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UV treatments
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Water Research 39 (2005) 1519–1526 www.elsevier.com/locate/watres
Inactivation of enteric microorganisms with chemical disinfectants, UV irradiation and combined chemical/UV treatments J. Koivunen, H. Heinonen-Tanski Department of Environmental Sciences, University of Kuopio, POB 1627, FIN-70211 Kuopio, Finland Received 31 August 2004; received in revised form 3 December 2004; accepted 25 January 2005 Available online 23 March 2005
Abstract The relative disinfection efficiencies of peracetic acid (PAA), hydrogen peroxide (H2O2) and sodium hypochlorite (NaOCl) against Escherichia coli, Enterococcus faecalis, Salmonella enteritidis and coliphage MS2 virus were studied in laboratory-scale experiments. This study also evaluated the efficiency of combined PAA/ultraviolet irradiation (UV) and H2O2/UV treatments to determine if the microbial inactivation was synergistic. Microbial cultures were added into a synthetic wastewater-like test medium and treated by chemical disinfectants with a 10 min contact time, UV irradiation or the combination of chemical and UV treatments. A peracetic acid dose of 3 mg/l resulted in approximately 2–3 log enteric bacterial reductions, whereas 7–15 mg/l PAA was needed to achieve 1–1.5 log coliphage MS2 reductions. Doses of 3–150 mg/l hydrogen peroxide achieved below 0.2 log microbial reductions. Sodium hypochlorite treatments caused 0.3–1 log microbial reductions at an 18 mg/l chlorine dose, while 2.6 log reductions of E. faecalis were achieved at a 12 mg/l chlorine dose. The results indicate that PAA could represent a good alternative to chlorine compounds in disinfection procedures, especially in wastewaters containing easily oxidizable organic matter. Hydrogen peroxide is not an efficient disinfectant against enteric microorganisms in wastewaters. The combined PAA/ UV disinfection showed increased disinfection efficiency and synergistic benefits with all the enteric bacteria tested but lower synergies for the coliphage MS2. This suggests that this method could improve the efficiency and reliability of disinfection in wastewater treatment plants. The combined H2O2/UV disinfection only slightly influenced the microbial reductions compared to UV treatments and showed some antagonism and no synergies. r 2005 Elsevier Ltd. All rights reserved. Keywords: Disinfection; Synergy; Peracetic acid; Hydrogen peroxide; Sodium hypochlorite; Ultraviolet irradiation
1. Introduction Municipal wastewaters typically contain pathogenic enteric bacteria, viruses and intestinal parasites. Although primary and secondary wastewater treatment Corresponding author. Tel.: +358 17 163164; fax: +358 17 163191. E-mail address: jari.koivunen@uku.fi (J. Koivunen).
processes eliminate 90–99.9% of enteric microorganisms and tertiary treatment, such as filtration, may further reduce 90–99% of these organisms, purified wastewaters can still contain high microbial numbers (Koivunen et al., 2003; Rajala et al., 2003). If a more efficient elimination of microorganisms is needed, disinfection of wastewater must be done. Chlorination is the traditional and most common wastewater disinfection method used around the world.
0043-1354/$ - see front matter r 2005 Elsevier Ltd. All rights reserved. doi:10.1016/j.watres.2005.01.021
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It is a quite efficient disinfectant against many enteric bacteria, but it has lower efficiency against viruses, bacterial spores and protozoan cysts (Tyrrell et al., 1995; Veschetti et al., 2003). The use of chlorination has been decreasing, mainly due to toxic, mutagenic and/or carcinogenic disinfection by-products (DBPs) and chlorine residuals formed in the disinfection process (Oppenheimer et al., 1997; Veschetti et al., 2003). Peracetic acid (PAA), CH3COOOH, is the peroxide of acetic acid. PAA has shown good disinfection efficiency against enteric bacteria in wastewaters, but viruses, bacterial spores and protozoan cysts are more resistant (Liberti and Notarnicola, 1999; Stampi et al., 2001, 2002; Salgot et al., 2002; Wagner et al., 2002; Veschetti et al., 2003). PAA disinfection produces no or only low amounts of DBPs into effluents, which is one of its main benefits (Booth and Lester, 1995; Liberti and Notarnicola, 1999; Collivignarelli et al., 2000; Monarca et al., 2000; Veschetti et al., 2003). In addition, disinfectant residues are readily decomposed into harmless compounds, acetic acid and oxygen. One of the components of peracetic acid products is hydrogen peroxide, which may also contribute to the disinfection action and bacteriostatic effects of PAA product (Liberti and Notarnicola, 1999). Hydrogen peroxide has not been widely used as a sole disinfectant for water or wastewater treatment, mainly due to its slow disinfection action and low disinfection efficiency (Liberti et al., 2000; Wagner et al., 2002). UV irradiation is an important physical procedure for water and wastewater disinfection; the number of plants using UV disinfection applications has been increasing in recent years. UV disinfection typically eliminates efficiently enteric bacteria, viruses, bacterial spores and parasite cysts, without producing DBPs or other chemical residues (Oppenheimer et al., 1997; Lazarova et al., 1998; Liberti and Notarnicola, 1999; Collivignarelli et al., 2000; Liberti et al., 2000; Rajala et al., 2003). The disadvantage of this disinfection method is its lack of bacteriostatic effect and possibility for photoreactivation or dark repair of UV-damaged microorganisms, enabling regrowth of the microbial population under certain conditions (Baron and Bourbigot, 1996; Lazarova et al., 1998). The efficiency and reliability of UV disinfection is greatly dependent on water quality, placing large demands on the upstream treatment processes (Salgot et al., 2002). Recently, advanced oxidation processes (AOPs) for wastewater disinfection have been introduced (RajalaMustonen et al., 1997; Lubello et al., 2002; Caretti and Lubello, 2003). AOPs are based on the utilization of secondary oxidants, such as free hydroxyl radicals (dOH), which are typically generated by the interaction of UV irradiation with a chemical disinfectant capable of releasing radicals. Hydroxyl radicals are considered as
the most reactive oxidizing agents in water treatment and they can be used for the oxidation of organic and inorganic compounds or for disinfection purposes. Rajala-Mustonen et al. (1997) observed increased coliphage inactivation with combined PAA/UV treatment of secondary wastewater effluents. Lubello et al. (2002) and Caretti and Lubello (2003) have reported increased disinfection efficiency and a synergistic effect of combined PAA/UV treatment of wastewaters, while H2O2/UV treatments showed only slight synergistic benefits. Synergy means that the efficiency of combined disinfection method is greater than the efficiency achieved when summing the effects of individual disinfectants. In this study we investigated the relative disinfection efficiencies of peracetic acid, hydrogen peroxide and sodium hypochlorite treatments against E. coli, Enterococcus faecalis, Salmonella enteritidis and coliphage MS2 viruses in laboratory-scale experiments. We also investigated if simultaneous UV irradiation could increase the efficiency of PAA or H2O2 treatments through increased radical formation, and assessed the magnitude of any synergistic effects. While previous studies on combined disinfection treatments have focused on removal of bacteria, we have also investigated the effect of these treatments on viruses.
2. Materials and methods 2.1. Chemical disinfectants and UV source The chemical disinfectants used were peracetic acid (PAA equilibrium mixture: 15% PAA, 13–16% H2O2, 21–26% acetic acid, Kemirox peracetic acid 10–20%, Kemira Oyj, Kemwater), hydrogen peroxide (H2O2, Hydrogen peroxide 50%, Kemira Oyj, Kemwater) and sodium hypochlorite (NaOCl, 1 N, Merck KGaA, Darmstadt, Germany). UV disinfection treatments were carried out with a collimator device (Blatchley III 1996) and a low pressure mercury arc lamp (Osram HNS 30 W, light intensity 90 mW/cm2, l ¼ 253:7 nm) was used as an UV source. The UV intensity on the sample surface (0.22 mW/cm2) was measured by a Macam SR9910 spectroradiometer.
2.2. Test medium Peptone water (PW) was used as a test medium. It was prepared by dissolving 0.15 g proteose peptone (Difco, Detroit, MI, USA) into one liter of deionised water, adjusting the pH to 7 and sterilizing by autoclaving.
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2.3. Microbial strains and preparation of experimental solution Culture collection strains of microorganisms were used: E. coli (ATCC 15597), Enterococcus faecalis (ATCC 19433, obtained from the Hambi collection of University of Helsinki), a pathogenic bacterium Salmonella enteritidis (B678/95/1, obtained from the National Veterinary and Food Research Institute) and coliphage MS2 virus (ATCC 15597-B1). Bacterial cultures for disinfection experiments were prepared before every set of experiments by transferring bacterial stock culture into 100 ml of tryptone-yeast extract-glucose-broth (TYG) and incubating for 24 h at 37 1C. Coliphage MS2 culture was produced by adding coliphage stock solution into exponentially growing (2 h old) E. coli ATCC 15597 pure culture growing in a TYG-broth at 37 1C. After 5 h incubation, the microbial culture was centrifuged to remove host bacteria and the phage-count of the supernatant solution was determined as described below. The phage solution was stored at 4 1C and the same solution was used throughout the study. Prior to the disinfection treatments, prepared microbial culture was added into PW to yield the microbial numbers of approximately 105–107 CFU or PFU/ml. Then turbidity (Hach Ratio X/R Turbidimeter), UV-absorbance at 253.7 nm wavelength (Shimadzu UV-1201 UV-Vis Spectrophotometer) and pH (Knick Portamess 751 pH-meter) values of the test medium were measured (Table 1). The CODCr (chemical oxygen demand) and TOC (total organic carbon) values of PW containing microorganisms were 150 mg O2/l and 64 mg/l, respectively. 2.4. Disinfection experiments A 10 ml volume of PW containing microorganisms was transferred onto a glass Petri dish (diameter 6 cm) and mixing was started with a magnetic stirrer. In the chemical disinfection experiments, the disinfectant chemical (PAA, H2O2 or NaOCl) was added into
Table 1 Average values of some water quality parameters in disinfection experiments by using different microorganisms in peptone water (PW)
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experimental solution and 10 min contact time was measured. Contact time of 10 min was chosen, since preliminary experiments that were carried out, as well as previous studies (Rajala-Mustonen et al., 1997) have shown that the most significant microbial reductions typically occur during the first 10–15 min of contact time. Disinfectant dosages causing approximately 0–3 log microbial reductions (partial elimination of test microorganisms) were selected, to allow assessment of synergies in combined disinfection treatments. At the end of the contact time, the residual concentration of disinfectant in experimental solution was measured by commercially available analytical tests (Peracetic acid test 5–50 mg/l, Merckoquant, Merck; Hydrogen peroxide test 0.5–25 or 1–100 mg/l, Merckoquant, Merck; Chlorine test 0.1–2 mg/l, Aquamerck, Merck). PAA and chlorine residues in the experimental solution were quenched by the addition of sodium thiosulphate, while H2O2 residues were eliminated by catalase (Sigma Catalase). In the UV disinfection experiments, collimator device and low pressure mercury arc lamp were used as an UV source. The applied UV doses (mWs/cm2) in the disinfection treatments were calculated as a product of average UV intensity in the reactor (mW/cm2) and irradiation time (s). As a result of relatively low UV intensity on the sample surface (0.22 mW/cm2), long irradiation times (compared to UV disinfection devices in practical applications) had to be used to achieve appropriate UV doses. In combined PAA/UV and H2O2/UV treatments, the UV irradiation of the experimental solution was always started 30 s after the addition of disinfectant chemical and was stopped by shielding of collimating tube after the desired UV irradiation time. The rest of the experiment followed the methods described above. The disinfectant chemical was added before the UV irradiation to take advantage of possible increased radical formation due to UV action, which could result in synergy benefits. After the disinfection treatments and quenching of disinfectant residues, samples were stored at 4 1C and microbiological analyses were started within 4 h. Four replicate tests were carried out for each disinfection treatment. All the experiments were carried out at 2072 1C. During each set of experiments, two untreated samples were taken to determine the initial microbial numbers.
Microorganism in PW
Turbidity (NTU)
Transmittance 253.7 nm (%)
pH
2.5. Enumeration of microorganisms
E. faecalis E. coli S. enteritidis Coliphage MS2
2.5 0.8 1.3 0.1
60.1 59.1 58.8 69.3
6.6 6.5 6.7 6.7
E. coli, E. faecalis and S. enteritidis were cultured on TYG agar by using a spread-plate technique and incubated at 37 1C for 4875 h. The prolonged incubation was used in order to allow growth of injured bacteria. Coliphage MS2 was determined on phage
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TYG-agar by using a double-agar-layer-method, E. coli ATCC 15597 as the host bacterium and overnight incubation at 37 1C. After the incubation period, bacterial colonies and virus plaques were counted and the results calculated as CFU or PFU/ml.
When the value of synergy is positive, there exists a synergy benefit in combined disinfection treatment, while a negative value represents an antagonistic effect. The value of zero means that the efficiency of combined treatment is the same than the sum of the two individual treatments. Wilcoxon signed ranks test was used to test if the synergy values achieved in combined disinfection treatments were statistically significant.
2.6. Presentation of results The efficiency of disinfection in the different disinfection treatments was assessed by determining microbial reductions (log10 reductions, average and standard deviation) from microbial numbers before and after disinfection treatments. The synergy values of combined chemical/UV disinfection treatments were calculated by the following equation:
3. Results Approximately 2–3 log (99–99.9%) enteric bacterial reductions were achieved by using 3 mg/l PAA dose and 10 min contact time (Table 2). PAA doses of 7–15 mg/l caused approximately 1–1.5 log coliphage MS2 reductions. H2O2 disinfection achieved only less than 0.2 log microbial reductions even at a dose as high as 150 mg/l. Sodium hypochlorite disinfection caused below 1 log E. coli, S. enteritidis and coliphage MS2 reductions with a 18 mg/l chlorine dose, while 2.6 log E. faecalis reductions
Synergy ðlog unitsÞ ¼ log reduction by combined chemical=UV disinfection ðlog reduction by UV disinfection þ log reduction by chemical disinfectionÞ:
Table 2 Log10 reductions (average7standard deviation) of enteric microorganisms with different disinfection treatments in peptone water E. faecalis
E. coli
S. enteritidis
MS2 coliphage
Disinfection treatment
Dose
Log10 reduction Dose
Log10 reduction Dose
Log10 reduction Dose
Log10 reduction
PAA (mg/l)
0.5 1.0 1.5 3.0
0.1170.08 0.9770.17 1.7470.67 3.1270.23
1.0 2.0 3.0
0.0470.05 0.5770.19 2.8170.37
1.0 2.0 3.0
0.0570.08 0.3870.19 1.9370.53
3.0 7.0 15
0.2170.10 0.8670.16 1.2870.33
PAA/UV (mg/l)/(mWs/cm2) 0.5/8.0 1.0/8.0 1.5/8.0 0.5/10 1.0/10 1.5/10
1.0570.18 2.3270.50 4.5670.40 1.9970.27 3.0070.76 4.4070.79
1.0/10 2.0/10 3.0/10 1.0/14 2.0/14 3.0/14
1.0270.39 2.2970.32 5.5670.59 2.4170.43 4.6670.29 5.9770.34
1.0/6.0 2.0/6.0 3.0/6.0 1.0/10 2.0/10 3.0/10
0.9370.04 1.8070.21 3.5670.45 2.5670.09 4.2470.55 6.1670.56
3.0/22 7.0/22 15/22 3.0/38 7.0/38 15/38
1.2170.09 1.7270.06 1.9670.20 1.8170.14 2.3770.18 2.5870.23
H2O2 (mg/l)
0.0270.02 0.0370.04 0.1170.09
ND
ND
ND
ND
3.0 30 150
0.0770.08 0.0270.04 0.0670.10
H2O2/UV (mg/l)/(mWs/cm2) 3.0/8.0 30/8.0 150/8.0 3.0/10 30/10 150/10
0.4370.10 0.3970.05 0.3570.05 1.0070.22 1.1270.06 1.1870.15
ND
ND
ND
ND
3.0/22 30/22 150/22 3.0/38 30/38 150/38
0.9170.12 0.8770.07 1.1670.08 1.4370.19 1.5170.07 1.7470.09
Chlorine (mg/l)
12
2.6970.52
18
0.2870.11
18
0.4470.08
18
1.0370.05
8.0 10
0.6170.20 1.2070.18
10 14
0.5570.11 1.4470.12
6.0 10
0.8770.12 2.6170.35
22 38
0.7970.12 1.4070.15
3.0 30 150
2
UV (mWs/cm )
ND ¼ Not determined, since disinfection experiments by using E. faecalis and MS2 coliphage showed low disinfection efficiency of H2O2 and low synergies in combined H2O2/UV treatments. The results are based on four parallel experiments.
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were achieved with a 12 mg/l chlorine dose. UV doses of 6–18 mWs/cm2 caused approximately 1–3 log enteric bacterial reductions. Coliphage MS2 required UV doses of 22–38 mWs/cm2 to obtain 1–1.5 log reductions. The combination of PAA and UV disinfection improved microbial reductions (Table 2) and synergistic benefits were observed against all the enteric bacteria tested (Table 3). The highest synergy values reached 2 log units, when 1.5–3 mg/l PAA in combination with UV irradiation were used. The increase of UV or PAA dose, when keeping the other one constant, typically increased the disinfection efficiency and synergy values. PAA/UV treatments of coliphage MS2 showed lower synergy values. The combination of H2O2 and UV disinfection only slightly influenced the microbial reductions compared to microbial inactivation in UV treatments (Table 2) and no synergies were observed (Table 3). The synergy values in combined PAA/UV treatments were statistically significant for E. faecalis and E. coli, but not for S. enteritidis and MS2 coliphage (Table 3). The combined H2O2/UV treatments showed statistically significant antagonistic effects for E. faecalis and non-significant synergy for MS2 coliphage. Peracetic acid residues were typically 1–2 mg/l lower than the applied dose and they were present when disinfection treatments were carried out by using X1.5 mg/l PAA doses. Also hydrogen peroxide residues were typically present at the end of the contact time. Chlorine doses of 6–10 mg/l resulted in residual chlorine concentrations below the detection limit (0.1 mg/l), while chlorine doses of 12–18 mg/l caused 0.2–0.3 mg/l residual chlorine concentrations.
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4. Discussion Peracetic acid was demonstrated to be an efficient disinfectant against enteric bacteria. PAA doses of 1.5–3 mg/l and 10 min contact time resulted in approximately 2–3 log E. coli, E. faecalis and S. enteritidis reductions. Similar PAA disinfection efficiencies against enteric bacteria in wastewater have been previously reported (Collivignarelli et al., 2000; Stampi et al., 2001, 2002; Wagner et al., 2002). Coliphage MS2 virus was more resistant than enteric bacteria against PAA disinfection: A dose of 7–15 mg/l PAA was needed to achieve 1–1.5 log reductions. This finding is in agreement with the results of previous studies (Salgot et al., 2002; Veschetti et al., 2003). Viruses are known to exhibit greater resistance also to other disinfection methods. The disinfection efficiency of H2O2 was found to be low, as has been reported also in previous studies (Liberti et al., 2000; Lubello et al., 2002; Wagner et al., 2002). Below 0.2 log microbial reductions were achieved even at the highest tested dose of 150 mg/l H2O2. Thus it can be concluded that the disinfection action of PAA product is mainly due to PAA, the role of hydrogen peroxide being less important. The difference in disinfection efficiencies of PAA and H2O2 may be explained by differences in disinfection mechanisms or by the higher reactivity of PAA. Peracetic acid is organic peroxide and may penetrate more efficiently into microbial cells than the hydrogen peroxide molecule, improving its disinfection properties such as disruption of cell membranes and blockage of enzymatic and transport systems in the microorganisms. Some micro-
Table 3 Average synergy values (log-units) achieved with the combined PAA/UV and H2O2/UV treatments in peptone water Disinfectant and dose Microorganism UV dose
H2O2 H2O2 WSRT PAA PAA PAA PAA PAA PAA PAA WSRT H2O2 0.5 mg/l 1.0 mg/l 1.5 mg/l 2.0 mg/l 3.0 mg/l 7.0 mg/l 15 mg/l p-value 3.0 mg/l 30 mg/l 150 mg/l p-value
E. faecalis UV 8.0 mWs/cm2 0.32 UV 10 mWs/cm2 0.67
0.74 0.82
2.21 1.46
ND
ND
ND
ND
0.031
0.21 0.22
0.25 0.11
0.38 0.13
E. coli UV 10 mWs/cm2 UV 14 mWs/cm2
0.42 0.93
ND
1.16 2.65
2.20 1.73
ND
ND
0.031
ND
ND
ND
0.01 0.11
ND
0.55 1.24
0.76 1.61
ND
ND
0.094
ND
ND
ND
ND
ND
ND
0.21 0.20
0.07 0.11
0.11 0.10
0.344
0.05 0.04
ND
S. enteritidis UV 6.0 mWs/cm2 ND UV 10 mWs/cm2 Coliphage MS2 UV 22 mWs/cm2 UV 38 mWs/cm2
ND
0.06 0.08
0.31 0.28
0.031a
0.063
ND ¼ Not determined; a ¼ antagonistic effect. The results are based on four parallel experiments. Statistical analyses have been done by Wilcoxon signed ranks test (WSRT).
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organisms may also be protected against hydrogen peroxide by their catalase enzyme activity. This enzyme does not protect against PAA, in fact this compound can also inactivate or inhibit catalase activity (Wagner et al., 2002). Inactivation of coliphages may be due to changes or damage to their surface structures, such as the protein coat or the attachment sites needed for infection of host cells. The efficiency of sodium hypochlorite disinfection against enteric microorganisms in PW was lower than that of PAA. Veschetti et al. (2003) have previously reported equal disinfection efficiencies of PAA and hypochlorite in the treatment of secondary wastewater effluents. The differences in results may be explained by differences in the qualities of water that were treated. The synthetic PW used in this study was probably more susceptible to chemical oxidation by disinfectants than some secondary treated wastewaters. In our experiments PAA and H2O2 showed lower reactivity for the water matrix than the chlorine. It suggests that the efficiency of PAA in treatment of lower quality wastewaters, such as secondary effluents containing organic matter, primary treated wastewaters and some industrial process waters, may be better than that of chlorine. This finding is supported by the results obtained by Baldry et al. (1991) and also by our results showing equal disinfection efficiencies of PAA and NaOCl when tap water with a very low organic matter content was used instead of peptone water as the test medium (results not shown). In our experiments, chemical and UV disinfection were combined to achieve synergistic benefits. UV irradiation was always done after the application of PAA or H2O2, to take advantage of increasing radical formation due to interaction of disinfectant chemical and UV. Combined PAA/UV disinfection treatment is more efficient if UV irradiation is done after application of PAA, compared to opposite order of disinfectants (Caretti and Lubello, 2003). In this study the combination of PAA and UV disinfection increased disinfection efficiency and showed synergistic benefits, the highest synergy values reaching 2 log units for enteric bacteria. This means that the microbial inactivation was 2 log units higher than could be expected by summing the microbial reductions achieved by using those two disinfectants separately. An increase in either the UV or the PAA dose increased the synergy values, probably due to increased radical formation. The combination of H2O2 and UV treatments showed no synergies. These findings are in agreement with the results obtained in previous studies (Lubello et al., 2002; Caretti and Lubello, 2003). In this study we found that despite of significant synergies in treatment of enteric bacteria, the combined disinfection treatments of coliphage MS2 did not achieve synergy benefits. It indicates higher resistance of coliphage viruses also against combined
disinfection treatments. Previously their higher resistance against single disinfectants has been described. The synergies achieved in PAA/UV treatments may be due to interaction between PAA and UV, producing reactive and microbicidal radicals due to the photolysis of PAA (Lubello et al., 2002; Caretti and Lubello, 2003). The lack of synergies in H2O2/UV treatments suggests that there are differences in the radical formation potential between PAA and H2O2, the potential and the resulting disinfection efficiency being higher for PAA. The mechanism of synergy could also be explained by a multiple damage mechanism: two different disinfection methods may cause different types of injuries for microorganisms. The principal targets of UV radiation are the nucleic acids, while chemical disinfectants, such as PAA and H2O2, is thought first to attack microbial cell walls, membranes and enzymatic or transport systems. As a result, the microbial repair mechanisms, required to repair minor damage, may become overloaded, leading to their inability to repair the injuries and subsequent death. In the case of single disinfectant, the damage may be smaller and susceptible to repair, which may not be possible in case of two disinfectants causing a greater variety of damages. The higher synergies achieved by using PAA/UV treatment compared to H2O2/UV treatment could thus be related to the greater disinfection efficiency of PAA compared to H2O2. PAA may also inactivate the catalase enzyme which is one mechanism involved in detoxifying free hydroxyl radicals. This would allow free radicals to more efficiently attack and inactivate microbial cells. Hydrogen peroxide may not have such an effect on catalase. The application of PAA/UV disinfection method in wastewater treatment plants could offer some advantages. It could allow lower disinfectant doses or lower contact times and thus allow smaller size of UV units and chemical disinfection basins, decreasing the costs of disinfection. The efficiency and reliability of disinfection in existing UV disinfection units could probably be improved by the addition of low PAA dose before the UV unit. This method could also improve the bacteriostatic effect and decrease the reactivation potential of microorganisms which had undergone the disinfection process. When PAA or UV treatments alone have been used for wastewater disinfection, viable but non-culturable bacteria and bacterial regrowth have been observed (Lazarova et al., 1995). The combination of two disinfection methods could also destroy a wider range of microorganisms than that achieved by a single disinfection method, since some microorganisms are UV resistant, but sensitive for chemical disinfectant, while others behave in an opposite way. It may be more efficient and economical to apply a combination of low chemical and UV doses, instead of a high dose of one disinfectant, to destroy these microorganisms. Further
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research must be conducted to verify these findings in pilot- and full-scale disinfection of wastewaters. Wastewater characteristics, such as suspended solids and other wastewater components, as well as characteristics and physiological state of microorganisms in real wastewaters may affect disinfection efficiency and synergies achievable by combined disinfection methods.
5. Conclusions Peracetic acid is an efficient chemical disinfectant against enteric bacteria. Low PAA doses (1.5–3 mg/l) were needed to achieve 2–3 log bacterial reductions. By using higher PAA doses (7–15 mg/l) coliphage MS2 could be inactivated. Hydrogen peroxide has low disinfection efficiency against enteric microorganisms and may not be a feasible disinfectant for wastewater disinfection. The disinfection action of the PAA product is mainly attributable to peracetic acid, while the effect of hydrogen peroxide seems to be low. The efficiency of sodium hypochlorite disinfection against enteric microorganisms in PW was low. PAA may be a more efficient disinfectant than chlorine for treatment of waters containing easily oxidizable organic matter. The combined PAA/UV disinfection treatments of enteric bacteria achieved significant synergy benefits, while the treatments of coliphage MS2 did not show significant synergies. The combined H2O2/UV disinfection only slightly influenced the microbial reductions compared to UV treatments and produced no synergies. The results clearly indicate higher resistance of coliphage viruses also against combined disinfection treatments.
Acknowledgments The authors wish to thank Kemira Oyj Kemwater for financial support. We also thank Ms Sirpa Martikainen and Mr Matti Pessi for their assistance in laboratory work.
References Baldry, M.G.C., French, M.S., Slater, D., 1991. The activity of peracetic acid on sewage indicator bacteria and viruses. Water Science and Technology 24 (2), 353–357. Baron, J., Bourbigot, M.-M., 1996. Repair of Escherichia coli and Enterococci in sea water after ultraviolet disinfection quantification using diffusion chambers. Water Research 30 (11), 2817–2821.
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Blatchley III, E.R., 1996. Numerical modelling of UV intensity: application to collimated-beam reactors and continuousflow systems. Water Research 31 (9), 2205–2218. Booth, R.A., Lester, J.N., 1995. The potential formation of halogenated by-products during peracetic acid treatment of final sewage effluent. Water Research 29 (7), 1793–1801. Caretti, C., Lubello, C., 2003. Wastewater disinfection with PAA and UV combined treatment: a pilot plant study. Water Research 37, 2365–2371. Collivignarelli, C., Bertanza, G., Pedrazzani, R., 2000. A comparison among different wastewater disinfection systems: experimental results. Environmental Technology 21, 1–16. Koivunen, J., Siitonen, A., Heinonen-Tanski, H., 2003. Elimination of enteric bacteria in biological-chemical wastewater treatment and tertiary filtration units. Water Research 37, 690–698. Lazarova, V., Janex, M., Fiksdal, L., Oberg, C., Barcina, I., Pommepuy, M., 1998. Advanced wastewater disinfection technologies: short and long term efficiency. Water Science and Technology 38 (12), 109–117. Liberti, L., Notarnicola, M., 1999. Advanced treatment and disinfection for municipal wastewater reuse in agriculture. Water Science and Technology 40 (4–5), 235–245. Liberti, L., Lopez, A., Notarnicola, M., Barnea, N., Pedahzur, R., Fattal, B., 2000. Comparison of advanced disinfection methods for municipal wastewater reuse in agriculture. Water Science and Technology 42 (1–2), 215–220. Lubello, C., Caretti, C., Gori, R., 2002. Comparison between PAA/UV and H2O2/UV disinfection for wastewater reuse. Water Science and Technology: Water Supply 2 (1), 205–212. Monarca, S., Feretti, D., Collivignarelli, C., Guzzella, L., Zerbini, I., Bertanza, G., Pedrazzani, R., 2000. The influence of different disinfectants on mutagenicity and toxicity of urban wastewater. Water Research 34 (17), 4261–4269. Oppenheimer, J.A., Jacangelo, J.G., Laıˆ ne´, J-M., Hoagland, J.E., 1997. Testing the equivalency of ultraviolet light and chlorine for disinfection of wastewater to reclamation standards. Water Environment Research 69 (1), 14–24. Rajala, R.L., Pulkkanen, M., Pessi, M., Heinonen-Tanski, H., 2003. Removal of microbes from municipal wastewater effluent by rapid sand filtration and subsequent UV irradiation. Water Science and Technology 47 (3), 157–162. Rajala-Mustonen, R.L., Toivola, P.S., Heinonen-Tanski, H., 1997. Effects of peracetic acid and UV irradiation on the inactivation of coliphages in wastewater. Water Science and Technology 35 (11–12), 237–241. Salgot, M., Folch, M., Huertas, E., Tapias, J., Avellaneda, D., Giro´s, G., Brissaud, F., Verge´s, C., Molina, J., Pigem, J., 2002. Comparison of different advanced disinfection systems for wastewater reclamation. Water Science and Technology: Water Supply 2 (3), 213–218. Stampi, S., De Luca, G., Zanetti, F., 2001. Evaluation of the efficiency of peracetic acid in the disinfection of sewage effluents. Journal of Applied Microbiology 91, 833–838. Stampi, S., De Luca, G., Onorato, M., Ambrogiani, E., Zanetti, F., 2002. Peracetic acid as an alternative wastewater disinfectant to chlorine dioxide. Journal of Applied Microbiology 93, 725–731.
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Tyrrell, S., Ripley, S.P., Watkins, W.D., 1995. Inactivation of bacterial and viral indicators in secondary sewage effluents, using chlorine and ozone. Water Research 29 (11), 2483–2490. Veschetti, E., Cutilli, D., Bonadonna, L., Briancesco, R., Martini, C., Cecchini, G., Anastasi, P., Ottaviani, M., 2003. Pilot-plant comparative study of peracetic acid and
sodium hypochlorite wastewater disinfection. Water Research 37, 78–94. Wagner, M., Brumelis, D., Gehr, R., 2002. Disinfection of wastewater by hydrogen peroxide or peracetic acid: development of procedures for measurement of residual disinfectant and application to a physicochemically treated municipal effluent. Water Environment Research 74 (1), 33–50.
Water Research 39 (2005) 1519–1526 www.elsevier.com/locate/watres
Inactivation of enteric microorganisms with chemical disinfectants, UV irradiation and combined chemical/UV treatments J. Koivunen, H. Heinonen-Tanski Department of Environmental Sciences, University of Kuopio, POB 1627, FIN-70211 Kuopio, Finland Received 31 August 2004; received in revised form 3 December 2004; accepted 25 January 2005 Available online 23 March 2005
Abstract The relative disinfection efficiencies of peracetic acid (PAA), hydrogen peroxide (H2O2) and sodium hypochlorite (NaOCl) against Escherichia coli, Enterococcus faecalis, Salmonella enteritidis and coliphage MS2 virus were studied in laboratory-scale experiments. This study also evaluated the efficiency of combined PAA/ultraviolet irradiation (UV) and H2O2/UV treatments to determine if the microbial inactivation was synergistic. Microbial cultures were added into a synthetic wastewater-like test medium and treated by chemical disinfectants with a 10 min contact time, UV irradiation or the combination of chemical and UV treatments. A peracetic acid dose of 3 mg/l resulted in approximately 2–3 log enteric bacterial reductions, whereas 7–15 mg/l PAA was needed to achieve 1–1.5 log coliphage MS2 reductions. Doses of 3–150 mg/l hydrogen peroxide achieved below 0.2 log microbial reductions. Sodium hypochlorite treatments caused 0.3–1 log microbial reductions at an 18 mg/l chlorine dose, while 2.6 log reductions of E. faecalis were achieved at a 12 mg/l chlorine dose. The results indicate that PAA could represent a good alternative to chlorine compounds in disinfection procedures, especially in wastewaters containing easily oxidizable organic matter. Hydrogen peroxide is not an efficient disinfectant against enteric microorganisms in wastewaters. The combined PAA/ UV disinfection showed increased disinfection efficiency and synergistic benefits with all the enteric bacteria tested but lower synergies for the coliphage MS2. This suggests that this method could improve the efficiency and reliability of disinfection in wastewater treatment plants. The combined H2O2/UV disinfection only slightly influenced the microbial reductions compared to UV treatments and showed some antagonism and no synergies. r 2005 Elsevier Ltd. All rights reserved. Keywords: Disinfection; Synergy; Peracetic acid; Hydrogen peroxide; Sodium hypochlorite; Ultraviolet irradiation
1. Introduction Municipal wastewaters typically contain pathogenic enteric bacteria, viruses and intestinal parasites. Although primary and secondary wastewater treatment Corresponding author. Tel.: +358 17 163164; fax: +358 17 163191. E-mail address: jari.koivunen@uku.fi (J. Koivunen).
processes eliminate 90–99.9% of enteric microorganisms and tertiary treatment, such as filtration, may further reduce 90–99% of these organisms, purified wastewaters can still contain high microbial numbers (Koivunen et al., 2003; Rajala et al., 2003). If a more efficient elimination of microorganisms is needed, disinfection of wastewater must be done. Chlorination is the traditional and most common wastewater disinfection method used around the world.
0043-1354/$ - see front matter r 2005 Elsevier Ltd. All rights reserved. doi:10.1016/j.watres.2005.01.021
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It is a quite efficient disinfectant against many enteric bacteria, but it has lower efficiency against viruses, bacterial spores and protozoan cysts (Tyrrell et al., 1995; Veschetti et al., 2003). The use of chlorination has been decreasing, mainly due to toxic, mutagenic and/or carcinogenic disinfection by-products (DBPs) and chlorine residuals formed in the disinfection process (Oppenheimer et al., 1997; Veschetti et al., 2003). Peracetic acid (PAA), CH3COOOH, is the peroxide of acetic acid. PAA has shown good disinfection efficiency against enteric bacteria in wastewaters, but viruses, bacterial spores and protozoan cysts are more resistant (Liberti and Notarnicola, 1999; Stampi et al., 2001, 2002; Salgot et al., 2002; Wagner et al., 2002; Veschetti et al., 2003). PAA disinfection produces no or only low amounts of DBPs into effluents, which is one of its main benefits (Booth and Lester, 1995; Liberti and Notarnicola, 1999; Collivignarelli et al., 2000; Monarca et al., 2000; Veschetti et al., 2003). In addition, disinfectant residues are readily decomposed into harmless compounds, acetic acid and oxygen. One of the components of peracetic acid products is hydrogen peroxide, which may also contribute to the disinfection action and bacteriostatic effects of PAA product (Liberti and Notarnicola, 1999). Hydrogen peroxide has not been widely used as a sole disinfectant for water or wastewater treatment, mainly due to its slow disinfection action and low disinfection efficiency (Liberti et al., 2000; Wagner et al., 2002). UV irradiation is an important physical procedure for water and wastewater disinfection; the number of plants using UV disinfection applications has been increasing in recent years. UV disinfection typically eliminates efficiently enteric bacteria, viruses, bacterial spores and parasite cysts, without producing DBPs or other chemical residues (Oppenheimer et al., 1997; Lazarova et al., 1998; Liberti and Notarnicola, 1999; Collivignarelli et al., 2000; Liberti et al., 2000; Rajala et al., 2003). The disadvantage of this disinfection method is its lack of bacteriostatic effect and possibility for photoreactivation or dark repair of UV-damaged microorganisms, enabling regrowth of the microbial population under certain conditions (Baron and Bourbigot, 1996; Lazarova et al., 1998). The efficiency and reliability of UV disinfection is greatly dependent on water quality, placing large demands on the upstream treatment processes (Salgot et al., 2002). Recently, advanced oxidation processes (AOPs) for wastewater disinfection have been introduced (RajalaMustonen et al., 1997; Lubello et al., 2002; Caretti and Lubello, 2003). AOPs are based on the utilization of secondary oxidants, such as free hydroxyl radicals (dOH), which are typically generated by the interaction of UV irradiation with a chemical disinfectant capable of releasing radicals. Hydroxyl radicals are considered as
the most reactive oxidizing agents in water treatment and they can be used for the oxidation of organic and inorganic compounds or for disinfection purposes. Rajala-Mustonen et al. (1997) observed increased coliphage inactivation with combined PAA/UV treatment of secondary wastewater effluents. Lubello et al. (2002) and Caretti and Lubello (2003) have reported increased disinfection efficiency and a synergistic effect of combined PAA/UV treatment of wastewaters, while H2O2/UV treatments showed only slight synergistic benefits. Synergy means that the efficiency of combined disinfection method is greater than the efficiency achieved when summing the effects of individual disinfectants. In this study we investigated the relative disinfection efficiencies of peracetic acid, hydrogen peroxide and sodium hypochlorite treatments against E. coli, Enterococcus faecalis, Salmonella enteritidis and coliphage MS2 viruses in laboratory-scale experiments. We also investigated if simultaneous UV irradiation could increase the efficiency of PAA or H2O2 treatments through increased radical formation, and assessed the magnitude of any synergistic effects. While previous studies on combined disinfection treatments have focused on removal of bacteria, we have also investigated the effect of these treatments on viruses.
2. Materials and methods 2.1. Chemical disinfectants and UV source The chemical disinfectants used were peracetic acid (PAA equilibrium mixture: 15% PAA, 13–16% H2O2, 21–26% acetic acid, Kemirox peracetic acid 10–20%, Kemira Oyj, Kemwater), hydrogen peroxide (H2O2, Hydrogen peroxide 50%, Kemira Oyj, Kemwater) and sodium hypochlorite (NaOCl, 1 N, Merck KGaA, Darmstadt, Germany). UV disinfection treatments were carried out with a collimator device (Blatchley III 1996) and a low pressure mercury arc lamp (Osram HNS 30 W, light intensity 90 mW/cm2, l ¼ 253:7 nm) was used as an UV source. The UV intensity on the sample surface (0.22 mW/cm2) was measured by a Macam SR9910 spectroradiometer.
2.2. Test medium Peptone water (PW) was used as a test medium. It was prepared by dissolving 0.15 g proteose peptone (Difco, Detroit, MI, USA) into one liter of deionised water, adjusting the pH to 7 and sterilizing by autoclaving.
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2.3. Microbial strains and preparation of experimental solution Culture collection strains of microorganisms were used: E. coli (ATCC 15597), Enterococcus faecalis (ATCC 19433, obtained from the Hambi collection of University of Helsinki), a pathogenic bacterium Salmonella enteritidis (B678/95/1, obtained from the National Veterinary and Food Research Institute) and coliphage MS2 virus (ATCC 15597-B1). Bacterial cultures for disinfection experiments were prepared before every set of experiments by transferring bacterial stock culture into 100 ml of tryptone-yeast extract-glucose-broth (TYG) and incubating for 24 h at 37 1C. Coliphage MS2 culture was produced by adding coliphage stock solution into exponentially growing (2 h old) E. coli ATCC 15597 pure culture growing in a TYG-broth at 37 1C. After 5 h incubation, the microbial culture was centrifuged to remove host bacteria and the phage-count of the supernatant solution was determined as described below. The phage solution was stored at 4 1C and the same solution was used throughout the study. Prior to the disinfection treatments, prepared microbial culture was added into PW to yield the microbial numbers of approximately 105–107 CFU or PFU/ml. Then turbidity (Hach Ratio X/R Turbidimeter), UV-absorbance at 253.7 nm wavelength (Shimadzu UV-1201 UV-Vis Spectrophotometer) and pH (Knick Portamess 751 pH-meter) values of the test medium were measured (Table 1). The CODCr (chemical oxygen demand) and TOC (total organic carbon) values of PW containing microorganisms were 150 mg O2/l and 64 mg/l, respectively. 2.4. Disinfection experiments A 10 ml volume of PW containing microorganisms was transferred onto a glass Petri dish (diameter 6 cm) and mixing was started with a magnetic stirrer. In the chemical disinfection experiments, the disinfectant chemical (PAA, H2O2 or NaOCl) was added into
Table 1 Average values of some water quality parameters in disinfection experiments by using different microorganisms in peptone water (PW)
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experimental solution and 10 min contact time was measured. Contact time of 10 min was chosen, since preliminary experiments that were carried out, as well as previous studies (Rajala-Mustonen et al., 1997) have shown that the most significant microbial reductions typically occur during the first 10–15 min of contact time. Disinfectant dosages causing approximately 0–3 log microbial reductions (partial elimination of test microorganisms) were selected, to allow assessment of synergies in combined disinfection treatments. At the end of the contact time, the residual concentration of disinfectant in experimental solution was measured by commercially available analytical tests (Peracetic acid test 5–50 mg/l, Merckoquant, Merck; Hydrogen peroxide test 0.5–25 or 1–100 mg/l, Merckoquant, Merck; Chlorine test 0.1–2 mg/l, Aquamerck, Merck). PAA and chlorine residues in the experimental solution were quenched by the addition of sodium thiosulphate, while H2O2 residues were eliminated by catalase (Sigma Catalase). In the UV disinfection experiments, collimator device and low pressure mercury arc lamp were used as an UV source. The applied UV doses (mWs/cm2) in the disinfection treatments were calculated as a product of average UV intensity in the reactor (mW/cm2) and irradiation time (s). As a result of relatively low UV intensity on the sample surface (0.22 mW/cm2), long irradiation times (compared to UV disinfection devices in practical applications) had to be used to achieve appropriate UV doses. In combined PAA/UV and H2O2/UV treatments, the UV irradiation of the experimental solution was always started 30 s after the addition of disinfectant chemical and was stopped by shielding of collimating tube after the desired UV irradiation time. The rest of the experiment followed the methods described above. The disinfectant chemical was added before the UV irradiation to take advantage of possible increased radical formation due to UV action, which could result in synergy benefits. After the disinfection treatments and quenching of disinfectant residues, samples were stored at 4 1C and microbiological analyses were started within 4 h. Four replicate tests were carried out for each disinfection treatment. All the experiments were carried out at 2072 1C. During each set of experiments, two untreated samples were taken to determine the initial microbial numbers.
Microorganism in PW
Turbidity (NTU)
Transmittance 253.7 nm (%)
pH
2.5. Enumeration of microorganisms
E. faecalis E. coli S. enteritidis Coliphage MS2
2.5 0.8 1.3 0.1
60.1 59.1 58.8 69.3
6.6 6.5 6.7 6.7
E. coli, E. faecalis and S. enteritidis were cultured on TYG agar by using a spread-plate technique and incubated at 37 1C for 4875 h. The prolonged incubation was used in order to allow growth of injured bacteria. Coliphage MS2 was determined on phage
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TYG-agar by using a double-agar-layer-method, E. coli ATCC 15597 as the host bacterium and overnight incubation at 37 1C. After the incubation period, bacterial colonies and virus plaques were counted and the results calculated as CFU or PFU/ml.
When the value of synergy is positive, there exists a synergy benefit in combined disinfection treatment, while a negative value represents an antagonistic effect. The value of zero means that the efficiency of combined treatment is the same than the sum of the two individual treatments. Wilcoxon signed ranks test was used to test if the synergy values achieved in combined disinfection treatments were statistically significant.
2.6. Presentation of results The efficiency of disinfection in the different disinfection treatments was assessed by determining microbial reductions (log10 reductions, average and standard deviation) from microbial numbers before and after disinfection treatments. The synergy values of combined chemical/UV disinfection treatments were calculated by the following equation:
3. Results Approximately 2–3 log (99–99.9%) enteric bacterial reductions were achieved by using 3 mg/l PAA dose and 10 min contact time (Table 2). PAA doses of 7–15 mg/l caused approximately 1–1.5 log coliphage MS2 reductions. H2O2 disinfection achieved only less than 0.2 log microbial reductions even at a dose as high as 150 mg/l. Sodium hypochlorite disinfection caused below 1 log E. coli, S. enteritidis and coliphage MS2 reductions with a 18 mg/l chlorine dose, while 2.6 log E. faecalis reductions
Synergy ðlog unitsÞ ¼ log reduction by combined chemical=UV disinfection ðlog reduction by UV disinfection þ log reduction by chemical disinfectionÞ:
Table 2 Log10 reductions (average7standard deviation) of enteric microorganisms with different disinfection treatments in peptone water E. faecalis
E. coli
S. enteritidis
MS2 coliphage
Disinfection treatment
Dose
Log10 reduction Dose
Log10 reduction Dose
Log10 reduction Dose
Log10 reduction
PAA (mg/l)
0.5 1.0 1.5 3.0
0.1170.08 0.9770.17 1.7470.67 3.1270.23
1.0 2.0 3.0
0.0470.05 0.5770.19 2.8170.37
1.0 2.0 3.0
0.0570.08 0.3870.19 1.9370.53
3.0 7.0 15
0.2170.10 0.8670.16 1.2870.33
PAA/UV (mg/l)/(mWs/cm2) 0.5/8.0 1.0/8.0 1.5/8.0 0.5/10 1.0/10 1.5/10
1.0570.18 2.3270.50 4.5670.40 1.9970.27 3.0070.76 4.4070.79
1.0/10 2.0/10 3.0/10 1.0/14 2.0/14 3.0/14
1.0270.39 2.2970.32 5.5670.59 2.4170.43 4.6670.29 5.9770.34
1.0/6.0 2.0/6.0 3.0/6.0 1.0/10 2.0/10 3.0/10
0.9370.04 1.8070.21 3.5670.45 2.5670.09 4.2470.55 6.1670.56
3.0/22 7.0/22 15/22 3.0/38 7.0/38 15/38
1.2170.09 1.7270.06 1.9670.20 1.8170.14 2.3770.18 2.5870.23
H2O2 (mg/l)
0.0270.02 0.0370.04 0.1170.09
ND
ND
ND
ND
3.0 30 150
0.0770.08 0.0270.04 0.0670.10
H2O2/UV (mg/l)/(mWs/cm2) 3.0/8.0 30/8.0 150/8.0 3.0/10 30/10 150/10
0.4370.10 0.3970.05 0.3570.05 1.0070.22 1.1270.06 1.1870.15
ND
ND
ND
ND
3.0/22 30/22 150/22 3.0/38 30/38 150/38
0.9170.12 0.8770.07 1.1670.08 1.4370.19 1.5170.07 1.7470.09
Chlorine (mg/l)
12
2.6970.52
18
0.2870.11
18
0.4470.08
18
1.0370.05
8.0 10
0.6170.20 1.2070.18
10 14
0.5570.11 1.4470.12
6.0 10
0.8770.12 2.6170.35
22 38
0.7970.12 1.4070.15
3.0 30 150
2
UV (mWs/cm )
ND ¼ Not determined, since disinfection experiments by using E. faecalis and MS2 coliphage showed low disinfection efficiency of H2O2 and low synergies in combined H2O2/UV treatments. The results are based on four parallel experiments.
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were achieved with a 12 mg/l chlorine dose. UV doses of 6–18 mWs/cm2 caused approximately 1–3 log enteric bacterial reductions. Coliphage MS2 required UV doses of 22–38 mWs/cm2 to obtain 1–1.5 log reductions. The combination of PAA and UV disinfection improved microbial reductions (Table 2) and synergistic benefits were observed against all the enteric bacteria tested (Table 3). The highest synergy values reached 2 log units, when 1.5–3 mg/l PAA in combination with UV irradiation were used. The increase of UV or PAA dose, when keeping the other one constant, typically increased the disinfection efficiency and synergy values. PAA/UV treatments of coliphage MS2 showed lower synergy values. The combination of H2O2 and UV disinfection only slightly influenced the microbial reductions compared to microbial inactivation in UV treatments (Table 2) and no synergies were observed (Table 3). The synergy values in combined PAA/UV treatments were statistically significant for E. faecalis and E. coli, but not for S. enteritidis and MS2 coliphage (Table 3). The combined H2O2/UV treatments showed statistically significant antagonistic effects for E. faecalis and non-significant synergy for MS2 coliphage. Peracetic acid residues were typically 1–2 mg/l lower than the applied dose and they were present when disinfection treatments were carried out by using X1.5 mg/l PAA doses. Also hydrogen peroxide residues were typically present at the end of the contact time. Chlorine doses of 6–10 mg/l resulted in residual chlorine concentrations below the detection limit (0.1 mg/l), while chlorine doses of 12–18 mg/l caused 0.2–0.3 mg/l residual chlorine concentrations.
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4. Discussion Peracetic acid was demonstrated to be an efficient disinfectant against enteric bacteria. PAA doses of 1.5–3 mg/l and 10 min contact time resulted in approximately 2–3 log E. coli, E. faecalis and S. enteritidis reductions. Similar PAA disinfection efficiencies against enteric bacteria in wastewater have been previously reported (Collivignarelli et al., 2000; Stampi et al., 2001, 2002; Wagner et al., 2002). Coliphage MS2 virus was more resistant than enteric bacteria against PAA disinfection: A dose of 7–15 mg/l PAA was needed to achieve 1–1.5 log reductions. This finding is in agreement with the results of previous studies (Salgot et al., 2002; Veschetti et al., 2003). Viruses are known to exhibit greater resistance also to other disinfection methods. The disinfection efficiency of H2O2 was found to be low, as has been reported also in previous studies (Liberti et al., 2000; Lubello et al., 2002; Wagner et al., 2002). Below 0.2 log microbial reductions were achieved even at the highest tested dose of 150 mg/l H2O2. Thus it can be concluded that the disinfection action of PAA product is mainly due to PAA, the role of hydrogen peroxide being less important. The difference in disinfection efficiencies of PAA and H2O2 may be explained by differences in disinfection mechanisms or by the higher reactivity of PAA. Peracetic acid is organic peroxide and may penetrate more efficiently into microbial cells than the hydrogen peroxide molecule, improving its disinfection properties such as disruption of cell membranes and blockage of enzymatic and transport systems in the microorganisms. Some micro-
Table 3 Average synergy values (log-units) achieved with the combined PAA/UV and H2O2/UV treatments in peptone water Disinfectant and dose Microorganism UV dose
H2O2 H2O2 WSRT PAA PAA PAA PAA PAA PAA PAA WSRT H2O2 0.5 mg/l 1.0 mg/l 1.5 mg/l 2.0 mg/l 3.0 mg/l 7.0 mg/l 15 mg/l p-value 3.0 mg/l 30 mg/l 150 mg/l p-value
E. faecalis UV 8.0 mWs/cm2 0.32 UV 10 mWs/cm2 0.67
0.74 0.82
2.21 1.46
ND
ND
ND
ND
0.031
0.21 0.22
0.25 0.11
0.38 0.13
E. coli UV 10 mWs/cm2 UV 14 mWs/cm2
0.42 0.93
ND
1.16 2.65
2.20 1.73
ND
ND
0.031
ND
ND
ND
0.01 0.11
ND
0.55 1.24
0.76 1.61
ND
ND
0.094
ND
ND
ND
ND
ND
ND
0.21 0.20
0.07 0.11
0.11 0.10
0.344
0.05 0.04
ND
S. enteritidis UV 6.0 mWs/cm2 ND UV 10 mWs/cm2 Coliphage MS2 UV 22 mWs/cm2 UV 38 mWs/cm2
ND
0.06 0.08
0.31 0.28
0.031a
0.063
ND ¼ Not determined; a ¼ antagonistic effect. The results are based on four parallel experiments. Statistical analyses have been done by Wilcoxon signed ranks test (WSRT).
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organisms may also be protected against hydrogen peroxide by their catalase enzyme activity. This enzyme does not protect against PAA, in fact this compound can also inactivate or inhibit catalase activity (Wagner et al., 2002). Inactivation of coliphages may be due to changes or damage to their surface structures, such as the protein coat or the attachment sites needed for infection of host cells. The efficiency of sodium hypochlorite disinfection against enteric microorganisms in PW was lower than that of PAA. Veschetti et al. (2003) have previously reported equal disinfection efficiencies of PAA and hypochlorite in the treatment of secondary wastewater effluents. The differences in results may be explained by differences in the qualities of water that were treated. The synthetic PW used in this study was probably more susceptible to chemical oxidation by disinfectants than some secondary treated wastewaters. In our experiments PAA and H2O2 showed lower reactivity for the water matrix than the chlorine. It suggests that the efficiency of PAA in treatment of lower quality wastewaters, such as secondary effluents containing organic matter, primary treated wastewaters and some industrial process waters, may be better than that of chlorine. This finding is supported by the results obtained by Baldry et al. (1991) and also by our results showing equal disinfection efficiencies of PAA and NaOCl when tap water with a very low organic matter content was used instead of peptone water as the test medium (results not shown). In our experiments, chemical and UV disinfection were combined to achieve synergistic benefits. UV irradiation was always done after the application of PAA or H2O2, to take advantage of increasing radical formation due to interaction of disinfectant chemical and UV. Combined PAA/UV disinfection treatment is more efficient if UV irradiation is done after application of PAA, compared to opposite order of disinfectants (Caretti and Lubello, 2003). In this study the combination of PAA and UV disinfection increased disinfection efficiency and showed synergistic benefits, the highest synergy values reaching 2 log units for enteric bacteria. This means that the microbial inactivation was 2 log units higher than could be expected by summing the microbial reductions achieved by using those two disinfectants separately. An increase in either the UV or the PAA dose increased the synergy values, probably due to increased radical formation. The combination of H2O2 and UV treatments showed no synergies. These findings are in agreement with the results obtained in previous studies (Lubello et al., 2002; Caretti and Lubello, 2003). In this study we found that despite of significant synergies in treatment of enteric bacteria, the combined disinfection treatments of coliphage MS2 did not achieve synergy benefits. It indicates higher resistance of coliphage viruses also against combined
disinfection treatments. Previously their higher resistance against single disinfectants has been described. The synergies achieved in PAA/UV treatments may be due to interaction between PAA and UV, producing reactive and microbicidal radicals due to the photolysis of PAA (Lubello et al., 2002; Caretti and Lubello, 2003). The lack of synergies in H2O2/UV treatments suggests that there are differences in the radical formation potential between PAA and H2O2, the potential and the resulting disinfection efficiency being higher for PAA. The mechanism of synergy could also be explained by a multiple damage mechanism: two different disinfection methods may cause different types of injuries for microorganisms. The principal targets of UV radiation are the nucleic acids, while chemical disinfectants, such as PAA and H2O2, is thought first to attack microbial cell walls, membranes and enzymatic or transport systems. As a result, the microbial repair mechanisms, required to repair minor damage, may become overloaded, leading to their inability to repair the injuries and subsequent death. In the case of single disinfectant, the damage may be smaller and susceptible to repair, which may not be possible in case of two disinfectants causing a greater variety of damages. The higher synergies achieved by using PAA/UV treatment compared to H2O2/UV treatment could thus be related to the greater disinfection efficiency of PAA compared to H2O2. PAA may also inactivate the catalase enzyme which is one mechanism involved in detoxifying free hydroxyl radicals. This would allow free radicals to more efficiently attack and inactivate microbial cells. Hydrogen peroxide may not have such an effect on catalase. The application of PAA/UV disinfection method in wastewater treatment plants could offer some advantages. It could allow lower disinfectant doses or lower contact times and thus allow smaller size of UV units and chemical disinfection basins, decreasing the costs of disinfection. The efficiency and reliability of disinfection in existing UV disinfection units could probably be improved by the addition of low PAA dose before the UV unit. This method could also improve the bacteriostatic effect and decrease the reactivation potential of microorganisms which had undergone the disinfection process. When PAA or UV treatments alone have been used for wastewater disinfection, viable but non-culturable bacteria and bacterial regrowth have been observed (Lazarova et al., 1995). The combination of two disinfection methods could also destroy a wider range of microorganisms than that achieved by a single disinfection method, since some microorganisms are UV resistant, but sensitive for chemical disinfectant, while others behave in an opposite way. It may be more efficient and economical to apply a combination of low chemical and UV doses, instead of a high dose of one disinfectant, to destroy these microorganisms. Further
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research must be conducted to verify these findings in pilot- and full-scale disinfection of wastewaters. Wastewater characteristics, such as suspended solids and other wastewater components, as well as characteristics and physiological state of microorganisms in real wastewaters may affect disinfection efficiency and synergies achievable by combined disinfection methods.
5. Conclusions Peracetic acid is an efficient chemical disinfectant against enteric bacteria. Low PAA doses (1.5–3 mg/l) were needed to achieve 2–3 log bacterial reductions. By using higher PAA doses (7–15 mg/l) coliphage MS2 could be inactivated. Hydrogen peroxide has low disinfection efficiency against enteric microorganisms and may not be a feasible disinfectant for wastewater disinfection. The disinfection action of the PAA product is mainly attributable to peracetic acid, while the effect of hydrogen peroxide seems to be low. The efficiency of sodium hypochlorite disinfection against enteric microorganisms in PW was low. PAA may be a more efficient disinfectant than chlorine for treatment of waters containing easily oxidizable organic matter. The combined PAA/UV disinfection treatments of enteric bacteria achieved significant synergy benefits, while the treatments of coliphage MS2 did not show significant synergies. The combined H2O2/UV disinfection only slightly influenced the microbial reductions compared to UV treatments and produced no synergies. The results clearly indicate higher resistance of coliphage viruses also against combined disinfection treatments.
Acknowledgments The authors wish to thank Kemira Oyj Kemwater for financial support. We also thank Ms Sirpa Martikainen and Mr Matti Pessi for their assistance in laboratory work.
References Baldry, M.G.C., French, M.S., Slater, D., 1991. The activity of peracetic acid on sewage indicator bacteria and viruses. Water Science and Technology 24 (2), 353–357. Baron, J., Bourbigot, M.-M., 1996. Repair of Escherichia coli and Enterococci in sea water after ultraviolet disinfection quantification using diffusion chambers. Water Research 30 (11), 2817–2821.
1525
Blatchley III, E.R., 1996. Numerical modelling of UV intensity: application to collimated-beam reactors and continuousflow systems. Water Research 31 (9), 2205–2218. Booth, R.A., Lester, J.N., 1995. The potential formation of halogenated by-products during peracetic acid treatment of final sewage effluent. Water Research 29 (7), 1793–1801. Caretti, C., Lubello, C., 2003. Wastewater disinfection with PAA and UV combined treatment: a pilot plant study. Water Research 37, 2365–2371. Collivignarelli, C., Bertanza, G., Pedrazzani, R., 2000. A comparison among different wastewater disinfection systems: experimental results. Environmental Technology 21, 1–16. Koivunen, J., Siitonen, A., Heinonen-Tanski, H., 2003. Elimination of enteric bacteria in biological-chemical wastewater treatment and tertiary filtration units. Water Research 37, 690–698. Lazarova, V., Janex, M., Fiksdal, L., Oberg, C., Barcina, I., Pommepuy, M., 1998. Advanced wastewater disinfection technologies: short and long term efficiency. Water Science and Technology 38 (12), 109–117. Liberti, L., Notarnicola, M., 1999. Advanced treatment and disinfection for municipal wastewater reuse in agriculture. Water Science and Technology 40 (4–5), 235–245. Liberti, L., Lopez, A., Notarnicola, M., Barnea, N., Pedahzur, R., Fattal, B., 2000. Comparison of advanced disinfection methods for municipal wastewater reuse in agriculture. Water Science and Technology 42 (1–2), 215–220. Lubello, C., Caretti, C., Gori, R., 2002. Comparison between PAA/UV and H2O2/UV disinfection for wastewater reuse. Water Science and Technology: Water Supply 2 (1), 205–212. Monarca, S., Feretti, D., Collivignarelli, C., Guzzella, L., Zerbini, I., Bertanza, G., Pedrazzani, R., 2000. The influence of different disinfectants on mutagenicity and toxicity of urban wastewater. Water Research 34 (17), 4261–4269. Oppenheimer, J.A., Jacangelo, J.G., Laıˆ ne´, J-M., Hoagland, J.E., 1997. Testing the equivalency of ultraviolet light and chlorine for disinfection of wastewater to reclamation standards. Water Environment Research 69 (1), 14–24. Rajala, R.L., Pulkkanen, M., Pessi, M., Heinonen-Tanski, H., 2003. Removal of microbes from municipal wastewater effluent by rapid sand filtration and subsequent UV irradiation. Water Science and Technology 47 (3), 157–162. Rajala-Mustonen, R.L., Toivola, P.S., Heinonen-Tanski, H., 1997. Effects of peracetic acid and UV irradiation on the inactivation of coliphages in wastewater. Water Science and Technology 35 (11–12), 237–241. Salgot, M., Folch, M., Huertas, E., Tapias, J., Avellaneda, D., Giro´s, G., Brissaud, F., Verge´s, C., Molina, J., Pigem, J., 2002. Comparison of different advanced disinfection systems for wastewater reclamation. Water Science and Technology: Water Supply 2 (3), 213–218. Stampi, S., De Luca, G., Zanetti, F., 2001. Evaluation of the efficiency of peracetic acid in the disinfection of sewage effluents. Journal of Applied Microbiology 91, 833–838. Stampi, S., De Luca, G., Onorato, M., Ambrogiani, E., Zanetti, F., 2002. Peracetic acid as an alternative wastewater disinfectant to chlorine dioxide. Journal of Applied Microbiology 93, 725–731.
ARTICLE IN PRESS 1526
J. Koivunen, H. Heinonen-Tanski / Water Research 39 (2005) 1519–1526
Tyrrell, S., Ripley, S.P., Watkins, W.D., 1995. Inactivation of bacterial and viral indicators in secondary sewage effluents, using chlorine and ozone. Water Research 29 (11), 2483–2490. Veschetti, E., Cutilli, D., Bonadonna, L., Briancesco, R., Martini, C., Cecchini, G., Anastasi, P., Ottaviani, M., 2003. Pilot-plant comparative study of peracetic acid and
sodium hypochlorite wastewater disinfection. Water Research 37, 78–94. Wagner, M., Brumelis, D., Gehr, R., 2002. Disinfection of wastewater by hydrogen peroxide or peracetic acid: development of procedures for measurement of residual disinfectant and application to a physicochemically treated municipal effluent. Water Environment Research 74 (1), 33–50.