Removal of viruses from surface water and secondary effluents by sand filtration

water research 43 (2009) 87–96

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Removal of viruses from surface water and secondary effluents by sand filtration Revital Aroninoa, Christina Dlugya, Elizabeth Arkhangelskya, Semion Shandalovb, Gideon Oronb, Asher Brennera, Vitaly Gitisa,* a

Unit of Environmental Engineering, Faculty of Engineering Sciences, Ben-Gurion University of the Negev, PO Box 653, Beer-Sheva 84105, Israel b Zuckerberg Institute for Water Research, Ben-Gurion University of the Negev, Kiryat Sede Boker 84990, Israel

article info

abstract

Article history:

The filtration of phi X 174, MS2, and T4 bacteriophages out of tap water and secondary

Received 24 July 2008

effluents was performed by rapid sand filtration. The viruses were characterized, and the

Received in revised form

influence of their microscopic characteristics on filterability was examined by comparing

2 October 2008

retention values, residence times, attachment, and dispersion coefficients calculated from

Accepted 7 October 2008

an advection–dispersion model and residence time variation. The only factor observed to

Published online 26 October 2008

influence retention was virus size, such that the larger the virus, the better the retention. The difference was due to the more effective transport of viruses inside the media, an

Keywords:

observation that runs counter to currently accepted filtration theory. Cake formation on

Granular filtration

top of the filter during the initial stages of secondary effluent filtration significantly

Virus

increased headloss, eventually resulting in shorter filtration cycles. However, deep filters

Municipal wastewater

contain buffering zones where the pressure drop is amortized, thus allowing for continued filtration. After the effluent passed through the buffer zone, regular filtration was observed, during which considerable virus retention was achieved. ª 2008 Elsevier Ltd. All rights reserved.

1.

Introduction

Wastewater reuse has a major impact on sustainability, as it reduces environmental damage and decreases the demand made on natural freshwater sources. In arid and semiarid regions, treated effluents constitute reliable resources for water production. Municipal wastewater is usually treated by a complex process that includes primary settling, biological degradation, and secondary clarification. The secondary effluents obtained are of good quality, and they can be used to irrigate a variety of food crops, excluding salad crops and vegetables that can be eaten uncooked. The most significant risk factor preventing unrestricted irrigation with secondary

effluents is the potential presence of pathogenic microorganisms (Blumenthal et al., 2000). Notable pathogens common in secondary wastewater effluents include the environmentally resistant oocysts of Cryptosporidium parvum, cysts of Giardia lamblia, and a variety of enteric pathogenic bacteria and viruses. Due to their small sizes (and potential mobility during advective groundwater flow) and their ability to resist disinfection via chlorination (Blatchley et al., 2007) and UV (Mamane et al., 2007), viruses were the focus of this study. The quality of secondary effluents is upgraded to a level suitable for unrestricted irrigation when they undergo tertiary treatment. Tertiary effluents can also substitute for freshwater sources in household and industrial uses. Tertiary

* Corresponding author. Tel.: þ972 8 6479031; fax: þ972 8 6479397. E-mail addresses: [email protected] (R. Aronino), [email protected] (C. Dlugy), [email protected] (E. Arkhangelsky), shandalo@ bgu.ac.il (S. Shandalov), [email protected] (G. Oron), [email protected] (A. Brenner), [email protected] (V. Gitis). 0043-1354/$ – see front matter ª 2008 Elsevier Ltd. All rights reserved. doi:10.1016/j.watres.2008.10.036

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treatment often involves a sequence of coagulation/flocculation and ultrafiltration (Goren et al., 2008) that together constitute a reliable alternative to rapid sand filtration applications. Despite the significant progress that has been made in the application of membrane technologies, rapid sand filtration is an old, well-known, and reliable water treatment process that is still applicable (Metcalf and Eddy et al., 2002). In terms of value for money, rapid sand filtration remains the cheapest and most reliable application for meeting the reuse criteria of secondary effluents. Many tertiary treatment units, from both new and veteran operations, continue to use rapid sand filtration technology, but most investigations are devoted to the membrane field. A number of studies have investigated the removal of pathogens by granular media filtration in water treatment applications, with some focusing specifically on the removal of the chlorine-resistant protozoan parasites Cryptosporidium and Giardia (Nieminski and Ongerth, 1995; Gitis et al., 2005). It has also been shown that optimized granular media filtration preceded by coagulation/flocculation can effectively remove viruses (Rao et al., 1988; Nasser et al., 1995) and viral indicators such as the bacteriophage MS2 (Huck et al., 2001, Gitis et al., 2002a–c) in surface water filtration. Chemical heterogeneity was shown to be one of important factors affecting virus attachment (Abudalo et al., 2005; Bradford et al., 2004). However, less attention was focused on the filtration of enteric viruses as a tertiary treatment, although the general applicability of granular filters for the treatment of secondary effluents has already been investigated (Brenner et al., 1994; Quanrud et al., 2003; Zanetti et al., 2006). The well-known and avoidable potential complications inherent in experimenting with enteric viruses dictated that they be simulated by the bacteriophages MS2, phi X-174, and T4. The similar behavioral traits of the viruses and the phages in porous media were reported by Grabow (2001). Among the largest of the phage DNA, that of T4 is 169–170 kilobase pairs (kbp) long and located in its icosahedral head. T4 is a tailed phage of Myoviridae family with linear double-stranded DNA. The overall size of T4 is 90 nm wide by 200 nm long. Much smaller than T4, phi X 174 is a small, icosahedral bacteriophage of Escherichia coli that is 26 nm in diameter and contains a single-stranded DNA genome. The genome consists of 11 genes spread across 5386 bases in a circular topology. Finally, MS2 is an icosahedral bacteriophage of Leviviridae family 32-nm long, comprising a maturation protein and a singlestranded RNA surrounded by a protein capsid. The bacteriophages chosen for the study exhibit different sizes (from 22 to 120 nm) and morphologies (icosahedral and tailed). This study focused on two previously unaddressed issues, the first of which entails identifying a possible link between the characteristics of enteric viruses and their transport and attachment in rapid sand filtration columns. Some preliminary data on electrokinetic properties of viruses and aggregative stability of viral suspension was found in the literature (Langlet et al., 2008; Han et al., 2006; Redman et al., 1999). Yet the effects of size and hydrophobicity were not fully addressed before. Based on current filtration theories (Tien, 1989; Vigneswaran and Ben Aim, 1989), size should significantly influence the viral transport and attachment abilities.

The second aim of this research was to evaluate the influence of possible cake formation on the filtration efficiency of viruses from secondary effluents. Filters are prone to cake formation, especially when secondary effluents of unstable quality are being filtered. In cases of effluents with high initial concentrations, the influent may clog the filter during the initial stages of filtration. The question is whether under these conditions the filter will at least partially retain the viruses, or whether immediate filter maintenance is needed. No answers were found in the scientific literature to the questions posed.

2.

Experimental

2.1.

Raw effluent quality

The study was carried out intermittently from July 2005 to December 2007 at the Beer-Sheva wastewater treatment plant (in southern Israel), which typically treats 41,000 m3/day of municipal wastewater by the activated sludge process. At present, no tertiary treatment is applied, and, by definition, the secondary effluent cannot be used for unrestricted irrigation. During the study period, average raw wastewater quality was characterized by a biological oxygen demand (BOD) of 400 mg/L and a total suspended solids (TSS) value of 420 mg/L. Secondary effluents had COD levels of 68  26 mg/L, TOC of 14.5  3.5 mg/L, turbidity of 14  2 NTU, and temperature of 25.5  7  C. Concerning regulatory demands, the pH of the secondary effluent (7.3–7.7) fell within the allowed range, and the average values of 11  6.6 mg/L BOD and 22  4.1 mg/L TSS were lower than those allowed by current Israeli legislation of 20 mg/L and 30 mg/L for BOD and TSS, respectively (Israeli Ministry of Health, 1992).

2.2.

Experimental setup

The experimental setup (Fig. 1) comprised an online, 500 L tank, filled with either secondary effluents or tap water, with a submersible, 1/15 hp pump. Cost considerations governed our choice of tap water over distilled water because relatively large volumes of water are required for the filter cycle trials. Water or secondary effluents were pumped from the feed tank to a head tank, from where it flowed by gravity into downward-sloping pipes that were loosely connected to filtration columns located 2.5 m below the head tank. Such a loose connection promoted a raising pressure above the filtration column and enabled each experiment to be performed at a constant rate. Filtration rate was monitored using a flow meter and a control valve on the effluent line, and it was controlled gravimetrically at least three times during the experiment. The sloping pipes were connected to the filter tops via Tygon silicone pipes with two-way valves used for spiking experiments with bacteriophages. To mix the suspension with injected viruses, an in-line static mixer was mounted after the valve. The filtration column was constructed from a transparent, 10-cm diameter acrylic pipe and had a total height of 2.2 m. Seventeen sampling ports and 17 pressure ports were arranged in pairs on opposite sides of the column.

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secondary effluents

0.13 m

0.1 m

4.95 m 4.80m

77

66

To drain

4.65 m

PC

5 12 8

10

2.6 m 44

To drain

11

To drain

2.65 m

Dataloger 1

1

9

Filtrate for backwash

air

13

2

0.15 m

3

Fig. 1 – Schematic of the filter column experimental setup: 1 [ filtration column, 2 [ sampling ports, 3 [ pressure ports, 4 [ dataloger for pressure control, 5 [ air bubbles release pipe, 6 [ feed tank, 7 [ head tank, 8 [ filtrate collectors, 9 [ filtrate flowmeters, 10 [ backwash flowmeter, 11 [ air flowmeter, 12 [ injection point and 13 [ static mixer.

Replaceable Teflon tubes were screwed into each monitored port, and upon sample collection, they were dipped isokinetically into 250-ml polypropylene particle-free beakers. Sampling ports were inserted 5 cm inside the column to avoid wall effect discrepancies that can lead to inaccurate measurements of filtrate quality. The pressure transmitters (STMA, Nuovafima, Italy) were constantly connected to the pressure ports and delivered continuous data to the PC. The filter was filled with 1.1 m of uniformly sized quartz sand (Haifa Bay) with effective size (d10) of 0.89 mm and a uniformity coefficient (UC) of 1.35. The measured filtration porosity was 0.44. The sand was soaked in 0.05 M HCl for 24 h, after which it was thoroughly washed with at least five bed volumes of tap water. That operation was performed to avoid geochemical heterogeneity that might influence filtration of viruses (Abudalo et al., 2005).

Filtration was performed at constant approach velocities of 5, 7.5, and 10 m/h. Turbidity was measured using a Hach 2100N turbidimeter (Hach Company, Loveland, CO). After each run the filter was backwashed for 1 min with air at 200 kN/m2 and for 10 min with Beer-Sheva tap water at 30 kg/m2 s flow rate. Prior to their introduction into the experimental systems, bacteriophages were kept at 4  C and spiked just before entering the static mixer, approximately 30 cm above the filter column. Samples from monitored ports were collected into 250-ml vials for 50 min after the spike. Monitored ports were located 2 cm above the filter media and at depths of 40 and 100 cm inside the filter bed. The samples were kept on ice and transported to the laboratory at Ben-Gurion University at the end of each experiment. In the laboratory, the samples were kept at 4  C until the analysis that, for most experiments, was performed the next morning. Organic content in the effluents

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was measured directly with a TOC analyzer (Apollo 9000 TOC analyzer, Tekmar Company, Cincinnati, OH) using 5310B combustion-infrared method (APHA, 1998).

2.3.

Bacteriophages, cultivation and enumeration

The bacteriophages T4, MS2, and phi X174 and their E. coli host cells were purchased from Deutsche Sammlung von Microorganismen und Zellkulturen GmbH (DSMZ, Germany). The concentration of E. coli was determined by plating 100 ml of culture solution on Lauria-Bertani (LB) agar and then counting colony forming units (cfu) after overnight incubation at 37  C. Initial concentration of E. coli cells was in the order of 103– 104 cfu/ml. The phages were added in 5 phages per cell ratio. Bacteriophages were propagated by inoculation of the LB medium with E. coli cells in the exponential growth phase (suspension turbidity between 0.2 and 0.3 OD), followed by incubation at 37  C for 24 h. Thereafter, 300 ml of chloroform was added to the medium to lyse the E. coli cells. The resulting solution was centrifuged at 6000 rpm for 5 min, the pellet was discarded, and the bacteriophage-containing supernatant was stored at 4  C. Bacteriophage concentration was determined by a plaque forming unit (pfu) assay, using the double-layer overlay method with the appropriate E. coli host (Adams, 1959). The bacteriophages obtained were characterized in terms of shape, size, and zeta potential. Transmission electron microscopy (TEM) was performed at 120 kV with a JEM-1230 equipped with a TemCam-F214 camera (TVIPS Company, Germany). To obtain hydrodynamic radii, the spectra were collected on a CGS-3 goniometer (ALV, Langen, Germany) equipped with a He–Ne 22 mW 632.8 nm laser, at angles ranging from 15 to 150 and in a pH range of 3–10. To reduce interference from multiple scattering, the measurements were performed at different concentrations. The autocorrelation function was calculated using an ALV/LSE 5003 multiple tau digital correlator (ALV, Langen, Germany). Each of the measurements, which were performed at 20  C, was a composite of 20 runs of 10 s each; runs with high baseline levels were disregarded. All correlogram data points were fitted to the CONTIN software package. Zeta potential was measured with a ZetaPlus (Brookhaven Instruments Corporation, Holtsville, NY, USA) zeta potential and particle size analyzer equipped with a 30 mW 657 nm laser (Hamamatsu Photonics K.K., Hamamatsu City, Japan). Vital hydrophobicity was determined in an adsorption glass column using Octyl Sepharose-4 fast flow resin (Sigma– Aldrich, Israel). Based on the procedure developed by Shields and Farrah (2002), 5 ml of adsorbing solution (10 mM imidazole, 4 M NaCl, pH 7) containing approximately 106 pfu of viruses was passed through the column at a rate of 2.5 ml/h f. The column was rinsed with two bed volumes of adsorbing solution, and the void volume and rinse solution were assayed to confirm virus adsorption.

2.4.

Data analysis

The residence time distribution (RTD) curves obtained for bacteriophages passing through the filtration columns were characterized by mean residence time t (Eq. (1)), its variance s2

(Eq. (2)), and the total amounts of accumulated viruses M (Eq. (3)) (Levenspiel, 1999). P ti Ci Dti t¼ P (1) Ci Dti P 2 t Ci Dti  t2 s2 ¼ P i Ci Dti

M¼

P

i ðCi Dti ÞQ

(2)

(3)

In Eqs. (1)–(3), t is the average retention time, t is the elapsed time since the injection i, Ci is the viral concentration, Q is the flow rate, and s2 is the retention time variance. Experimental curves were approximated from the theoretical curves obtained from a local mass balance equation as follows: u

vC v2 C þ kuC  3D 2 ¼ 0 vz vz

(4)

where C ¼ the mass concentration of particles in suspension, k ¼ the attachment rate coefficient, u ¼ the approach velocity, z ¼ the position in the column, measured as the distance from the entrance in the direction of the flow, 3D ¼ the effective dispersion, accounting for diffusion and hydrodynamic dispersion, and 3 stands for porosity. The equation was supplemented by a set of initial and boundary conditions (Eqs. (5)– (7)) and solved analytically to obtain Eq. (8). For the first boundary condition, at the entrance to the column, a specific, constant concentration of viruses in the feed suspension was assumed Cð0; tÞ ¼ C0 ¼ m=Q

(5)

The column was introduced as semi-infinite media CðN; tÞ ¼ 0

(6)

The filter bed was assumed to be clean prior to any experiment Cðz; 0Þ ¼ 0

(7)

The model variables were transformed into their natural forms, by definition independent of changes in either filter length or initial concentration of the suspension, and solved using dimensionless variables to obtain a residence time distribution (RTD) curve Eq: i h 2 expðktÞexp  ð1qÞ D 4qðuLÞ CðtÞ qffiffiffiffiffiffiffi Eq ¼ (8) C0 D 2 puL where q ¼ t=t (Polyanin, 2001). The theoretical curves were used to fit the experimental data using the best fit method. The attachment coefficient k was the only independent variable, and it changed in the range between 0 and 0.02. The Peclet number Pe ¼ uL=D was found from tracer studies using the inert dye KMnO4 in a solution for small extents of dispersion (Levenspiel, 1999): rffiffiffiffiffiffi i h Pe (9) C¼ exp  0:25Peð1  qÞ2 4p Average Peclet numbers were 23.8 for 10 m/h and 11.9 for 5 m/h.

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Results and discussion

3.1. Size, shape, surface charge and hydrophobicity of the phages Comprehensive phage characterization included the determination of hydrodynamic radii and zeta potential changes as a function of pH. Hydrodynamic radii measurements of T4 (Fig. 2a) showed that the radius changes as a function of pH. Measurements performed at ionic strengths of 2  103, 2.6, and 7.6 mM KCl showed that larger T4 sizes, with radii from 100 to 140 nm, were observed at the lower pH of 3. In the more alkaline pH range of 4–8, T4 radii decreased to 70 nm. Some increase in the hydrodynamic radius was observed at pH values between 8 and 10. The observed hydrodynamic radius of T4 is smaller than its geometrical dimensions, and unexpectedly depends on solution pH. One possible explanation of the observed phenomenon is the formation of agglomerates of T4 viruses at pH values close to the isoelectric point, the determination of which was performed with zeta potential measurements. Phage zeta potential was measured as a function of pH, and the data obtained correspond to the ionic strength values of 2  103, 2.6, and 7.7 mM KCl (Fig. 2b). For the low ionic strength of 2  103, the zeta potential of T4 was positive at pH 2, and it became more acidic and negative in the more alkaline range above pH 2. The observed isoelectric point at pH 2 was

Hydrodynamic radii, nm

160

Ionic strength

a

0.002 mM 2.6 mM 7.6 mM

140

120

100

80

60

similar to that reported by Herath et al. (1999). Isoelectric points for experiments performed at higher ionic strengths were not observed, mainly because of the technical difficulties associated with measuring in superacid conditions. The general trend of increasing negative zeta potentials at more alkaline pH values held for all three ionic strengths. The values plateaued at pH 8 and higher, and the absolute values of 38, 36 and 27 mV were observed for the ionic strengths of 2  103, 2.6 and 7.7 mM, respectively. The observed tendency is not surprising considering the compensation of the electrical double-layer at smaller distances for suspensions with higher concentrations of cations. Measurements of the hydrodynamic radii of phi X 174 (Fig. 3a) showed that its radius changes as a function of pH. Measurements performed at ionic strengths of 2  103, 2.4, and 8.1 mM consistently returned hydrodynamic radius values of 60–80 nm, which were not affected by changes in pH. A single measurement point at pH 4, however, showed consistently higher values of 180 nm, possibly because the point of zero charge (pzc) of phi X 174 occurs at the same pH value of 4. Zeta potential measurements as a function of pH (Fig. 3B) were positive at the acidic pH of 2 and negative at the more alkaline pH values above 3. The pzc was observed at pH 2.6, which is much lower than the previously reported value of pH 6.6 (Dowd et al., 1998). A feasible explanation of the observed discrepancy lies in the different ionic strength values, or the different subtypes of the phi X 174 phage.

Ionic strength

200

Hydrodynamic radii, nm

3.

a

0.002 mM 2.4 mM 8.1 mM

180 160 140 120 100 80 60

2

4

6

8

10

12

2

3

4

5

pH Ionic strength

b

pH Zeta potential, mV

0

0

2

4

6

0.002 mM 2.6 mM 7.7 mM 8

10

10

7

8

9

10

-10

-20

0

2

4

6

8

10

pH -10

-20

-30

-30

-40

-40

Fig. 2 – Values of hydrodynamic radii (a) and zeta potential (b) as functions of pH for the T4 bacteriophage.

b

0

Zeta potential, mV

10

6

pH

0.002 mM 3.2 mM 7.6 mM

Ionic strength

Fig. 3 – Values of hydrodynamic radii (a) and zeta potential (b) as functions of pH for the phi X 174 bacteriophage.

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water research 43 (2009) 87–96

3.2.

Bacteriophages filtration from tap water

The effects of microscopic parameters on the transport and the attachment of viruses to porous media were assessed in filtration experiments. The bacteriophages were introduced in spikes into a porous filter after 2 and 4 h of operation. Tap water and secondary effluents were filtered separately. The turbidity level of tap water in filter influent was 1.2 NTU, and after filtration was so low as to be close to the turbidimeter’s minimum detection level of 0.03 NTU. The results of turbidity measurements, presented as the turbidity residual ratio, show averaged 95% removal (Fig. 4) indicating that for the duration of the experiments the filter functioned well, following general trends for the removal of inorganic colloids. A bacteriophage spike was introduced after 2 h of filtration, when filtrate turbidity was 0.25 NTU. Turbidity levels of the secondary effluents were in the range of 12–14 NTU. The turbidity removal ratio associated with secondary effluents was lower, and it was not stable throughout the experiments. After 2 h of filtration, retention reached 50%, which was an average removal level. Headloss profiles for the filtration of tap water and secondary effluents are plotted for 30, 55, 90, and 120 min of filtration (Fig. 5a and b). The profiles are presented in plots of

pressure difference as functions of filter depth for each time period (Brenner et al., 1994). The profiles depicted were calculated as the pressure differences between two consecutive pressure measurement ports, a data presentation technique that allows filter layers with significant pressure jumps to be singled out. Such layers are usually associated with the areas of the filter most directly involved in the filtration process. Head losses associated with the filtration of tap water (Fig. 5a) are usually low (less than 3 cm) and do not vary significantly between the layers. Typical for all the experiments performed with tap water, values of 1–2 cm were measured, and they did not change significantly during the 2 h course of the filtration experiment. Initial headloss values of 1–2 cm measured during the filtration of secondary effluents were similar to those for tap water. Greater head loss measurements of 5 cm after 90 min and 33 cm after 2 h were observed in the top 10 cm of the filter. Pressure losses at other depths were as low as 0–1 cm. Such behavior is typical of cake formation on top of the filter, and hence, a comparison of turbidity and head loss profiles helps to distinguish between two different filtration situations. Tap water filtration is the filtration of viruses using pure filter media in which the media grains are essentially deposit-free. In contrast, secondary effluent filtration entails cake filtration associated with the build-up of cake on top or within the top layer of filter media.

a

Head loss, cm 0

10

20

30

0 30 min 55 min 90 min 120 min

20

Filter depth, cm

Bacteriophages exhibited a general tendency to be more negatively charged at higher pH values. At pH 4 the average zeta potential was 10 mV, but at pH 8 that value decreased to 30 mV. No significant difference between zeta potential values measured for different ionic strengths was observed. Zeta potential measurements of MS2 as a function of pH were reported (Gitis et al., 2002a). Measurements of the hydrodynamic radius (data not shown) showed trends similar to those observed with phi X 174. In the pH range of 3–9, a constant radius of 50 nm was observed, although there was an consistent jump up to 80 nm at a pH of 8, a phenomenon for which we found no explanation in the scientific literature. Measurements of hydrophobicity were performed at pH 7.2 and showed that MS2 and T4 are hydrophobic viruses (5.3 and 6 log removal, respectively), and phi X 174 is a hydrophilic one (0.26 log removal).

40 60

tap water 5 m/h

80 100 120

40

-5

0.4

20 0.2 spike

0.0

0

100

200

300

10

0 400

20

Filter depth, cm

0.3

0

5

10

15

20

25

30

0

30

0.1

Head loss, cm

b

Turbidity Head loss

Headloss, cm

Turbidity residual ratio, C/C0

0.5

40

30 min 55 min 90 min 120 min

60 80

secondary effluent 5 m/h

100

Filter run, min Fig. 4 – Turbidity measurements at a depth of 100 cm. Experimental conditions: tap water, 5 m/h approach velocity, pH 7.2, T 23 8C.

120

Fig. 5 – Head loss during filtration of tap water (a) and secondary effluents (b).

35

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water research 43 (2009) 87–96

0.012 0.010

phi X 174 MS2 T4

tap water 5 m/h phiX 174< MS2 < T4

C/C0, -

0.008

5000

Virus concentration, pfu/ml

The filtration of viruses from tap water (Fig. 6) is depicted as RTD curves of the bacteriophages phi X 174, MS2, and T4 at a depth of 100 cm and with a filtration velocity of 5 m/h. Bacteriophages were spiked after 2 h of tap water filtration, and filtrate samples were collected for 30 min after the spike for further microbiological analysis under laboratory conditions. Differences in initial spike concentrations were offset by presenting the data as the virus relative residual ratio C/C0 as a function of time after the spike. All three curves exhibited Gaussian distributions of filtrate virus concentrations, indicating either low dispersion numbers or the prevalence of inertial forces over those of diffusion. The absolute values of virus removal were calculated from RTD curves using Eq. (2). Viral percentage reduction from filtered tap water was 52, 55, and 74% for phi X 174, MS2, and T4, respectively. Viral size was positively correlated with better removal. Although the retention values obtained were somewhat lower than what is expected from filtration in granular media, the filtration was performed without the addition of coagulants or flocculants that can significantly increase the efficiency with which pathogenic microorganisms are retained (Nasser et al., 1995; Gitis et al., 2002c). No correlation between the average retention times and the surface charges or hydrophobicities of the viruses was found. In general, the smaller mean residence time and retention level of phi X 174 can be attributed to its hydrophilicity, but the insignificant differences between phi X 174 and MS2 for the same variables indicate a greater dependence on size than on hydrophobicity (MS2 is hydrophobic). Mean residence times increased linearly with the increase in virus size, measuring 13.7, 14.5, and 16.64 min for phi X 174 (25 nm), MS2 (32 nm), and T4 (200 nm), respectively. Bigger virus sizes usually mean higher dispersion coefficients (Levenspiel, 1999), but the calculated values of 7.14  105 m2/s for phi X 174, 1.4  105 m2/s for MS2, and 3  105 m2/s for T4 showed no relation between the two variables. For tap water filtration, mean residence time is linearly correlated with filter depth. RTD curves for MS2 in tap water plotted for depths of 0, 40, and 100 cm revealed mean residence times of 11.4, 15, and 16 min, respectively (Fig. 7). Assuming a closed plug-flow reactor, a simple mean time distribution in a typical filter column volume divided by flow

4000 3000

Filter depth

MS2 0 cm 40 cm 100 cm

2000 1000 0

0

5

10

15

20

25

Retention time, min Fig. 7 – RTD curves for tap water filtration of MS2 at different filter depths.

rate is 13.2, 14.5, and 16.6 min for 0, 40, and 100 cm depths. Calculated for all three filtration depths, dispersion coefficients were in the range of 1.35  105 m2/s. Removal ratios were calculated as 0.49 and 0.55 for 40 and 100 cm depths, respectively. The observed values indicated that filtration occurred mainly in the upper 40 cm of filter, where the removal ratio changed from 0 to 0.49, compared to a ratio of 0.06 for polishing in the next 60 cm. Residence time changed linearly over the depth range of 0–100 cm, showing its most significant increases in the upper 40 cm of the filter. All the tendencies described are typical of deep bed filtration and are in general agreement with current filtration theories. The filtration data for T4 filtration velocities of 5, 7, and 10 m/h in tap water exhibited expected trends based on general filtration theories (Fig. 8). Higher filtration velocities mean typically shorter residence times, and therefore, the mean retention times for 7 m/h were 10.2, 10, and 9.1 min. The values of t were close to the theoretical values of 9.2, 10.1 and 11.5 min for 0, 40, and 100 cm depths, averaging 2 min less than the residence times for 5 m/h. Such an insignificant rise in filtration velocity was critical for removal purposes. Using the best fit method, absolute values of virus retention were compared by finding the attachment coefficient k (Eq. (8)), which increased from 0 L/min for 5 m/h to 0.001 L/min for 7 m/h to 0.015 L/min for 10 m/h, showing that T4 removal efficiency improved with increasing filtration velocity.

3.3.

Bacteriophage removal from secondary effluents

0.006 0.004

spike

0.002 0.000

0

5

10

15

20

25

30

Retention time, min Fig. 6 – Bacteriophage retention times at a depth of 100 cm in tap water.

Virus concentrations expressed as plaque forming units per ml of influent (PFU/ml) during phi X 174 filtration from secondary effluents are plotted against residence times after a viral spike (Fig. 9). The RTD curves are for depths of 0, 40, and 100 cm. Secondary effluent filtration tendencies differ from those observed during tap water filtration. The mean residence times of 12.2, 12.62, and 14.9 min for depths of 0, 40, and 100 cm were calculated from RTD curves. For all three filtration depths the dispersion coefficient was about 7.7  105 m2/ s. Removal ratios were calculated as 0.04 and 0.47 for depths of 40 and 100 cm, respectively. Similar experiments performed

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1.8 1.6

0.010 5 m/h 7 m/h 10 m/h

T4 tap water

1.4

0.008 0.006

1.0

C/C0, -

RTD, -

1.2

secondary effluents 5 m/h phiX 174= MS2 = T4

phiX 174 MS2 T4

0.8 0.6

0.004 spike 0.002

0.4 0.2

0.000

0.0 0

1

2

3

4

0

5

θ

with T4 and MS2 showed mean residence times (for the same filter depths) of 10.9, 11.12, and 12.7 min for T4, and 11.47, 11.7, and 13.5 min for MS2. Calculated for both viruses and all three filtration depths, dispersion coefficients clustered around 6.2  105 m2/s. A comparison of RTD curves of all three viruses at a depth of 100 cm with a 5 m/h approach velocity is depicted on Fig. 10. Trends in MS2 removal resembled those for phi X 174, for which minimal removal ratios of 0.07 and 0.58 were observed for depths of 40 cm and 100 cm, respectively. T4 filtration from secondary effluents, however, was unsuccessful. Virus residual ratios increased from 0.78 for 5 m/h to 1 for 7 m/h to 1.05 for 10 m/h. Virus concentration above the filter was 103 PFU/ml and remained constant throughout the sampling collection, which continued for 2.5 h at 10 min intervals. At a depth of 40 cm, the initial concentration was 800  78 PFU/ml, and that grew to 1400  120 PFU/ ml after running the filter 1.5 h. The viral concentration at 100 cm depth changed from 1000  90 PFU/ml to 1800  85 PFU/ml 2 h after the filter run started. Some of the observed tendency can be attributed to experimental uncertainty yet

Virus concentration, pfu/ml

Filter depth

phi X 174

5000

0 cm 40 cm 100 cm

4000 3000 2000 1000 0 0

5

10

10

15

20

25

30

Retention time, min

Fig. 8 – RTD curves for the filtration from tap water of T4 at approach velocities of 5, 7, and 10 m/h.

6000

5

15

20

25

30

Retention time, min Fig. 9 – RTD curves of phi X 174 for different filter depths during secondary effluent filtration at an approach velocity of 5 m/h.

Fig. 10 – Bacteriophage retention times in secondary effluents at a depth of 100 cm.

the observed trend suggests that viral growth occurs during phage passage through the filter, probably due to their cultivation inside hosting cells during filtration. Since the average hydraulic residence time is 24 min, and the T4 lytic cycle is approximately 20 min (Stanier et al., 1986), it is entirely possible that upon phage insertion into the filter, they undergo full scale replication, and what is actually seen at the filter exit is the concentration of spiked and replicated viruses. This tendency was especially prevalent for T4 viruses, but less so for MS2 and phi X 174, due to the prolonged lytic and lysogenic cycles of MS2 and phi X 174, respectively. The phenomenon was observed only when filtering secondary effluents, which provided the necessary host cells. One solution to the problem is to use dyed bacteriophages (Gitis et al., 2002b) that have been inactivated but that can still be quantified using fluorimetric techniques.

3.4.

Discussion

The luxury of a changeable suspension source for the same filtration column created an interesting but usually unachievable situation in which two different types of filtration were modeled for the same set of filtration variables. Bacteriophage retention from tap water and from secondary effluents was compared based on residence time, retention value, and degree of dispersion. Tap water is usually characterized by lower loads of colloidal and organic material, and therefore, currently existing filtration theories can generally be used to explain the filtration of viruses out of tap water. Secondary effluents were associated with higher colloidal and organic loads, and under the same filtration conditions, they created a cake layer on top of the filter. Formation of the filter cake was not expected to change the kinetics of the filtration process, and in that sense the filtration of viruses from secondary effluent deserved a closer look. Regarding the virus microscopic characteristics and a possible link with their filterability, virus size was the only influential factor. The surface charges of all three viruses at the pH of water was very negative, in values of 38, 30

water research 43 (2009) 87–96

and 40 mV for T4, phi X 174 and MS2, respectively, and therefore, no differences in attachment due to zeta potential were expected. Despite that T4 and MS2 are hydrophobic and phi X 174 is hydrophilic, hydrophobicity was not shown to affect either virus retention or the residence time. Classic deep bed filtration theories (Yao et al., 1971; O’Melia and Ali, 1978) propose that the removal of entities is governed by transport and attachment mechanisms, the latter being a sum of the electrostatic, hydrophilic, and steric interactions between the viruses and the media itself. The negative charges of the viruses and quartz sand and the hydrophobic character of T4 and MS2 vis-a`-vis the hydrophilic nature of the sand, and the tailed structure of T4 were all indicators of potential attachment difficulties due to steric interactions. Accordingly, we expected phi X 174 to exhibit a certain level of attachment while T4 and MS2 would show no attachment. The same applies to transport efficiency. The minimum particle size in filter removal capacity was postulated to fall in the 1 mm range. As such particles bigger than 1 mm will be transported by interception, settling, and inertia, particles smaller than 1 mm will be transported by Brownian motion. Filtration efficiency decreases as particle size approaches the 1 mm barrier from either side. At the same time the attachment efficiency should remain unchanged assuming similar surface charge and hydrophobicity, and therefore the lower filtration efficiency is attributed to inefficient transport of the viruses toward media grains. The experimental results deviated from the expected: T4 was removed better than both MS2 and phi X 174, despite its large size of about 200 nm vs. 26– 33 nm for the latter two. Its superior removal cannot be attributed to surface charge, hydrophobicity, or shape. The better transport of T4 is clearly reflected in the virus residence times: the larger residence times suggest more effective lateral movement of the viruses into and out of the media grains. The formation of a cake layer on top of the filter suggests a different filtration scenario. The cake layer prolongs the stay of the viruses in the top layer of the filter, leading to a higher degree of retention. At the same time the build-up in headloss caused by the cake layer results in a higher pressure difference across the filter. For the same influent, such a pressure loss leads to the formation of dead filtration areas and highspeed channels (initially named worm-channels by Baylis, 1937). We postulate that after cake formation, no virus attachment occurs as the presence of the cake layer leads to a higher interstitial velocity inside the filter, which enhances the transport of viruses inside the pores. Correspondingly increased Reynolds numbers suggest the increased inertia of the viruses, resulting in minimal attachment. Measured retention times and retention values show that the phages are flowing through the top 40 cm of the filter at very high velocity using worm-like channels associated with dead areas inside the filter that are not used for filtration. Filtration and retention occur in the lower 60 cm of the filter where both differences in retention times and residual concentrations become significant (Figs. 9 and 10). The probable reason for such behavior is the pressure difference between the top and second layers inside the filter. As a function of cake layer build-up in the top layer of filter, the headloss became so high that the influent runs through the top filter layers at high interstitial velocities that do not allow for particle attachment.

95

That pressure difference becomes smaller inside the filter where, at a depth of 60 cm, it functions as a regular filter.

4.

Conclusions

Cake formation on top of a filter during the initial stages of filtration causes a significant pressure drop that eventually results in shorter filtration cycles. However, in filters deep enough to contain a buffer zone where the pressure drop is amortized, filtration can continue unabated. After the effluent crossed the buffer zone, regular filtration was observed, during which considerable virus retention was achieved. The current research described using a 40 cm deep buffer zone during the filtration of secondary effluents. Since the buffer zone occupied the upper 40 cm of the filter, actual effluent filtration occurred only in the lower 60 cm of the filter bed. Size was the only microscopic characteristic that influenced virus retention: bigger viruses were retained better than smaller ones. That observation, however, runs counter to the currently accepted filtration theory, since the bigger viruses are in the size range of 200 nm, dimensions that approach minimum transport efficiency (Yao et al., 1971; O’Melia and Ali, 1978). Without flocculation, the transport mechanism operating in filtration is the opposite of that previously reported, and the presence of bigger viruses may be less problematic than smaller ones.

Acknowledgements This research was supported by THE ISRAEL SCIENCE FOUNDATION (grant No. 1184/06) with VG. The help of Mrs. Vanounou from R. Stadler Minerva center for Mesoscale Macromolecular Engineering at BGU with dynamic light scattering experiments is gratefully acknowledged. Special thanks are due to Mrs. I. Mureinik and Mr. Patrick Martin for scientific editing of the manuscript.

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