Ultrafiltration MembranesIncorporating Amphiphilic CombCopolymer Additives PreventIrreversible Adhesion of BacteriaA T A R A D O U T , † S E O K T A E K A N G , ‡

A Y S E A S A T E K I N , § A N N E M . M A Y E S , | A N DM E N A C H E M E L I M E L E C H * , †

Department of Chemical Engineering, EnvironmentalEngineering Program, Yale University, New Haven,Connecticut 06520-8286, Department of Civil andEnvironmental Engineering, University of Alberta, Edmonton,Alberta, Canada T6G 2W2, and Department of ChemicalEngineering and Department of Materials Science andEngineering, Massachusetts Institute of Technology,Cambridge, Massachusetts 02139

Received September 24, 2009. Revised manuscript receivedJanuary 19, 2010. Accepted February 10, 2010.

We examined the resistance to bacterial adhesion of a novelpolyacrylonitrile (PAN) ultrafiltration membrane incorporating theamphiphilic comb copolymer additive, polyacrylonitrile-graft-polyethylene oxide (PAN-g-PEO). The adhesion of bacteria (E.coli K12) and the reversibility of adhered bacteria weretested with the novel membrane, and the behavior wascompared to a commercial PAN ultrafiltration membrane.Under static (no flow) bacterial adhesion tests, we observedno bacterial adhesion to the PAN/PAN-g-PEO membrane at allionic strengths tested, even with the addition of calciumions. In contrast, significant adhesion of bacterial cells wasobserved on the commercial PAN membrane, with increasedcell adhesion at higher ionic strengths and in the presence ofcalcium ions. Under crossflow filtration conditions, initialbacterial deposition rate increased with ionic strength andwith addition of calcium ions for both membranes, with generallylower bacterial deposition rate with the PAN/PAN-g-PEOmembrane. However, deposited bacteria were readily removed(between 97 and 100%) from the surface of the PAN/PAN-g-PEO membrane upon increasing the crossflow and eliminatingthe permeate flow (i.e., no applied transmembrane pressure),suggesting reversible adhesion of bacteria. In contrast, bacterialadhesion on the commercial PAN membrane was irreversible,with approximately 50% removal of adhered bacteria atmoderate ionic strengths (10 and 30 mM) and less than 25%removalathigh ionicstrength(100mM).Theresistancetobacterialadhesion of the PAN/PAN-g-PEO membrane was furtheranalyzed via measurement of interaction forces with atomicforce microscopy (AFM). No adhesion forces were detectedbetween a carboxylated colloid probe and the PAN/PAN-g-PEOmembrane, while the probe exhibited strong adhesion to the

commercial PAN membrane, consistent with the bacterialadhesion tests. The exceptional resistance of the PAN/PAN-g-PEO membrane to bacterial adhesion is attributable to stericrepulsion imparted by the dense brush layer of polyethylene oxide(PEO) chains.

IntroductionA major challenge in long-term operation of membranefiltration systems is the growth of biofilm on membranesurfaces, or biofouling (1). Biofouling leads to the use of higheroperating pressure, more frequent chemical cleaning, andshorter membrane life. Designing membranes that resistbiofouling is, therefore, of utmost importance for sustainableuse of membrane technology in water and wastewatertreatment as well as desalination and wastewater reuse.

The key stages in biofilm formation are bacterial deposi-tion and irreversible adhesion, formation of microcolonies,and biofilm maturation (2, 3). Most of biofouling controltechniques have been developed to prevent or retard one ormore of these stages. For example, membrane surfaces havebeen modified to be hydrophilic, negatively charged, and/orsmooth to minimize the initial adhesion of bacteria (4-7).To inactivate irreversibly adhered microorganisms andprevent the formation of microcolonies, various antimicrobialstrategies have been investigated, including coatings thatincorporate Ag or photobactericidal metal oxide nanopar-ticles (8-11), self-assembling peptides that disrupt bacterialmembranes (12, 13), and quorum quenching enzymes thatobstruct microcolony signaling linked to biofilm growth (14).Bacterial deposition in membrane systems is inevitable dueto the ubiquitous convective permeate flow and resultingpermeation drag force during membrane filtration. Whilecoatings that employ natural and engineered nanomaterialswith antimicrobial activity show promise for reducingbacterial adhesion (9, 15), such agents cannot maintain theirantimicrobial activity after adsorption of organic foulantssuch as natural organic matter and soluble microbial products(9). An ideal antibiofouling membrane should not onlyprovide effective hindrance of bacterial adhesion and cellinactivation, but also ensure that the deposition of micro-organisms on the membrane surface is reversible.

To minimize the strength of microbial adhesion and toensure that microbial adhesion to the membrane surface isreversible, various types of polymers have been grafted onmembrane surfaces, including poly(ethylene oxide) (PEO),poly(2-hydroxyethyl methacrylate), poly(2-hydroxyethyl acry-late), poly(acrylic acid), poly(acrylamide), poly(N,N-dim-ethylacrylamide), phosphorylcholine (PC), poly(2-acrylamido-2-methyl-1-propanesulfonic acid), and poly(2-(dimethylamino)ethyl methacrylate) (7, 16, 17). Among thesepolymers, PEO has been shown to be a very effective materialto prevent adhesion of biomacromolecules due to itshydrophilicity, large excluded volume, and unique ability tocoordinate surrounding water molecules in an aqueousmedium (18-20). For example, we have investigated the useof amphiphilic graft/comb copolymers having hydrophobicbackbones (polyvinylidene fluoride, PVDF) and hydrophilicPEO side chains (poly(oxyethylene) methacrylate) as surface-modifying membrane coatings (21). More recently, theantifouling property of a novel polyacrylonitrile (PAN)ultrafiltration (UF) membrane incorporating the amphiphiliccomb copolymer additive, polyacrylonitrile-graft-poly-ethylene oxide (PAN-g-PEO), has been successfully dem-onstrated with bovine serum albumin, sodium alginate, andhumic acid as model organic foulants (19, 22).

* Corresponding author phone: (203)432-2789; e-mail:menachem.elimelech@yale.edu.

† Yale University.‡ University of Alberta.§ Department of Chemical Engineering, MIT.| Department of Materials Science and Engineering, MIT.

Environ. Sci. Technol. 2010, 44, 2406–2411

2406 9 ENVIRONMENTAL SCIENCE & TECHNOLOGY / VOL. 44, NO. 7, 2010 10.1021/es902908g 2010 American Chemical SocietyPublished on Web 03/01/2010

In this study, we examined the resistance of the PAN/PAN-g-PEO ultrafiltration membrane to bacterial adhesionand, more importantly, the reversibility of bacterial adhesionto the membrane. A Gram negative bacterial strain, Escheri-chia coli K12 MG1655, chromosomally tagged with greenfluorescence protein, was used as a model bacterium. Staticadhesion tests and crossflow membrane filtration experi-ments with a system that allows direct observation of celldeposition on the membrane surface demonstrated thesuperb resistance of the membrane to bacterial adhesion.Measurements of interaction forces with atomic forcemicroscopy (AFM) were used to elucidate the mechanismsgoverning the resistance of the PAN/PAN-g-PEO membraneto bacterial adhesion.

Materials and MethodsUltrafiltration Membrane. A novel fouling resistant ultra-filtration membrane, denoted PAN/PAN-g-PEO, was fabri-cated following two steps. First, a polyacrylonitrile-graft-poly(ethylene oxide) (PAN-g-PEO) comb copolymer wassynthesized by free radical polymerization of acrylonitrileand poly (ethylene glycol) methyl ether acrylate (PEGA,Mn)454 g/mol). Next, the PAN/PAN-g-PEO blend membranewas prepared from a mixture of polyacrylonitrile (PAN) andPAN-g-PEO solutions by precipitation followed by annealingin a water bath. Details on the preparation and characteriza-tion of the PAN/PAN-g-PEO membrane are given elsewhere(19, 22).

A commercial PAN ultrafiltration membrane (Osmonics,Minnetonka, MN), with a similar hydraulic permeability asthat of the PAN/PAN-g-PEO blend membrane, was selectedfor comparison. The commercial PAN membrane is madefrom polyacrylonitrile that has been modified to possess ahydrophilic surface with minimal surface roughness (23).The manufacturer rates the commercial PAN membrane ashaving a nominal molecular weight cutoff of approximately45 kDa at 90% retention of dextran.

Measurement of Membrane Contact Angle and ZetaPotential. Contact angle measurements were performedusing a VCA-2500 system (AST Products, Bellerica, MA). Priorto measurement, the membrane coupons were air-driedovernight. The dried membrane coupon was then adheredto a glass slide by a double-sided tape and was brought nearthe tip of the syringe. DI water was injected with the syringeuntil a water droplet formed on the membrane surface (∼2.5µL per droplet). About 10 contact angle measurements wereperformed across the membrane coupon at equally spacedintervals. Each measurement provided left and right contactangles from a cross-sectional view of a water droplet placedon the membrane surface. There was no difference betweenthe left and right contact angles, and the average of 10measurements (20 values) was calculated.

An asymmetric clamping cell was employed to measurethe streaming potential of the membranes (EKA, BrookhavenInstruments, Holtsville, NY). Streaming potential measure-ments were taken 8 times with alternating flow direction ofthe 10 mM NaCl solution (pH∼5.5, unadjusted). Detailedexperimental procedure and the method to calculate the zetapotential from the measured streaming potential are givenelsewhere (24).

Model Bacteria. Escherichia coli K12 MG1655 was usedas a model bacterial cell (25). The strain was kindly receivedfrom S. Molin, the Technical University of Denmark. To allowlive cell detection under fluorescent microscopy, bacterialstrains were tagged with a plasmid coding for green fluo-rescent protein (MG1655 was introduced with the suicideplasmid pSM1696). The E. coli cells were incubated andharvested at midexponential growth phase in Luria Bertani(LB) broth with 50 mg/L kanamycin at 37 °C. The bacterialsuspension was centrifuged (Sorvall SS-34) at 15,000 rpm for

3 min and then washed/resuspended with 154 mM NaClsolution. The centrifugation and resuspension were repeatedthree times to remove organic and inorganic impurities. Afterthe last centrifugation, the bacterial cell pellet was resus-pended in the electrolyte solution which was used in theexperiment and was vortexed shortly.

The electrophoretic mobility of the bacterial cells wasmeasured as a function of ionic strength at ambient pH (∼5.5)and room temperature (22 °C) using ZetaPALS (BrookhavenInstruments, Holtsville, NY). Electrophoretic mobilities werethen converted to zeta potentials using the Smoluchowskiequation because of the relatively large size of the bacterialcells and the ionic strength used.

Solution Chemistries. Four different electrolyte solutionswere used for the bacterial adhesion and deposition experi-ments: 10, 30, and 100 mM NaCl, and a combination of 27mM NaCl and 1 mM CaCl2 (total ionic strength of 30 mM).Chemicals were ACS grade (Fisher Scientific, Pittsburgh, PA).The electrolyte solutions were prepared with deionized water(Millipore, MA). Solutions were used without pH adjustment,with the ambient pH ranging from 5.5 to 5.7.

Static Bacterial Adhesion Tests. Two membrane coupons(each with dimensions of 1 cm by 1 cm) were placed in 20mL cell suspension (4 × 107 cells/mL) at the desired electrolytesolution. The cells and the membranes were incubated in ashaker (Lab-line 4631 Maxi Rotator) at 20 rpm and roomtemperature (22 °C) for 1 h. The membrane coupons werethen rinsed gently with a bacteria-free electrolyte solutionhaving the same concentration as that used for the adhesiontest to remove weakly bound cells. Membrane coupons werethen observed under a fluorescent microscope (OlympusBX41, Japan), and at least 10 images were taken across themembrane surface. The average number of cells on themembrane was normalized by the observed membrane area(0.145 mm2).

Direct Microscopic Observation of Bacterial Depositionand Reversibility. The crossflow membrane filtration unitfor direct observation of bacterial deposition is depicted inFigure S1. We have used such a system previously for studyingprotein fouling of ultrafiltration membranes (19). Briefly, thefeed solution (2 L) was pressurized to 150 kPa (1.5 bar) andcirculated by a gear pump (Cole-Parmer, Vernon Hills, IL)at a fixed crossflow velocity of 10 cm/s in a crossflow channel(9 cm long, 3 cm wide, and 0.1 cm high). The permeate fluxwas kept constant at 3 × 10-5 m/s (108 L/m2 ·h) during theruns by an 8-roller digital peristaltic pump (Cole-Parmer,Vernon Hills, IL) mounted on the permeate line. Themembrane was first compacted and equilibrated for 50 minwith bacteria-free electrolyte solution that had solutionchemistry identical to that used in the subsequent foulingruns (10, 30, and 100 mM NaCl, and 27 mM NaCl plus 1 mMCaCl2). An appropriate amount of bacterial cells was thenadded to the feed tank to achieve cell concentration of ∼1.3× 105 cells/mL. The bacterial deposition experiments werecarried out for 30 min at a constant permeate flux, with animage of the membrane taken every 5 min. The depositionrate coefficient of bacterial cells was calculated from the slopeof the curve plotting the number of deposited cells per unitmembrane area versus time divided by the number con-centration of cells in the influent (26). Bacterial depositionruns were carried out at least three times for each electrolytecondition at an ambient pH (unadjusted, pH of 5.5-5.7) anda temperature of 22 °C.

At the conclusion of the fouling runs, physical cleaningexperiments were preformed by increasing the crossflowvelocity from 10 to 150 cm/s while turning off the permeatepump (relaxing the transmembrane pressure) for 10 min,without changing the feed solution or the pressure (150 kPa)in the system. After 10 min of physical cleaning, images ofthe membrane were taken. For cases where we had less than

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∼20 cells in the image, another 7 images at different locationsof the membrane were taken. Cleaning efficiency wascalculated as the percentage of the number of cells remainingon the membrane after the 10 min of cleaning compared tothe number of cells on the membrane after the 30 mindeposition run (just before starting the cleaning). All re-versibility tests were performed at least three times.

Interaction Force Measurements. A Nanoscope IIIaMultimode atomic force microscope (AFM) (Digital Instru-ments, Santa Barbara, CA) was used to quantify the interac-tion forces between foulant and membrane surfaces. Acarboxylate-modified latex (CML) particle (Interfacial Dy-namics Corp., Portland, OR), 3.9 µm in diameter, was gluedto the end of a commercial 0.06 N/m SiN tipless cantilever(Veeco Metrology Group, Santa Barbara, CA). The particlewas used as a surrogate for the bacteria cells because it carriesnegative carboxylic functional groups similar to biomacro-molecules (19, 22, 27) and bacterial cells (28). The AFM wasoperated in force mode, with an approach/retraction speedof 1 µm/s and 1 µm of piezo-movement. The experimentswere performed in a liquid cell, and the system was allowedto equilibrate for 60 min for each experimental condition.Raw data were converted from cantilever deflection andz-piezo position into force-distance curves. The force wasthen normalized to the radius of the CML particle, R. Thedetailed experimental procedures for using the AFM colloid-probe technique to quantify interaction forces relevant toorganic fouling are given in our previous publications(29-31). Force measurements were performed at fourdifferent locations on the membrane surface, with 20measurements at each location to minimize inherent vari-ability in the force data. The solution chemistries of the testsolutions in the liquid cell were the same as those used inthe adhesion and deposition experiments.

Results and DiscussionBacterial Electrokinetic Properties. The electrophoreticmobility (EPM) and corresponding calculated zeta potentialfor the E. coli cells as a function of solution ionic strengthand the presence of divalent (calcium) cations are presentedin Figure 1. Measured electrophoretic mobilities (EPM)followed expected behavior of decreasing in magnitude withincreasing ionic strength due to compression of the electricdouble layer. Addition of divalent Ca2+ ions further decreasedthe magnitude of bacterial cell EPM, likely due to specificinteraction with bacterial surface functional groups andcharge neutralization. The relatively high residual electro-phoretic mobility at high ionic strength is indicative of thepresence of the “soft” polyelectrolyte layer at the bacterialsurface (32).

Bacterial Adhesion and Reversibility. Under static, batchconditions (no pressure, no flow), there is a significantdifference in the affinity of the bacterial cells to the PAN/PAN-g-PEO and the commercial PAN membranes (Figure2). The number of attached bacterial cells on the commercialPAN membrane increased as the ionic strength of the solutionincreased. Similarly, the addition of Ca2+ ions resulted in anincrease of the number of attached cells to the commercialmembrane, compared to the case with no Ca2+ but a similartotal ionic strength (30 mM). The observed behavior withthe commercial PAN membrane agrees with the trendsobserved with the EPM of E. coli cells (Figure 1), therebysuggesting that electrostatic interactions govern the celladhesion behavior. In a stark contrast, no bacteria attachedto the PAN/PAN-g-PEO membrane at any of the solutionconditions investigated. This observation suggests that thePAN/PAN-g-PEO membrane has strong antifouling proper-ties that prevent irreversible adhesion of bacteria at thesurface. Further discussion on the bacterial adhesion mech-anism, supported by AFM measurements, is given later inthe paper.

Bacterial Deposition Rate and Reversibility in CrossflowFiltration. The bacterial deposition rate coefficients on bothmembranes, determined from the crossflow filtration ex-periments, are presented in Figure 3a. For both the com-mercial and the PAN/PAN-g-PEO membranes, the depositionrate increases continuously with increasing ionic strengthand following the addition of 1 mM of calcium ions at a fixedtotal ionic strength of 30 mM. The deposition rate on thecommercial PAN membrane is higher than that on the PAN/PAN-g-PEO membrane at ionic strengths of 10 and 30 mM,but attains similar values at high ionic strength (100 mM).

The deposition of bacterial cells on the membrane surfaceis determined by the interplay between permeation dragresulting from the convective permeate flow and the repulsiveinteractions between the bacteria and the membrane surfaceat close separations. The latter interactions include elec-trostatic interactions as well as steric forces for the PAN/PAN-g-PEO membrane as discussed in detail later. As theionic strength increases, the range and magnitude of theelectrostatic repulsion is decreased, and bacterial cells caneither attach to the membrane or be held very close to themembrane surface by the permeate drag force that actscontinuously on the cells. Note that the permeation flow inultrafiltration is relatively high compared to reverse osmosisor nanofiltration membranes (3 × 10-5 m/s or 108 L m2- h-1

in our case), resulting in a significant permeation drag force.The deposition or accumulation of bacteria on the UF

membrane surface is inevitable because of the ubiquity ofthe permeation drag force during membrane filtration. Akey aspect to investigate is the reversibility of bacterialdeposition on the membrane surface. Because UF membrane

FIGURE 1. Electrophoretic mobility (EPM) and calculated zetapotential of E. coli cells at different ionic strengths andcomposition. Experimental conditions: unadjusted (ambient) pHof 5.5-5.7, temperature of 22 °C.

FIGURE 2. Number of E. coli cells attached on the unitmembrane surface (per mm2) at various solution ionic strengths.Membrane coupons were incubated for 1 h with ∼4 × 107

cells/mL. Experimental conditions: unadjusted (ambient) pH of5.5-5.7, temperature of 22 °C. No cell attachment to the PAN/PAN-g-PEO membrane was observed.

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systems involve frequent physical cleaning of the membrane(with no chemicals), bacteria that reversibly deposit on themembranes will be readily washed away, thus preventingbacterial growth and subsequent biofilm formation. Figure3b presents the percent of deposited cells that were removedfrom the surface when subjected to “physical cleaning” atelevated crossflow (150 cm/s) with no permeate flow (orapplied transmembrane pressure). We observe relatively lowremoval of deposited bacteria from the commercial mem-brane (at most ∼50%) compared to above 97% and oftenclose to 100% for the PAN/PAN-g-PEO membrane. Further-more, while the removal efficiency from the commercial PANmembrane is dependent on ionic strength, dropping to lessthan 25% at 100 mM, the removal of deposited bacteria fromPAN/PAN-g-PEO membrane is independent of ionic strength.As we discuss later, bacteria attach irreversibly on thecommercial PAN membrane in deep energy wells becauseno repulsive forces exist at very close separations betweenthe bacteria and the membrane surface. On the other hand,strong repulsive forces between the bacterial cells and thePAN/PAN-g-PEO membrane exist very close to the membranesurface, and once the permeation drag force is eliminated(when permeate flow vanishes), the bacterial cells near themembrane surface are washed away by the crossflow.

Why Is Bacterial Adhesion to the PAN/PAN-g-PEOMembrane Negligible? As our previous studies demonstrated(19, 29, 31), atomic force microscopy (AFM) allows bothquantitative measurement of adhesion forces and mecha-nistic understanding of foulant-membrane interaction. Wehave also shown that F/R, that is the adhesion (pull-off) force(F) normalized by the radius of the colloid/foulant probe(R), serves as an indicator for the fouling propensity ofpolymeric membranes. Bacterial adhesion behavior observed

in the static adhesion tests (Figure 2) and the measuredadhesion forces (Figure 4) are in general agreement with ourprevious findings. The results imply that the presence ofadhesive forces (F/R < 0) between the model colloid probeand the membranes is an indicator for bacterial adhesionpotential, as we showed earlier for adhesion of biomacro-molecules (19, 31, 33). For the commercial PAN membrane,relatively strong adhesion forces (i.e., negative values of F/R)are measured for the entire range of ionic strength inves-tigated (Figures S2 and S3), thereby explaining the highnumber of attached cells on the membrane surface. Incontrast, no adhesion forces (i.e., no negative values of F/R)were detected between the CML probe and the PAN/PAN-g-PEO membrane (Figure 4, Figures S2 and S3). As shown inFigure 4, F/R values (or adhesion events) were distributedbetween zero to positive values for all solution chemistriestested, with or without calcium, which explains the absenceof cell attachment on the PAN/PAN-g-PEO membranesurface. Note that the positive adhesion force values in Figure4 correspond to the repulsive force at zero separation, as nonegative (adhesion) events were detected.

The observed cell deposition kinetics of E. coli cells undercrossflow membrane filtration conditions were similar forthe commercial PAN membrane and PAN/PAN-g-PEO blendmembrane (Figure 3a). This observation is attributable tothe permeation drag force which acts on the cells when thefiltration system is under pressure. Cells are transported fromthe bulk suspension toward the membrane by the convectivepermeate flow and subsequently deposit on the membranesurface. On the commercial PAN membrane surface, thedeposition of cells resulted in irreversible attachment (Figure3b), supported by the strong adhesion forces measured withAFM. On the other hand, on the PAN/PAN-g-PEO membranesurfaces, cells are only held on the membrane surface by thepermeation drag force. Consequently, when the permeationflow is stopped (by relaxing the applied transmembranepressure), the deposited E. coli cells diffuse back to the bulksolution because only repulsive forces exist at contact withthe membrane surface (Figure 4). Figure 3b demonstratesthe near complete removal (detachment) of E. coli cells fromthe membrane when subjected to elevated crossflow velocity(150 cm/s) for 10 min with no permeation flow (or appliedtransmembrane pressure). For the commercial membrane,removal of deposited cells at higher crossflow velocity wasnot effective (<50%), indicating irreversible adhesion ofdeposited cells.

FIGURE 3. (a) Observed cell deposition rate coefficients of E.coli during 30 min of crossflow filtration. Deposition experimentswere carried out at a crossflow velocity of 10 cm/s, permeationvelocity of 30 µm/s, and cell concentration of ∼1.3 × 105

cells/mL at a constant pressure of 150 kPa (1.5 bar). (b) Percentremoval of E. coli cells from the membrane after 10 min ofrinsing with the same solution at 150 cm/s cross-flow velocityand with no permeate flow at 150 kPa (1.5 bar).

FIGURE 4. Distributions of adhesion forces (at contact or zeroseparation) between the AFM colloid probe and the PAN/PAN-g-PEO membrane. Solution conditions are the same asthose used in the corresponding cross-flow cell deposition andstatic adhesion experiments. Solid lines represent the fit ofGaussian distribution.

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Retraction force profiles for the PAN/PAN-g-PEO mem-brane greatly differ from those of the commercial PANmembrane (Figure S2), although the contact angle (35 ( 5°for the commercial PAN and 35 ( 6° for the PAN/PAN-g-PEO membrane) and zeta potential (-11.3 mV for bothmembranes) were similar for both membranes. This obser-vation suggests that the resistance to bacterial adhesion ofthe PAN/PAN-g-PEO membrane cannot be explained byelectrostatic or hydrophilic/hydrophobic interactions (19, 34).To gain insight into the antibiofouling mechanism of thePAN/PAN-g-PEO membrane, we analyzed the approach forceprofiles between the AFM colloid probe and the membrane.The approach force curves between the model colloid probeand the PAN/PAN-g-PEO membrane surface are purelyrepulsive and perfectly match the profile of retraction(adhesion) force curves (Figure S4). Moreover, semilog plotsof the approach force profiles under various solutionchemistries show that the range or the decay length of theinteraction forces (i.e., the distance where the force decaysto near zero) is much greater than that expected forelectrostatic repulsion between charged surfaces (Figure 5).For instance, the decay length for electrostatic repulsion(characterized by the Debye screening length) varies from10 to 1 nm when the ionic strength increases from 1 mM to100 mM, respectively, whereas the observed range ofinteraction for the PAN/PAN-g-PEO membrane is much largerand remains unchanged up to 25 nm at all ionic strengthsinvestigated. These observations suggest that the interactionbetween model colloid probe and the PAN/PAN-g-PEOmembrane is not electrostatic and that other forces, namelysteric repulsion, govern the interaction with the membrane.In contrast, the decay lengths for the commercial PANmembrane closely follow the calculated Debye lengthsexpected for electrostatic repulsion, thus supporting theobserved increase in bacterial adhesion with increasing ionicstrength (Figure 2).

Steric Repulsion Governs the Resistance of the PAN-g-PEO Membrane to Bacterial Adhesion. Grafting of PEOto surfaces has been used extensively to reduce organicadsorption primarily through its hydrophilicity, large ex-cluded volume, electroneutrality, and unique ability tocoordinate surrounding water molecules in aqueous media(18, 19, 34). These features of the PEO brushlike layer providea strong steric barrier for adsorption of bacteria. Our AFMinteraction force analysis strongly supports the presence ofa steric barrier for bacterial adsorption imparted by the PAN-g-PEO amphiphilic comb copolymer. As discussed above,we observe no adhesion forces between the colloid probe

and the PAN/PAN-g-PEO membrane (Figure 4 and FiguresS2 and S3), with the adhesion curves displaying strongrepulsion at contact (Figure S4). Second, the interaction forceprofiles for the PAN/PAN-g-PEO membrane are similar at allionic strengths and display long-range repulsive forces underall solution chemistries tested (Figure S2). During the castingof the PAN/PAN-g-PEO membrane, the high molecularweight (∼170 kDa) PAN-g-PEO additive segregates to forma dense PEO brush layer on membrane surfaces and pores.X-ray photoelectron spectroscopy (XPS) has verified that thePEO content on the membrane surface is much higher thanthe overall PEO content of the blend (22). The dense PEOchains stretch into the surrounding solution and provide asteric barrier that prevents attachment of bacteria. Thecompression of the PEO brush layer by bacteria approachingthe PAN/PAN-g-PEO membrane surface leads to an increasein the local concentration of PEO, which, in turn, leads to anexponential increase of the repulsive interaction (Figure S4).The AFM approach curves clearly indicate that, under allionic strengths investigated, bacterial cells experience repul-sion at a distance of ∼20 nm from the surface at which theattractive van der Waals interaction force is almost negligible(Figure S2).

AcknowledgmentsWe thank S. Molin from the Technical University of Denmarkfor sending plasmid pSM1696. The work was supported bythe WaterCAMPWS, a Science and Technology Center ofAdvanced Materials for the Purification of Water with Systemsunder the National Science Foundation Grant CTS-0120978.

Supporting Information AvailableSchematic diagrams of the crossflow membrane cell andclosed-loop direct microscopic observation crossflow mem-brane filtration system (Figure S1), representative retractionforce curves for the PAN/PAN-g-PEO and commercial PANmembranes (Figure S2), average values of normalized adhe-sion forces (F/R) (Figure S3), and representative approachand retraction force curves for the PAN/PAN-g-PEO mem-brane (Figure S4). This material is available free of charge viathe Internet at http://pubs.acs.org.

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