Znd. Eng. Chem. Res. 1995,34, 4106-4109
4106
Disinfection of Water by N-Halamine Biocidal Polymers Gang Sun, Leslie C. Allen, E. Paige Luckie, Walter B. Wheatley, and S. Davis Worley* Department of Chemistry, Auburn University, Auburn, Alabama 36849
Two novel polymers have been tested in a filter application for biocidal efficacy for flowing aqueous solutions of microorganisms. A laboratory water pump was employed in the experiments to provide contact times on the order of 1-2 s/mL. The two polymers are N-chlorinated and N-brominated derivatives of a polystyrene hydantoin. The variables of pH, temperature, flow rate, and dilution with inert sand have been examined. The N-bromamine polymer (Poly-IB) was generally more effective than the N-chloramine polymer (Poly-I), probably because N-bromamines and free bromine are more biocidal under a given set of conditions than are their N-chloramine and free chlorine analogs. Once the biocidal efficacies of the polymers were exhausted due to chemical reaction with microorganisms or organic load, they could be regenerated by exposure to circulated free chlorine or bromine. This demonstrated that the polymer. materials were not permanently damaged by the exhaustion process.
A recent paper in this journal described the preparation and preliminary test results for a novel N-chloramine biocidal polymer, poly(l,3-dichloro-5-methyl-5-(4'vinylpheny1)hydantoin)(Poly-I),which was insoluble in water, and since it showed biocidal efficacy, it was suggested that it possessed potential as a biocidal water filter (Sun et al., 1994). The polymer was synthesized by performing a Friedel-Crafts acylation reaction on commercial polystyrene using acetyl chloride in the presence of aluminum chloride in carbon disulfide solvent to form poly(4-vinylacetophenone), followed by conversion to poly(5-methyld-(4'-vinylphenyl)hydantoin) using potassium cyanide and ammonium carbonate in acetamide solvent at 150 "C and 10 atm and then chlorination in an aqueous basic suspension to produce Poly-I. Complete details concerning the synthesis of Poly-I have been presented recently (Sun et al., 1994, 1995a). It has been demonstrated that Poly-I inactivates a variety of microorganisms in flowing water in gravity-feed experiments, i.e., rather long contact times of ca. 1 m i d m l (Sun et al., 19941, and it leaches very low quantities of free chlorine (less than 0.5 mg/L) and potentially harmful organic materials such as trihalomethanes (less than 20 pg/L) into the water (Sun et al., 1995b). The current work was performed in order to challenge Poly-I filters with Staphylococcus aureus, Pseudomonas aeruginosa, Escherichia coli, and Candida albicans in flowing water a t short contact times using a laboratory water pump as well as to examine the variables of pH, temperature, and concentration of Poly-I in mixtures with sand. Data will also be presented concerning exhaustion of the polymer filters in the presence of high volumes of bacterial solution and organic load. In addition, the performance in similar experiments of the brominated analog of the N-chlorinated polystyrene hydantoin (Poly-IB) will be addressed. Experimental Methods Samples of Poly-I were prepared in overall 87% yield for the three-step synthesis as described by Sun et al. (1995a). Poly-IB was prepared by an analogous procedure in 81% yield; in this case, bromine was slowly
* Author to whom correspondence should be addressed; e-mail address:
[email protected];fax: 334-844-6980.
added to the aqueous basic suspension of the unhalogenated polystyrene hydantoin at 10 "C with isolation of the product by suction filtration. Filter units were constructed from 1.11cm i.d., 1.27 cm 0.d. Pyrex tubing containing a stopcock at one end to control flow rates. The granular solids (particle mesh size 35-60) were loaded into the tubes bounded by sterile glass-wool plugs. Control filters consisted of unhalogenated precursor polystyrene hydantoin polymer of the same particle size. A peristaltic pump (Gelman Sciences, Ann Arbor, MI) was used to pump aqueous solutions of microorganisms of measured colony concentrations through the filter. The tubing connected to the filters had a 6 mm i.d. Before each experiment, demand-free water of an appropriate pH was pumped through the filters until the free halogen concentration in the effluent was less than 0.5 mgL for chlorine and 2.0 mg/L for bromine. Then solutions of the microorganisms containing about lo6 cfu/mL were pumped through the filters with the flow rates carefully measured. Aliquots from the effluent were quenched with 0.02 N sodium thiosulfate (to terminate any disinfection by free halogen) and plated out on Tryptic Soy Agar. The plates were incubated a t 37 "C for 24-48 h and examined for viable colonies. The detailed procedures used for microbiological analyses in these laboratories have been documented (Williams et al., 1987). Samples removed from the inlet t o the filters were used to measure the concentration of the microorganisms before disinfection. After each experiment, all tubing was disinfected with free chlorine or bromine, and the polymer filters were exposed to aqueous solutions of free chlorine or free bromine to regenerate any halogen lost by the materials. This was accomplished by circulating 500 mgL free chlorine (bleach solution) or 0.01 N bromine water through the filters for 30 min. The experiments were generally performed at least in duplicate and in some cases in triplicate. For the Poly-I experiments, 6.3 g of material was used in the filters, which provided a column length of 15.2 cm; for Poly-IB, the sample weight was 2.3 g, which provided a column length of 5.1 cm. In some experiments, sterilized sea sand was either mixed with the biocidal polymers or maintained in a layer above o r below the polymers. The column filters were held in vertical position with the direction of flow being from bottom to top. Temperatures of the solutions pumped
0888-588519512634-4106$09.00/0 0 1995 American Chemical Society
Ind. Eng. Chem. Res., Vol. 34, No. 11, 1995 4107 Table 1. Biocidal Effects of Poly-I and Poly-IB against Four Microorganisms in 1-L Samples of Demand-Free Water at DH 7.0 and 22 "C av concn of av concn of organism organism av flow a t inlet a t exit time for (cfdmL) (cfdmL) organism polymer 100 mL (s) I 4.9 x 106 0 S. aureus 71 I 71 P. aeruginosa 3.3 x 106 0 3.1 x lo8 70 E. coli I 0 82 C. albicans 3.3 x 106 I 0 131 1.5 x lo6 IB 0 S. aureus P. aeruginosa 6.1 105 0 72 IB 1.2 x 106 0 110 IB E. coli 1.1 x 106 C. albicans IB 0 79
through the filters were controlled by maintaining the solution reservoir at a given temperature by immersion in a temperature-controlled water bath. In general, 1 L of solution was pumped through the columns for each run. The microorganisms employed in this study were purchased from the American Type Culture Collection (Rockville, MD) (S. aureus ATCC 6538, P. aeruginosa ATCC 27853, E. coli ATCC 2666, C. albicans ATCC 44506). The aqueous solutions of organisms were prepared immediately before each experiment. For most experiments, buffered demand-free water a t pH 4.5, 7.0, or 9.5 was used, but for a few experiments a "worst case water" (WCW) containing a 10%by volume aqueous solution of 375 mg/L for each of the salts calcium chloride, magnesium chloride, potassium chloride, and sodium chloride; 50 mg/L Bentonite clay; 30 mg/L humic acid; 0.01%concentration of heat-treated horse serum; and 5 x lo5 celldmL heat-killed Saccharomyces cerevisiae buffered to pH 7.0 was employed.
Results and Discussion Table 1 illustrates the performance of the two polymer filters against the four microorganisms used in this study; the inoculated solutions were prepared from demand-free water buffered to pH 7.0 and held a t a temperature of 22 "C. As can be seen, both of the polymers completely inactivated all four of the microorganisms under these conditions, the inactivation ranging from 5 to 8 logs depending upon the initial colony concentrations. The contact times ranged from 1.2 to 2.2 s/mL aqueous inoculum. It can be concluded from this data that both polymers function very effectively against a variety of microorganisms in pH 7.0 demand-free water a t ambient temperature. Control experiments in which the solutions were pumped through unhalogenated precursor polymer with the same particle size distribution as Poly-I and Poly-IB indicated no filtration effects, i.e., the colony concentrations were virtually the same a t the inlet and the exit of the column. Table 2 shows the effect of pH variation a t 22 "C for one of the microorganisms (E. coli) in demand-free water. The two polymers functioned effectively a t all three pH's studied. There is no indication that the two polymers are less effective at alkaline pHs, as is the case for free chlorine (Worley and Williams, 1988). Tables 3 and 4 present data concerning temperature variation for E. coli in demand-free water a t pH 7.0. Table 3 shows that Poly-IB was effective at producing a 6-log inactivation at all temperatures evaluated. However, the data in Table 4 indicate that Poly-I was definitely less effective against E. coli as the tempera-
Table 2. Inactivation of E . coli by Poly-I and Poly-IB in 1-L Samples of Demand-Free Water at 22 "C as a Function of DH
pH
4.5 7.0 9.5 4.5 7.0 9.5
polymer I I I IB IB IB
av flow time for 100mL(s) 72 70 78 76 110 106
av concn at inlet (cfdmL) 6.4 x lo6 3.1 x lo8 5.2 x lo6 1.1 x 106 1.2 x 106 3.8 x 106
av concn of E. coli at exit (cfu/mL) 0 0 0 0 0 0
Table 3. Inactivation of E. coli by Poly-IB in 1-L Samples of Demand-Free Water at pH 7.0 as a Function of TemDerature av flow av concn of av concn of temp time for E. coli at inlet E. coli at exit (cfd mL) (cfu/mL) ("0 100 mL ( 8 ) 4 114 4.1 x lo6 0 10 100 2.4 x lo6 0 15 96 4.8 x 106 0 22 110 1.2 x 106 0 37 90 4.3 x 106 0 Table 4. Inactivation of E . coli by Poly-I in Demand-Free Water at pH 7.0 as a Function of Temperature av flow av concn of av vol of inoculum E. coli at inlet in which inactivation temp time for was complete (mL) ("C) 100 mL (s) (cfdmL) 8.0 x lo6 250 4 101 10 73 6.6 x lo6 450 833 15 89 1.0 x 107 3.1 x lo8 1410 22 70 37 83 5.0 x lo6 'lOOOa a Total inactivation was obtained for 1000 mL; larger volumes were not tested.
ture of the water was reduced. At the higher temperatures (22 and 37 "C), the Poly-I still provided a complete inactivation after over 1 L of E . coli was pumped through the column; whereas, a t 4 "C complete inactivation (ca. 7 logs) was only obtained for the first 250 mL of E . coli solution. After 250 mL of bacterial solution had been pumped through the filter a t 4 "C, viable colonies were detected thereafter in the eflluent. We believe that the temperature-dependent behavior of the Poly-I material is a result of lesser amounts of free chlorine hydrolyses from the polymer a t low temperatures. Poly-IB does not appear to be temperature dependent because the free bromine hydrolyses from it will always be more significant than that for free chlorine from Poly-I due to the increased lability of the N-Br bond relative t o the N-C1 bond. Prior work in these laboratories has established the fact that organic N-bromamines are more effective against bacteria than are analogous organic N-chloramines, just as is the case for free bromine as compared to free chlorine (Williams et al., 1988). Thus, the temperature effect was not observed for Poly-IB (Table 3) due to the conditions of the experiment. Undoubtedly Poly-IB is also less effective a t low temperatures than at higher ones because of less hydrolyses of the N-Br functional groups a t low temperatures. Free chlorine and free bromine are more effective against most microorganisms at all temperatures than are combined N-halamine compounds. Experiments were also performed in which the maximum flow rate that could be provided by the laboratory water pump was employed. The inoculum was E . coli at pH 7.0 and a temperature of 22 "C. In the case of Poly-I, the maximum flow rate attainable was 100 mL/
4108 Ind. Eng. Chem. Res., Vol. 34, No. 11, 1995 Table 5. Inactivation of 4.5 x lo6 cWmL of E . coli in Demand-Free Water at pH 7.0 and 22 "Cas a Function of Flow Volume by Poly1 time (min) vol (mL) c f d m l a t exit 0 15 30 45 60 75 90 a
0 1084 2169 3253 4337 5422 6506
0 0 280 680 TNTC" TNTC TNTC
TNTC = too numerous to count.
31 s (contact time of 0.31 s/mL); the value for Poly-IB was 100 mu10 s (contact time of 0.1 s/mL). In both cases, a complete 6-log inactivation of E. coli was obtained for 1 L of inoculum solution. These results indicate that at ambient temperature both of the polymers are effective as biocides even at rapid pumping rates. Attempts were made to evaluate the longevities of the two polymer materials. In the case of Poly-I, large volumes of E. coli (4.5 x lo6 cfdmL) in pH 7.0 demandfree water a t 22 "C were pumped through the filter at an average rate of 100 m u 8 4 s. Samples of effluent were removed and tested for viable bacteria a t 15-min intervals. The data in Table 5 show that complete inactivation occurred up to 15 min, and partial inactivation was observed up until the 45-60-min interval, after which the filter became ineffective. However, the filter again provided complete inactivation following rechlorination, indicating that the chemical reaction occurring between the polymer and the bacteria did not permanently deteriorate the material. It should also be noted that a column of Poly-I generally shows biocidal activity following exhaustion by large doses of bacteria after standing overnight, indicating that the chlorine diffuses slowly from within the granular particles to the particle surfaces where it rechlorinates nitrogen-receptor sites. In the case of Poly-IB, a quantity of 15 L of E. coli (lo6 cfu/mL) in demand-free water at pH 7.0 and 22 "C was pumped through the column over a period of 5 days (3 L during each day) at which time the experiment was terminated. A complete 6-log inactivation was observed throughout the entire experiment. This was indeed impressive as 1.5 x 1O1O cfu ofE. coli were killed during the experiment without rebromination of the column being necessary. In a second type of exhaustion experiment, E . coli at a concentration of 3.6 x lo6 cfdmL in 1L of ''worst case water" (see Experimental Section) at pH 7.0 and 22 "C was pumped through the Poly-IB column at an average flow rate of 100 m u 9 8 s. Complete inactivation in this water containing heavy halogen demand was obtained throughout the experiment. In a similar experiment for Poly-I, a 3-log reduction of E. coli was obtained for 1 L of WCW. However, in this case the WCW solution was 10 times more concentrated with the additives than was the case for the Poly-IB experiment. In any case, following the WCW experiment, the column of Poly-I was rechlorinated, and it then provided a 6-log reduction of E. coli in demand-free water. This shows that Poly-I was not permanently damaged by chemical reaction with the constituents in WCW. Finally, experiments were performed designed to demonstrate the efficacies of the two biocidal polymers in the presence of sand as a diluent. The purpose of these experiments was 2-fold. First, a sancUpolymer mixture should provide a less expensive filter. Second,
the abrasive nature of sand could damage cell walls of microorganisms rendering them less resistant to the N-halamine disinfectants. In fact, it has been demonstrated that sand filtration does weaken the cell walls of Cryptosporidium oocysts, which are normally extremely resistant to disinfectants such as free-chlorine (Parker and Smith, 1993). Two types of experiments were performed for Poly-I. In the first experiment, 7.0 g of Poly-I was mixed homogeneously with varying weights of sterile sand. Complete inactivation of lo6 cfdmL of E. coli in pH 7.0 demand-free water at 22 "C at flow rates in the range of 100 mW120-150 s were obtained for a 1:l mixture of the materials. Partial inactivation was obtained at flow rates of 100 mLJ90100 s for 1:2, 15, and 1:lO dilutions, the degree of inactivation deteriorating as the proportion of sand was increased as expected. In the second experiment, 2.0 g of Poly-I was mixed with 6.0 g of sand in two layers. The E. coli solution was pumped through the sand layer first at an average rate of 100 mu85 s. A complete 6-log inactivation was obtained from an entire 1L of solution. Thus, it would appear that Poly-I functions more effectively when mixed with sand in a layered mode than in a homogeneous mode. A complete inactivation of E. coli (6 logs) was also obtained at a flow rate of 100 mW73 s for 2.3 g of Poly-IB and an equal weight of sand in a layered filter, the direction of flow being through the polymer first. It is evident that the two biocidal polymers can be employed in conjunction with sand filtration; additional work along these lines will soon be performed in these laboratories using solutions of resistant organisms such as Cryptosporidium paruum and Giardia lamblia.
Conclusions Small quantities of Poly-I and Poly-IB are effective at inactivating a variety of microorganisms in flowing water when employed in a filter application. The two materials function satisfactorily in the pH range 4.59.5 and at temperatures between 4 and 37 "C. Poly-IB appears to be more effective than Poly-I, probably due to the fact that bromamines and free bromine are more biocidal than their chlorine counterparts. When the biocidal action of the polymers is exhausted due to excessive exposure to microorganisms or halogen demand, the activity can be regenerated by rechlorination or rebromination using solutions of free chlorine or free bromine, respectively, indicating that the polymer materials are not degraded by chemical reaction with the microorganisms or halogen demand. We remain convinced that these materials are potentially excellent candidates for use in biocidal water filters.
Acknowledgment The authors acknowledge support of this work by the Department of the Interior, US. Geological Survey through the Alabama Water Resources Research Institute at Auburn University. The contents of this publication do not necessarily reflect the views and policies of the Department of the Interior, nor does mention of trade names or commercial products constitute their endorsement by the U.S. Government.
Nomenclature Poly-I = poly(l,3-dichloro-5-methyl-5-(4'-vinylphenyl)hy dantoin)
Ind. Eng. Chem. Res., Vol. 34,No. 11, 1995 4109 Poly-IB = poly(1,3-dibromo-5-rnethyl-5-(4'-vinylphenyl)hydantoin) i.d. = inside diameter 0.d. = outside diameter N = normal (normality) ATCC = American Type Culture Collection WCW = worst case water cfu = colony-forming units TNTC = too numerous to count
Sun, G.; Habercom, M. S.; Wheatley, W. B.; Worley, S. D. Performance of a New Polymeric Water Disinfectant. Water Res. Bull. 199Sb,submitted for publication. Williams, D. E.; Worley, S. D.; Barnela, S. B.; Swango, L. J. Bactericidal Activities of Selected Organic N-Halamines. Appl. Environ. Microbiol. 1987,53, 2082-2089. Williams, D. E.; Elder, E. D.; Worley, S. D. Is Free Halogen Necessary for Disinfection? Appl. Environ. Microbiol. 1988,54, 2583-2585. Worley, S.D.; Williams, D. E. Halamine Water Disinfectants. Crit. Rev. Environ. Contrl. 1988,18, 133-175.
Literature Cited Parker, J. F. W.; Smith, H. V. Destruction of Oocysts of Cryptosporidium parvum by Sand and Chlorine. Water Res. 1993,27, 727-731. Sun, G.; Wheatley, W. B.; Worley, S. D. A New Cyclic N-Halamine Biocidal Polymer. Znd. Eng. Chem. Res. 1994,33,168-170. Sun, G.; C!hen, T. Y.;Sun, W.; Wheatley, W. B.; Worley, S. D. Preparation of Novel Biocidal N-Halamine Polymers. J.Bioact. Compat. Polym. 1995a,10, 135-144.
Received for review April 3, 1995 Accepted July 11, 1995@ IE9502190
Abstract published in Advance ACS Abstracts, September 15, 1995. @
