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Separation and Purification Technology 62 (2008) 619–628

Contents lists available at ScienceDirect

Separation and Purification Technology

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ffect of transmembrane pressure on rejection of viruses by

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ion oplanaof viread trs bec. It is

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ultrafiltration membranes

Elizabeth Arkhangelsky, Vitaly Gitis ∗

Unit of Environmental Engineering, Ben-Gurion University of the Negev, POB 653, Beer-

a r t i c l e i n f o

Article history:Received 14 October 2007Received in revised form 9 March 2008Accepted 11 March 2008

Keywords:MS2T4phi X174E. coliPES membrane

a b s t r a c t

Although partial penetratthere is no satisfactory exby studying the retentionpredictions, higher TMPs lthat the penetration occuof abnormally large poreshigh virus retention levels

1. Introduction

Many water treatment facilities use ultrafiltration (UF) mem-

ranes for the removal of mineral and organic particles, especiallyathogenic microorganisms such as protozoa, bacteria, and viruses.etention of microorganisms is expressed in log removal valuesLRV), that is the logarithm to base 10 of the ratio of viral con-entrations in the feed to those in the permeate. UF technologyas been shown to deliver at least a 6-log attenuation of protozoand bacteria [1,2]. As a result, the UF technology is recognizeds a single-step process able to meet high regulation standardsor removal of protozoa and bacteria. For viruses, however, theituation is different: although UF membranes can potentiallyetain 99.999% of the feed viruses, the technology has been grantednly a 4-log removal credit in four states of the USA and 1- to 2-logemoval credit in three states. Indeed, in 43 of the 50 states of theSA, UF membrane technologies have not been granted any log

emoval credits for viruses [3].Two explanations may be offered for the refusal of so many

tates to grant log removal credits for viruses. The first, and mostmportant, is that the existing methods are simply incapable ofetecting virus-sized pores in the membrane active layer [4].irect pore size tests based on bubble point measurements are notble to detect pores below the range of 2–3 �m [3], a size rangeuch larger than the average virus size. Protocols for indirect

∗ Corresponding author. Tel.: +972 8 6479031; fax: +972 8 6472983.E-mail address: gitis@bgu.ac.il (V. Gitis).

383-5866/$ – see front matter © 2008 Elsevier B.V. All rights reserved.oi:10.1016/j.seppur.2008.03.013

84105, Israel

f ultrafiltration membranes by viruses and bacteria is well documented,tion for the phenomenon. The current study seeks a possible explanationuses at different transmembrane pressures (TMPs). In contrast to previouso reduced virus retention levels. Based on indirect evidences it is proposedause of the formation during operation, rather than the initial presence,

therefore suggested that since pore enlargement is induced by high TMPs,be obtained only at low TMP values.

© 2008 Elsevier B.V. All rights reserved.

measurement of pore size are based on measuring rejections ofcompounds that can be indicative of virus removal. In all thesetests, changes in filtrate quality are monitored by comparing testprobe levels with a previously established baseline level. The testsare not sufficiently sensitive to detect virus-size particles, andthe interpretation of their results in terms of viral permeabilityis questionable. The only test that might somehow indicate thelikelihood of virus penetration is based on filtration of organic

molecules, usually dextrans or polyethylene glycols (PEG), as afunction of their molecular weights. With the latter methodologies,pore size is determined using an empirical relation [5–7] betweenmolecular weights and the hydraulic radii of the molecules, andtherefore, there is little correlation between pore size and virusremoval. During routine operation, due to aging or as a result ofexposure to incompatible chemicals [4,8,9], the membrane porestend to expand, spawning the fear that viruses will pass throughthe membrane barrier. In the absence of reliable monitoringtechniques, current water regulation policies require a significantreduction in virus concentration by disinfection rather than by UF, amultibarrier approach that entails additional operational expenses.

The second reason that log removal credits are not granted forviruses lies in differences in reported virus removal levels. AlthoughUF membranes consistently exhibit high retention levels of 6- to8-log removal for bacteria, the reports on virus removal levels varybetween less than 2- and up to 5-log. The complete removal ofpoliovirus [10] and more than 6-log removal of MS2 virus [11,12]were obtained with 30- and 100-kDa molecular weight cut-off(MWCO) membranes, respectively, whereas retention of Q� and T4viruses was incomplete (2.5-log removal) with UF membranes in

620 E. Arkhangelsky, V. Gitis / Separation and Puri

Nomenclature

Ak membrane surface porosityCi,p concentration of polymer fraction i in the permeate

(mg/L)Ci,0 concentration of polymer fraction i in the feed

(mg/L)d hydraulic diameter of polymers (m)dm.p. mean pore diameter (m)dp pore diameter (m)DAB diffusivity of macromolecule in the water (m2/s)f(dp) solute separation

2 2

J flux (L/(h m bar)) or L/(h m Pa)k Boltzmann constant (J/K)Lp hydraulic permeability (L/(h m2 Pa2))�m permeate weight difference (kg)MA molecular weight of the large molecule (Da)P transmembrane pressure (bar or Pa)r solute particle radii (m)R rejection (%)S area of active membrane surface (m2)�t frequency interval (h)T absolute temperature (K)Vv void volume in membrane (m3)Vt total volume of membrane (m3)�x membrane thickness (m)

Greek symbols� water viscosity (Pa s)�p geometric mean pore diameter (m)�p geometric standard deviation of the membrane

the 30-kDa range [13,14]. In addition, some reports claiming com-plete removal were based on low initial concentrations of virusesin the feed, so the actual virus retention level could be even higher.

To date, no clear correlations have been established betweenvirus removal and any of the UF operating parameters, such asmembrane material and MWCO, transmembrane pressure (TMP),or operating mode. The present study was initiated to investigatethe relationship between TMP and virus retention. Despite grow-ing interest in utilizing UF membranes for virus filtration, thereare no published studies that investigate how TMP affects virus

removal levels. However, three explanations for the partial passageof viruses through UF membranes have been proposed. One pop-ular explanation cited throughout scientific literature posits thepresence of abnormally large pores that are not included in themain pore size distribution [13,14]. Such abnormal pores result fromimperfections in the polymerization process. A second hypothe-sis focuses on failed sealing and a lack of membrane integrity dueto the presence of pinholes [15,16]. The third explanation involvesviral decomposition or a change in the shape of the viruses as theypass through the membrane. The three explanations outlined abovewere examined in the current study.

Existing evidence indicates that higher TMPs lead to a denser,compacted membrane structure with smaller pores and lowermembrane flux. Numerous studies [17–19] show that the com-paction is more pronounced in reverse osmosis processes, wherethe membrane is under high pressure (10–100 bar; [20]). Morerecent studies [21] demonstrated that even under relatively lowapplied pressures, UF porous membranes still tend to compact,leading to the hypothesis that higher TMP will cause higher viralretention rates and lower fluxes [22–24]. That point was thoroughlychecked under laboratory conditions.

fication Technology 62 (2008) 619–628

2. Experimental

2.1. Viruses, cultivation and characterization

T4, MS2 and phi X174 bacteriophages and their Escherichiacoli host cells were purchased from Deutsche Sammlung vonMicroorganismen und Zellkulturen GmbH (DSMZ, Germany). Theconcentration of E. coli was determined by plating 100 �L ofculture solution on Lauria–Bertani (LB) agar and colony form-ing units (cfu) after overnight incubation at 37 ◦C. Bacteriophageswere propagated by inoculation of the LB medium with E.coli cells in the exponential growth phase (suspension turbid-ity between 0.2 and 0.3 OD), followed by incubation at 37 ◦Cfor 24 h. Thereafter, 300 �L of chloroform was added to themedium to lyse the E. coli cells. The resulting solution was cen-trifuged at 6000 rpm for 5 min, the pellet was discarded, and thebacteriophage-containing supernatant was stored at 4 ◦C. Bacterio-phage concentration was determined by a plaque forming unit (pfu)assay, using the double-layer overlay method with the appropriateE. coli host.

The cultures obtained were characterized in terms of shape,size and zeta potential. Visualization was performed with an epi-fluorescent microscope (Axioskop 2 plus, Carl Zeiss Jena GmBH,Germany) equipped with a Nikon DXM 1200F (Nikon Inc., Japan)digital camera. Atomic force microscopy (AFM) was performedunder ambient conditions using a Digital Instrument Dimension3100 (Digital Instruments, Santa-Barbara, CA) mounted on anactive anti-vibration table. About 20 �L of suspension was placedonto freshly cleaved mica and dried in air [25]. A 100 �m scan-ner was used to take 512 × 512 pixel images in tapping modewith a scan size of up to 5 �m at a scan rate of 1 Hz. Transitionelectron microscopy (TEM) was performed at 120 kV with a JEM-1230 equipped with a TemCam-F214 (TVIPS Company, Germany)camera.

To obtain hydrodynamic radii, the spectra [26] were collected ona CGS-3 goniometer (ALV, Langen, Germany equipped with a He–Ne22 mW 632.8 nm laser) at angles ranging from 15◦ to 150◦ and ata pH range of 3–10. To reduce interference from multiple scatter-ing, the measurements were performed at different concentrations.The autocorrelation function was calculated using an ALV/LSE 5003multiple tau digital correlator (ALV, Langen, Germany). Measure-ments were performed at 20 ◦C, with each measurement being acomposite of 20 runs of 10 s each; runs with high baseline levelswere disregarded. All correlogram data points were fitted to the

CONTIN model [27].

Zeta potential was measured with a ZetaPlus (BrookhavenInstruments Corporation, Holtsville, NY, USA) zeta potential andparticle size analyzer equipped with a 30 mW 657 nm laser (Hama-matsu Photonics K.K., Hamamatsu City, Japan).

2.2. Membranes and filtration experiments

Commercially available 20-kDa MWCO UF membranes (Ster-litech Corporation, Kent, WA, USA) made of polyethersulfone(PES-20) were used. The membranes were supplied as flat sheetsand were stored dry under ambient conditions. Nominal MWCO,contact angle, and permeability of the membranes, have beenreported previously [28], as was a detailed description of the lab-scale 150-ml stirred cell used for the experiments. In brief, piecesof membrane taken from a flat sheet were cut into round shapeswith cross-sectional areas of 0.0017 m2. The membrane pieces werethoroughly rinsed with deionized water at 30 ◦C by shaking at100 rpm on a mechanical shaker for 1 h before an experiment, afterwhich the membrane sheets were placed in the cell. Membranecompaction experiments were performed by filtration of deion-

d Puri

E. Arkhangelsky, V. Gitis / Separation an

ized water for 30 min at 2 bars N2 (99.99% purity) pressure. In theseexperiments, membrane flux was calculated gravimetrically as:

J = �m

�S�tP(1)

where �m is the permeate weight difference (kg), �t is the fre-quency interval (h), S is the active membrane surface area (m2), Pis TMP (bar), and � is the permeate density (kg/m3).

Measured membrane permeability values were50 ± 2 (L/m2 h bar). Membrane samples that showed signifi-cant deviations from the measured average permeability valueswere discarded.

The filtration experiments were performed at 20 ± 1 ◦C and pH7 with 2 × 108 cfu of E. coli or with 5 × 109 pfu of bacteriophagesdiluted in 100 mL of sterile deionized water. The solution waspressed through the membrane by nitrogen for time periods suffi-cient to allow filtration of 50 mL of the feed suspension. The TMPwas altered over the range 0.5–5 bars.

2.3. Streaming potential

The streaming potential was measured in the stirred cell bypumping a KCl solution through the membrane pores, i.e., the flowwas directed perpendicular to the membrane active layer. To thisend, the membrane cell was remodeled to include a salt bridgebetween the electrodes on the feed and the permeate sides of amembrane cell, in a setup similar to that used by Pontie et al. [29].Detailed setup of the remodeled cell can be found elsewhere [8].The membrane sample was placed above a PTFE spacer (0.2 mm inthickness) with the membrane skin-layer facing the flow channel(200 mm × 74 mm). The electrolyte solution was forced through themembrane by static pressure given by nitrogen. Gas pressure wasset by a precision regulator equipped with a digital pressure dis-play (IR2000-FO2 and ISE40, respectively, SMC Corporation, TokyoJapan) at a range of 0–2 bar and with a 0.01 bar accuracy. The elec-trical potential difference was measured between the feed andpermeate by opening the back-pressure valve, thus allowing thefeed electrolyte to vacuum soak into a vial containing an electrode.Such a setup allows the electrodes to be kept separate from the celland prevents the electrokinetic effects that arise from the convec-tive hydrodynamic movement of the electrolyte near the electrodesurface.

For streaming potential measurements, the membrane was

equilibrated by soaking it in a 0.001 mol/L KCl solution overnight.Measurements were performed for 10 mM KCl at pressures of 0.5and 1 bar in the pH range 3–10.5 (at pH values higher than 10.5, theelectrode is unstable; at pH measurements lower than 3, solutionconductivity will be too large and will affect the measurement).Measurements were conducted with Ag/AgCl reference electrodes(model 723/733, Metrohm Ltd., Switzerland) and a high impedancedigital multimeter (EDM 2347, Escort, Taiwan). The asymmetrypotential of the electrode pair was less than 1 mV.

The streaming potential values for pristine and fouled mem-branes were calculated from four measurements for eachKCl strength/pressure combination by applying the Helmholtz–Smoluchowski equation:

� = �E

�P

�k

ε0ε(2)

where �E is the potential difference across the membrane, �Pthe applied pressure, � the dynamic viscosity of the electrolyte,k the conductivity of bulk electrolyte, ε the dielectric permittivityof water, and ε0 the permittivity of vacuum.

fication Technology 62 (2008) 619–628 621

2.4. Pore size determination

Pore size and pore size distribution were determined by AFM,and tests of solute transport and water permeability. Based on acapillary pore diffusion model [30], the water permeability testenables a determination of mean pore size diameter using theHagan–Poiseuille equation:

dp = 2

√8��xM

Ak(3)

where � is the viscosity of water, �x is the membrane thickness, Akis the membrane surface porosity (0.19, determined by weighing ofdry and wet samples), and M is the membrane permeability.

Solute transport tests were performed in separate experimentswith PEG and polyethylene oxide (PEO) polymers (Fluka GmbH,Germany) of various molecular weights. Seven PEG (0.3, 0.6, 3.35,6, 10, 20 and 35 kDa molecular masses) and three PEO (100, 200 and600 kDa) polymers were used. The broad molecular weight distri-bution selected for the PEG and PEO solutions (ranging from 0.3 to600 kDa) was chosen to correspond to rejection coefficients rang-ing from 0 to 100%. The effluent concentration was determined by acombustion-infrared method using a total organic carbon analyzer(Apollo 9000 TOC analyzer, Tekmar Company, Cincinnati, OH). Theinfluent PEG solution was prepared at a concentration of 3 g/L ofdeionized water (MilliQ quality). The rejection (%) for each polymerwas calculated using the formula:

R =(

1 − Ci,p

Ci,0

)× 100% (4)

where Ci,p is the concentration of polymer fraction i in the permeate(mg/L), and Ci,0 is the concentration of polymer fraction i in the feed(mg/L).

The rejection coefficient values obtained were plotted againstthe hydrodynamic radii of the polymers to obtain the MWCO,usually described as the upper molecular weight limit of trans-port, above which less than 10% of the particles are transportedthrough the membrane [31]. Molecular weights were convertedinto hydrodynamic radii by comparing the diffusivities of thespheres obtained from the Stokes–Einstein equation (Eq. (5)) withthe diffusivities of organic molecules with molecular masses above1000 Da, as suggested by Polson (Eq. (6)) [7]:

DAB = kT

6r�(5)

where DAB is the diffusivity of a perfect sphere in water (m2/s), k isthe Boltzmann constant (J/K), T is the absolute temperature (K), r isthe radius of the sphere (m), and � is the water viscosity (Pa s).

DAB = 9.4 × 10−15T

�(MA)1/3(6)

where MA is the molecular weight of the large molecule (Da). Thehydraulic diameter of the polymers (m) was calculated by combin-ing Eqs. (5) and (6):

d = 0.156 M0.33A (7)

Pore size distribution was obtained using a log-normal proba-bility density function [6]:

f (dp) = 1

dp ln �p√

2exp

[−1

2

(ln(dp/�p)

ln �p

)2]

(8)

where dp is the pore diameter (m), f(dp) is solute separation, �p

is geometric mean pore diameter (m), and �p is the geometricstandard deviation of the membrane. The function parameters

622 E. Arkhangelsky, V. Gitis / Separation and Puri

were determined from the rejection coefficient-hydraulic radiusplot, where �p was calculated as ds corresponding to f = 50% and�p = ds90/ds50.

3. Results and discussion

Prior to the UF experiments both the membrane and the viruseswere thoroughly characterized.

3.1. Membrane characterization

Fig. 1 shows the AFM micrographs of pristine PES membranes,together with their line profiles, from which pore size diametercan be determined. According to the AFM measurements, themembrane pore diameter varies from 15 to 75 nm, a significantly

Fig. 1. The AFM images of pristine PES-20 membrane. The cross-sections in the bottom papore openings.

fication Technology 62 (2008) 619–628

higher diameter range than that specified by the manufacturer.We attribute the difference to the limited suitability of AFM asa technique for determining pore size. The valleys seen in themembrane profile are probably sinks leading to the membranepores, rather than the membrane pores themselves. The AFMmeasurements were completed by solute transport tests and waterpermeability measurements.

Hydraulic radii were calculated using Eq. (7) and are presentedas diameters in Table 1. Organic solute retention was then plot-ted as a function of solute hydraulic diameter at TMPs of 1–5 bars(Fig. 2). Pearson’s coefficients were surprisingly low, with R2 rang-ing from 0.5 to 0.67. The lack of correlation was attributed to anabsence of PEGs with molecular masses greater than 35 kDa and aneed for PEOs for complete membrane characterization. The rejec-tion rates of PEOs of 100, 200, and 600 kDa were lower than those

rt of each picture were taken along the indicated lines and include profiles of typical

E. Arkhangelsky, V. Gitis / Separation and Puri

Table 1Relationship between the molecular weight of PEG/PEO and the hydraulic diameter

Molecular weight, Da Hydraulic diameter (d), nm

200 0.90300 1.02600 1.293350 2.276000 2.7510,000 3.2620,000 4.1035,000 4.93100,000 6.97200,000 8.76600,000 12.59

for PEGs of 35 kDa. Furthermore, solute rejection was independentof molecular weight, as all three PEO polymers were retained withsimilar effectiveness, and the evaluation of membrane pore sizewith polymer probes was pressure-dependent. The MWCO of the

Fig. 2. Solute separation curves (solute diameter vs. their sep

fication Technology 62 (2008) 619–628 623

same membrane varied from 9.82 to 10.56 nm for pressures of 1and 5 bars, respectively.

The plots in Fig. 2 were used to obtain the MWCO values, thegeometric mean pore diameter (�p), geometric standard devia-tion (�p), finite pore diameter and hydraulic mean pore diameter(Table 2). The MWCO determined by the polymer sampling methodranged from 28 kDa obtained for 1 bar to 35 kDa for 5 bars indicat-ing that pore size expands as a function of the applied pressure. Thefinite pore diameter, i.e., the pore diameter corresponding to 100%retention, varied from 11.06 nm for 1 bar to 11.72 nm for 5 bars.Although the data in all five cases shows a sigmoidal curve, linearmodel was implemented. The reason is the current method ofMWCO determination requires a linear fit and not a best fit possible.

The pore size distribution of the PES membrane at differentTMPs was plotted in Fig. 3 using the log-normal distributionfunction (Eq. (8)). Although a general Gaussian distribution wasmaintained, the graph depicts a shift toward bigger pore sizesas a function of applied pressure. The mode pore diameter

aration): 1 bar (a), 2 bar (b), 3 bar (c), 4 bar (d), 5 bar (e).

624 E. Arkhangelsky, V. Gitis / Separation and Puri

Table 2Geometric mean pore diameter (�p) and the geometric standard deviation (�p) calculate

TMP, atm MWCO, kDa Geometric mean porediameter (�p), nm

Geodev

1 28 4.88 2.02 33 4.9 2.13 31 5.6 1.84 30 5.58 1.75 35 5.93 1.7

Fig. 3. Probability density function curves.

Table 3Mode pore diameter of PES-20 membrane as the function of TMP

TMP, bar Mode pore diameter, nm

1 2.752 2.753 4.14 4.15 4.93

increased from 2.75 nm for 1 bar to 4.93 nm for 5 bars (Table 3,Fig. 3.).

Table 4 presents the calculations of membrane mean porediameter based on water permeability measurements. The porediameters calculated with Eq. (2) varied from 7.67 nm for 1 barto 19.54 nm for 5 bars TMP, despite the fact that inclusion of themembrane permeability term M in the equation should alleviatethe effect of TMP. The general trend of expansion of pore size withhigher TMP, depicted with AFM measurements and solute trans-port tests, remained. The observed trend is strikingly opposite topreviously reported trend of graduate increase in cut-off values astransmembrane pressure decreases [32].

Fig. 4. Streaming potential–pH dependence for PES membrane in 10 mM KClsolution.

fication Technology 62 (2008) 619–628

d from separation data

metric standardiation (�p)

Finite porediameter, nm

Hydraulic meanpore diameter, nm

1 9.76 9.822 9.8 10.381 11.2 10.119 11.16 9.998 11.86 10.56

Contact angle and streaming potential measurements werealso used to characterize the membranes. The tests provide anindication of hydrophobic interactions and electrostatic repulsion,two other mechanisms currently known to affect the retention ofviruses by UF membranes. The contact angle measurements gavea value 62 ± 1.6◦, indicating that the PES membrane is relativelyhydrophobic.

Streaming potential measurements were performed at pH val-ues ranging from 3 to 10.5 (Fig. 4). At that pH range PES membranesamples were negatively charged, and as the pH became more alka-line, the absolute values of the streaming potential measured in10 mM KCl increased from −5 mV at pH 3 to −12 mV at pH 10.5.Since PES has no dissociated functional groups, specific ion adsorp-tion is the only possible process for the formation of surface charge,i.e., the initial charge can be attributed to the adsorption of hydroxylions on the PES membrane surface.

3.2. Virus characterization

While the T4 and phi X174 bacteriophages are considered to behydrophilic, MS2 is hydrophobic [33]. Based on the hydrophobic-ity measurements, for similar sizes and zeta potential values, it isexpected that the degree of MS2 retention will be higher than thatfor both T4 and for phi X174.

Zeta potential values for T4, MS2, phi X174 and E. coli in the pH

range of 2–10 are shown in Fig. 5. A point of zero charge (pzc) wasobserved at pH 3.2 for T4, pH 2.6 for MS2 and pH 2.4 for X-174. Belowpzc X-174 and MS2 were positively charged. As the pH increased,the phage charges tended toward electroneutrality, with absolutezeta potential values in the range of 5 mV at pH 3. As the pH becameeven more alkaline, the phages usually took on a negative charge.At pH 7, the zeta potential values of the phages were −38, −30, and−27 mV for T4, MS2, and phi X174, respectively. The zeta potentialof E. coli, on the other hand, varied from −24 to −55 mV, whichis lower (vs. −50 to −75 mV) than that previously observed [34].Based on the highly negative zeta potential of E. coli at pH 7, itspzc is probably located in the hyperacidic environment, which thecurrently available instrumentation is incapable of measuring.

Fig. 6 shows AFM and TEM images of the T4, MS2, and phi X174bacteriophages. In the AFM images of the T4 bacteriophage, thetypical icosahedral structures of the head [35,36] and the tail wereclearly visible (Fig. 6a). The measured size of the phage was 200 nm,slightly smaller than the previously reported values of 220 and250 nm [35]. The size difference was attributed to the flattening ofthe head during drying on the mica surface prior to AFM microgra-phy. The flattening effect was more pronounced for the head than

Table 4Mean pore diameter determined by water permeability method

TMP, bar Mean pore diameter (dm.p.), nm

1 7.672 11.783 14.774 17.485 19.54

E. Arkhangelsky, V. Gitis / Separation and Purification Technology 62 (2008) 619–628 625

MS2 (

Fig. 5. Zeta potential of T4 (a),

for the tail, probably due to the tail’s increased compactness andflattening resistance [25]. The TEM images of the MS2 and phi X174bacteriophages showed that these phages have spherical shapeswith diameters of 30 and 26 nm, respectively (Fig. 6b and c), valuesthat are somewhat lower than those reported earlier [38]. In con-trast to MS2, phi X174 exhibited spikes that were observed alongthe periphery of the virion.

Plots of the hydrodynamic radii of the T4, MS2 and phi X174 bac-teriophages as functions of pH showed peaks around pH 3–4, wherethe hydrodynamic radii jumped to 140, 180, and 60 nm for T4, MS2,and phi X174, respectively (Fig. 7). A small increase in hydrody-namic radii of T4, although occurring at pzc pH of 3.2, was compara-ble to values of 100 ± 20 nm measured at other pH values. The mea-sured values were those of a single T4 (200 nm size) indicating thatthe virus does not undergo an aggregation. The aggregation wasclearly observable for MS2 with 170 nm measured at pH 3 whereelectrophoretic mobility equals zero. A shift toward alkaline condi-tions causes a reduction in hydrodynamic radii values down to indi-vidual MS2 radius of 15 nm observed at pH 5 and higher. Althoughpzc of phi X-174 was measured at pH 2.4, a peak in virus size wasobserved at pH 4. The discrepancy may be related to differencesbetween static and dynamic light scattering measurement methodsused for determination of zeta potential and virus size, respectively.The virus radii lowered to 40 nm for acidic conditions and to 20 nmfor alkaline conditions. For pH higher than pH 6 measured hydro-dynamic radii of X-174 was equal to 26 nm, a size of a single virus.

An estimation of E. coli dimensions, based on a digital pic-ture from an epifluorescence microscope, gave the width to be

b), phi X174 (c) and E. coli (d).

1 �m and the length 2–4 �m. The hydrodynamic radius of E.coli at pH 7 was 776 nm, a measurement consistent with micro-scope observations. The observed values, however, are somewhathigher than those reported earlier [37]. We attribute the observeddifferences to a stationary phase of bacterial growth in ourmeasurements.

3.3. Rejection prediction

Our membrane analysis indicates that the UF membrane shouldform an efficient barrier against the penetration of bacteriophagesand E. coli. The dimensions of the microorganisms used in thestudy were at least twice the average membrane pore size (10 nm)of the 20-kDa MWCO PES-20 membrane. We should rememberthat our membrane retention measurements were performedwith linear, water-soluble molecules (PEG and PEO), which aremore easily able to pass through membrane pores than non-linearmolecules. Therefore, the membrane rejection levels for non-linearmolecules of the same molecular weights as our linear polymersshould, in fact, be much higher, i.e., the difference between thesize of the microorganism and pore sizes is actually much larger.We may thus assume that the PES-20 membrane constitutes analmost absolute barrier to the passage of the phages used in thisstudy. The validity of this assumption gains additional strengthfrom the zeta potential values. The values of −20 mV and higherfor E. coli and bacteriophages in the typical tap water range of pH6–8.5 vs. a streaming potential of −15 mV for the membrane willresult in a strong repulsion. Consequently, the retention of E. coli

626 E. Arkhangelsky, V. Gitis / Separation and Purification Technology 62 (2008) 619–628

Fig. 6. The microscope image of bacteriophages: AFM image of T4 (a); TEM imageof MS2 (b) and phi X174 (c).

and bacteriophages by a 20-kDa MWCO membrane is expected tobe very efficient or even unlimited.

3.4. Filtration experiments

The changes in flux with time for a PES-20 membrane used for UFof T4, MS2, and phi X174 at TMPs of 1–5 bars are presented in Fig. 8.For all the viruses, the extent of fouling increased with increasedTMP. Additionally, fouling was shown to be dependent based onboth virus size and virus hydrophobicity/hydrophilicity: althoughMS2 is smaller than the relatively hydrophobic T4 and bigger thanthe relatively hydrophilic phi X174, membranes exposed to MS2were more severely fouled than membranes exposed to the othertwo viruses. For MS2, a 50% drop in flux was observed after as littleas 5 min of operation at a TMP of 5 bars. The smallest drop in fluxwas obtained for the smallest virus, phi X174, i.e., only 30% duringthe first 5 min of exposure. For T4, a 40% drop in flux was observed

Fig. 7. Size measurement of bacteriophage T4 (a), MS2 (b) and phi X174 (c).

for the same time period. Furthermore, although the drop in fluxfor T4 or phi X174 plateaued after 10 or 15 min of operation, thefouling of MS2-exposed membranes steadily increased in severity.

The first filtration test was performed with E. coli to test theoverall setup and to verify the membrane’s ability to retain microor-ganisms. UF of E. coli showed a 6-log removal of the bacteria,consistent with previous reports of a 7- to 8-log retention [1,2]. Theresults indicate that size exclusion is the main mechanism for filtra-tion of bacteria by UF membranes. Thereafter, the suitability of UFfor virus retention was examined as a function of TMP. LRV vs. TMPwas plotted for the three phages at TMP values of 1–5 bars (Fig. 9).As TMP was increased from 1 to 5 bars, the observed LRV for T4

E. Arkhangelsky, V. Gitis / Separation and Purification Technology 62 (2008) 619–628 627

Fig. 8. Effect of TMP on flux drop for T4 (a), MS2 (b) and phi X174 (c) bacteriophages.

fell from 3.83 to 3.35 (a half-log difference), for phi X174 from 3.15to 2.35 (also a half-log difference), and for MS2 from 3.82 to 2.76(>1-log removal difference). Despite the significant size differencesbetween the viruses, similar LRVs were observed for a TMP of 1 bar.At higher TMPs, however, the LRVs of the smaller viruses droppedfaster than those for T4. For example, at a TMP of 2 bars, T4 retentiondropped from 3.8 to 3.7 while phi X174 retention fell from 3.1 to 2.4.At TMPs > 2, the LRVs of MS2 and phi X174 remained almost con-stant, fluctuating only slightly. Despite the significant zeta potentialvalues of the bacteriophages and the membrane, the LRVs obtainedwere smaller than those previously reported and therefore, elec-trostatic repulsion probably did not significantly influence virus

Fig. 9. Retention of bacteriophages as a function of applied TMP.

removal. Hydrophobicity, however, does appear to be important,at least for low TMP. The size and shape of MS2 and phi X174 aresimilar, but their corresponding virus retention levels differ signif-icantly: MS2 is retained more efficiently than phi X174, an observa-tion that may be attributed to the hydrophobicity of the MS2 virus.

The above notwithstanding, size can have an effect on the degreeof retention: at all pressures, the degree of retention was foundto behave according to the following order: T4 > MS2 > phi X174.Hence, it will be probably possible to assess the degree of retentionof viruses by the smaller viruses available assuming that degree of

retention of bigger viruses will be at least as big as the removal ofthe smaller representatives. At the same time MS2 probably is notthe best choice due to high hydrophobicity and therefore enhancedremoval at lower TMP.

3.5. Discussion

It is likely that partial penetration of UF membranes by virusesis facilitated by enlargement of the membrane pores rather thanby “shrinkage” of virus particles, since elastic changes in the viralforms have never been reported.

Two hypotheses have been put forward to explain the phe-nomenon of selective solute removal by UF membranes. Thehypothesis of Urase et al. [13,14] states simply that the membraneshave abnormally large pores that are not included in the main poresize distribution of UF membranes, but there is insufficient directevidence to support this hypothesis. In addition, the hypothesisdoes not explain why the removal of bacteria and protozoa is incom-plete despite pore sizes small enough to retain viruses. The secondhypothesis rests on the presence of physical defects and the lack ofmembrane integrity manifested as pinholes in the membrane [15].

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628 E. Arkhangelsky, V. Gitis / Separation an

Although the physical defects and a lack of membrane integritymight occur, that cannot be taken as a general phenomenon. Lastly,physical defects linked to the seal material are unlikely to signifi-cantly impair virus removal rates [12,16].

In an effort to find a more satisfying explanation, the currentstudy focused on the retention of viruses as a function of TMP.Previous studies indicated that increases in TMP led to mem-brane compaction, ultimately resulting in the formation of a densermembrane with smaller pores, but this was not the case for themembranes employed in the current study. However, it is likelythat the flux deterioration associated with increased TMP is linkedto virus hydrophobicity rather than to shrinking pores. Our studydid not show the expected increase in particle retention as TMP wasincreased. One possible explanation is a pressure-induced enlarge-ment of membrane pores with time, which enables viruses topenetrate through the membrane matrix. That this process is TMP-dependent, such that the greater the increase in pressure the moreenlarged the pores become, appears to apply equally to virusesand to organic materials. Measurements performed using threeunrelated techniques for pore size determination and distributionindicated clearly that pore sizes in the membrane matrix increasedwith increasing TMP. The evidence thus indicates that viral penetra-tion of the membrane is linked to the formation with time, ratherthan to the initial presence, of abnormally large pores. The totalabsence of viruses in the permeate of diffusion-driven membraneslends support to the pore-formation hypothesis. No direct proofsfor the pressure-driven changes in membrane pore size can be pre-sented yet. The indirect evidences along with deficiencies of otherexplanations point on an additional retention mechanism that joinsthe well-known mechanisms of size based retention, charge repul-sion and hydrophobic fouling.

Assessing virus retention rates via tests that utilize polymermaterials is so far an unproven approach. The prediction of mem-brane viral retention capacity without filtration experiments withviruses may, therefore, be very difficult or even impossible. Theresults of the current research indicate the importance of develop-ing new and better nanometric prediction tools for the safe use ofUF in water treatment processes.

4. Conclusions

Recent studies indicate that viruses and bacteria may permeatepolymeric membranes and reach water streams despite the factthat they are too large to pass through the membrane pores. One

possible mechanism of penetration has its origins in TMP: the pres-sure causes enlargement of pores, with time enabling the virusesto pass through them. Complete virus retention cannot be obtainedwith pressure-driven membrane processes, and therefore, in watertreatment applications requiring the removal of viruses, optimalTMPs must be determined.

Acknowledgements

This project was partially supported by a grant from The Stephenand Nancy Grand Water Research Institute (GWRI). E.A. thanks theRieger Foundation for a Rieger-JNF fellowship in environmentalstudies. Special thanks are due to Mrs. I. Mureinik and Mr. P. Martinfor scientific editing of the manuscript.

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