to be inactivated after free chlorine concentration is stabilized. For E. coli and S. epidermidis, cells were inactivated rapidly and continued chlorine demand was attributed to oxidation of dead cellular material including leaked intracellular macromolecules. The converse trend was
observed for M. aurum, where chlorine demand was rapid and the inactivation of the cells continued after the free chlorine concentration stabilized. Taylor et al. (2000) report that Mycobacterium grown in media tend to form aggregates that may make quantification by spread plate difficult due to inadequate disaggregation during plate preparation. This is not likely a factor in the results shown in Fig. 2 as a clear trend was observed for all initial cell concentrations between survivability and initial free chlorine concentration.
3.4.
Organism comparison
Due to differences in the maximum stationary phase concentration of each of the organisms, only a single common cell concentration was considered for comparison (105 CFU/mL). Fig. 3 shows the chlorine demand (left) and cell
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Fig. 3 – Chlorine demand and survivability of each organism at initial free chlorine concentrations of (a and b) 0.20 mg/L, (c and d) 0.40 mg/L, and (e and f) 0.80 mg/L, and initial cell concentrations of 1.34 105 CFU/mL (E. coli, m), 2.81 105 CFU/mL (S. epidermidis, ’), and 4.43 105 CFU/mL (M. aurum, E).
survival fraction (right) for each organism at an initial cell concentration of approximately 105 CFU/mL and initial free chlorine concentrations of 0.20, 0.40, and 0.80 mg/L. Fig. 3 clearly shows that the chlorine demand of microbial suspensions is species specific. Of the organisms studied, ultimate chlorine demand was greatest in M. aurum, followed by S. epidermidis and E. coli. Further, it is shown that, at 0.40 and 0.80 mg/L, chlorine demand of M. aurum continues to increase after 120 min, while the other species have reached their ultimate chlorine demand. Cell survivability is also strongly species dependent. E. coli is most sensitive to free
chlorine, followed by S. epidermidis. M. aurum is shown to be very resistant to free chlorine. In Figs. 3b and d, cell survivability is only shown to 5 min in order to capture the survival curves for S. epidermidis, and E. coli, but M. aurum survived for the entire 120-min assay. A trend that was common among all species was that chlorine demand was linearly dependent on initial free chlorine concentration. Fig. 4 shows that all organisms had the greatest chlorine demand at 0.80 mg/L. This result is significant because it shows the importance of maintaining higher residual free chlorine concentrations in the
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Fig. 4 – Chlorine demand versus initial free chlorine concentration for (a) E. coli, (b) S. epidermidis, and (c) M. aurum at each initial cell concentration.
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distribution system. In order for free chlorine sensors to have utility in distribution systems, chlorine demand signals from contamination events must be detected within the natural variability of free chlorine concentrations. Maintenance of higher free chlorine residuals at sensor locations will magnify the chlorine demand signal of microbial contamination events and minimize the false alarm rate.
3.5.
CT comparison
Observed chlorine demand for each organism followed the biphasic demand kinetics predicted and observed previously in the literature for pure microbial cultures (Shang and Blatchley, 2001; Virto et al., 2004; Virto et al., 2005a) and sewage effluents (Lin and Evans, 1974; Haas and Karra, 1984). Chlorine demand curves were modeled with a biphasic exponential decay model: C ¼ ð1 QÞek1 t þ Qek2 t ,
(1)
where C is the free chlorine fraction at time t defined as (Ct/C0); Ct is the free chlorine concentration at time t; C0 is the initial free chlorine concentration; (1Q) is the free chlorine fraction demanded in the initial phase of chlorine demand; Q is the free chlorine fraction demanded in the second phase of chlorine demand; k1 is the chlorine decay kinetic coefficient for the initial phase of chlorine demand; and k2 is the chlorine decay kinetic coefficient for the second phase of chlorine demand. The parameters Q, k1, and k2 are dependent on initial free chlorine concentration (C0), initial cell concentration (X0), and cell species. Observed free chlorine data for each species were fitted using the biphasic exponential decay model by nonlinear regression (Microsoft Office Excel 2003, Solver) to minimize the root mean squared error between the observed chlorine fraction and the model predicted chlorine fraction (results not shown). Using both the chlorine demand and cell survival data collected for each organism, a quantification of each organism’s ability to resist free chlorine can be calculated. This quantification is the organism’s chlorine contact time (CT) and, for this analysis, was calculated as the CT required to produce a three log (3-log CT) inactivation in cell concentration. In being consistent with recent literature on calculation of CT values (Haas et al., 1996), the time-dependent residual biphasic exponential decay model (Eq. (1)) was used to improve the accuracy of the CT value. Interpolations of the cell survival curves were used to determine the amount of time required for a 3-log inactivation (t3-log inactivation) of each organism under each set of initial conditions. The fitted biphasic decay model can be integrated to yield k t e 1 ek2 t þQ , (2) C ¼ ð1 QÞ k1 k2 which can then be evaluated from t0 to t3-log inactivation to yield the 3-log CT for each organism in units of (mg min/L) interpreted as the concentration of free chlorine required to reduce the active population by 3-logs over a 1-min duration. The calculated CT values for E. coli, S. epidermidis, and M. aurum are 0.03270.009, 0.22170.080, and 42.972.71 mg min/L, respectively. These values show that there is a direct relationship between the 3-log CT values of the organisms studied
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and their ultimate chlorine demand. It is thus hypothesized that chlorine demand may be related to characteristics of the cell that aid in resistance to chlorine. This hypothesis may be tested in future work with additional organisms.
4.
Conclusions
The objective of this work was to investigate the limitations of free chlorine sensors as a surrogate monitor for microorganisms by investigating the chlorine demand of different organisms under different initial free chlorine concentrations. The persistence of each organism under oxidative stress was also investigated. It was determined that microorganisms do exert a chlorine demand in proportion to their concentration. Chlorine-resistant organisms also demand chlorine, and their resistance mechanisms are hypothesized to be attributed to extracellular material sacrificially reacting with free chlorine to reduce the concentration at the cell membrane or wall. In the organisms studied, chlorineresistant organisms demanded more chlorine than more sensitive organisms. Chlorine demand also increased with increasing free chlorine concentration. Maintenance of higher chlorine residuals in the distribution system will produce greater chlorine demand signals during contamination events and a greater chance of detecting biological contamination. For E. coli and S. epidermidis, chlorine demand approached zero at initial cell concentrations of 105 and 104 CFU/mL, respectively. Contamination at concentrations at or below these levels will not be detectable by free chlorine sensors. However, these organisms were inactivated below the assay detection limits within 30 s for all initial conditions, so it is not likely that a concentration of these organisms grown under these experimental conditions could survive without producing a detectable signal. M. aurum demanded chlorine at all concentrations studied. They also remained viable for long periods, indicating that M. aurum could persist in water distribution systems, but free chlorine sensors should detect chlorine demand signals generated by the presence of M. aurum.
Acknowledgement This work was funded by the National Science Foundation Sensors Program through the Division of Biological and Environmental Engineering under grant BES-0329549. R E F E R E N C E S
Abid, N., Maalej, S., Rouis, S., 2004. Morphological and physiological changes of staphylococcus aureus exposed to hypochlorous acid. Lett. Appl. Microbiol. 38 (3), 245–250. APHA AWWA WEF, 2005. Standard Methods for the Examination of Water and Wastewater, 21st ed. APHA AWWA WEF, Washington, DC. Blackburn, B., Craun, G., Yoder, J., Hill, V., Calderon, R., Chen, N., Lee, S., Levy, D., Beach, M., 2004. Surveillance for waterborne-disease outbreaks associated with drinking
ARTICLE IN PRESS 4434
WAT E R R E S E A R C H
41 (2007) 4424– 4434
water—United States, 2001–2002. Morbid. Mortal. Wkly. Rep. 53 (SS-08), 23–45. Byer, D.C.K., 2005. Real time detection of intentional chemical contamination in the distribution system. J. Am. Water Works Assoc. 97. Cloete, T.E., 2003. Resistance mechanisms of bacteria to antimicrobial compounds. Int. Biodeterior. Biodegrad. 51 (4), 277–282. Corso, P., Kramer, M., Blair, K., Addiss, D., Davis, J., Haddix, A., 2003. Cost of illness in the 1993 waterborne cryptosporidium outbreak, Milwaukee, Wisconsin. Emerging Infect. Dis. 9 (4), 426–431. Daly, B., Betts, W.B., Brown, A.P., O’neill, J.G., 1998. Bacterial loss from biofilms exposed to free chlorine. Microbios 96 (383), 7–21. Deisingh, A.T.M., 2004. Biosensors for the detection of bacteria. Can. J. Microbiol. 50, 69–77. Eichler, S., Christen, R., Holtje, C., Westphal, P., Botel, J., Brettar, I., Mehling, A., Hofle, M.G., 2006. Composition and dynamics of bacterial communities of a drinking water supply system as assessed by RNA- and DNA-based 16s rRNA gene fingerprinting. Appl. Environ. Microbiol. 72 (3), 1858–1872. Fass, S., Dincher, L., Reasoner, D., Gatel, D., Block, J., 1996. Fate of Escherichia coli experimentally injected in a drinking water distribution pilot system. Water Res. 30 (9), 2215–2221. Haas, C., Karra, S., 1984. Kinetics of wastewater chlorine demand and exertion. J. Water Pollut. Control Fed. 56, 170–173. Haas, C.N., Jacangelo, J.G., Bishop, M.M., Cameron, C.D., Chowdhury, Z.K., Connell, G.F., Doty, G.A., Finch, G.R., Gates, D.J., Greenberg, A.E., Hoehn, R.C., Huebner, W.B., Jensen, J.N., Lange, A.L., Long, B.W., Moyer, N.P., Nagel, W.H., Noran, P.F., Palin, A.T., Regli, S.E., Routt, J.C., Symons, J.M., Thompson, C.K., Voyles, C.F., 1992. Survey of water utility disinfection practices. J. Am. Water Works Assoc. 84 (9), 121–128. Haas, C.N., Joffe, J., Anmangandla, U., Jacangelo, J., Heath, M., 1996. Water quality and disinfection kinetics. J. Am. Water Works Assoc. 88 (3), 95–103. Keinanen, M.M., Korhonen, L.K., Lehtola, M.J., Miettinen, I.T., Martikainen, P.J., Vartiainen, T., Suutari, M.H., 2002. The microbial community structure of drinking water biofilms can be affected by phosphorus availability. Appl. Environ. Microbiol. 68 (1), 434–439. Khan, A.S., Swerdlow, D.L., Juranek, D.D., 2001. Precautions against biological and chemical terrorism directed at food and water supplies. Public Health Rep. 116 (1), 3–14. Le Dantec, C., Duguet, J.P., Montiel, A., Dumoutier, N., Dubrou, S., Vincent, V., 2002. Chlorine disinfection of atypical mycobacteria isolated from a water distribution system. Appl. Environ. Microbiol. 68 (3), 1025–1032. Lin, S., Evans, R., 1974. Chlorine demand study of secondary sewage effluents. Water Sewage Works 121 (1), 35–44. Lisle, J.T., Broadaway, S.C., Prescott, A.M., Pyle, B.H., Fricker, C., Mcfeters, G.A., 1998. Effects of starvation on physiological
activity and chlorine disinfection resistance in Escherichia coli O157: H7. Appl. Environ. Microbiol. 64 (12), 4658–4662. Luthy, R.G., 2002. Bioterrorism and water security. Environ. Sci. Technol. 36 (7), 123A. Maalej, S., Dammak, I., Dukan, S., 2006. The impairment of superoxide dismutase coordinates the derepression of the perr regulon in the response of Staphylococcus aureus to Hocl stress. Microbiology 152, 855–861. Rodriguez-Mozaz, S., Marco, M., Lopez de Alda, M., Barcelo, D., 2004. Biosensors for environmental applications: future development trends. Pure Appl. Chem. 76 (4), 723–752. Ryu, J.H., Beuchat, L.R., 2004. Factors affecting production of extracellular carbohydrate complexes by Escherichia coli O157: H7. Int. J. Food Microbiol. 95 (2), 189–204. Schmeisser, C., Stockigt, C., Raasch, C., Wingender, J., Timmis, K.N., Wenderoth, D.F., Flemming, H.C., Liesegang, H., Schmitz, R.A., Jaeger, K.E., Streit, W.R., 2003. Metagenome survey of biofilms in drinking-water networks. Appl. Environ. Microbiol. 69 (12), 7298–7309. Shang, C.B.E., Blatchley, E.R., 2001. Chlorination of pure bacterial cultures in aqueous solution. Water Res. 35 (1), 244–254. Taylor, R.H., Falkinham, J.O., Norton, C.D., Lechevallier, M.W., 2000. Chlorine, chloramine, chlorine dioxide, and ozone susceptibility of Mycobacterium Avium. Appl. Environ. Microbiol. 66 (4), 1702–1705. United States Environmental Protection Agency, 2005. WaterSentinel Online Water Quality Monitoring as an Indicator of Drinking Water Contamination. December 12. /http:// www.epa.gov/safewater/watersecurity/pubs/watersentinel_ wq_monitoring.pdfS. Venkobachar, C., Iyengar, L., Rao, A., 1977. Mechanism of disinfection: effect of chlorine on cell membrane functions. Water Res. 11, 727–729. Virto, R., Alvarez, I., Condon, S., Raso, J., 2004. Relationship between inactivation kinetics of a Listeria moncytogenes suspension by chlorine and its chlorine demand. J. Appl. Microbiol. 97, 1281–1288. Virto, R., Alvarez, I., Condon, S., Raso, J., 2005a. Modeling the effect of initial concentration of Escherichia coli suspensions on their inactivation by chlorine. J. Food Saf. 25 (2), 120–129. Virto, R., Manas, P., Alvarez, I., Condon, S., Raso, J., 2005b. Membrane damage and microbial inactivation by chlorine in the absence and presence of a chlorine-demanding substrate. Appl. Environ. Microbiol. 71 (9), 5022–5028. Whitman, C.T. Strategic Plan for Homeland Security. September 2002. /http://www.epa.gov/epahome/downloads/epa_ homeland_security_strategic_plan.pdfS. Williams, M.M., Braun-Howland, E.B., 2003. Growth of Escherichia coli in model distribution system biofilms exposed to hypochlorous acid or monochloramine. Appl. Environ. Microbiol. 69 (9), 5463–5471.