Chlorination of pure bacterial cultures in aqueous solution

PII: S0043-1354(00)00248-7

Wat. Res. Vol. 35, No. 1, pp. 244±254, 2001 7 2000 Elsevier Science Ltd. All rights reserved Printed in Great Britain 0043-1354/00/$ - see front matter

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CHLORINATION OF PURE BACTERIAL CULTURES IN AQUEOUS SOLUTION CHII SHANGM and ERNEST R. BLATCHLEY III*M School of Civil Engineering, Purdue University, West Lafayette, IN 47907-1284, USA (First received 1 May 1999; accepted in revised form 1 April 2000) AbstractÐThe fate and distribution of chlorine in aqueous solutions containing four pure bacterial cultures was studied. Solutions were subjected to chlorination at di€erent initial free chlorine concentrations. Resulting concentrations of residual chlorine were determined by both DPD/FAS titration and membrane introduction mass spectrometry (MIMS). In all cases, false-positive breakpoint chlorination curves, probably attributable to the formation of chloroorganic-N compounds, were observed by DPD/FAS titration, while little or no inorganic residual chloramine was found by MIMS. Free chlorine was observed in similar quantities by both methods after chlorine demand by bacterial cellular materials in solution was satis®ed. These results indicated the residual chloramines existed in the form of organic chloramines; these compounds are generally recognized as being poor antimicrobial agents. Further investigation con®rmed that the bacterial cells were the source of organic-N compounds. The kinetics of chlorination of pure bacterial suspensions was also studied. The pattern of residual chlorine decay following chlorination of the bacterial suspensions indicated rapid initial free chlorine consumption, followed by slow free chlorine consumption, with trace quantities of inorganic chloramine being formed. 7 2000 Elsevier Science Ltd. All rights reserved Key wordsÐbacteria, chlorine, chloramine, DPD/FAS, MIMS, organic-N

INTRODUCTION

Chlorination is widely practiced as a disinfection process for microbial control in water and wastewater. When properly designed and operated, the process is well-developed, inexpensive, and ecient. However, several drawbacks of chlorine-based disinfection systems have been identi®ed, including the formation of (potentially) hazardous disinfection by-products and the discovery of waterborne microbial pathogens that are relatively resistant to chlorine inactivation. These factors de®ne the risks to be balanced in the design of disinfection systems, and dictate (new) optimization strategies for chlorination (Bryant et al., 1992). As a means of simplifying the interpretation of experimental results, pure bacterial suspensions in high cell density studies have often been used to examine the antibacterial ecacy of disinfectants. These experiments allow the examination of large extents of inactivation under tightly-controlled laboratory conditions so that interference by the complex environment of natural water and wastewater can be avoided (Halaby, 1998; Shang et al., 1996; Kouame and Haas, 1991; Benabdesselam et al., *Author to whom all correspondence should be addressed. Tel.: +1-765-494-0316; fax: +1-765-496-1107; e-mail: [email protected] 244

1987; Haas et al., 1984). Under these conditions, dose-response behavior can be established for microorganism-disinfectant pairs by analyzing the extent of inactivation as a function of the time-integral of disinfectant concentration (intensity) over the period of exposure. In most experiments of this type, a pure bacterial culture, from pure bacterial stock has been inoculated in a growth medium for a given set of incubation conditions. Cells are then separated from the growth medium and resuspended in nutrient-free solution. In this manner, the organic materials of the growth medium, which might interfere or otherwise interact with disinfectants, are separated from the organisms of interest, thereby facilitating analyses of organism:disinfectant interactions. However, solutions containing these ``washed'' bacterial suspensions at high cell density have been found to exert high chlorine demand. Signi®cant consumption of free chlorine and shifts of chlorine to chloramines have been observed when residual chlorine concentrations were de®ned by DPD/FAS, even in the ``washed'' bacterial suspensions (Shang et al., 1996). Therefore, point-wise measurements of disinfectant concentration alone cannot be used for accurate predications of disinfection ecacy due to the complexity of chlorine reaction kinetics. Similar phenomena have been observed by Haas et al. (1984), wherein no quantitative residual free chlorine was measured in chlori-

Aqueous bacterial suspension chlorination

nated Tetrahymena pyriformis (a vegetative protozoan) solution at practical chlorine doses. Therefore, net inactivation by free chlorine, which could not be directly measured, was hypothesized to be represented by the di€erence between total inactivation and inactivation attributable to chloramine exposure, as determined from a pre-chloramination study. However, the form and distribution of chlorine in both studies and the validity of the hypothesis remain unclear. To understand the fate and chemistry of chlorine as a result of contact with bacterial suspensions, it is essential to understand the mechanisms of inactivation of bacteria by chlorination. The mechanisms by which bacteria are inactivated by free and combined chlorine have been studied in the literature (Bryant et al., 1992; Dukan and Touati, 1996). One of the reported mechanisms of inactivation of bacteria by chlorination, wherein cell membrane permeability is altered, can lead to an explanation of the fate and chemistry of chlorine after contact with bacterial suspensions. However, no signi®cant change in Zeta potential and no gross disruption of the cellular envelopes was observed from chlorination of bacterial suspensions at concentrations employed in water and wastewater disinfection (Venkobachar et al., 1977; Jacangelo et al., 1991). Cytoplasmic materials, such as proteins and nucleic acid, were released from bacterial cells after chlorine exposure (Venkobachar et al., 1977; Haas and Engelbrecht, 1980a). Amino acids and nucleic acid bases were also found to be released following chlorination of bacterial suspensions (Benabdesselam et al., 1987). These simple organic nitrogen substances ejected by the bacteria were quickly eliminated by subsequent reactions with excess free chlorine in solution. After satisfying the chlorine demand by the growth medium and bacteria, a small portion of (+1-valent) residual chlorine was observed to have permeated through the cell (Haas and Engelbrecht, 1980b) or to have caused DNA damage (Dukan and Touati, 1996). The causative agents in chlorine-induced DNA damage were hypothesized to be derivatives such as inorganic chloramines, since HOCl did not readily di€use into cells. With respect to residual chlorine analysis, conventional analytical methods for measurement of residual chlorine concentrations represent an important yet often overlooked limitation of disinfection systems. For example, continuous titrators are commonly used to control residual chlorine concentrations in contact chambers, yet these instruments are subject to considerable analytical interference in the residual chlorine measurements they conduct. While conventional analytical methods (usually based on titration) can be used to di€erentiate and quantify the various forms of +1valent chlorine in some simple aquatic matrixes, they are of questionable usefulness as tools for

245

characterization of chlorine residuals in ``realworld'' disinfection applications (White, 1992; Jenson and Johnson, 1990; Scully et al., 1984). In particular, these methods are unable to distinguish organic and inorganic chloramines, yet these forms of residual chlorine are vastly di€erent in terms of their ability to inactivate microorganisms and form disinfection by-products. The role of organic chloramines in the determination of residual chlorine, has been addressed in recent studies by Shang and Blatchley (1999) and Shang et al. (2000). In these studies, a new analytical approach based on membrane introduction mass spectrometry (MIMS) has been used to di€erentiate and quantify inorganic chlorine compounds in the presence of chloroorganic-N interference. By using the MIMS and DPD/FAS titrimetric methods in parallel, it has been possible to study the fate of residual chlorine in aqueous solutions containing model organic-N compounds at environmentallyrelevant concentrations. An ``apparent'' breakpoint chlorination shape curve, which was mainly attributable to the formation of chloroorganic-N compounds, was observed by DPD/FAS titration, while little or no inorganic chloramine was detected by MIMS. The data from these experiments demonstrate the magnitude and importance of chloroorganic-N compound formation, and clearly illustrate a lack of inorganic chloramine formation. In the research described herein, the MIMS and DPD/FAS titrimetric methods were applied in parallel to examine the chlorination of four pure bacterial suspensions. As such, it was possible to examine: (1) the ``breakpoint'' behavior of di€erent bacterial cultures; (2) the sources of organic and inorganic nitrogen compounds; and (3) the kinetics and fate of free chlorine in contact with bacteria suspensions. Additionally, an empirical expression, of the general form proposed by Taras (1950), was applied to time-course residual chlorine concentration data following chlorination of dense bacterial suspensions for purposes of evaluating free chlorine demand dynamics attributable to bacteria: Dt ˆ ktn where Dt is the chlorine demand (mg/l as Cl2)=(chlorine doseÿchlorine residual); t is the chlorine contact time (h); k the constant, equal to chlorine demand after 1 h (mg/l as Cl2); and n the slope of curve in log t vs log D plot. EXPERIMENTAL MATERIALS AND METHODS

General In all cases, solutions were prepared from reagent grade chemicals (sodium hypochlorite, 4%, from Aldrich) or stock solutions. Dilution to target aqueous-phase concentration was accomplished with distilled, deionized water. Free chlorine stock solutions and standards of inorganic mono-, di-, and tri-chloramines for calibration curve

Rod 0.5±1.0 by 1.5±4.0 Soil and aquatic habitats, human infections Infection of burns, wounds, and urinary tracts. Rod 0.4±1.5 by 1.0±6.9 Colon of humans and warm-blooded animals Mostly innocuous, some cause ``tourist diarrhea'' and infections of the digestive and urinary tracts and central nervous system.

6538 Gram-positive, Cocci Faculatively anaerobic, preferably aerobic 15,442 Gram-negative, Respiratory Aerobic 11,229 Gram-negative, Enteric Faculatively anaerobic

Staphylococcus aureus Pseudomonas aeruginosa Escherichia coli

establishment were prepared as described in Shang and Blatchley (1999). Pure culture preparation Four pure cultures of bacteria were selected and subjected to chlorination. Bacteria were selected on the basis of their relevance to aqueous disinfection processes and for purposes of examining organisms with a broad spectrum of characteristics (see Table 1). Bacterial cell pellets (originally from ATCC) were re-grown in 10 ml TryptonYeast-Extract (TYE) broth (which contained 10 g/l trypton, 5 g/l yeast extract, and 5 g/l sodium chloride) for 24 h, then were distributed to cyrogenic vials and stored at 48C as stocks. Fresh bacterial cultures used in these experiments were developed prior to experimentation by adding one loop of a culture stock to 20 ml TYE broth and incubating with aeration at 378C for 24 h. After incubation, the nutrient-rich bacterial solutions were centrifuged at 14,000 g for 10 min to isolate the cell pellet from the broth. The liquid nutrient-rich broth was then removed and the cell pellet was rinsed and re-suspended in 20 ml phosphate bu€er solution (0.01 M, pH 7). This washing procedure was repeated three times to minimize the concentrations of non-cell associated constituents in solution that could react with chlorine. Both TYE broth and phosphate bu€er solution were sterilized at 1208C and 15 psi for 15 min prior to their use. The target number concentration of viable bacteria in suspension after incubation was approximately 108±109 cfu per ml. After ®nal re-suspension, the bacterial suspensions were diluted 50-fold to achieve a ®nal number concentration of approximately 107 bacteria per ml. In the ®ltered bacterial solution experiments, these solutions were ®ltered through a 0.45 mm membrane ®lter. The MIMS method The system was based on a modi®cation of an HP 5892 bench-top GC/MS (Hewlett-Packard Co.) containing an HP 5972A Mass Selective Detector (MSD) that was equipped with electron (70 eV) ionization (EI). Selected ion monitoring (SIM) mode was used for quanti®cation of inorganic chlorine residuals. Details of the con®guration and set-up of the MIMS system, and other operational conditions can be obtained from Shang and Blatchley (1999). Concentrations of inorganic chlorine residuals were determined by comparison of ion abundance measurements with those developed from a series of standard solutions. Inorganic chloramines were directly analyzed while free chlorine was estimated by the di€erence of inorganic monochloramine concentrations before and after sample ammoni®cation (Shang and Blatchley, 1999). Ions at m/z 53, 87, and 119 were selected and monitored for quanti®cation of inorganic mono-, di-, and trichloramine, respectively.

Pathogenesis

Morphology Size, mm Habitat

ATCC] Group Metabolism

Experimental procedures

Species

Table 1. Information of bacterial species used in this study (Shang et al., 1996; Halaby, 1998)

6569 Gram-positive, Cocci Faculatively anaerobic, typically fermentative Spherical Ovoid 0.5±1.5 1.4±1.7 by 1.4±2.1 Humans and warm-blooded animals Intestines of humans and warm blooded animals Cause infections in any part of the body. Subacute bacterial endocarditis, Cause boils, abscesses, pyemia, meningitis, cystitis, and urinary tract infections. gastroenteritis, and food poisoning.

Chii Shang and Ernest R. Blatchley III

Streptococcus faecalis

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Chlorination experiments were carried out in well-mixed glass stoppered ¯asks in the dark containing 200 ml of 0.01 M phosphate bu€er solution. For each experiment of the breakpoint chlorination study, un®ltered or ®ltered bacterial suspensions (200 ml) were chlorinated for 30 min by addition of aliquots of standardized free chlorine (NaOCl) stock solution. The experiments were repeated at initial chlorine concentrations ranging from 1.0 to 5.0 mg/ l (as Cl2). Concentrations of residual chlorine were determined 30 min after chlorine addition by DPD/FAS titration and MIMS. DPD/FAS titration of free chlorine was completed within 1 min of sample collection, as recommended by Jensen and Johnson (1990), to minimize interference by chloramines. For each experiment of the chlorination demand study, un®ltered bacterial suspensions were chlorinated as

Aqueous bacterial suspension chlorination described above at initial chlorine concentrations of 1.0, 3.0, 5.0 mg/l (as Cl2). The suspensions were continuously pumped to the MIMS system to monitor the concentrations of inorganic chloramines. In this manner, concentrations of residual chloramines were measured in real time throughout the entire 30 min of chlorination. Free chlorine was periodically determined by DPD/FAS titration. RESULTS AND DISCUSSION

Breakpoint study of bacteria chlorination Chlorination of bacterial suspensions was performed at di€erent initial free chlorine concentrations to evaluate the chemical interactions between free chlorine and bacteria. Residual chlorine was measured by DPD/FAS and MIMS after 30 min of exposure to free chlorine. Results of these experiments are summarized in Fig. 1. In all cases, the residual chlorine measured by

247

DPD/FAS indicated the formation of titrable ``chloramines'' responding predominately in the form of dichloramine. However, only relatively small quantities of inorganic chloramines were detected by MIMS. These results suggested falsepositive measurements of residual chlorine concentration by DPD/FAS titration. These ®ndings are consistent with the results of previous work with model organic-N compounds wherein chlorination of aqueous solutions of amino acids and nucleic acid bases yielded ``apparent'' chloramines by DPD/FAS titration, with little or no chloramine formation being found in the MIMS analyses (Shang et al., 2000). The ``apparent'' chloramines measured by DPD/FAS titration were presumed to be predominately attributable to the formation of chloroorganic-N compounds that displayed the same behavior as inorganic chloramines in DPD/ FAS titration (i.e., the ability to oxidize Iÿ). Since

Fig. 1. Residual chlorine measurements of bacterial suspensions after exposure to free chlorine at various concentrations for 30 min at pH=7.0 by DPD/FAS titration (left bar) and MIMS (right bar).

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Chii Shang and Ernest R. Blatchley III

chlorinated organic-N compounds are believed to be essentially non-microbiocidal and only small quantities of inorganic chloramines were present in solution, it could be concluded that the vast majority of microbial inactivation was attributable to residual free chlorine, even though substantial quantities of (organic) combined chlorine were implied by the DPD/FAS data. These ®ndings are in disagreement with those of Haas et al. (1984), wherein it was conjectured that both free chlorine and combined chlorine were actively involved in chlorination of bacterial suspensions under similar conditions. The conclusions of the work by Haas et al. (1984) were based on residual chlorine analyses by forward amperometric titrationÐunlike MIMS, this method cannot di€erentiate organic and inorganic chloramines. For the purpose of discussion, the term ``breakpoint'' will be de®ned as the initial free chlorine concentration above which free residual chlorine is measured in solution. At initial chlorine concentrations below the breakpoint, it is assumed that all residual free chlorine is ``consumed'' through substitution or oxidation/reduction reactions. In all cases investigated herein, the ``breakpoints'' of these bacterial suspensions were found to be similar. At very low chlorine addition (1 mg/l as Cl2), free chlorine reactions yielded predominately organic chloramines; these compounds responded as a mixture of ``apparent'' mono- and dichloramine in the DPD/ FAS titrations. In all suspensions except Streptococcus faecalis, small quantities of NH2Cl were also detected by MIMS. At free chlorine addition of 2 mg/l (as Cl2) and higher, free residual chlorine was observed while ``apparent'' dichloramine, believed to be attributable to chloroorganic-N compounds, was predominately observed in DPD/FAS analysis. The quantities of the ``apparent'' dichloramine tended to level o€ at free chlorine additions of 3.0 mg/l (as Cl2) and higher. These observations are believed to be attributable to a limit of organic-N sources in bacterial solution that completely reacted with chlorine within 30 min under the reaction conditions used in this research. Free chlorine measurements by DPD/FAS and MIMS were virtually identical in all samples. These ®ndings suggest that interference of free chlorine determination by chloroorganic-N compounds was negligible in either method. Figure 1 illustrates some variation in chlorine consumption among the four bacterial suspensions. Escherichia coli and Pseudomonas aeruginosa, both of which are gram-negative bacteria, exerted a somewhat smaller chlorine demand and lower formation of ``apparent'' chloramines in DPD/FAS data than S. faecalis and Staphylococcus aureus, which are gram-positive bacteria. Though many factors could contribute to this behavior, it is conjectured that the bacterial cell wall structure may have played an important role. Gram-negative bacteria

are characterized by a thin peptidoglycan cell wall and an outer lipopolysaccharide-protein membrane layer, which provides an extra barrier to the outer environment, whereas gram-positive bacteria have only a peptidoglycan cell wall (no outer membrane) (Madigan et al., 1997). As such, when chlorine was contacted with bacterial cells, the gram-positive bacteria would be expected to be more vulnerable to alterations in membrane permeability and subsequent release of intracellular materials to the solution; these released constituents would be expected to react rapidly with chlorine, thereby resulting in chlorine demand. Additionally, the cross-linkage in the peptidoglycan cell wall of gram-positive bacteria is usually by a peptide interbridge, while that of gram-negative bacteria is mainly by direct linkage. As such, this cross-linkage provides more amino acid sources in gram-positive bacteria than in gramnegative bacteria. To investigate whether the source of reactive nitrogen was cellular materials or residual dissolved constituents in solution after the washing-procedure, washed bacterial suspensions were ®ltered through a 0.45 mm membrane; the ®ltrates (now free of bacterial cells and debris) were then subjected to chlorination in the same manner as the un®ltered solutions. Figure 2 presents the residual chlorine analyses by both DPD/FAS titration and MIMS for E. coli ®ltrate solutions after 30 min of exposure to free chlorine for initial free chlorine additions of 1±5 mg/l as Cl2. The results indicated that relatively small quantities of free chlorine were consumed; likewise, only small quantities of residual chloramines were detected after 30 min of chlorination by the DPD/FAS and MIMS methods. Moreover, the residual chloramine concentrations determined by both methods were largely in agreement. These ®ndings suggested the presence of small quantities of residual nitrogen in the form of ammonia after sample pretreatment. In comparing the DPD/FAS data for the chlorinated E. coli suspensions and their ®ltrates, a signi®cant decrease in ``apparent'' dichloramine is evident. This di€erence is likely attributable to greater formation of chloroorganic-N compounds in the bacterial suspensions than in their corresponding ®ltrates. These data also suggest that the reactive organic-N was largely attributable to the cellular materials of the bacteria. This observation supports the ®ndings of Benabdesselam et al. (1987) and Haas and Engelbrecht (1980a) wherein it was found that chlorination of E. coli suspensions caused changes in the cell membrane, thereby promoting the release of some cellular materials to the solution. These materials further reacted with free chlorine to cause the depletion of free chlorine concentration in solution for chlorine addition equal or greater than the 2-h chlorine demand. The inorganic chloramine concentrations in the MIMS data were essentially identical in the ®ltered

Aqueous bacterial suspension chlorination

and un®ltered E. coli solutions. This ®nding suggests that the precursor to inorganic chloramine formation (ammonia) was intimately associated with the residual in solution itself instead of with bacterial cells. However, the relative distribution of residual chloramines was shifted in the MIMS data between the ®ltered and un®ltered solutions. In particular, the inorganic chloramines in the ®ltrates showed a bias toward higher-chlorinated compounds. This was attributed to the increase of free chlorine residual (higher Cl:NH3 ratio) due to the absence of bacterial cells. Figure 3 presents the results of residual chlorine measurements by DPD/FAS and MIMS 30 min after chlorination of four di€erent bacterial ®ltrates at an initial free chlorine concentration of 2.0 mg/l as Cl2. Similar residual chlorine concentrations were observed in all cases. In particular, the chlorine residuals (as determined by both methods) were predominantly free chlorine, with small quantities (typically 1 15% of total residual reported as Cl2) in the form of chloramines. The fact that the chloramine measurements by both methods were in general agreement suggests that the ``chloramine'' signals were probably attributable to inorganic chloramine compounds (i.e., NH2Cl, NHCl2, and NCl3) rather than compounds that could provide a false positive signal (e.g., organic-N chloramines). Interestingly, the data from these measurements indicate a bias toward trichloramine; NCl3 would be expected to dominate the chlorine residual under

249

conditions of high Cl:NH3 ratio. Collectively, these data imply the presence of a small quantity of exocellular NH3 in the ®ltrate samples. The data from these experiments indicate that cellular constituents represent the primary precursor to chloramine formation following chlorination of bacterial suspensions (as opposed to excreted soluble materials). Bacterial cell membranes are composed of phospholipids and proteins. Phospholipid, which has no amine group, and protein, which has been reported to be relatively resistant to chlorine (Taras, 1950), are not likely to contribute substantially in the formation of ``apparent'' chloramine in 30 min. On the other hand, bacterial cell walls contain amino acid linkages that may provide a source of reactive nitrogen. However, the quantities of amino acids associated with extracellular materials represent only about 10% of the total amino acid content of most bacteria (Madigan et al., 1997). It is likely that intracellular materials, such as amino acids, peptides, and nucleic acids are important sources of nitrogen for these reactions. Cellular carbon, present in both cell membranes and cell walls, is hypothesized to be a target for oxidation by free chlorine, thereby resulting in ``consumption'' of free chlorine; these reactions may cause damage to external cell structures, thereby resulting in release of intracellular materials. Kinetic study of bacterial solution chlorination A study of chlorine decay attributable to con-

Fig. 2. Residual chlorine measurements of Escherichia coli ®ltrates after exposure to free chlorine at various concentrations for 30 min at pH=7.0 by DPD/FAS titration (left bar) and MIMS (right bar).

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Chii Shang and Ernest R. Blatchley III

tact with bacteria was performed to assess the dynamic behavior of residual inorganic chlorine and its possible in¯uence on bacterial inactivation. Previous experiments revealed that free chlorine concentration measurements by DPD/ FAS titration and MIMS were quite similar, thereby suggesting that both methods are accurate and largely free of interference; on the other hand, inorganic chloramine measurements by DPD/FAS are subject to substantial interference by organic chloramines, while MIMS is apparently free of such interference. Therefore, DPD/ FAS titration was used for measurement of residual free chlorine concentration, while the concentrations of inorganic chloramine compounds were measured by MIMS. This analytical protocol allowed frequent sample collection and titration for free chlorine to be conducted in parallel with continuous measurements of inorganic chloramines by MIMS. For each of the four bacterial suspensions, a range of initial free chlorine concentrations (1, 3, and 5 mg/l as Cl2) was applied. Figure 4 presents the results of chlorination of E. coli at initial free chlorine concentrations of 1, 3, and 5 mg/l as Cl2. In all cases, free chlorine demonstrated a pattern of rapid decay (®rst few minutes) followed by an extended period of slow decay. Only small quantities of inorganic chloramines were found, and in all cases the absolute and relative concentrations of these compounds were quite similar. These data indicate that only a small fraction of the free chlorine ``decay'' could be

attributed to the formation of inorganic chloramines. As such, the majority of the free chlorine decay was attributable to the oxidation-reduction and substitution reactions between chlorine and cellular materials. As an indication of the relative importance of oxidation-reduction reactions, measurements of Clÿ concentration were conducted. The concentrations of chloride ion were measured by the argentometric method (APHA, AWWA, WEF, 1995) at 1, 4, 8, 13, and 30 min after the addition of free chlorine. Virtually no change in chloride ion concentration in solution was observed after the ®rst minute of reaction time. These data indicate that the oxidation-reduction reactions involving +1-valent chlorine took place within the ®rst minute for chlorination, thereby resulting in a substantial drop in free chlorine concentration. Subsequently, the decay of free chlorine by bacteria in suspension was attributable to substitution reactions between free chlorine and bacterial cellular components. The constraints of the argentometric method prevent its practical and reliable application for contact times of less than 1 min. It is likely that the reactions between chlorine and carbon (or other easily oxidized constituents) in the extracellular materials were responsible for reduction of free chlorine to chloride. Variation in chloride formation was observed for di€erent initial free chlorine concentrations. This ®nding suggests the formation of Clÿ can be attributed to chlorine addition (Fig. 4a and b). Nevertheless, the di€erence becomes less substantial at higher concentrations of chlorine ad-

Fig. 3. Residual chlorine measurements of bacterial ®ltrates after exposure to free chlorine at 2 mg/l (as Cl2) for 30 min at pH=7.0 by DPD/FAS titration (left bar) and MIMS (right bar).

Aqueous bacterial suspension chlorination

Fig. 4. Residual chlorine and chloride ion measurements for kinetic study of chlorination of Escherichia coli suspensions at: a, 1.0 mg/l; b, 3.0 mg/l; c, 5.0 mg/l as Cl2.

251

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Chii Shang and Ernest R. Blatchley III

dition (Fig. 4b and c), thereby indicating an approach to the maximum Clÿ formation due to the limitation of oxidizable constituents in the bacterial suspension. Inorganic chloramine dynamics resulting from chlorination of other bacterial suspensions were quite similar to the data illustrated in Fig. 4 (not shown); therefore, the e€ect of inorganic chloramine on inactivation could be assumed negligible for samples that have been well pretreated (washed). The dynamic behavior of free residual chlorine upon chlorination of all four bacterial suspensions is summarized in Fig. 5. Qualitatively similar chlorine decay kinetics were observed in all cases. At low initial chlorine concentration (ca. 1 mg/l as Cl2), free chlorine was completely consumed by the bacterial cell suspension in less than 10 min. At higher

initial free chlorine concentration, the rapid demand took place in the ®rst few minutes, followed by a slow demand at higher contact time. As described previously, some quantitative di€erences in chlorine decay kinetics were observed among the four bacterial suspensions. The gramnegative bacteria (E. coli and P. aeruginosa ) displayed slower chlorine consumption than the grampositive bacteria (S. aureus and S. faecalis ). These di€erences in chlorine consumption kinetics, which were most evident in the ®rst few minutes following chlorination, provide additional support to the hypothesis that the di€erences in extracellular material (cell membrane and cell walls) between gram(+) and gram(ÿ) bacteria may have accounted for the di€erences in chlorine decay dynamics between the cultures. However, the data provided by the analyti-

Fig. 5. Free chlorine decay kinetics of chlorination of bacterial suspensions at various initial free chlorine additions: * 1.0 mg/l as Cl2; Q 3.0 mg/l as Cl2; R 5.0 mg/l as Cl2.

Aqueous bacterial suspension chlorination

cal method used herein do not provide conclusive proof of this point. To quantitatively describe free chlorine demand in aqueous bacterial suspensions, the method originally proposed by Taras (1950) for evaluation of chlorine demand in wastewater and aqueous solutions of model organic-N compounds was used. In this approach, the chlorine demand (de®ned as CltÿCl0, where Cli=residual free chlorine concentration at t=i ) of an experimental matrix was plotted with time on a log±log basis (see Fig. 6). In contrast to the ®ndings of Taras (1950), Feben and Taras (1951), and Lin and Evans (1974), the data reveal the existence of a bi-linear trend in all cases, with a downward-bending ``elbow'' (discontinuity) being present at a contact time of approximately 5± 7 min. Regression analysis of the log-transformed data was used to estimate the slope (n ) of these bi-

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linear relationships before and after the ``elbow.'' The value of n is an index of the rate of chlorine demand expression, with large values of n being indicative of rapid chlorine decay. In the experiments conducted with initial chlorine concentrations of 1.0 mg/l as Cl2, data at chlorine demand equal to chlorine addition were excluded from the regressions since free chlorine was completely consumed (see Fig. 6). It is hypothesized that this bi-linear behavior was attributable to the existence of two distinct reaction types in the chlorination of bacteria. Before the elbow, relatively rapid decay (n = 0.30±0.40) was observed. This would be consistent with a relatively complex matrix of materials in bacterial suspension with which chlorine could react. It is during this period when the most rapid changes in the exterior components of the bacterial cell (e.g., cell wall)

Fig. 6. Free chlorine demand kinetics of chlorination of bacterial suspensions at various initial free chlorine additions: * 1.0 mg/l as Cl2; Q 3.0 mg/l as Cl2; R 5.0 mg/l as Cl2.

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Chii Shang and Ernest R. Blatchley III

would be expected to take place. Therefore, it is reasonable to expect intracellular materials, such as nucleic acids and amino acids, to be released from bacterial cells during this time (Benabdesselam et al., 1987; Haas and Engelbrecht, 1980a). These compounds have been shown to react rapidly with free chlorine and would be expected to be ``consumed'' rapidly. In the second phase, a slower decay rate (n = 0.10±0.16) was observed. This lower rate of demand expression implied the existence of a less-reactive matrix of materials in bacterial suspensions after the ``elbow''. It is also possible that ``demand'' in the second phase was attributable to di€usion of chlorine into the bacterial cells, a process that would also be expected to be quite slow. The ``pre-elbow'' and ``post-elbow'' slopes were quite similar in all cases, regardless of initial chlorine concentration or the bacterial species in suspension. These data suggest similarity among the bacterial suspensions in terms of the processes that were responsible for free chlorine decay. CONCLUSIONS

The consumption of free chlorine and the possible formation of chlorinated organic-N compounds, as indicated by the presence of ``apparent'' chloramines, were observed from chlorination of pure bacterial suspensions. The reactive organic-N was found to be intimately associated with the bacterial cells, and not present in solution. Small quantities of inorganic chloramine were also observed upon chlorination of washed bacterial suspensions and were attributed to trace quantities of residual ammonia in aqueous solution after the washing procedure. Similar patterns of residual chlorine distribution were found in the chlorine-decay kinetic portion of the study. Small di€erences in chlorine decay kinetics among gram(+) and gram(ÿ) bacterial suspensions were attributed to di€erences in their respective cell wall structures. The expression of chlorine demand was demonstrated to follow a consistent bi-linear (log±log) behavior, irrespective of initial chlorine concentration or the speci®c bacterium. As such, these data suggested the existence of two general types of chlorine demand expression by bacterial cells. REFERENCES

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