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Removal of viruses and bacteriophages from drinking water using zero-valent iron
Separation and Purification Technology 84 (2012) 72–78
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Removal of viruses and bacteriophages from drinking water using zero-valent iron Chunjian Shi a, Jie Wei b, Yan Jin c, Kalmia E. Kniel b, Pei C. Chiu a,⇑ a
Department of Civil and Environmental Engineering, University of Delaware, Newark, DE 19716, United States Department of Animal and Food Sciences, University of Delaware, Newark, DE 19716, United States c Department of Plant and Soil Sciences, University of Delaware, Newark, DE 19716, United States b
a r t i c l e
i n f o
Article history: Available online 2 July 2011 Keywords: Zero-valent iron Virus Water treatment Adenovirus Aichi virus Drinking water
a b s t r a c t Zero-valent iron (ZVI) has been shown to remove and inactivate bacteriophages in synthetic groundwater, which suggests that it may be potentially useful for drinking water treatment. As an effort to assess this possibility, we assessed the effectiveness of ZVI to remove pathogenic viruses from two water treatment plant samples (from Allentown, PA and Newark, DE) through a series of column experiments. Removal of Aichi virus (AiV), Adenovirus 41 (Ad41), and the bacteriophages MS2 and /X174 in columns packed with clean sand only and sand with a layer of ZVI-sand mix (1:1, v/v) was examined through pulse tests under saturated flow conditions. With the exception of Ad41, removal of all viruses by sand was zero or limited in all cases, regardless of the water used. In contrast, ZVI-containing columns gave removal efficiencies between 4.5 and 6 logs for all viruses and all waters studied. Ad41 was retained markedly by sand and even more strongly (by 2–3 logs) by ZVI and did not breakthrough fully from either column even after 32 pore volumes. The significant retardation of Ad41 is ascribed to the relatively high isoelectric points of the long and short fibers of Ad41. Our results, combined with studies elsewhere that demonstrate organic matter removal by ZVI and iron oxides, suggest ZVI may potentially offer a simple and low-cost option to help utilities control microorganisms, disinfection byproducts, and chlorine residuals simultaneously. Possible applications of ZVI in water treatment plants are proposed and the potential benefits are discussed. Ó 2011 Elsevier B.V. All rights reserved.
1. Introduction Quantity and quality of drinking water have been recognized as increasingly critical issues for the coming decades. Among the factors that contribute to the looming water crisis are continued population growth and urbanization, deteriorating water infrastructure, increasing influence of wastewater and biosolids on drinking water sources, growing number of emerging contaminants, and uncertain future water availability and quality due to climate change. It has been questioned recently [1] whether the conventional water treatment processes, which are based on rapid sand filtration and chlorination and have stayed largely unchanged for decades, can adequately remove the many chemical and microbial contaminants simultaneously. Among the problems facing water utilities and regulators, one particularly daunting challenge is how to control microbial pathogens, disinfection by-products (DBPs) and residual disinfectants simultaneously – and to do so at an acceptable cost. On the one hand, high dosage of chlorine can produce high chlorine residuals and high levels of DBPs, including trihalomethanes, haloacetic acids, and haloacetonitriles [2]. On the other hand, low chlorine ⇑ Corresponding author. Tel.: +1 302 831 3104; fax: +1 302 831 3640. E-mail address: [email protected] (P.C. Chiu). 1383-5866/$ - see front matter Ó 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.seppur.2011.06.036
dosage may provide insufficient protection of distribution systems and human health against microbial pathogens. While alternative disinfection methods are available; e.g., UV, membrane processes (e.g., reverse osmosis and nano-filtration), ozone, and chloramines, some of them require significant capital investment and plant modifications or are expensive to install and/or difficult to operate. Ozone and chloramines also produce DBPs of great health concerns, including N-nitrosodimethylamine, bromate, and bromoand iodo-acids [3]. It is thus highly desirable to develop a simple, effective, inexpensive, and non-oxidant-based method to help control microorganisms, DBPs, and chlorine residuals in drinking water – simultaneously. This is particularly important for midand small-size treatment plants, which may not have the necessary resources to readily invest in or migrate to another disinfection method in response to increasing regulatory demand and/or deteriorating source water quality. One method that may potentially achieve these multiple goals is zero-valent iron (ZVI). ZVI has been used to remediate contaminated groundwater for over 15 years [4] and has been shown to remain reactive in anaerobic subsurface for multiple years [5]. While initially used to treat chlorinated solvents and hexa-valent chromium, ZVI has also been shown over the last two decades to remove a broad range of chemical contaminants, from heavy metals and radionuclides to agrochemicals and explosives. To date,
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application of ZVI has been limited to treating chemical pollutants in groundwater. For drinking water, however, the contaminants of primary concern are microbial. In 2005, You et al. [6] demonstrated that ZVI, when added to a sand filter, removed the bacteriophages MS2 and /X174 at efficiencies between 5 and 6 logs. The removal was ascribed to adsorption of these phages to iron (hydr)oxides formed via anaerobic corrosion of ZVI. You et al. also showed that removal of these viruses by ZVI was rapid (most likely mass transfer-limited), and that most of the viruses removed were either inactivated or irreversibly bound. In addition, ZVI is capable of removing many other pollutants relevant to drinking water, including arsenic [7], lead [8], and natural organic matter (NOM), a major precursor of DBPs [9]. These findings suggest ZVI may be useful for drinking water treatment and can potentially offer multiple benefits. For example, if used in a water treatment plant ZVI can remove NOM, arsenic and microorganisms before chlorine addition – a strategy that may help meet multiple treatment goals while preventing excessive DBP formation. The work by You et al. [6] was conducted with two model viruses in synthetic groundwater. To further assess the feasibility of ZVI for drinking water treatment, this study was carried out to evaluate the ability of ZVI to remove pathogenic viruses in real water treatment plant samples. Viruses were targeted because they are particularly problematic due to their small size (20–150 nm), high mobility in porous media, and resistance to filtration and chlorination relative to bacteria [10,11]. Viruses are believed to be responsible for the majority of the disease outbreaks for which the infectious agents are identifiable [12]. Viruses are also the target microorganisms of the Ground Water Rule, which took effect in 2007 [13]. In this study, we performed a series of flow-through column experiments to examine the ability of ZVI to remove two pathogenic viruses, Aichi virus and Adenovirus 41, and two bacteriophages /X174 and MS2, from water samples collected from Newark Water Treatment Plant (NWTP) in Newark, DE and Allentown Water Filtration Plant (AWFP) in Allentown, PA. These viruses have been linked to many water- and food-borne disease outbreaks or used as surrogates for human enteric viruses. We will discuss the results and their implications for water treatment and propose potential applications of ZVI in water treatment plants. 2. Materials and methods 2.1. Materials Virus removal experiments were conducted using pairs of acrylic columns. Eighteen identical columns (3.8 cm i.d. 10 cm, bed volume = 113 cm3) were fabricated at the University of Delaware in the Engineering Machine Shop. Each column was wet-packed with sand only (‘‘sand column’’) or both sand and ZVI (‘‘ZVI column’’). The ZVI used for all experiments was commercial iron granules ETI850/50 from Peerless Metal Powders and Abrasive (Detroit, MI). The specific surface area of the Peerless iron was 1.67 m2/g, as measured by the Brunauer–Emmett–Teller (BET) adsorption method with N2. The iron was sieved, and the size fraction 60–35 mesh (250–500 lm) was used for column packing. We used Accusand (Unimin, Le Sueur, MN) with the following size distribution: 9% 0.1–0.25 mm, 70% 0.25–0.5 mm, and 21% 0.5– 1.0 mm. The sand was treated with sodium citrate and dithionite to remove organic and metal oxide coatings before use [14]. 2.2. Viruses The viruses used for this study were Aichi virus (AiV), Adenovirus 41 (Ad41), and the bacteriophages MS2 and /X174. AiV, a
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member of the family Picornaviridae, was first isolated in 1989 in Aichi, Japan [15]. AiV is prevalent in wastewater and river water [16] and has caused many cases of illness in Europe, Asia and South/Central America [17]. Adenoviruses are emerging contaminants on US EPA’s Contaminant Candidate List (CCL3). Ad41 is representative of the approximately 42 different serotypes of adenoviruses causing human disease from conjunctivitis and respiratory infection to hepatic disease and gastroenteritis (e.g., infantile diarrhea). In addition, we included two bacteriophages, MS2 and /X174, in this study. These phages structurally resemble many human enteric viruses and have been widely used as surrogates [18–20]. MS2 is an icosahedral, single-stranded RNA phage with a diameter of 26 nm [21] and an isoelectric point (pHisp) of 3.9 [22]. /X174 is a single-stranded DNA phage, 23 nm in diameter, with a pHisp of 6.6 [23]. AiV was cultured on Vero cells (ATCC # CCL-81TM) originated from Africa green monkey kidney in Eagle’s minimal medium (Mediatech, VA) containing either 2% (for maintenance) or 10% (for growth) fetal bovine serum (FBS, Hyclone, TX), 2% sodium bicarbonate, 1% sodium pyruvate, 1% penicillin/streptomycin/ ampicillin (Mediatech), and 1% non-essential amino acids (Mediatech). AiV in infected cell lysates was purified by one freeze–thaw cycle, followed by centrifugation at 2500g for 15 min. The upper aqueous layer was recovered and stored at 80 °C for analysis. Virus infectivity was determined using the tissue culture infectious dose (TCID50) assay. AiV was inoculated into cell mono-layers grown in 96 wells plates, serially diluted with Hank’s Buffered Salt Solution (HBSS, Mediatech) and incubated for 2 h before applying medium containing 2% FBS. The plates were incubated for 3–5 days for cytopathic effects, and virus titer was obtained according to the Reed and Meunch calculation. Ad41 (ATCC # VR-930™) was obtained from ATCC and cultured on human embryonic kidney (HEK) 293 cells (ATCC # CRL-1573) in Eagle’s Minimal Medium containing 10% fetal bovine calf serum. Ad41 in infected cell lysates were purified by one freeze–thaw cycle followed by centrifugation at 2500g for 15 min. The upper aqueous layer was recovered and stored at 80 °C for further analysis. The titer of Ad41 was estimated by quantal endpoint assay employing 10-fold serial dilutions and using PCR to amplify viral DNA to obtain the most probable number (MPN) [24]. The Ad41 stock titer was approximately 6.7 105 viral particles/mL. Ad41 was measured by quantitative PCR (qPCR). The primer pair for qPCR, Ad41F/R, was designed to target the hexon region, amplifying a 135-bp fragment of adenoviruses of species F (Ad40 and Ad41) [25]. The sequences of Ad41F and Ad41R are 50 -GGACGCCTCGGAGTACTGA30 and 50 -CGCTGIGACCIGTCTGTGG-30 , respectively. To generate a standard curve, 1 mL Ad41 (6.7 105 VP/mL) was added to AGW and vortexed. The solution was then 10-fold diluted serially with HBSS to 67 VP/mL, and DNA was extracted from all dilutions using a DNeasy Tissue Kit following the manufacturer’s instructions (Qiagen, CA). The DNA was applied to qPCR with Qiagen SYBR Green (Qiagen, CA) to generate the standard curve. The qPCR was performed in 25-lL volumes containing 12.5 lL of 2x SYBR Green Mix, 1.5 lL of primer Ad41F/R, 4 lL of cDNA, and 5.5 lL of H2O, using ABI 7900HT system (Applied Biosystems, CA). The amplification cycle was 95 °C for 15 min, 40 cycles of 94 °C for 15 s, 60 °C for 30 s, and 72 °C for 30 s, followed by a dissociation step of 95 °C for 15 s, 60 °C for 15 s, and 95 °C for 15 s. MS-2 was obtained from the American Type Culture Collection (ATCC 15597B1) and grown on bacterial lawns of Escherichia coli (ATCC 15597). /X174 was grown on an E. coli host (ATCC 13706). MS2 and /X174 were assayed using the double-agar overlay method [26]. Briefly, 1 mL of host culture and 0.5 mL of diluted virus sample were added to a trypticase soy agar (TSA) tube and the mixture poured onto a TSA plate. The plates were solidified for 15 min and placed in a 37 °C incubator for 5 h and 12 h for /X174 and
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MS2, respectively. Viable (i.e., infective) phage concentrations were determined by counting the plaques in the host lawn and reported as plaque-forming units per mL (pfu/mL). Only dilutions that result in between 10 and 300 plaques per plate were accepted/used (i.e., limit of quantification = 10 pfu/plate or 20 pfu/mL). All assays were run in duplicates, and data were accepted only if the duplicates agreed within 20%. 2.3. Water samples The waters tested in this study included samples from Newark Water Treatment Plant (NWTP) in Newark, DE and Allentown Water Filtration Plant (AWFP) in Allentown, PA. The NWTP and AWFP samples, both collected in late November, 2008, were effluents from filtration tanks prior to final chlorination. These samples were used shortly after collection and were stored in a 5 °C constant-temperature room before use and between experiments. Because AWFP pre-chlorinates their water while NWTP does not, the AWFP water contained a low level of chlorine. Table 1 gives water quality of the treatment plant samples. For comparison, artificial groundwater (AGW) was also used in this study. The AGW contained 0.075 mM CaCl2, 0.082 mM MgCl2, 0.051 mM KCl, and 1.5 mM NaHCO3, giving an ionic strength of 2 mM. AGW was prepared fresh, autoclaved, vacuum-degassed, and pH-adjusted to between 7.0 and 7.4. Virus input solution for each experiment was prepared by diluting an aliquot of virus stock of desirable titer in a treatment plant water or AGW to yield the target virus concentration. 2.4. Experimental methods In a typical experiment, a pair of columns were wet-packed through incremental addition of sand (and ZVI) in AGW. This was done carefully to ensure that columns were free of air bubbles. The sand column (control) was packed with cleaned sand only, whereas the ZVI column consisted of a 3-cm layer of 1:1 (v/v) ZVI-sand mix sandwiched by two 3.5-cm layers of cleaned sand, giving an overall ZVI content of 15% (v/v, solid basis). The pore volume (PV) of each column was calculated by subtracting the volume of sand/ZVI from the total void volume or estimated based on the volume of water used during packing. The PVs of typical sand and ZVI columns were 38.5 ± 0.5 and 45.0 ± 1.0 mL, respectively. Given the column bed volume of 113.4 cm3, the corresponding porosities were approximately 34% for sand columns and 40% for ZVI columns. Columns were operated under saturated and continuous up-flow conditions at a rate of 0.73 ± 0.03 mL/min regulated by a peristaltic pump. The columns were first flushed with 10 PVs of background solution (a treatment plant water or AGW) at 0.5 mL/min; the flow rate was then increased to 0.73 mL/min for another hour to establish a steady flow prior to virus introduction. Based on the PVs, flow rate, and ZVI content, the effective iron contact time in the ZVI column was 9.3 ± 0.3 min. Uniform flow conditions for columns prepared using these procedures have been verified by bromide tracer tests [6]. Fig. 1 illustrates the set-up for a typical column experiment. To initiate a virus removal experiment, the influent was switched at time zero from a virus-free background solution to a
Table 1 Properties of the NWTP and AWFP water samples.
NWTP AWFP
pH
TOC (mg/L)
EC (ms/cm)
Alkalinity (mg/L)
Total chlorine (mg/L)
7.20 7.72
1.50 1.02
4.5 6.4
58 190
– 0.99
virus input solution. Virus solution was introduced into both sand and ZVI columns for 5 PVs (based on the ZVI column), the input was switched back to the virus-free solution, and pumping was continued for another 5–27 PVs depending on the rate of virus breakthrough. Starting at time zero, sand and ZVI column effluents were collected in 5-mL glass tubes using fraction collectors (Fig. 1). A typical experiment for which 10 PVs of effluent was collected would generate 90–100 tubes of samples from each column. Samples were analyzed for target virus, using the infectivity or qPCR assays described above to obtain a virus breakthrough curve for each column. Samples were analyzed on the same day or as soon as feasible and were stored in a freezer before analysis. Additional samples of the virus input solution were collected at the end of experiment for analysis to ensure minimal virus inactivation during experiment. 3. Results and discussion 3.1. Effects of chlorine and Na2SO3 on MS2, /X174 and AiV Before starting column experiments, a preliminary test was performed to determine whether the AWTP sample should be dechlorinated before use. This was because the residual chlorine (1 ppm) in the sample might cause excessive inactivation of viruses and/or increase ZVI oxidation [27]. To evaluate the effects of chlorine and the dechlorinating agent, Na2SO3 on /X174, MS2, and AiV, an aliquot of each virus stock was added separately to AGW, as-received AWFP water, AWFP water with a low dose of Na2SO3 (stoichiometric to chlorine), and AWFP water with a high dose of Na2SO3 (2x stoichiometric dose). The samples were incubated at 5 °C and analyzed for the viruses after 20 min and 8 h of incubation. Result of this test is summarized in Table 2. The three viruses were stable at 5 °C in AGW even after hours. In contrast, the phages and AiV were completely and moderately inactivated, respectively, in the AWFP water. When the water was dechlorinated with stoichiometric amount of Na2SO3, limited or no virus inactivation was observed. This suggests that the residual chlorine was responsible for the observed inactivation. It also indicates that after Na2SO3 addition, the AWFP sample would be suitable for the study as excessive background inactivation of viruses would not be a concern. Finally, when Na2SO3 was added in excess, it did not appear to have any adverse effect on the three viruses, suggesting that any residual Na2SO3, if present in dechlorinated AWFP water, would not influence virus removal results. Based on the data in Table 2, all AWFP water was pretreated with Na2SO3 before use. 3.2. Removal of MS2, /X174, and AiV by sand and ZVI Separate pairs of sand and ZVI columns were set up to assess removal of viruses in the NWTP water, the AWFP water, and AGW. To establish a baseline for comparison, we first ran two pulse tests with /X174 and MS2 in AGW. The removal efficiencies for these phages by the ZVI column were 5.80 logs (99.9998%) and 5.60 logs (99.9997%), respectively, consistent with the results of You et al. [6]. Additional pulse tests were conducted with these phages in the NWTP sample, and similar removal by an ZVI column – 5.92 logs for /X174 and 5.67 logs for MS2 – was obtained. This indicates that ZVI can remove these indicator viruses from real water treatment plant samples at high efficiencies within 9–10 min of iron contact time. High removal by ZVI was also observed for dechlorinated AWFP water: 5.22 logs for /X174 and 5.16 logs for MS2. In all the above experiments, virtually no virus removal was observed for any of the sand columns (Table 3), and hence the removal could be attributed entirely to the 15% iron in each ZVI column. An
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Fig. 1. Setup of a typical virus removal experiment. Influent (AGW or a water treatment plant sample) was supplied by a peristaltic pump to both the sand (left) and ZVI (right) columns in an upward flow direction. Effluents from these columns were collected separately by two fraction collectors, for 10 PVs for /X174, MS2, and AiV, and for 10–32 PVs for Ad41.
Table 2 Effects of chlorine and Na2SO3 on virus infectivity in AWFP water sample. [Cl2] (mg/L)
[Na2SO3] (mg/L)
– 0.99 – –
0 0 1.76 3.52
[/X174] (pfu/mL)
[MS2] (pfu/mL)
[AiV] (TCID50/mL)
525,000 0 589,000 427,000
21,400 6200 23,700 28,800
20 min AGW AWFP Low SO3 High SO3
577,000 0 555,000 545,000
[/X174] (pfu/mL)
[MS2] (pfu/mL)
[AiV] (TCID50/mL)
479,000 0 501,000 363,000
19,100 3800 17,500 21,400
8h
example illustrating how these removal efficiencies were calculated is described below. Fig. 2(a) shows a typical virus breakthrough curve for a sand column, exemplified by /X174 in AWFP water. The effluent virus concentration reached the input concentration slightly after 1 PV and remained constant for approximately 5 PVs before declining. The virus removal efficiency was taken to be one minus the ratio of the integrated area under the breakthrough curve (on a normal–normal plot) to the total virus count in 5 PVs of input solution. For this and all other sand column breakthrough experiments with /X174 and MS2 in AGW, the AWFP water, and the NWTP water, the calculated virus removal was very close to zero (Table 3). That is, clean sand was feckless at removing these phages for all three waters tested. Fig. 2(a) also contains four data points of non-zero
589,000 0 563,000 554,000
(albeit below detection limit) /X174 concentrations in the ZVI column effluent. All other ZVI effluent samples yielded no plaque. In fact, in all the phage removal tests performed using any of the three water matrices, the majority of ZVI effluent samples gave either zero or below-detection-limit plaque counts. Since in most cases there were insufficient countable plates to construct a full breakthrough curve, removal efficiencies for ZVI columns were calculated using either actual plaque counts of all readable effluent samples or the detection limit (i.e., 20 pfu/mL) over 10 PVs. Table 3 shows the two sets of ZVI column removal efficiencies, which differ only by 0.3 log or less in most cases. The phage removal experiments were followed by an AiV pulse test in the AWFP water. The AiV stock was concentrated to remove organic substrates in the medium by centrifugation at 13,000g for
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Table 3 Log removal of /X174, MS2 and AiV from AGW and NWTP and AWFP waters. The negative sand column removal efficiencies (in parentheses) are due to slightly smaller integrated area under sand column breakthrough curve than the calculated total virus input and are set to be zero. The two sets of ZVI column efficiencies were calculated based on actual infectivity data of all readable effluent samples (efficiency 1) and the detection limit (20 pfu/mL or 10 TCID50/mL, efficiency 2).
/X174 (AGW) MS2 (AGW) /X174 (NWTP) MS2 (NWTP) /X174 (AWFP) MS2 (AWFP) AiV (AWFP) /X174 (AWFP) MS2 (AWFP) *
Input concentration (pfu/mL or TCID50/mL)
Sand column efficiency
ZVI column efficiency 1
ZVI column efficiency 2
6.03 106 4.27 106 7.94 106 4.68 106 2.51 106 1.23 106 0.98 106 1.02 106 1.05 106
0 (0.03) 0 (0.02) 0 (0.04) 0 (0.01) 0 0 0.83 0.02 0.06
5.80 5.60 5.92 5.67 5.22 5.16 –* 5.01 5.02
5.48 5.33 5.60 5.37 5.10 4.79 4.99 4.71 4.72
A log removal could not be calculated since the efficiency was 100% based on actual assay data (i.e., all ZVI column effluents samples were non-infective).
Fig. 2. Breakthrough concentrations of (a) /X174 in sand and ZVI column effluents and (b) AiV in sand column effluent. The water matrix was dechlorinated AWFP sample in both cases. The detection limits were 10 pfu/plate (or 20 pfu/mL) for /X174 and 10 TCID50/mL for AiV. No AiV was detected in any of the ZVI column effluent samples (thus no data are shown in (b)).
10 min using 100,000-MW centricon (Millipore, MA). The filtrate was discarded and AiV was recovered from the filter membrane
with HBSS. The AiV breakthrough curve for the sand column is shown in Fig. 2(b). Interestingly, in contrast to the results for the
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100000
Effluent Ad41 Concentration (VP/mL)
two phages, the sand column was able to remove 0.83 log of AiV in the AWFP water. The higher removal might be due to stronger electrostatic interactions between AiV and sand surface; however, this cannot be confirmed as the pHisp of AiV has not been reported to our knowledge. In contrast to the sand column result, no viable AiV was detected in the ZVI column effluent (i.e., all effluent samples were non-infective). Using the conservative assumption that the AiV concentrations in all ZVI effluent samples were equal to the detection limit (10 PV/mL), we obtained a removal efficiency of 5.0 logs for AiV in the AWFP sample. Virus removal efficiencies for the experiments described above and additional experiments are summarized in Table 3. The sand column efficiencies for /X174, MS2, and AiV were limited (<1 log) in all cases, consistent with a recent study [28]. In contrast, the ZVI column efficiency was between 4.5 and 6 logs in all three water matrices tested, regardless of whether the calculation was based on actual plaque counts or detection limit. It should be noted that, due to limited breakthrough and low (often below-detection) effluent concentrations, the calculated ZVI column efficiencies depended strongly on the input concentration; i.e., the removal efficiencies would have been greater had higher influent virus titers been used.
Sand Column ZVI Column
10000
Detection Limit (5 VP/mL) Input Conc. (74,000 VP/mL)
1000
100
10
1
0
1
2
3
4
5
6
7
8
9
10
Pore Volume (PV) Fig. 3. Elution concentration profiles of Ad41 for sand and ZVI columns. The upper dash line corresponds to the input concentration of Ad41, 74,000 VP/mL. The lower dash line represents the qPCR detection limit of 5 PV/mL.
3.3. Removal of Ad41 by sand and ZVI Similar experiments were also conducted with Ad41, but the results are not included in Table 3, because the removal efficiencies could not be obtained in the same manner. Unlike /X174, MS2 and AiV, Ad41 exhibited a very different breakthrough behavior for both sand and ZVI columns, as shown in Fig. 3. In contrast to the other viruses, Ad41 was retarded more strongly in the sand column and did not break through fully within 10 PVs. The effluent concentration started to rise at approximately 1 PV and continued to increase gradually but never reached the input concentration (74,000 VP/mL). The mass recovery, based on the integrated area underneath the sand column breakthrough curve, was only 7.5%. By comparison, the higher removal of Ad41 by ZVI was clear. The Ad41 concentration in ZVI column effluent remained low throughout the experiment, hovering over the detection limit (5 VP/mL). Despite the incomplete breakthrough and low mass recovery, we made an attempt to estimate Ad41 removal by ZVI. Based on the ratio of the two integrated areas (i.e., virus counts) under the ZVI and sand column breakthrough curves over 10 PVs, the efficiency of Ad41 removal by the 15% iron in the ZVI column was 2.6 and 2.7 logs, as estimated using the actual qPCR data and the detection limit, respectively. These numbers are consistent with a visual inspection of the data in Fig. 3, where the difference in eluted Ad41 concentrations from the ZVI and sand columns was between 2 and 3 logs. Because of the unusual breakthrough pattern and slow and incomplete elution of Ad41, we repeated the experiment using a similar influent concentration (75,500 VP/mL) but collected 32 PVs of effluent samples instead of 10. The same breakthrough pattern was observed as in the first experiment: Ad41 was retarded by sand and even more markedly by ZVI, and its elution from both columns was slow and incomplete, even after 32 PVs (35 h). The pronounced retention/removal of Ad41 by sand has important implications for its fate and transport in the subsurface and in treatment systems. Although Ad41 is larger than /X174 and MS2 (90 nm vs. 23–26 nm), it is still far smaller than the pores between sand and ZVI grains, which are on the order of a few hundred lm; therefore, the retention mechanism was most likely chemical rather than physical. While the pHisp values of the core and hexon proteins of Ad41 are unavailable, its penton base protein (located at the 12 vertices of the capsid) has a pHisp of 5.5 [29], which does not explain the strong retention of Ad41 by sand
as both would be negatively charged at circum-neutral pH. However, in contrast to the phages, Ad41 also contains long (34 ± 1.3 nm) and short (20 ± 1.3 nm) fibers that mediate virus attachment to the host cell [29]. Both the long and short fibers of Ad41 have relatively high pHisps (7.51 and 9.13, respectively), which were suggested to be responsible for an erratic behavior of the fiber protein on denaturing polyacrylamide gels [29]. It is plausible that these fibers, rather than the capsid, control how Ad41 particles interact with surfaces. The positively charged fibers might interact favorably with sand and iron surfaces, resulting in the significant retention and slow elution of Ad41 we observed.
4. Conclusions and implications This study extends the work of You et al. [6] by including pathogenic viruses and real water treatment plant samples in the assessment of ZVI for water treatment purposes. The main results are summarized as follows: 1. At ppm levels, chlorine effectively inactivated MS2 and /X174, and to a lesser extent AiV, while Na2SO3 had no observable adverse effects on these viruses. 2. Clean sand alone achieved limited or no removal of MS2, /X174, and AiV, regardless of whether AGW, AWFP water, or NWTP water was used. 3. In contrast, when a sand column was amended with 15% ZVI, removal efficiencies of 4.5–6 logs were achieved for all three viruses and three waters tested. 4. Ad41 exhibited a markedly different elution pattern than the other viruses; it was retained by clean sand and even more strongly by ZVI, resulting in 2–3 orders of magnitude lower eluted concentrations of Ad41. The data illustrate some differences between the viruses and phages; e.g., in chlorine sensitivity and transport behavior through sandy media. While MS2 and /X174 have been shown to be representative or conservative surrogates for viral pathogens in certain treatment processes such as (enhanced) coagulation [30,31] their suitability as surrogates may require further investigation with respect to chlorination and sand filtration and may also depend on the target virus in question.
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The results demonstrate clearly that ZVI could remove pathogenic viruses and phages from real drinking water at high efficiencies. This finding, together with the fact that ZVI and its corrosion products (i.e., iron oxides) can remove natural organic matter from water [9,32], suggests that ZVI may be used in water treatment plants to help control microbe and DBP simultaneously. As a specific example, ZVI particles may be added to filtration basins as another granular medium (in addition to sand, anthracite coal, and gravel). This simple application could enhance removal of viruses and bacteria [33] as well as chemical pollutants such as arsenic and lead [7,8]. And if filtration with ZVI takes place prior to chlorination or chloramination, ZVI may also help to remove organic precursors of DBPs, an approach that is considered most effective to prevent DBP formation [34]. Furthermore, via organic precursor removal, the proposed ZVI application may offer the added benefits of sustained chlorine residuals and lower DBP levels in distribution systems [34]. As another example, treatment plants that chlorinate groundwater directly without coagulation may derive similar benefits by passing the water through a granular filter containing ZVI first before chlorination. These applications would not require high capital investments and would reduce both microbes and toxic byproducts. Thus, ZVI may offer a simple, inexpensive, and low-risk alternative (to UV, ozone, and membrane filtration) to help water utilities meet the multiple treatment goals – an option that may be particularly attractive to medium- and small-size plants. This study provides a basis for subsequent, larger-scale studies that are necessary to fully evaluate/demonstrate the feasibility and cost-effectiveness of using ZVI in water treatment plants. Acknowledgments The authors thank Water Research Foundation (WateRF) for funding this study. Thanks also go to Liping Zhang, Jennifer Handlin, Ramesh Attinti, and Jodie Han for their work and technical assistance. We appreciate Drs. Hsiao-wen Chen, Nicholas Ashbolt, Vincent Hill, and Timothy Straub for their helpful suggestions, and Peerless Metal Powders and Abrasive for providing iron samples for this study. Finally, we thank Allentown Water Filtration Plant (PA), Newark Water Treatment Plant (DE), and Los Angeles County Sanitation Districts (CA) for providing facility tours, water samples, and water quality data. References [1] T.A. Bartrand, M. Weir, C.N. Haas, Advancing the quality of drinking water: expert workshop to formulate a research agenda, Environ. Eng. Sci. 24 (2007) 863–872. [2] S.E. Hrudey, Chlorination disinfection by-products, public health risk trade-offs and me, Water Res. 43 (2009) 2057–2092. [3] S.W. Krasner, H.S. Weinberg, S.D. Richardson, S.J. Pastor, R. Chinn, M.J. Sclimenti, G.D. Onstad, A.D. Thruston Jr., Occurrence of a new generation of disinfection byproducts, Environ. Sci. Technol. 40 (2006) 7175–7185. [4] Remediation Technologies Development Forum (RTDF). (accessed 24.03.11). [5] A.D. Henderson, A.H. Demond, Long-term performance of zero-valent iron permeable reactive barriers: a critical review, Environ. Eng. Sci. 24 (2007) 401– 423. [6] Y. You, J. Han, P.C. Chiu, Y. Jin, Removal and inactivation of waterborne viruses using zero-valent iron, Environ. Sci. Technol. 39 (2005) 9263–9269. [7] N. Melitas, M. Conklin, J. Ferrell, Electrochemical study of arsenate and water reduction on iron media used for arsenic removal from potable water, Environ. Sci. Technol. 36 (2002) 3188–3193.
[8] S.M. Ponder, J.G. Darab, T.E. Mallouk, Remediation of Cr(VI) and Pb(II) aqueous solutions using supported, nanoscale zero-valent iron, Environ. Sci. Technol. 34 (2000) 2564–2569. [9] A.B.M. Giasuddin, S.R. Kanel, H. Choi, Adsorption of humic acid onto nanoscale zerovalent iron and its effect on arsenic removal, Environ. Sci. Technol. 41 (2007) 2022–2027. [10] C.P. Gerba, J.B. Rose, Viruses in source and drinking water, in: G.A. McFeters (Ed.), Drinking Water Microbiology: Progress and Recent Developments, Springer, New York, 1990, pp. 380–396. [11] Y. Jin, M. Flury, Fate and transport of viruses in porous media, Adv. Agron. 77 (2002) 39–102. [12] J.N. Ryan, R.W. Harvey, D. Metge, M. Elimelech, T. Navigato, A.P. Pieper, Field and laboratory investigations of inactivation of viruses (PRD1 and MS-2) attached to iron oxide-coated quartz sand, Environ. Sci. Technol. 36 (2002) 2403–2413. [13] US Environmental Protection Agency, National Primary Drinking Water Regulations: Ground Water Rule, Final Rule, Federal Register 71/216, EPA 40 CFR Parts 9, 141 and 142, Washington, DC, November 2006. [14] O.P. Mehra, M.L. Jackson, Iron oxide removal from soils and clays by a dithionite-citrate system buffered with sodium bicarbonate, in: Clays and Clay Minerals, Proceedings of the Seventh National Congress, Washington, DC, Pergamon, London, 1960, pp. 317–327. [15] T. Yamashita, K. Sakae, H. Tsuzuki, Y. Suzuki, N. Ishikawa, N. Takeda, T. Miyamura, S. Yamazaki, Complete nucleotide sequence and genetic organization of Aichi virus, a distinct member of the Picornaviridae associated with acute gastroenteritis in humans, J. Virol. 72 (1998) 8408–8412. [16] M. Kitajima, E. Haramoto, C. Phanuwan, H. Katayama, Prevalence and genetic diversity of Aichi viruses in wastewater and river water in Japan, Appl. Environ. Microbiol. 77 (2011) 2184–2187. [17] D.Y. Oh, P.A. Silva, B. Hauroeder, S. Diedrich, D.D.P. Cardoso, E. Schreier, Molecular characterization of the first Aichi viruses isolated in Europe and in South America, Arch. Virol. 151 (2006) 1199–1206. [18] J.A. Snowdon, D.O. Cliver, Coliphages as indicators of human enteric viruses in groundwater, Crit. Rev. Environ. Control 19 (1989) 231–249. [19] IAWPRC Study Group on Health Related Water Microbiology, Bacteriophages as model viruses in water quality control, Water Res. 25 (1991) 529–545. [20] P. Gehringer, H. Eschweiler, H. Leth, W. Pribil, S. Pfleger, A. Cabaj, T. Haider, R. Sommer, Bacteriophages as viral indicators for radiation processing of water: a chemical approach, Appl. Radiat. Isot. 58 (2003) 651–656. [21] J. Vanduin, The single-stranded RNA bacteriophages, in: R. Calendar (Ed.), The Bacteriophages, vol. 1, Plenum, New York, 1988, pp. 117–167. [22] K.S. Zerda, Adsorption of viruses to charge-modified silica, Ph.D. Diss., Baylor College of Medicine, Houston, TX, 1982. [23] H.W. Ackerman, M.S. Dubow, Viruses of Prokaryotes, vol. 2, CRC, Boca Raton, FL, 1987. pp. 190–191. [24] G. Ko, N. Jothikumar, V.R. Hill, M.D. Sobsey, Rapid detection of infectious Adenoviruses by mRNA real-time RT-PCR, J. Virol. Methods 127 (2005) 148– 153. [25] G. Ko, T.L. Cromeans, M.D. Sobsey, UV inactivation of Adenovirus type 41 measured by cell culture mRNA RT-PCR, Water Res. 39 (2005) 3643– 3649. [26] M.H. Adams, Bacteriophages, Interscience Publishers, New York, 1959. pp. 1–4. [27] M. Bedner, W.A. MacCrehan, G.R. Helz, Making chlorine greener: investigation of alternatives to sulfite for dechlorination, Water Res. 38 (2004) 2505–2514. [28] R. Attinti, J. Wei, K. Kniel, J.T. Sims, Y. Jin, Virus’ (MS2, /X174, and Aichi) attachment on sand measured by atomic force microscopy and their transport through sand columns, Environ. Sci. Technol. 44 (2010) 2426–2432. [29] A.-L. Favier, G. Schoehn, M. Jaquinod, C. Harsi, J. Chroboczek, Structural studies of human enteric Adenovirus type 41, Virology 293 (2002) 75–85. [30] B.K. Mayer, H. Ryu, M. Abbaszadegan, Treatability of US Environmental Protection Agency contaminant candidate list viruses: removal of Coxsackievirus and Echovirus using enhanced coagulation, Environ. Sci. Technol. 42 (2008) 6890–6896. [31] M. Abbaszadegan, B.K. Mayer, H. Ryu, N. Nwachuku, Efficacy of removal of CCL viruses under enhanced coagulation conditions, Environ. Sci. Technol. 41 (2007) 971–977. [32] B. Gu, J. Schmitt, Z. Chen, L. Liang, J.F. McCarth, Adsorption and desorption of natural organic matter on iron oxide: mechanisms and model, Environ. Sci. Technol. 28 (1994) 38–46. [33] A. Derevianko, Effect of zero-valent iron on the removal of Escherichia coli O157:H7 from water, M.S. Thesis, University of Delaware, Newark, DE, 2008. [34] I. Kristiana, C. Joll, A. Heitz, Powdered activated carbon coupled with enhanced coagulation for natural organic matter removal and disinfection by-product control: application in a western Australian water treatment plant, Chemosphere 83 (2011) 661–667.
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Separation and Purification Technology journal homepage: www.elsevier.com/locate/seppur
Removal of viruses and bacteriophages from drinking water using zero-valent iron Chunjian Shi a, Jie Wei b, Yan Jin c, Kalmia E. Kniel b, Pei C. Chiu a,⇑ a
Department of Civil and Environmental Engineering, University of Delaware, Newark, DE 19716, United States Department of Animal and Food Sciences, University of Delaware, Newark, DE 19716, United States c Department of Plant and Soil Sciences, University of Delaware, Newark, DE 19716, United States b
a r t i c l e
i n f o
Article history: Available online 2 July 2011 Keywords: Zero-valent iron Virus Water treatment Adenovirus Aichi virus Drinking water
a b s t r a c t Zero-valent iron (ZVI) has been shown to remove and inactivate bacteriophages in synthetic groundwater, which suggests that it may be potentially useful for drinking water treatment. As an effort to assess this possibility, we assessed the effectiveness of ZVI to remove pathogenic viruses from two water treatment plant samples (from Allentown, PA and Newark, DE) through a series of column experiments. Removal of Aichi virus (AiV), Adenovirus 41 (Ad41), and the bacteriophages MS2 and /X174 in columns packed with clean sand only and sand with a layer of ZVI-sand mix (1:1, v/v) was examined through pulse tests under saturated flow conditions. With the exception of Ad41, removal of all viruses by sand was zero or limited in all cases, regardless of the water used. In contrast, ZVI-containing columns gave removal efficiencies between 4.5 and 6 logs for all viruses and all waters studied. Ad41 was retained markedly by sand and even more strongly (by 2–3 logs) by ZVI and did not breakthrough fully from either column even after 32 pore volumes. The significant retardation of Ad41 is ascribed to the relatively high isoelectric points of the long and short fibers of Ad41. Our results, combined with studies elsewhere that demonstrate organic matter removal by ZVI and iron oxides, suggest ZVI may potentially offer a simple and low-cost option to help utilities control microorganisms, disinfection byproducts, and chlorine residuals simultaneously. Possible applications of ZVI in water treatment plants are proposed and the potential benefits are discussed. Ó 2011 Elsevier B.V. All rights reserved.
1. Introduction Quantity and quality of drinking water have been recognized as increasingly critical issues for the coming decades. Among the factors that contribute to the looming water crisis are continued population growth and urbanization, deteriorating water infrastructure, increasing influence of wastewater and biosolids on drinking water sources, growing number of emerging contaminants, and uncertain future water availability and quality due to climate change. It has been questioned recently [1] whether the conventional water treatment processes, which are based on rapid sand filtration and chlorination and have stayed largely unchanged for decades, can adequately remove the many chemical and microbial contaminants simultaneously. Among the problems facing water utilities and regulators, one particularly daunting challenge is how to control microbial pathogens, disinfection by-products (DBPs) and residual disinfectants simultaneously – and to do so at an acceptable cost. On the one hand, high dosage of chlorine can produce high chlorine residuals and high levels of DBPs, including trihalomethanes, haloacetic acids, and haloacetonitriles [2]. On the other hand, low chlorine ⇑ Corresponding author. Tel.: +1 302 831 3104; fax: +1 302 831 3640. E-mail address: [email protected] (P.C. Chiu). 1383-5866/$ - see front matter Ó 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.seppur.2011.06.036
dosage may provide insufficient protection of distribution systems and human health against microbial pathogens. While alternative disinfection methods are available; e.g., UV, membrane processes (e.g., reverse osmosis and nano-filtration), ozone, and chloramines, some of them require significant capital investment and plant modifications or are expensive to install and/or difficult to operate. Ozone and chloramines also produce DBPs of great health concerns, including N-nitrosodimethylamine, bromate, and bromoand iodo-acids [3]. It is thus highly desirable to develop a simple, effective, inexpensive, and non-oxidant-based method to help control microorganisms, DBPs, and chlorine residuals in drinking water – simultaneously. This is particularly important for midand small-size treatment plants, which may not have the necessary resources to readily invest in or migrate to another disinfection method in response to increasing regulatory demand and/or deteriorating source water quality. One method that may potentially achieve these multiple goals is zero-valent iron (ZVI). ZVI has been used to remediate contaminated groundwater for over 15 years [4] and has been shown to remain reactive in anaerobic subsurface for multiple years [5]. While initially used to treat chlorinated solvents and hexa-valent chromium, ZVI has also been shown over the last two decades to remove a broad range of chemical contaminants, from heavy metals and radionuclides to agrochemicals and explosives. To date,
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application of ZVI has been limited to treating chemical pollutants in groundwater. For drinking water, however, the contaminants of primary concern are microbial. In 2005, You et al. [6] demonstrated that ZVI, when added to a sand filter, removed the bacteriophages MS2 and /X174 at efficiencies between 5 and 6 logs. The removal was ascribed to adsorption of these phages to iron (hydr)oxides formed via anaerobic corrosion of ZVI. You et al. also showed that removal of these viruses by ZVI was rapid (most likely mass transfer-limited), and that most of the viruses removed were either inactivated or irreversibly bound. In addition, ZVI is capable of removing many other pollutants relevant to drinking water, including arsenic [7], lead [8], and natural organic matter (NOM), a major precursor of DBPs [9]. These findings suggest ZVI may be useful for drinking water treatment and can potentially offer multiple benefits. For example, if used in a water treatment plant ZVI can remove NOM, arsenic and microorganisms before chlorine addition – a strategy that may help meet multiple treatment goals while preventing excessive DBP formation. The work by You et al. [6] was conducted with two model viruses in synthetic groundwater. To further assess the feasibility of ZVI for drinking water treatment, this study was carried out to evaluate the ability of ZVI to remove pathogenic viruses in real water treatment plant samples. Viruses were targeted because they are particularly problematic due to their small size (20–150 nm), high mobility in porous media, and resistance to filtration and chlorination relative to bacteria [10,11]. Viruses are believed to be responsible for the majority of the disease outbreaks for which the infectious agents are identifiable [12]. Viruses are also the target microorganisms of the Ground Water Rule, which took effect in 2007 [13]. In this study, we performed a series of flow-through column experiments to examine the ability of ZVI to remove two pathogenic viruses, Aichi virus and Adenovirus 41, and two bacteriophages /X174 and MS2, from water samples collected from Newark Water Treatment Plant (NWTP) in Newark, DE and Allentown Water Filtration Plant (AWFP) in Allentown, PA. These viruses have been linked to many water- and food-borne disease outbreaks or used as surrogates for human enteric viruses. We will discuss the results and their implications for water treatment and propose potential applications of ZVI in water treatment plants. 2. Materials and methods 2.1. Materials Virus removal experiments were conducted using pairs of acrylic columns. Eighteen identical columns (3.8 cm i.d. 10 cm, bed volume = 113 cm3) were fabricated at the University of Delaware in the Engineering Machine Shop. Each column was wet-packed with sand only (‘‘sand column’’) or both sand and ZVI (‘‘ZVI column’’). The ZVI used for all experiments was commercial iron granules ETI850/50 from Peerless Metal Powders and Abrasive (Detroit, MI). The specific surface area of the Peerless iron was 1.67 m2/g, as measured by the Brunauer–Emmett–Teller (BET) adsorption method with N2. The iron was sieved, and the size fraction 60–35 mesh (250–500 lm) was used for column packing. We used Accusand (Unimin, Le Sueur, MN) with the following size distribution: 9% 0.1–0.25 mm, 70% 0.25–0.5 mm, and 21% 0.5– 1.0 mm. The sand was treated with sodium citrate and dithionite to remove organic and metal oxide coatings before use [14]. 2.2. Viruses The viruses used for this study were Aichi virus (AiV), Adenovirus 41 (Ad41), and the bacteriophages MS2 and /X174. AiV, a
73
member of the family Picornaviridae, was first isolated in 1989 in Aichi, Japan [15]. AiV is prevalent in wastewater and river water [16] and has caused many cases of illness in Europe, Asia and South/Central America [17]. Adenoviruses are emerging contaminants on US EPA’s Contaminant Candidate List (CCL3). Ad41 is representative of the approximately 42 different serotypes of adenoviruses causing human disease from conjunctivitis and respiratory infection to hepatic disease and gastroenteritis (e.g., infantile diarrhea). In addition, we included two bacteriophages, MS2 and /X174, in this study. These phages structurally resemble many human enteric viruses and have been widely used as surrogates [18–20]. MS2 is an icosahedral, single-stranded RNA phage with a diameter of 26 nm [21] and an isoelectric point (pHisp) of 3.9 [22]. /X174 is a single-stranded DNA phage, 23 nm in diameter, with a pHisp of 6.6 [23]. AiV was cultured on Vero cells (ATCC # CCL-81TM) originated from Africa green monkey kidney in Eagle’s minimal medium (Mediatech, VA) containing either 2% (for maintenance) or 10% (for growth) fetal bovine serum (FBS, Hyclone, TX), 2% sodium bicarbonate, 1% sodium pyruvate, 1% penicillin/streptomycin/ ampicillin (Mediatech), and 1% non-essential amino acids (Mediatech). AiV in infected cell lysates was purified by one freeze–thaw cycle, followed by centrifugation at 2500g for 15 min. The upper aqueous layer was recovered and stored at 80 °C for analysis. Virus infectivity was determined using the tissue culture infectious dose (TCID50) assay. AiV was inoculated into cell mono-layers grown in 96 wells plates, serially diluted with Hank’s Buffered Salt Solution (HBSS, Mediatech) and incubated for 2 h before applying medium containing 2% FBS. The plates were incubated for 3–5 days for cytopathic effects, and virus titer was obtained according to the Reed and Meunch calculation. Ad41 (ATCC # VR-930™) was obtained from ATCC and cultured on human embryonic kidney (HEK) 293 cells (ATCC # CRL-1573) in Eagle’s Minimal Medium containing 10% fetal bovine calf serum. Ad41 in infected cell lysates were purified by one freeze–thaw cycle followed by centrifugation at 2500g for 15 min. The upper aqueous layer was recovered and stored at 80 °C for further analysis. The titer of Ad41 was estimated by quantal endpoint assay employing 10-fold serial dilutions and using PCR to amplify viral DNA to obtain the most probable number (MPN) [24]. The Ad41 stock titer was approximately 6.7 105 viral particles/mL. Ad41 was measured by quantitative PCR (qPCR). The primer pair for qPCR, Ad41F/R, was designed to target the hexon region, amplifying a 135-bp fragment of adenoviruses of species F (Ad40 and Ad41) [25]. The sequences of Ad41F and Ad41R are 50 -GGACGCCTCGGAGTACTGA30 and 50 -CGCTGIGACCIGTCTGTGG-30 , respectively. To generate a standard curve, 1 mL Ad41 (6.7 105 VP/mL) was added to AGW and vortexed. The solution was then 10-fold diluted serially with HBSS to 67 VP/mL, and DNA was extracted from all dilutions using a DNeasy Tissue Kit following the manufacturer’s instructions (Qiagen, CA). The DNA was applied to qPCR with Qiagen SYBR Green (Qiagen, CA) to generate the standard curve. The qPCR was performed in 25-lL volumes containing 12.5 lL of 2x SYBR Green Mix, 1.5 lL of primer Ad41F/R, 4 lL of cDNA, and 5.5 lL of H2O, using ABI 7900HT system (Applied Biosystems, CA). The amplification cycle was 95 °C for 15 min, 40 cycles of 94 °C for 15 s, 60 °C for 30 s, and 72 °C for 30 s, followed by a dissociation step of 95 °C for 15 s, 60 °C for 15 s, and 95 °C for 15 s. MS-2 was obtained from the American Type Culture Collection (ATCC 15597B1) and grown on bacterial lawns of Escherichia coli (ATCC 15597). /X174 was grown on an E. coli host (ATCC 13706). MS2 and /X174 were assayed using the double-agar overlay method [26]. Briefly, 1 mL of host culture and 0.5 mL of diluted virus sample were added to a trypticase soy agar (TSA) tube and the mixture poured onto a TSA plate. The plates were solidified for 15 min and placed in a 37 °C incubator for 5 h and 12 h for /X174 and
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MS2, respectively. Viable (i.e., infective) phage concentrations were determined by counting the plaques in the host lawn and reported as plaque-forming units per mL (pfu/mL). Only dilutions that result in between 10 and 300 plaques per plate were accepted/used (i.e., limit of quantification = 10 pfu/plate or 20 pfu/mL). All assays were run in duplicates, and data were accepted only if the duplicates agreed within 20%. 2.3. Water samples The waters tested in this study included samples from Newark Water Treatment Plant (NWTP) in Newark, DE and Allentown Water Filtration Plant (AWFP) in Allentown, PA. The NWTP and AWFP samples, both collected in late November, 2008, were effluents from filtration tanks prior to final chlorination. These samples were used shortly after collection and were stored in a 5 °C constant-temperature room before use and between experiments. Because AWFP pre-chlorinates their water while NWTP does not, the AWFP water contained a low level of chlorine. Table 1 gives water quality of the treatment plant samples. For comparison, artificial groundwater (AGW) was also used in this study. The AGW contained 0.075 mM CaCl2, 0.082 mM MgCl2, 0.051 mM KCl, and 1.5 mM NaHCO3, giving an ionic strength of 2 mM. AGW was prepared fresh, autoclaved, vacuum-degassed, and pH-adjusted to between 7.0 and 7.4. Virus input solution for each experiment was prepared by diluting an aliquot of virus stock of desirable titer in a treatment plant water or AGW to yield the target virus concentration. 2.4. Experimental methods In a typical experiment, a pair of columns were wet-packed through incremental addition of sand (and ZVI) in AGW. This was done carefully to ensure that columns were free of air bubbles. The sand column (control) was packed with cleaned sand only, whereas the ZVI column consisted of a 3-cm layer of 1:1 (v/v) ZVI-sand mix sandwiched by two 3.5-cm layers of cleaned sand, giving an overall ZVI content of 15% (v/v, solid basis). The pore volume (PV) of each column was calculated by subtracting the volume of sand/ZVI from the total void volume or estimated based on the volume of water used during packing. The PVs of typical sand and ZVI columns were 38.5 ± 0.5 and 45.0 ± 1.0 mL, respectively. Given the column bed volume of 113.4 cm3, the corresponding porosities were approximately 34% for sand columns and 40% for ZVI columns. Columns were operated under saturated and continuous up-flow conditions at a rate of 0.73 ± 0.03 mL/min regulated by a peristaltic pump. The columns were first flushed with 10 PVs of background solution (a treatment plant water or AGW) at 0.5 mL/min; the flow rate was then increased to 0.73 mL/min for another hour to establish a steady flow prior to virus introduction. Based on the PVs, flow rate, and ZVI content, the effective iron contact time in the ZVI column was 9.3 ± 0.3 min. Uniform flow conditions for columns prepared using these procedures have been verified by bromide tracer tests [6]. Fig. 1 illustrates the set-up for a typical column experiment. To initiate a virus removal experiment, the influent was switched at time zero from a virus-free background solution to a
Table 1 Properties of the NWTP and AWFP water samples.
NWTP AWFP
pH
TOC (mg/L)
EC (ms/cm)
Alkalinity (mg/L)
Total chlorine (mg/L)
7.20 7.72
1.50 1.02
4.5 6.4
58 190
– 0.99
virus input solution. Virus solution was introduced into both sand and ZVI columns for 5 PVs (based on the ZVI column), the input was switched back to the virus-free solution, and pumping was continued for another 5–27 PVs depending on the rate of virus breakthrough. Starting at time zero, sand and ZVI column effluents were collected in 5-mL glass tubes using fraction collectors (Fig. 1). A typical experiment for which 10 PVs of effluent was collected would generate 90–100 tubes of samples from each column. Samples were analyzed for target virus, using the infectivity or qPCR assays described above to obtain a virus breakthrough curve for each column. Samples were analyzed on the same day or as soon as feasible and were stored in a freezer before analysis. Additional samples of the virus input solution were collected at the end of experiment for analysis to ensure minimal virus inactivation during experiment. 3. Results and discussion 3.1. Effects of chlorine and Na2SO3 on MS2, /X174 and AiV Before starting column experiments, a preliminary test was performed to determine whether the AWTP sample should be dechlorinated before use. This was because the residual chlorine (1 ppm) in the sample might cause excessive inactivation of viruses and/or increase ZVI oxidation [27]. To evaluate the effects of chlorine and the dechlorinating agent, Na2SO3 on /X174, MS2, and AiV, an aliquot of each virus stock was added separately to AGW, as-received AWFP water, AWFP water with a low dose of Na2SO3 (stoichiometric to chlorine), and AWFP water with a high dose of Na2SO3 (2x stoichiometric dose). The samples were incubated at 5 °C and analyzed for the viruses after 20 min and 8 h of incubation. Result of this test is summarized in Table 2. The three viruses were stable at 5 °C in AGW even after hours. In contrast, the phages and AiV were completely and moderately inactivated, respectively, in the AWFP water. When the water was dechlorinated with stoichiometric amount of Na2SO3, limited or no virus inactivation was observed. This suggests that the residual chlorine was responsible for the observed inactivation. It also indicates that after Na2SO3 addition, the AWFP sample would be suitable for the study as excessive background inactivation of viruses would not be a concern. Finally, when Na2SO3 was added in excess, it did not appear to have any adverse effect on the three viruses, suggesting that any residual Na2SO3, if present in dechlorinated AWFP water, would not influence virus removal results. Based on the data in Table 2, all AWFP water was pretreated with Na2SO3 before use. 3.2. Removal of MS2, /X174, and AiV by sand and ZVI Separate pairs of sand and ZVI columns were set up to assess removal of viruses in the NWTP water, the AWFP water, and AGW. To establish a baseline for comparison, we first ran two pulse tests with /X174 and MS2 in AGW. The removal efficiencies for these phages by the ZVI column were 5.80 logs (99.9998%) and 5.60 logs (99.9997%), respectively, consistent with the results of You et al. [6]. Additional pulse tests were conducted with these phages in the NWTP sample, and similar removal by an ZVI column – 5.92 logs for /X174 and 5.67 logs for MS2 – was obtained. This indicates that ZVI can remove these indicator viruses from real water treatment plant samples at high efficiencies within 9–10 min of iron contact time. High removal by ZVI was also observed for dechlorinated AWFP water: 5.22 logs for /X174 and 5.16 logs for MS2. In all the above experiments, virtually no virus removal was observed for any of the sand columns (Table 3), and hence the removal could be attributed entirely to the 15% iron in each ZVI column. An
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Fig. 1. Setup of a typical virus removal experiment. Influent (AGW or a water treatment plant sample) was supplied by a peristaltic pump to both the sand (left) and ZVI (right) columns in an upward flow direction. Effluents from these columns were collected separately by two fraction collectors, for 10 PVs for /X174, MS2, and AiV, and for 10–32 PVs for Ad41.
Table 2 Effects of chlorine and Na2SO3 on virus infectivity in AWFP water sample. [Cl2] (mg/L)
[Na2SO3] (mg/L)
– 0.99 – –
0 0 1.76 3.52
[/X174] (pfu/mL)
[MS2] (pfu/mL)
[AiV] (TCID50/mL)
525,000 0 589,000 427,000
21,400 6200 23,700 28,800
20 min AGW AWFP Low SO3 High SO3
577,000 0 555,000 545,000
[/X174] (pfu/mL)
[MS2] (pfu/mL)
[AiV] (TCID50/mL)
479,000 0 501,000 363,000
19,100 3800 17,500 21,400
8h
example illustrating how these removal efficiencies were calculated is described below. Fig. 2(a) shows a typical virus breakthrough curve for a sand column, exemplified by /X174 in AWFP water. The effluent virus concentration reached the input concentration slightly after 1 PV and remained constant for approximately 5 PVs before declining. The virus removal efficiency was taken to be one minus the ratio of the integrated area under the breakthrough curve (on a normal–normal plot) to the total virus count in 5 PVs of input solution. For this and all other sand column breakthrough experiments with /X174 and MS2 in AGW, the AWFP water, and the NWTP water, the calculated virus removal was very close to zero (Table 3). That is, clean sand was feckless at removing these phages for all three waters tested. Fig. 2(a) also contains four data points of non-zero
589,000 0 563,000 554,000
(albeit below detection limit) /X174 concentrations in the ZVI column effluent. All other ZVI effluent samples yielded no plaque. In fact, in all the phage removal tests performed using any of the three water matrices, the majority of ZVI effluent samples gave either zero or below-detection-limit plaque counts. Since in most cases there were insufficient countable plates to construct a full breakthrough curve, removal efficiencies for ZVI columns were calculated using either actual plaque counts of all readable effluent samples or the detection limit (i.e., 20 pfu/mL) over 10 PVs. Table 3 shows the two sets of ZVI column removal efficiencies, which differ only by 0.3 log or less in most cases. The phage removal experiments were followed by an AiV pulse test in the AWFP water. The AiV stock was concentrated to remove organic substrates in the medium by centrifugation at 13,000g for
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Table 3 Log removal of /X174, MS2 and AiV from AGW and NWTP and AWFP waters. The negative sand column removal efficiencies (in parentheses) are due to slightly smaller integrated area under sand column breakthrough curve than the calculated total virus input and are set to be zero. The two sets of ZVI column efficiencies were calculated based on actual infectivity data of all readable effluent samples (efficiency 1) and the detection limit (20 pfu/mL or 10 TCID50/mL, efficiency 2).
/X174 (AGW) MS2 (AGW) /X174 (NWTP) MS2 (NWTP) /X174 (AWFP) MS2 (AWFP) AiV (AWFP) /X174 (AWFP) MS2 (AWFP) *
Input concentration (pfu/mL or TCID50/mL)
Sand column efficiency
ZVI column efficiency 1
ZVI column efficiency 2
6.03 106 4.27 106 7.94 106 4.68 106 2.51 106 1.23 106 0.98 106 1.02 106 1.05 106
0 (0.03) 0 (0.02) 0 (0.04) 0 (0.01) 0 0 0.83 0.02 0.06
5.80 5.60 5.92 5.67 5.22 5.16 –* 5.01 5.02
5.48 5.33 5.60 5.37 5.10 4.79 4.99 4.71 4.72
A log removal could not be calculated since the efficiency was 100% based on actual assay data (i.e., all ZVI column effluents samples were non-infective).
Fig. 2. Breakthrough concentrations of (a) /X174 in sand and ZVI column effluents and (b) AiV in sand column effluent. The water matrix was dechlorinated AWFP sample in both cases. The detection limits were 10 pfu/plate (or 20 pfu/mL) for /X174 and 10 TCID50/mL for AiV. No AiV was detected in any of the ZVI column effluent samples (thus no data are shown in (b)).
10 min using 100,000-MW centricon (Millipore, MA). The filtrate was discarded and AiV was recovered from the filter membrane
with HBSS. The AiV breakthrough curve for the sand column is shown in Fig. 2(b). Interestingly, in contrast to the results for the
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100000
Effluent Ad41 Concentration (VP/mL)
two phages, the sand column was able to remove 0.83 log of AiV in the AWFP water. The higher removal might be due to stronger electrostatic interactions between AiV and sand surface; however, this cannot be confirmed as the pHisp of AiV has not been reported to our knowledge. In contrast to the sand column result, no viable AiV was detected in the ZVI column effluent (i.e., all effluent samples were non-infective). Using the conservative assumption that the AiV concentrations in all ZVI effluent samples were equal to the detection limit (10 PV/mL), we obtained a removal efficiency of 5.0 logs for AiV in the AWFP sample. Virus removal efficiencies for the experiments described above and additional experiments are summarized in Table 3. The sand column efficiencies for /X174, MS2, and AiV were limited (<1 log) in all cases, consistent with a recent study [28]. In contrast, the ZVI column efficiency was between 4.5 and 6 logs in all three water matrices tested, regardless of whether the calculation was based on actual plaque counts or detection limit. It should be noted that, due to limited breakthrough and low (often below-detection) effluent concentrations, the calculated ZVI column efficiencies depended strongly on the input concentration; i.e., the removal efficiencies would have been greater had higher influent virus titers been used.
Sand Column ZVI Column
10000
Detection Limit (5 VP/mL) Input Conc. (74,000 VP/mL)
1000
100
10
1
0
1
2
3
4
5
6
7
8
9
10
Pore Volume (PV) Fig. 3. Elution concentration profiles of Ad41 for sand and ZVI columns. The upper dash line corresponds to the input concentration of Ad41, 74,000 VP/mL. The lower dash line represents the qPCR detection limit of 5 PV/mL.
3.3. Removal of Ad41 by sand and ZVI Similar experiments were also conducted with Ad41, but the results are not included in Table 3, because the removal efficiencies could not be obtained in the same manner. Unlike /X174, MS2 and AiV, Ad41 exhibited a very different breakthrough behavior for both sand and ZVI columns, as shown in Fig. 3. In contrast to the other viruses, Ad41 was retarded more strongly in the sand column and did not break through fully within 10 PVs. The effluent concentration started to rise at approximately 1 PV and continued to increase gradually but never reached the input concentration (74,000 VP/mL). The mass recovery, based on the integrated area underneath the sand column breakthrough curve, was only 7.5%. By comparison, the higher removal of Ad41 by ZVI was clear. The Ad41 concentration in ZVI column effluent remained low throughout the experiment, hovering over the detection limit (5 VP/mL). Despite the incomplete breakthrough and low mass recovery, we made an attempt to estimate Ad41 removal by ZVI. Based on the ratio of the two integrated areas (i.e., virus counts) under the ZVI and sand column breakthrough curves over 10 PVs, the efficiency of Ad41 removal by the 15% iron in the ZVI column was 2.6 and 2.7 logs, as estimated using the actual qPCR data and the detection limit, respectively. These numbers are consistent with a visual inspection of the data in Fig. 3, where the difference in eluted Ad41 concentrations from the ZVI and sand columns was between 2 and 3 logs. Because of the unusual breakthrough pattern and slow and incomplete elution of Ad41, we repeated the experiment using a similar influent concentration (75,500 VP/mL) but collected 32 PVs of effluent samples instead of 10. The same breakthrough pattern was observed as in the first experiment: Ad41 was retarded by sand and even more markedly by ZVI, and its elution from both columns was slow and incomplete, even after 32 PVs (35 h). The pronounced retention/removal of Ad41 by sand has important implications for its fate and transport in the subsurface and in treatment systems. Although Ad41 is larger than /X174 and MS2 (90 nm vs. 23–26 nm), it is still far smaller than the pores between sand and ZVI grains, which are on the order of a few hundred lm; therefore, the retention mechanism was most likely chemical rather than physical. While the pHisp values of the core and hexon proteins of Ad41 are unavailable, its penton base protein (located at the 12 vertices of the capsid) has a pHisp of 5.5 [29], which does not explain the strong retention of Ad41 by sand
as both would be negatively charged at circum-neutral pH. However, in contrast to the phages, Ad41 also contains long (34 ± 1.3 nm) and short (20 ± 1.3 nm) fibers that mediate virus attachment to the host cell [29]. Both the long and short fibers of Ad41 have relatively high pHisps (7.51 and 9.13, respectively), which were suggested to be responsible for an erratic behavior of the fiber protein on denaturing polyacrylamide gels [29]. It is plausible that these fibers, rather than the capsid, control how Ad41 particles interact with surfaces. The positively charged fibers might interact favorably with sand and iron surfaces, resulting in the significant retention and slow elution of Ad41 we observed.
4. Conclusions and implications This study extends the work of You et al. [6] by including pathogenic viruses and real water treatment plant samples in the assessment of ZVI for water treatment purposes. The main results are summarized as follows: 1. At ppm levels, chlorine effectively inactivated MS2 and /X174, and to a lesser extent AiV, while Na2SO3 had no observable adverse effects on these viruses. 2. Clean sand alone achieved limited or no removal of MS2, /X174, and AiV, regardless of whether AGW, AWFP water, or NWTP water was used. 3. In contrast, when a sand column was amended with 15% ZVI, removal efficiencies of 4.5–6 logs were achieved for all three viruses and three waters tested. 4. Ad41 exhibited a markedly different elution pattern than the other viruses; it was retained by clean sand and even more strongly by ZVI, resulting in 2–3 orders of magnitude lower eluted concentrations of Ad41. The data illustrate some differences between the viruses and phages; e.g., in chlorine sensitivity and transport behavior through sandy media. While MS2 and /X174 have been shown to be representative or conservative surrogates for viral pathogens in certain treatment processes such as (enhanced) coagulation [30,31] their suitability as surrogates may require further investigation with respect to chlorination and sand filtration and may also depend on the target virus in question.
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The results demonstrate clearly that ZVI could remove pathogenic viruses and phages from real drinking water at high efficiencies. This finding, together with the fact that ZVI and its corrosion products (i.e., iron oxides) can remove natural organic matter from water [9,32], suggests that ZVI may be used in water treatment plants to help control microbe and DBP simultaneously. As a specific example, ZVI particles may be added to filtration basins as another granular medium (in addition to sand, anthracite coal, and gravel). This simple application could enhance removal of viruses and bacteria [33] as well as chemical pollutants such as arsenic and lead [7,8]. And if filtration with ZVI takes place prior to chlorination or chloramination, ZVI may also help to remove organic precursors of DBPs, an approach that is considered most effective to prevent DBP formation [34]. Furthermore, via organic precursor removal, the proposed ZVI application may offer the added benefits of sustained chlorine residuals and lower DBP levels in distribution systems [34]. As another example, treatment plants that chlorinate groundwater directly without coagulation may derive similar benefits by passing the water through a granular filter containing ZVI first before chlorination. These applications would not require high capital investments and would reduce both microbes and toxic byproducts. Thus, ZVI may offer a simple, inexpensive, and low-risk alternative (to UV, ozone, and membrane filtration) to help water utilities meet the multiple treatment goals – an option that may be particularly attractive to medium- and small-size plants. This study provides a basis for subsequent, larger-scale studies that are necessary to fully evaluate/demonstrate the feasibility and cost-effectiveness of using ZVI in water treatment plants. Acknowledgments The authors thank Water Research Foundation (WateRF) for funding this study. Thanks also go to Liping Zhang, Jennifer Handlin, Ramesh Attinti, and Jodie Han for their work and technical assistance. We appreciate Drs. Hsiao-wen Chen, Nicholas Ashbolt, Vincent Hill, and Timothy Straub for their helpful suggestions, and Peerless Metal Powders and Abrasive for providing iron samples for this study. Finally, we thank Allentown Water Filtration Plant (PA), Newark Water Treatment Plant (DE), and Los Angeles County Sanitation Districts (CA) for providing facility tours, water samples, and water quality data. References [1] T.A. Bartrand, M. Weir, C.N. Haas, Advancing the quality of drinking water: expert workshop to formulate a research agenda, Environ. Eng. Sci. 24 (2007) 863–872. [2] S.E. Hrudey, Chlorination disinfection by-products, public health risk trade-offs and me, Water Res. 43 (2009) 2057–2092. [3] S.W. Krasner, H.S. Weinberg, S.D. Richardson, S.J. Pastor, R. Chinn, M.J. Sclimenti, G.D. Onstad, A.D. Thruston Jr., Occurrence of a new generation of disinfection byproducts, Environ. Sci. Technol. 40 (2006) 7175–7185. [4] Remediation Technologies Development Forum (RTDF).
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