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Free Chlorine and Monochloramine Application to Nitrifying Biofilm: Comparison of Biofilm Penetration, Activity, and Viability Woo Hyoung Lee,†,‡ David G. Wahman,§ Paul L. Bishop,† and Jonathan G. Pressman*,§ †
School of Energy, Environmental, Biological and Medical Engineering, University of Cincinnati, Cincinnati, Ohio 45221, United States National Risk Management Research Laboratory, U.S. Environmental Protection Agency, Cincinnati, Ohio 45268, United States
§
bS Supporting Information ABSTRACT: Biofilm in drinking water systems is undesirable. Free chlorine and monochloramine are commonly used as secondary drinking water disinfectants, but monochloramine is perceived to penetrate biofilm better than free chlorine. However, this hypothesis remains unconfirmed by direct biofilm monochloramine measurement. This study compared free chlorine and monochloramine biofilm penetration into an undefined mixed-culture nitrifying biofilm by use of microelectrodes and assessed the subsequent effect on biofilm activity and viability by use of dissolved oxygen (DO) microelectrodes and confocal laser scanning microscopy (CLSM) with LIVE/DEAD BacLight. For equivalent chlorine concentrations, monochloramine initially penetrated biofilm 170 times faster than free chlorine, and even after subsequent application to a monochloramine penetrated biofilm, free chlorine penetration was limited. DO profiles paralleled monochloramine profiles, providing evidence that either the biofilm was inactivated with monochloramine’s penetration or its persistence reduced available substrate (free ammonia). While this research clearly demonstrated monochloramine’s greater penetration, this penetration did not necessarily translate to immediate viability loss. Even though free chlorine’s penetration was limited compared to that of monochloramine, it more effectively (on a cell membrane integrity basis) inactivated microorganisms near the biofilm surface. Limited free chlorine penetration has implications when converting to free chlorine in full-scale chloraminated systems in response to nitrification episodes.
’ INTRODUCTION Biofilm in drinking water distribution systems may degrade water quality (e.g., disinfectant depletion, coliform occurrences) and cause compliance issues (e.g., violation of the Surface Water Treatment Rule, Total Coliform Rule, or Lead and Copper Rule). Several reports describe biofilm microorganism resistance to disinfection when compared with similar suspended cells.1-3 Effective biofilm control is desirable to maintain public health, with free chlorine and monochloramine commonly used as secondary disinfectants. Because monochloramine forms lower levels of regulated disinfectant by-products (DBPs), many utilities are expected to switch from free chlorine to monochloramine for regulatory compliance.4,5 However, monochloramine use may lead to nitrification caused by ammonia addition for chloramine formation and subsequent release during chloramine decay.6,7 It is well-known that nitrifying biofilm [e.g., ammonia-oxidizing bacteria (AOB), nitriteoxidizing bacteria (NOB)] are involved in distribution system nitrification.8,9 Nitrification reduces water quality, causes difficulties maintaining adequate disinfectant residual, and poses public health concerns, including nitrite, nitrate, and opportunistic pathogenic microorganism exposure.9 In practice, free chlorine is commonly used to recover from nitrification episodes (e.g., break point chlorination) but may not prevent nitrification from reoccurring.9,10 The basis for free chlorine biofilm resistance is not completely understood;1 however, its impeded biofilm penetration has been shown.11-13 In comparison, monochloramine is perceived to better penetrate biofilm than free chlorine because of its decreased reactivity and thus greater persistence.3,14 LeChevallier et al.3 suggested that r 2011 American Chemical Society
monochloramine and free chlorine act differently at biofilm surfaces by use of plate counts. However, monochloramine’s greater penetration has not been proven by direct measurement and therefore not directly compared with free chlorine because a chloramine-sensitive microelectrode did not previously exist. The present research used a recently developed chloramine-sensitive microelectrode15 to provide disinfectant penetration comparisons. This study’s primary objective was to measure free chlorine and monochloramine penetration into nitrifying biofilm by use of chlorine/chloramine-sensitive microelectrodes and to evaluate the impact on activity and viability by use of dissolved oxygen (DO) microelectrodes and confocal laser scanning microscopy (CLSM) with LIVE/DEAD BacLight.
’ EXPERIMENTAL SECTION Biofilm Growth and Development. Annular biofilm reactors (BioSurface Technologies Corp., Bozeman, MT; model 1320 LJ) containing 20 removable polycarbonate slides were inoculated with an undefined mixed nitrifying culture obtained from a chloraminated drinking water distribution system experiencing nitrification. The reactors were operated at 100 rpm to simulate shear stress associated with 30.5 cm/s (1 ft/s) flow in 10.2-cm (4-in.) pipe.16 Initially, reactors were operated in batch mode to develop nitrifying biofilm attached to polycarbonate slides with air-saturated dechlorinated tap water that contained Received: October 19, 2010 Accepted: December 23, 2010 Revised: December 23, 2010 Published: January 12, 2011 1412
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Environmental Science & Technology 250 mg N/L ammonia and adjusted to pH 8.0 with 5% (w/v) NaHCO3 at 23 °C. After nitrification establishment (ammonia below 50 mg N/L after 1 week of batch operation), operation was changed to continuous mode (Figure S1, Supporting Information) with tap water (dechlorinated by use of a granular activated carbon column) supplemented with growth media (described in Supporting Information), resulting in feed concentrations of 200 mg N/L ammonia, 12 mg P/L phosphorus, and 8 mg C/L organic carbon to promote an appropriate biofilm thickness (>200 μm) for microprofiling. The reactor hydraulic residence time (HRT) was maintained at 6 h to promote biofilm over suspended nitrifier growth. During the reactor operations, active nitrification within the reactors was demonstrated by weekly pH, DO, ammonia, nitrite, and nitrate measurements in the bulk (data not shown). The reactors were operated for 6 months at 23 °C, pH 7.5-8.0 (maintained by NaHCO3 addition), and 1-3 mg O2/L DO concentration. Microelectrode Preparation and Microprofiling. Free chlorine and total chlorine microelectrodes (5-10 μm tip size) were used to measure free chlorine and monochloramine at applied potentials of þ200 mV11 and þ150 mV vs Ag/AgCl, respectively. The previous applied potential of monochloramine measurement15 was changed to þ150 mV to reduce DO interference during monochloramine measurements. Details on total chlorine microelectrode validation at þ150 mV are described in the Supporting Information (Figures S2-S5). DO microelectrodes17 were used to investigate the aerobic microbial activity during free chlorine and monochloramine application, and ammonium microelectrodes18 were fabricated to measure the ammonium concentration microprofiles. Microelectrodes were mounted in a microprofiling experimental apparatus15 (Figure S6a, Supporting Information) and controlled by a three-dimensional micromanipulator (World Precision Instrument, M3301). Microelectrodes were calibrated in the same media used for microprofiling. A used microelectrode was used to measure biofilm thickness.15 A separate medium (described in Supporting Information) was prepared for initial DO and ammonium microprofiling before disinfection. The tip of the microelectrode was initially positioned at 1000 μm above the biofilm-water interface. To measure the initial DO biofilm microprofiles before disinfection, the biofilm sample was acclimated in the flow chamber (Figure S6b, Supporting Information) for 1 h (15 mL/min, 23 °C, and 8.3 mg/L DO), to ensure that initial steady-state profiles were obtained.19 By controlling the micromanipulator and using a microscope, the microelectrode was positioned at the center of a well-shaped biofilm structure that was selected for microprofiling (Figure S6c, Supporting Information). All microprofile measurements were performed in a flow chamber. The flow rate to the flow chamber was continuous at 15 mL/min, resulting in a hydraulic residence time in the flow chamber of 2 min and an average surface velocity of 7.5 cm/min. A single microprofile was completed typically in 2-4 min ( 48 h) with the same monochloramine concentration (Figure 4d) or a greater monochloramine concentration 1415
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Figure 3. DO, monochloramine and free chlorine concentration microprofiles measured during experiment 2: (a) phase 1 DO, (b) phase 1 monochloramine, and (c) phase 2a free chlorine. Both phases were conducted under the same conditions (5 mM buffer solution, pH 8.0, 23 °C, 8.3 mg/L DO, and 2.7-2.8 mg Cl2/L) except disinfectant (phase 1, 2.7 mg Cl2/L monochloramine (4:1 Cl2:N); phase 2a, 2.8 mg Cl2/L free chlorine). The biofilm surface depth was defined as 0 μm.
(e.g., >10 mg Cl2/L; Figure S11, Supporting Information) was required to inactivate the biofilm near the surface. During monochloramine exposure, no apparent biofilm sloughing occurred, while only very limited random biofilm sloughing was observed during free chlorine exposure. However, because the sloughing was minor, biofilm removal or thickness changes were not considered to affect subsequent concentration profiles. Free Chlorine Penetration after Monochloramine Exposure. After monochloramine exposure, the feed solution was changed to free chlorine (experiment 2, phase 2a) at an equivalent concentration (2.8 mg Cl2/L). DO profiles continued to penetrate the biofilm similar to phase 1 (data not shown). Free chlorine microprofiles differed from those of monochloramine (Figure 3c). Even though monochloramine reached 97% of the bulk concentration (250 μm biofilm depth) during phase 1, free chlorine reached only 8% of the bulk concentration (250 μm biofilm depth) after a subsequent 20 h exposure. Because monochloramine and free chlorine have similar diffusion coefficients in water at 25 °C [chlorine diffusion coefficient (Dchl) = 1.44 10-5 cm2/s 11 and monochloramine diffusion coefficient (Dmono) = 1.6610-5 cm2/s 24], the measured free chlorine microprofiles detail the differences in reactivity between free chlorine and monochloramine with biofilm constituents. Even after monochloramine had essentially fully penetrated the biofilm, subsequent addition of free chlorine showed an impeded penetration, presumably from free chlorine reacting faster than it could diffuse.
Figure 4 panels d and e compare the difference between free chlorine and monochloramine (experiment 2, phases 2a and 2b) on cell viability. Nonviable cells (fluorescent red) at the end of phase 2a showed that free chlorine was more effective near the biofilm surface (Figure 4e) than continuing with a similar monochloramine application (phase 2b) (Figure 4d), highlighting that free chlorine is a stronger disinfectant where it can penetrate. Free Chlorine and Monochloramine Comparison and Implications. To quantify free chlorine and monochloramine penetration, two values were determined from the microprofiles (Cz and Czmax). Cz (mg Cl2) is the total mass delivered to a biofilm unit area (cm2) throughout the biofilm depth (z, cm) at a given time, and Czmax (mg Cl2) is the maximum Cz (i.e., the mass delivered to the biofilm when full penetration is achieved). From this, Cz/Czmax represents the percentage of maximum mass delivery, with 0% and 100% representing no biofilm and complete biofilm penetration, respectively. Figure 5a displays Cz/Czmax over time for each experimental condition. For equivalent chlorine concentrations (2.6-2.7 mg Cl2/L), monochloramine penetration was more rapid than that of free chlorine and quickly approached full penetration (Figure 5a), but free chlorine showed limited temporal biofilm penetration. The initial mass delivery rate was determined for the first 2 and 20 h for monochloramine and free chlorine, respectively, where Cz/Czmax has a linear trend (Figure 5b). Because free chlorine 1416
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Figure 4. Progression of biofilm CLSM images during experiment 2. Progression of biofilm CLSM images is shown during experiment 2 phase 1 (panels a-c) plus phase 2b (panel d) with an additional 24 h of monochloramine application: biofilm (a) before disinfection and (b) 2 h, (c) 24 h, and (d) 48 h after monochloramine disinfection. Panel e shows 24 h of chlorination (phase 2a) after 24 h of monochloramine disinfection. Each Z stack has a solid blue line where the XY displayed image was acquired.
Figure 5. Comparison of monochloramine and free chlorine penetration. (a) Calculated free chlorine and monochloramine Cz/Czmax profiles. (b) Penetration/ mass delivery rate comparison between monochloramine and free chlorine for the first 2 and 20 h for monochloramine and free chlorine, respectively.
and monochloramine were applied to the same biofilm [i.e., free chlorine (2.8 mg Cl2/L) after monochloramine (2.7 mg Cl2/L)] in experiment 2 (phases 1 and 2a), comparison between the closed and open circles in Figure 5b represents the best direct penetration comparison between free chlorine and monochloramine, removing possible effects from biofilm differences. Also, because this comparison used a biofilm with full monochloramine penetration, it represents a “best-case” scenario for free chlorine penetration. For this biofilm experiment, the initial rate of Cz/Czmax for monochloramine was 170 times greater than for free chlorine.
When determining inactivation, exposure time must be considered in addition to disinfectant concentration. Concentration time (Ct) profiles (Figure 6) were calculated on the basis of experimental profiles for free chlorine (Figure 1b) and monochloramine (Figure 3b). Additional details on Ct profile generation are provided in the Supporting Information. To evaluate expected biofilm inactivation, the delayed Chick-Watson model20 for monochloramine and the Chick-Watson model25 for free chorine were used to determine Ct values for 2 and 4 log reductions. For calculations, kinetic parameters were taken from 1417
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Figure 6. Calculated (a) free chlorine and (b) monochloramine concentration time (Ct) profiles.
the literature, using disinfection kinetics determined for a suspended ammonia-oxidizing bacterium (Nitrosomonas europaea) with cell-membrane integrity techniques. Even though using kinetic parameters determined for a pure suspended culture may underpredict Ct values (and overpredict disinfection) for an undefined biofilm, they provide a reference point for discussion purposes. For monochloramine, 490 (mg Cl2) 3 min/L for the lag coefficient (b) and 2.8 10-3 L/(mg Cl2) 3 min for the Chick-Watson disinfection rate constant (k) were used.20 For free chlorine at pH 8.0, the rate constant was determined by assuming that the biocidal potency of HOCl:OCl-:NH2Cl is approximately 200:2.5:1,26 resulting in a k of 0.14 L/(mg Cl2) 3 min, using the previous monochloramine rate constant for scaling and a pKa of 7.5 for free chlorine. The predicted Ct profiles and resulting inactivation (Figure 6) qualitatively correlate with the last CLSM image from phase 1 (Figure 2c for free chlorine and Figure 4c for monochloramine). A 4 log reduction would mean that only 0.01% of the image remains green (i.e., it is essentially all red) based on starting with an all-green (i.e., 100% viable) CLSM image. The free chlorine image shows essentially all inactivated (red) cells, which corresponds to the calculated greater than 4 log inactivation. The monochloramine image shows cells that are mostly inactive (red) with some viable (green), which corresponds with the calculated inactivation between 2 and 4 log. Overall, the CLSM images and the Ct profiles qualitatively agreed. It also helps explain why nitrification is so hard to stop once it has started because Ct is too low in biofilm if residual cannot be maintained. Two results from these experiments may have practical implications for chloraminating drinking water utilities that conduct free chlorine application periods (i.e., free chlorine burns) to prevent and/or control nitrification: 1. Free chlorine application after full monochloramine biofilm penetration resulted in minimal free chlorine penetration 2. Bulk chemical concentrations provided no information on biofilm penetration Together, these results imply that even if “target” free chlorine concentrations are maintained in the bulk water system, they may provide little indication on biofilm penetration and subsequent inactivation, which depends on several additional factors (e.g., substratum, DO, hydraulic residence time, penetration time, and biofilm thickness). In addition, these experiments were conducted with a nonreactive substratum (i.e., polycarbonate slides). If the substratum was reactive to either monochloramine or free chlorine (e.g., iron pipe), the reactive substratum would further
decrease the biofilm chemical concentrations, reducing the effectiveness of either disinfectant. If utilities are finding inadequate nitrification control from periodic free chlorine application, they should consider increased free chlorine concentrations and/or increased application times to overcome the reactive biofilm components. Either of these implementations may have unintended consequences (i.e., increased DBPs and/or lead release) and must be carefully evaluated before and during application.
’ ASSOCIATED CONTENT
bS
Supporting Information. Eleven figures and additional text with information on reactor operation, media solution, microelectrode optimization, microprofiling apparatus, replication, profiles and CLSM images, and Ct profile generation. This material is available free of charge via the Internet at http:// pubs.acs.org.
’ AUTHOR INFORMATION Corresponding Author
*Phone: (513) 569-7625; fax: (513) 487-2543; e-mail: Pressman.
[email protected]. Present Addresses ‡
Oak Ridge Institute for Science and Education Post-Doctoral Fellow at U.S EPA, Cincinnati, Ohio 45268.
’ ACKNOWLEDGMENT The U.S. Environmental Protection Agency, through its Office of Research and Development, funded and collaborated in the research described herein (Contract EP-C-05-056/WA 3-47). It has been reviewed and approved for publication. Any opinions expressed are those of the authors and do not necessarily reflect the views of the agency, therefore, no official endorsement should be inferred. Any mention of trade names or commercial products does not constitute endorsement or recommendation for use. ’ REFERENCES (1) Stewart, P. S.; Rayner, J.; Roe, F.; Rees, W. M. Biofilm penetration and disinfection efficacy of alkaline hypochlorite and chlorosulfamates. J. Appl. Microbiol. 2001, 91, 525–532. (2) Gilbert, P.; Brown, M. R. W. Mechanisms of the protection of bacterial biofilms from antimicrobial agents. In Microbial Biofilms; 1418
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