Peracetic acid as an alternative wastewater disinfectant to chlorine dioxide

AIMS: The aim of this study was to compare the efficiency of peracetic acid with that of chlorine dioxide in the disinfection of wastewater from a sewage treatment plant (serving about 650 000 inhabitants) that has been using peracetic acid as a disinfectant since 1998. METHODS AND RESULTS: A total of 23 samplings were made, each consisting of three samples: from secondary effluent, effluent disinfected with 2 mg l(-1) of peracetic acid and effluent disinfected with 2.2 mg l(-1) of chlorine dioxide (contact time 20 min). For each sample, measurements were made of the heterotrophic plate count at 36 degrees C, total and faecal coliforms, Escherichia coli, enterococci, pH, suspended solids and chemical oxygen demand (COD). During the first phase of the experiment the peracetic acid was seen to be less efficient than chlorine dioxide. To improve the disinfectant action a system of mechanical agitation was added which led to a greater efficiency in the inactivation of bacteria of faecal origin. CONCLUSIONS: Both products were found to be influenced by the level of microbial contamination, the amount of suspended solids and COD but not by the pH of the effluent before disinfection. The immediate mixing of the wastewater and disinfectant caused a greater reduction in enterococci. SIGNIFICANCE AND IMPACT OF THE STUDY: Since peracetic acid was seen to produce a high abatement of micro-organisms, it can be considered as a valid alternative to chlorine dioxide in the disinfection of wastewaters.     

Relationship between inactivation kinetics of a Listeria monocytogenes suspension by chlorine and its chlorine demand

AIMS: Chlorine demand by Listeria monocytogenes cells and inactivation of L. monocytogenes by chlorine (0.6-1.0 mg l(-1)) at different temperatures (4, 20 and 30 degrees C) have been investigated in a batch reactor. METHODS AND RESULTS: Chlorine demand depended on the microbial concentration and was independent on the initial chlorine concentration and temperature. Chlorine decay was modelled by the addition of two first-order decay equations. Inactivation of L. monocytogenes by chlorine depended on the initial microbial concentration, initial chlorine concentration and temperature. A mathematical model based on a biphasic inactivation properly described survival curves of L. monocytogenes and a tertiary model was developed that satisfactorily predicted the inactivation of L. monocytogenes by different concentrations of initial chlorine at different temperatures. CONCLUSIONS: Both available chlorine decay and inactivation of L. monocytogenes by chlorine were biphasic and can be modelled by a two-term exponential model. SIGNIFICANCE AND IMPACT OF THE STUDY: The biphasic nature of survival curves of L. monocytogenes did not reflect the effect of a change of available chlorine concentration during the treatment. The microbial inactivation was caused by successive reactions that occur after the consumption of the chlorine by the bacterial cell components.     

Amoebae in domestic water systems: resistance to disinfection treatments and implication in Legionella persistence

AIMS: Monitoring of microbial changes during and after application of various disinfection treatments in a model domestic water system. METHODS AND RESULTS: A pilot-scale domestic water system consisting of seven galvanized steel re-circulation loops and copper dead legs was constructed. Culture techniques, confocal laser scanning microscopy after fluorescent in situ hybridization and viability staining with the BacLight LIVE/DEAD kit were used for planktonic and biofilm flora monitoring. Before starting the treatments, the system was highly contaminated with Legionella pneumophila and biofilm populations mainly consisted of beta-proteobacteria. In the water and the biofilm of the loops, continuous application of chlorine dioxide (0.5 mg l(-1)), or chlorine (2.5 mg l(-1)) were very effective in reducing the microbial flora, including L. pneumophila. Heterotrophic bacteria, although strongly reduced, were still detectable after ozone application (0.5 mg l(-1)), whereas with monochloramine (0.5 mg l(-1)) and copper-silver ionization (0.8/0.02 mg l(-1)), the contamination remained significantly higher. Monochloramine and copper-silver did not remove the biofilm. During copper-silver application, Legionella re-growth was observed. Only chlorine dioxide led to detectable effects in the dead leg. Amoebae could not be eliminated, and after interrupting the treatments, L. pneumophila quickly recovered their initial levels, in all cases. CONCLUSIONS: Chlorine dioxide, applied as a continuous treatment, was identified in this study as the most efficient for controlling L. pneumophila in a domestic water system. Chlorine dioxide showed a longer residual activity, leading to improved performance in the dead leg. Amoebae resisted to all the treatments applied and probably acted as reservoirs for L. pneumophila, allowing a quick re-colonization of the system once the treatments were interrupted. SIGNIFICANCE AND IMPACT OF THE STUDY: Control of microbial contamination requires maintenance of a constant disinfectant residual throughout the water system. Treatment strategies targeting free-living amoebae should lead to improved control of L. pneumophila. Such treatment strategies still have to be investigated.     

Efficacy of copper and silver ions and reduced levels of free chlorine in inactivation of Legionella pneumophila.

Water disinfection systems utilizing electrolytically generated copper and silver ions (200 and 20, 400 and 40, or 800 and 80 micrograms/liter) and low levels of free chlorine (0.1 to 0.4 mg/liter) were evaluated at room (21 to 23 degrees C) and elevated (39 to 40 degrees C) temperatures in filtered well water (pH 7.3) for their efficacy in inactivating Legionella pneumophila (ATCC 33155). At room temperature, a contact time of at least 24 h was necessary for copper and silver (400 and 40 micrograms/liter) to achieve a 3-log10 reduction in bacterial numbers. As the copper and silver concentration increased to 800 and 80 micrograms/liter, the inactivation rate significantly (P less than or equal to 0.05) increased from K = 2.87 x 10(-3) to K = 7.50 x 10(-3) (log10 reduction per minute). In water systems with and without copper and silver (400 and 40 micrograms/liter), the inactivation rates significantly increased as the free chlorine concentration increased from 0.1 mg/liter (K = 0.397 log10 reduction per min) to 0.4 mg/liter (K = 1.047 log10 reduction per min). Compared to room temperature, no significant differences were observed when 0.2 mg of free chlorine per liter with and without 400 and 40 micrograms of copper and silver per liter was tested at 39 to 40 degrees C. All disinfection systems, regardless of temperature or free chlorine concentration, showed increase inactivation rates when 400 and 40 micrograms of copper and silver per liter was added; however, this trend was significant only at 0.4 mg of free chlorine per liter.     

Inactivation of biofilm bacteria

The current project was developed to examine inactivation of biofilm bacteria and to characterize the interaction of biocides with pipe surfaces. Unattached bacteria were quite susceptible to the variety of disinfectants tested. Viable bacterial counts were reduced 99% by exposure to 0.08 mg of hypochlorous acid (pH 7.0) per liter (1 to 2 degrees C) for 1 min. For monochloramine, 94 mg/liter was required to kill 99% of the bacteria within 1 min. These results were consistent with those found by other investigators. Biofilm bacteria grown on the surfaces of granular activated carbon particles, metal coupons, or glass microscope slides were 150 to more than 3,000 times more resistant to hypochlorous acid (free chlorine, pH 7.0) than were unattached cells. In contrast, resistance of biofilm bacteria to monochloramine disinfection ranged from 2- to 100-fold more than that of unattached cells. The results suggested that, relative to inactivation of unattached bacteria, monochloramine was better able to penetrate and kill biofilm bacteria than free chlorine. For free chlorine, the data indicated that transport of the disinfectant into the biofilm was a major rate-limiting factor. Because of this phenomenon, increasing the level of free chlorine did not increase disinfection efficiency. Experiments where equal weights of disinfectants were used suggested that the greater penetrating power of monochloramine compensated for its limited disinfection activity. These studies showed that monochloramine was as effective as free chlorine for inactivation of biofilm bacteria. The research provides important insights into strategies for control of biofilm bacteria.     

Inactivation of biofilm bacteria.

The current project was developed to examine inactivation of biofilm bacteria and to characterize the interaction of biocides with pipe surfaces. Unattached bacteria were quite susceptible to the variety of disinfectants tested. Viable bacterial counts were reduced 99% by exposure to 0.08 mg of hypochlorous acid (pH 7.0) per liter (1 to 2 degrees C) for 1 min. For monochloramine, 94 mg/liter was required to kill 99% of the bacteria within 1 min. These results were consistent with those found by other investigators. Biofilm bacteria grown on the surfaces of granular activated carbon particles, metal coupons, or glass microscope slides were 150 to more than 3,000 times more resistant to hypochlorous acid (free chlorine, pH 7.0) than were unattached cells. In contrast, resistance of biofilm bacteria to monochloramine disinfection ranged from 2- to 100-fold more than that of unattached cells. The results suggested that, relative to inactivation of unattached bacteria, monochloramine was better able to penetrate and kill biofilm bacteria than free chlorine. For free chlorine, the data indicated that transport of the disinfectant into the biofilm was a major rate-limiting factor. Because of this phenomenon, increasing the level of free chlorine did not increase disinfection efficiency. Experiments where equal weights of disinfectants were used suggested that the greater penetrating power of monochloramine compensated for its limited disinfection activity. These studies showed that monochloramine was as effective as free chlorine for inactivation of biofilm bacteria. The research provides important insights into strategies for control of biofilm bacteria.     

Chlorine decay and bacterial inactivation kinetics in drinking water in the tropics

The decay of free chlorine (Cl₂) and combined chlorine (mostly monochloramine: NH₂Cl) and the inactivation of bacteria was examined in Dar es Salaam, Tanzania. Batch experiments, pilot-scale pipe experiments and full-scale pipe experiments were carried out to establish the kinetics for both decay and inactivation, and to compare the two disinfectants for use under tropical conditions. The decay of both disinfectants closely followed first order kinetics, with respect to the concentration of both disinfectant and disinfectant-consuming substances. Bacterial densities exhibited a kinetic pattern consisting of first order inactivation with respect to the density of the bacteria and the concentration of the disinfectant, and first order growth with respect to the bacterial density. The disinfection kinetic model takes the decaying concentration of the disinfectant into account. The decay rate constant for free chlorine was 114 lg^-1h^-1, while the decay rate constant for combined chlorine was 1.84 lg^-1h^-1 (1.6% of the decay rate for free chlorine). The average concentration of disinfectant consuming substances in the water phase was 2.6 mg Cl₂/l for free chlorine and 5.6 mg NH₂Cl/l for combined chlorine. The decay rate constant and the concentration of disinfectant consuming substances when water was pumped through pipes, depended on whether or not chlorination was continuous. Combined chlorine especially could clean the pipes of disinfectant consuming substances. The inactivation rate constant , was estimated at 3.06×10⁴ lg^-1h^-1. Based on the inactivation rate constant, and a growth rate constant determined in a previous study, the critical concentration of free chlorine was found to be 0.08 mg Cl₂/l. The critical concentration is a value below which growth rates dominate over inactivation.The authors are with the Technical University of Denmark, IMT, CDC, Build. 208, DK-2800 Lyngby, Denmark     

Determinants of the microbiological characteristics of South Australian swimming pools.

A survey of 100 swimming pools has been conducted to assess the effectiveness of disinfection practices against various microorganisms and to check compliance with recommended chlorine levels and pH. Microbiological quality was assessed from densities of total coliforms, Escherichia coli, and Pseudomonas aeruginosa, total colony counts, and the presence or absence of amoebae, including the pathogen Naegleria fowleri. Although a free chlorine residual of 1.0 mg/liter and a pH range of 7.0 to 7.6 are recommended by local health authorities, 41 pools had a lower free chlorine residual and 37 had a pH outside this range. Multiple logistic regression analysis was used to test the association of field measurements with the microbiological data. The analysis demonstrated a strong positive association of free chlorine with bacteriological quality and the absence of Naegleria spp. No other field measurement was predictive in this regard, although the absence of all amoebae was associated with a relatively low water temperature and pH. Using a mathematical model derived from this analysis, it was estimated that 99% of pools would have acceptable bacteriological quality and 94% would be free of Naegleria spp. at a free chlorine residual of 1.0 mg/liter. However, at the mean water temperature (23 degrees C) and pH (7.5) seen in this study, other amoebae would still be detectable in 500-ml samples taken from 40% of pools at this chlorine level.     

Determinants of the microbiological characteristics of South Australian swimming pools

A survey of 100 swimming pools has been conducted to assess the effectiveness of disinfection practices against various microorganisms and to check compliance with recommended chlorine levels and pH. Microbiological quality was assessed from densities of total coliforms, Escherichia coli, and Pseudomonas aeruginosa, total colony counts, and the presence or absence of amoebae, including the pathogen Naegleria fowleri. Although a free chlorine residual of 1.0 mg/liter and a pH range of 7.0 to 7.6 are recommended by local health authorities, 41 pools had a lower free chlorine residual and 37 had a pH outside this range. Multiple logistic regression analysis was used to test the association of field measurements with the microbiological data. The analysis demonstrated a strong positive association of free chlorine with bacteriological quality and the absence of Naegleria spp. No other field measurement was predictive in this regard, although the absence of all amoebae was associated with a relatively low water temperature and pH. Using a mathematical model derived from this analysis, it was estimated that 99% of pools would have acceptable bacteriological quality and 94% would be free of Naegleria spp. at a free chlorine residual of 1.0 mg/liter. However, at the mean water temperature (23 degrees C) and pH (7.5) seen in this study, other amoebae would still be detectable in 500-ml samples taken from 40% of pools at this chlorine level.     

Stability of green fluorescent protein (GFP) in chlorine solutions of varying pH

Green fluorescent protein (GFP) is an excellent biosensor as a result of its ability to be easily monitored in a wide variety of applications. Enzymes and proteins have been used as biological indicators to evaluate the immediate efficacy of industrial procedures, such as blanching, pasteurization, and disinfection treatments, as well as to monitor the satisfactory preservation of a product subjected to disinfection or sterilization. The purpose of this work was to study GFP stability in chlorinated water for injection (WFI) and chlorinated buffered solutions at various pH ranges, with and without agitation, to evaluate the exposure time required for chlorine to decrease 90% of its fluorescence intensity (decimal reduction time, D-value, min, 25 degrees C). Fluorescence intensity (Ex/Emmax = 394/509 nm) was measured immediately after the addition of GFP (8.0-9.0 microg/mL) into buffered or unbuffered chlorine solutions with or without constant stirring. With solutions constantly stirred, GFP fluorescence decreased abruptly on contact with chlorine in concentrations greater than 150 ppm, with D-values between 1.3 min (147 ppm chlorine) and 1.7 min (183 ppm chlorine). In phosphate buffered chlorine solutions (pH = 7.15 +/- 0.08), GFP retained its structure between 52 and 94 ppm, but protein stability decreased 10-fold when exposed to 110 ppm chlorine. The recovery of GFP fluorescence intensity due to renaturation was observed between 30 and 100 ppm chlorine in WFI (final pH = 11.01 +/- 0.23) without stirring. Stirring enhanced the contact between GFP and chlorine throughout the assay and provided a more accurate D-value evaluation. GFP performed as a suitable fluorescent marker for monitoring disinfection effectiveness.     

Assessment of bismuth thiols and conventional disinfectants on drinking water biofilms

AIMS: Biofilms in water distribution systems represent a far more significant reservoir of micro-organisms than the water phase. Biofilms are (i) resistant to disinfectants, (ii) nuclei for microbial regrowth, (iii) a refuge for pathogens, (iv) accompanied by taste and odour problems, and (v) corrode surfaces. The effects of the current strategies for disinfection of drinking water systems in large buildings (chlorination, copper and silver ionization, and hyper-heating) were compared with a new generation of bismuth thiol (BT) biocides. METHODS AND RESULTS: Multispecies biofilms were treated with 0.8 mg l(-1) of free chlorine, 400 and 40 microg l(-1) of copper and silver ions, respectively, at 55 and 70 degrees C, and bismuth-2,3-dimercaptopropanol (BisBAL). Furthermore, the effect of combined heat and BisBAL on planktonic cell viability was examined in monoculture using Escherichia coli suspensions. Inactivation rates for BisBAL were similar to copper-silver ions, where the effects were slower than for free chlorine or temperature. The BisBAL effect on E. coli monocultures was augmented greatly by increasing temperatures. CONCLUSIONS: Like copper-silver ions, BTs show more persistent residual effects than chlorine and hyper-heating in water systems. BT efficiency increased with temperature. Like copper-silver ions, BT action is relatively slow. SIGNIFICANCE AND IMPACT OF THE STUDY: BT presents a new approach to containing water biofilms. BT action is not as rapid, but is more thorough than chlorine, and less caustic. BTs may also be more efficacious in hot water systems. At sub-minimum inhibition concentration levels, BTs uniquely inhibit bacterial exopolysaccharide, thereby retarding biofilm formation. Thus, the combination of bactericidal and residual effects may prevent slime build-up in hot water systems.     

A spore counting method and cell culture model for chlorine disinfection studies of Encephalitozoon syn. Septata intestinalis

The microsporidia have recently been recognized as a group of pathogens that have potential for waterborne transmission; however, little is known about the effects of routine disinfection on microsporidian spore viability. In this study, in vitro growth of Encephalitozoon syn. Septata intestinalis, a microsporidium found in the human gut, was used as a model to assess the effect of chlorine on the infectivity and viability of microsporidian spores. Spore inoculum concentrations were determined by using spectrophotometric measurements (percent transmittance at 625 nm) and by traditional hemacytometer counting. To determine quantitative dose-response data for spore infectivity, we optimized a rabbit kidney cell culture system in 24-well plates, which facilitated calculation of a 50% tissue culture infective dose (TCID(50)) and a minimal infective dose (MID) for E. intestinalis. The TCID(50) is a quantitative measure of infectivity and growth and is the number of organisms that must be present to infect 50% of the cell culture wells tested. The MID is as a measure of a system's permissiveness to infection and a measure of spore infectivity. A standardized MID and a standardized TCID(50) have not been reported previously for any microsporidian species. Both types of doses are reported in this paper, and the values were used to evaluate the effects of chlorine disinfection on the in vitro growth of microsporidia. Spores were treated with chlorine at concentrations of 0, 1, 2, 5, and 10 mg/liter. The exposure times ranged from 0 to 80 min at 25 degrees C and pH 7. MID data for E. intestinalis were compared before and after chlorine disinfection. A 3-log reduction (99.9% inhibition) in the E. intestinalis MID was observed at a chlorine concentration of 2 mg/liter after a minimum exposure time of 16 min. The log(10) reduction results based on percent transmittance-derived spore counts were equivalent to the results based on hemacytometer-derived spore counts. Our data suggest that chlorine treatment may be an effective water treatment for E. intestinalis and that spectrophotometric methods may be substituted for labor-intensive hemacytometer methods when spores are counted in laboratory-based chlorine disinfection studies.     

Effects of Assimilable Organic Carbon and Free Chlorine on Bacterial Growth in Drinking Water

Assimilable organic carbon (AOC) is one of the most important factors affecting the re-growth of microorganisms in drinking water. High AOC concentrations result in biological instability, but disinfection kills microbes to ensure the safety of drinking water. Free chlorine is an important oxidizing agent used during the disinfection process. Therefore, we explored the combined effects of AOC and free chlorine on bacterial growth in drinking water using flow cytometry (FCM). The initial AOC concentration was 168 μg.L^-1 in all water samples. Without free chlorine, the concentrations of intact bacteria increased but the level of AOC decreased. The addition of sodium hypochlorite caused an increase and fluctuation in AOC due to the oxidation of organic carbon. The concentrations of intact bacteria decreased from 1.1×10⁵ cells.mL^-1 to 2.6×10⁴ cells.mL^-1 at an initial free chlorine dose of 0.6 mg.L^-1 to 4.8×10⁴ cells.mL^-1 at an initial free chlorine dose of 0.3 mg.L^-1 due to free chlorine originating from sodium hypochlorite. Additionally, free chlorine might be more obviously affected AOC concentrations than microbial growth did. These results suggested that AOC and free chlorine might have combined effects on microbial growth. In this study, our results showed concentrations determined by FCM were higher than those by HPC, which indicated that some E. coli detected by FCM might not be detected using HPC in drinking water. The level of free chlorine might restrain the consumption of AOC by inhibiting the growth of E. coli; on the other hand, chlorination might increase the level of AOC, thereby increase the potential for microbial growth in the drinking water network.     

Assessment of the bacteriological activity associated with granular activated carbon treatment of drinking water

Bacteriological analyses were performed on the effluent from a conventional water treatment pilot plant in which granular activated carbon (GAC) had been used as the final process to assess the impact of GAC on the microbial quality of the water produced. Samples were collected twice weekly for 160 days from the effluents of six GAC columns, each of which used one of four different empty-bed contact times (7.5, 15, 30, and 60 min). The samples were analyzed for heterotrophic plate counts and total coliforms. Effluent samples were also exposed to chloramines and free chlorine for 60 min (pH 8.2, 23 degrees C). Bacterial identifications were performed on the disinfected and nondisinfected effluents. Additional studies were conducted to assess the bacteriological activity associated with released GAC particles. The results indicated that heterotrophic plate counts in the effluents from all columns increased to 10(5) CFU/ml within 5 days and subsequently stabilized at 10(4) CFU/ml. The heterotrophic plate counts did not differ at different empty-bed contact times. Coliforms (identified as Enterobacter spp.) were recovered from the nondisinfected effluent on only two occasions. The disinfection results indicated that 1.5 mg of chloramines per liter inactivated approximately 50% more bacteria than did 1.0 mg of free chlorine per liter after 1 h of contact time. Chloramines and chlorine selected for the development of different bacterial species--Pseudomonas spp. and Flavobacterium spp., respectively.(ABSTRACT TRUNCATED AT 250 WORDS)     

Assessment of the bacteriological activity associated with granular activated carbon treatment of drinking water.

Bacteriological analyses were performed on the effluent from a conventional water treatment pilot plant in which granular activated carbon (GAC) had been used as the final process to assess the impact of GAC on the microbial quality of the water produced. Samples were collected twice weekly for 160 days from the effluents of six GAC columns, each of which used one of four different empty-bed contact times (7.5, 15, 30, and 60 min). The samples were analyzed for heterotrophic plate counts and total coliforms. Effluent samples were also exposed to chloramines and free chlorine for 60 min (pH 8.2, 23 degrees C). Bacterial identifications were performed on the disinfected and nondisinfected effluents. Additional studies were conducted to assess the bacteriological activity associated with released GAC particles. The results indicated that heterotrophic plate counts in the effluents from all columns increased to 10(5) CFU/ml within 5 days and subsequently stabilized at 10(4) CFU/ml. The heterotrophic plate counts did not differ at different empty-bed contact times. Coliforms (identified as Enterobacter spp.) were recovered from the nondisinfected effluent on only two occasions. The disinfection results indicated that 1.5 mg of chloramines per liter inactivated approximately 50% more bacteria than did 1.0 mg of free chlorine per liter after 1 h of contact time. Chloramines and chlorine selected for the development of different bacterial species--Pseudomonas spp. and Flavobacterium spp., respectively.(ABSTRACT TRUNCATED AT 250 WORDS)     

Biofilm penetration and disinfection efficacy of alkaline hypochlorite and chlorosulfamates

AIMS: The purpose of this study was to compare the efficacy, in terms of bacterial biofilm penetration and killing, of alkaline hypochlorite (pH 11) and chlorosulfamate (pH 5.5) formulations. METHODS AND RESULTS: Two species biofilms of Pseudomonas aeruginosa and Klebsiella pneumoniae were grown by flowing a dilute medium over inclined stainless steel slides for 6 d. Microelectrode technology was used to measure concentration profiles of active chlorine species within the biofilms in response to treatment at a concentration of 1000 mg total chlorine l(-1). Chlorosulfamate formulations penetrated biofilms faster than did hypochlorite. The mean penetration time into approximately 1 mm-thick biofilms for chlorosulfamate (6 min) was only one-eighth as long as for the same concentration of hypochlorite (48 min). Chloride ion penetrated biofilms rapidly (5 min) with an effective diffusion coefficient in the biofilm that was close to the value for chloride in water. Biofilm bacteria were highly resistant to killing by both antimicrobial agents. Biofilms challenged with 1000 mg l(-1) alkaline hypochlorite or chlorosulfamate for 1 h experienced 0.85 and 1.3 log reductions in viable cell numbers, respectively. Similar treatment reduced viable numbers of planktonic bacteria to non-detectable levels (log reduction greater than 6) within 60 s. Aged planktonic and resuspended laboratory biofilm bacteria were just as susceptible to hypochlorite as fresh planktonic cells. CONCLUSION: Chlorosulfamate transport into biofilm was not retarded whereas hypochlorite transport clearly was retarded. Superior penetration by chlorosulfamate was hypothesized to be due to its lower capacity for reaction with constituents of the biofilm. Poor biofilm killing despite direct measurement of effective physical penetration of the antimicrobial agent into the biofilm demonstrates that bacteria in the biofilm are protected by some mechanism other than simple physical shielding by the biofilm matrix. SIGNIFICANCE AND IMPACT OF THE STUDY: This study lends support to the theory that the penetration of antimicrobial agents into microbial biofilms is controlled by the reactivity of the antimicrobial agent with biofilm components. The finding that chlorine-based biocides can penetrate, but fail to kill, bacteria in biofilms should motivate the search for other mechanisms of protection from killing by antimicrobial agents in biofilms.     

Chlorine disinfection of atypical mycobacteria isolated from a water distribution system

We studied the resistance of various mycobacteria isolated from a water distribution system to chlorine. Chlorine disinfection efficiency is expressed as the coefficient of lethality (liters per minute per milligram) as follows: Mycobacterium fortuitum (0.02) > M. chelonae (0.03) > M. gordonae (0.09) > M. aurum (0.19). For a C.t value (product of the disinfectant concentration and contact time) of 60 mg.min.liter(-1), frequently used in water treatment lines, chlorine disinfection inactivates over 4 log units of M. gordonae and 1.5 log units of M. fortuitum or M. chelonae. C.t values determined under similar conditions show that even the most susceptible species, M. aurum and M. gordonae, are 100 and 330 times more resistant to chlorine than Escherichia coli. We also investigated the effects of different parameters (medium, pH, and temperature) on chlorine disinfection in a chlorine-resistant M. gordonae model. Our experimental results follow the Arrhenius equation, allowing the inactivation rate to be predicted at different temperatures. Our results show that M. gordonae is more resistant to chlorine in low-nutrient media, such as those encountered in water, and that an increase in temperature (from 4 degrees C to 25 degrees C) and a decrease in pH result in better inactivation.     

A direct viable count method for the enumeration of attached bacteria and assessment of biofilm disinfection

This report describes the adaptation of an in situ direct viable count (in situ DVC) method in biofilm disinfection studies. The results obtained with this technique were compared to two other enumeration methods, the plate count (PC) and conventional direct viable count (c-DVC). An environmental isolate (Klebsiella pneumoniae Kp1) was used to form biofilms on stainless steel coupons in a stirred batch reactor. The in situ DVC method was applied to directly assess the viability of bacteria in biofilms without disturbing the integrity of the interfacial community. As additional advantages, the results were observed after 4 h instead of the 24 h incubation time required for colony formation and total cell numbers that remained on the substratum were enumerated. Chlorine and monochloramine were used to determine the susceptibilities of attached and planktonic bacteria to disinfection treatment using this novel analytical approach. The planktonic cells in the reactor showed no significant change in susceptibility to disinfectants during the period of biofilm formation. In addition, the attached cells did not reveal any more resistance to disinfection than planktonic cells. The disinfection studies of young biofilms indicated that 0.25 mg/l free chlorine (at pH 7.2) and 1 mg/l monochloramine (at pH 9.0) have comparable disinfection efficiencies at 25 degrees C. Although being a weaker disinfectant, monochloramine was more effective in removing attached bacteria from the substratum than free chlorine. The in situ DVC method always showed at least one log higher viable cell densities than the PC method, suggesting that the in situ DVC method is more efficient in the enumeration of biofilm bacteria. The results also indicated that the in situ DVC method can provide more accurate information regarding the cell numbers and viability of bacteria within biofilms following disinfection.     

Note: susceptibility to chlorine of Aeromonas hydrophila strains

The susceptibility of five Aeromonas hydrophila strains and one Escherichia coli strain to chlorine was studied under carefully controlled laboratory conditions. Of the Aer. hydrophila strains, two were from untreated water, two from tap water (immediately downstream of a water treatment plant) and one from the DSM collection. The study included disinfectant concentration (0.1, 0.2 and 0.5 mg l-1), pH (6, 7 and 8) and temperature (4, 21 and 32 degrees C) as controlled variables. The results indicated that the untreated water strains, the DSM strain and the E. coli strain were inactivated within 1 min of chlorine treatment. The strains from chlorinated water (TW11 and TW27) showed a different susceptibility to chlorine disinfection, the rate of inactivation being greater at pH6 than at pH8 for both strains. Under the standard conditions of temperature 21 degrees C, pH7 and chlorine concentration 0.2 mg l-1, an increase or decrease of approximately 1 log unit in the number of bacteria did not affect the kill rate of the strains TW11 and TW27.     

Inactivation of bacteria by Purogene

The bacteriocidal efficacy of Purogene, a stabilized aqueous solution of chlorine dioxide (ClO₂) was examined using bacteria of concern to public health. The organisms tested were: Escherichia coli, Pseudomonas aeruginosa, Yersinia enterocolitica, Klebsiella pneumoniae, Streptococcus pyogenes Group A, Salmonella typhimurium and Bacillus subtilis . The test organisms responded differently to inactivation by Purogene. At least a 4 log reduction in bacterial counts was noted when Purogene was applied at a concentration of 0.75 mg/l. Since Purogene is a stabilized complex, it was necessary to provide a chemical environment suitable for the release of ClO₂ in this solution. This was done by varying the pH of Purogene from 3.5 to 8.6 (pH of Purogene is 8.6) while keeping the pH of the experimental medium constant (pH 7.0). The results showed that Purogene was most efficacious at the lowest pH tested (pH 3.5). This indicates that as chlorine dioxide solutions were reduced to chlorite (which predominates at pH 8.6), their bacteriocidal efficacy was reduced, suggesting free chlorine dioxide as the active disinfecting species.