Article pubs.acs.org/est

Effect of Chemical Oxidation on the Sorption Tendency of Dissolved Organic Matter to a Model Hydrophobic Surface Teng Zeng,†,§ Corey J. Wilson,† and William A. Mitch*,§ †

Department of Chemical and Environmental Engineering, Yale University, 9 Hillhouse Avenue, New Haven, Connecticut 06520, United States § Department of Civil and Environmental Engineering, Stanford University, 473 Via Ortega, Stanford, California 94305, United States S Supporting Information *

ABSTRACT: The application of chemical oxidants may alter the sorption properties of dissolved organic matter (DOM), such as humic and fulvic acids, proteins, polysaccharides, and lipids, affecting their fate in water treatment processes, including attachment to other organic components, activated carbon, and membranes (e.g., organic fouling). Similar reactions with chlorine (HOCl) and bromine (HOBr) produced at inflammatory sites in vivo affect the fate of biomolecules (e.g., protein aggregation). In this study, quartz crystal microbalance with dissipation monitoring (QCM-D) was used to evaluate changes in the noncovalent interactions of proteins, polysaccharides, fatty acids, and humic and fulvic acids with a model hydrophobic surface as a function of increasing doses of HOCl, HOBr, and ozone (O3). All three oxidants enhanced the sorption tendency of proteins to the hydrophobic surface at low doses but reduced their sorption tendency at high doses. All three oxidants reduced the sorption tendency of polysaccharides and fatty acids to the hydrophobic surface. HOCl and HOBr increased the sorption tendency of humic and fulvic acids to the hydrophobic surface with maxima at moderate doses, while O3 decreased their sorption tendency. The behavior observed with two water samples was similar to that observed with humic and fulvic acids, pointing to the importance of these constituents. For chlorination, the highest sorption tendency to the hydrophobic surface was observed within the range of doses typically applied during water treatment. These results suggest that ozone pretreatment would minimize membrane fouling by DOM, while chlorine pretreatment would promote DOM removal by activated carbon.

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desalination, and drinking water treatment reduces water flux and increases energy consumption and operational costs.9−12 DOM molecules sorb to hydrophobic membrane surfaces, promoting fouling.13 Recent research has focused on decreasing membrane surface hydrophobicity to attenuate fouling by DOM.13 However, chlorine or chloramines often are applied upstream of membrane processes employed for municipal wastewater recycling or seawater desalination to minimize biofouling.14−19 Recently, ozone has been pilot-tested as an alternative oxidant for the mitigation of organic fouling on RO membranes in a municipal wastewater reclamation system.20,21 To understand the impacts of changes in oxidative treatments on membrane fouling, it would be useful to evaluate the effects of different oxidants on the sorption tendency of DOM to hydrophobic surfaces. Oxidative modifications of biomolecules also carry important implications in the etiology and pathogenesis of human diseases. Hypochlorous acid and hypobromous acid are produced by neutrophils and eosinophils, respectively, as part

INTRODUCTION The sorption properties of dissolved organic matter (DOM) constituents affect their fate both within water treatment plants and at inflammatory sites in vivo. Traditionally, humic and fulvic acids, deriving from the degradation of plant-based biopolymers (e.g., lignin), are considered as the predominant dissolved organic constituents in pristine drinking water sources.1 Faced with rising populations, drinking water utilities are increasingly evaluating water supplies impaired by upstream wastewater discharges or algal blooms, the intentional recycling of municipal wastewater for potable uses, and even seawater desalination.2 In addition to humic and fulvic acids, these waters feature elevated concentrations of biomolecules including proteins, polysaccharides, and lipids.3,4 The sorption properties of these DOM constituents govern their potential to sorb to other organic components, to activated carbon,5,6 and to membranes.7,8 Covalent modifications to DOM molecules caused by reactions during chemical oxidation are likely to alter their sorption properties, and thus their removal by different treatment processes. For example, organic fouling in pressuredriven membrane separation processes (e.g., microfiltration, ultrafiltration, nanofiltration, and in particular reverse osmosis (RO)) employed for wastewater reclamation, seawater © 2014 American Chemical Society

Received: Revised: Accepted: Published: 5118

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of the immune response to pathogen infections.22,23 Nevertheless, overproduction of these oxidants can damage host biomolecules, including proteins, carbohydrates, and lipids.24 Elevated oxidant production has been associated with the progression of various inflammatory diseases, such as atherosclerosis, cystic fibrosis, asthma, arthritis, and Alzheimer’s disease.24 Because of their prevalence in plasma and cells and their high reactivity with oxidants, significant attention has been paid to proteins as a major target for hypohalous acid reactions.25 Covalent modifications to amino acid side chains by oxidants promote the progressive unfolding and aggregation of proteins,26 which not only contributes to pathogen inactivation, but also disease manifestations.24 For example, protein aggregation mediated by oxidants is associated with increasing cellular dysfunction and aging.26,27 Noncovalent interactions play a key role in the initial stages of accumulation and aggregation of oxidized proteins.26,27 The goal of this work was to quantify changes in the sorption tendency of proteins, polysaccharides, fatty acids, and humic and fulvic acids to a model hydrophobic surface upon oxidation by chlorine (HOCl), bromine (HOBr), or ozone (O3). Although relative retention on reversed-phase high-performance liquid chromatography has been used to infer the apparent hydrophobicity of humic and fulvic acids,28 the prolonged contact with organic solvents and the column stationary phase causes protein denaturation and an artifactual increase in hydrophobicity resulting from the exposure of the hydrophobic protein core.29 Fluorescence spectroscopy employing hydrophobic dyes offers a rapid and sensitive measure to probe the hydrophobicity of proteins under organic solvent-free conditions,30 but this technique cannot be applied to other DOM constituents. Additionally, residual oxidants, including organic chloramines and bromamines, may alter the fluorescence properties of the dyes. Size exclusion chromatography can fractionate DOM constituents31,32 but cannot distinguish aggregates resulting from covalent cross-linkages or noncovalent interactions. Herein, we applied quartz crystal microbalance with dissipation monitoring (QCM-D) to compare variations in the sorption tendency of proteins, polysaccharides, fatty acids, and humic and fulvic acids to a hydrophobic surface before and after exposure to different doses of HOCl, HOBr, or O3. For QCM-D, a laminar-flow stream of aqueous solution containing the oxidized DOM constituents is passed over an ultrasensitive quartz mass sensor crystal, which monitors the accumulation of mass on the surface via the change in the resonant frequency of the crystal to a resolution of 1 ng/cm2.33,34 We functionalized gold-coated quartz sensor crystals with a homogeneous hydrophobic 1-octadecanethiol self-assembled monolayer (SAM).35 Attachment of DOM molecules to the sensor crystal surface results from noncovalent interactions, not covalent cross-linkages. Although QCM-D has been used to track the kinetics, extent, and reversibility of DOM sorption to a variety of SAMs representative of membrane surface functionalities relevant for water treatment,36−39 this technique has not previously been applied to evaluate changes in the sorption properties of DOM resulting from oxidative treatments. Accordingly, two independent techniques, sorption of a hydrophobic fluorescent dye to oxidized proteins and sorption of oxidized DOM to sheet graphite, were used to validate results obtained via QCM-D.

Article

MATERIALS AND METHODS

Materials and Samples. Chemical sources and purities are provided in the Supporting Information (SI). HOCl, HOBr, and O3 working solutions were freshly prepared and standardized according to published procedures (see SI section S1 for details). Note that HOCl and HOBr were used to represent the sum of all free chlorine and bromine species, respectively.40 Five proteins, two polysaccharides, two fatty acids, two humic acids, and two fulvic acids were chosen as model DOM for use in the oxidation-attachment experiments. Selected characteristics of model DOM are summarized in SI Tables S1−S3. Model proteins were selected to cover a range of molecular weights and numbers of oxidizable residues, and because we had previously evaluated the oxidation of their specific residues and loss of secondary structure upon exposure to HOCl and HOBr.40 Bovine serum albumin (BSA; ≥99%), human serum albumin (HSA; ≥99%), and lysozyme (LYZ; ≥99%) were obtained from Sigma-Aldrich. Adenylate kinase (ADK) and ribose-binding protein (RBP) were expressed and purified as described before.40 HSA is the most abundant carrier protein in human plasma and plays an essential physiological role in the transport and disposition of endogenous ligands and xenobiotics, as well as the regulation of osmotic pressure and pH in the bloodstream.41 BSA is a transport protein bearing an overall 75.6% sequence identity to HSA42 and is frequently used as a generic protein in environmental QCM-D studies.37,38,43 LYZ is a glycoside hydrolase that catalyzes the hydrolysis of the peptidoglycan architecture in bacterial cell walls.44 ADK is a phosphotransferase that catalyzes the interconversion of adenine nucleotides and modulates cellular energy homeostasis.45 RBP is a carbohydrate binding protein that acts as the primary receptor for extracellular solutes in Gram-negative bacteria.46 Model polysaccharides, including alginate (ALG; a β-(1,4)-linked nonrepeating heteropolymer consisting of mannuronic and guluronic acids) and dextran (DEX; a α(1,6)-linked and α-(1,3)-branched homopolymer consisting of glucose), were obtained from Sigma-Aldrich. Model fatty acids, including oleic acid (OA; cis-9-octadecenoic acid, ≥99%) and linoleic acid (LOA; cis-,cis-9,12-octadecenoic acid, ≥98%), were obtained as sodium salts from Sigma-Aldrich. Model humic and fulvic acids, including Leonardite humic acid (LHA), Pahokee Peat humic acid (PPHA), Nordic Lake fulvic acid (NLFA), and Suwannee River fulvic acid (SRFA), were obtained from the International Humic Substances Society. The dissolved organic carbon (DOC) content of DOM stock solutions was measured on a Shimadzu TOC-V CPH total organic carbon analyzer. Two water samples were collected for use in the oxidationattachment experiments. Selected properties of water samples are provided in SI Table S4. Sample KY was a primary sedimentation effluent collected from a water utility located in Kentucky, whose source water was impacted by upstream municipal wastewater discharges. Sample CT was raw water collected from the Mill River in Connecticut, a river impacted by urban stormwater and agricultural runoff. Both samples were sequentially vacuum-filtered through precombusted 0.7-μm Whatman glass fiber filters and 0.45-μm Millipore polycarbonate filters, and refrigerated at 4 °C in the dark until use. DOM Oxidation Experiments. All DOM oxidation experiments were conducted using Eppendorf LoBind polypropylene vials to minimize adsorptive losses of DOM substrates, particularly proteins.43,47 Control experiments indicated no significant reaction of the oxidants with the 5119

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were analyzed using the QTools modeling software for the initial attachment rate of DOM kDOM (ng·cm−2·min−1) by fitting the linear increase in Δm from 1 to 3 min after the DOM-containing solution or water sample was introduced into the flow chamber.37,51,52 The first minute of data was excluded to ensure calculated rates were obtained when the bulk fluid DOM concentration reached a steady state.52 To quantitatively compare different DOM-oxidation scenarios, the initial attachment rate of the oxidized DOM was normalized against that of the unoxidized DOM to determine the relative attachment efficiency αR (eq 2):

vials. Each set of vials contained the same volume of DOM stock solutions that were supplemented with oxidants and buffered at pH 7.4 using 5 mM phosphate buffer to target a constant final volume. With this strategy, the DOC content of reaction solutions was maintained at 2 mgC/L. Oxidation was initiated by spiking in HOCl, HOBr, or O3 working solutions to achieve a DOC-normalized dose range of 0 to 1.0 mmol of oxidant per mmol of carbon (i.e., 0−1.0 mmoloxidant/mmolC), which covered doses commonly used for water treatment. Upon oxidant addition, the vials were immediately capped, briefly vortex-mixed, and then incubated in the dark at 23 ± 1 °C for 24 h. After the incubation, no residual ozone was detectable. The maximum total residual chlorine or bromine was 10 μM, but a significant fraction of this total likely existed in the form of organic chloramines or bromamines. No oxidant quenching agent was added to avoid unintended alterations of sample composition.48 Water samples were treated following the same oxidation protocols after adjustment of the initial pH and DOC levels to match those of the model DOM solutions. DOM Attachment Experiments. All DOM attachment experiments were conducted on a Q-Sense D300 quartz crystal microbalance (Q-Sense AB, Sweden) equipped with a radial stagnation point, temperature-controlled flow chamber. A second identical module was run in parallel for duplicate measurements. Prior to use, gold-coated AT-cut quartz sensor crystals were functionalized with a 1-octadecanethiol selfassembled monolayer as previously described.35 Further details regarding the preparation and characterization of the model hydrophobic surface are given in the SI (see section S2). Upon attachment of DOM molecules onto the crystal surface, the QCM-D can simultaneously monitor shifts in the resonance frequency (Δf) and the energy dissipation (ΔD) of the crystal at its fundamental (4.95 MHz) and oscillation overtones.49 For thin, rigid adlayers (the ΔD/Δf ratio ≪ 0.4 × 10−6),34 an increasing total sensed mass, including sorbed DOM and hydrodynamically coupled water molecules, is proportional to a decreasing resonance frequency as described by the Sauerbrey equation (eq 1):50 Δm = C

αR =

kDOM,unoxidized

(2)

Under this definition, an αR value greater than one indicates an increased sorption tendency toward the hydrophobic surface relative to the native DOM molecule. The minimum discernible αR that could be differentiated from the background signal noise was determined to be 0.007. This value was derived by dividing the largest slope of the 10 min of Δf n/n data immediately prior to the introduction of DOM to the flow chamber by kDOM,unoxidized.52 At the end of each QCM-D run, used sensor crystals were replaced with recoated crystals and the flow chamber was thoroughly rinsed by a 2% Hellmanex solution and ultrapure water to avoid cross contamination. One set of example DOM mass accumulation profiles for protein ADK are provided in SI Figure S4. Complementary Techniques for Evaluating DOM Sorption Tendency. Changes in the mass accumulation rate with oxidant dose measured by QCM-D may reflect either changes in the sorption properties of DOM molecules or changes in their water contents. Two complementary techniques were employed to distinguish these two possibilities. Bis-ANS is a fluorescent dye that can bind to hydrophobic patches in protein molecules and has long been used for protein characterization.30,53 Experimental details and results for the application of this dye to O3-treated BSA are given in the SI (see section S3). Additionally, the tendency of oxidized DOM to sorb to nonporous sheet graphite, another model hydrophobic surface, was investigated through batch sorption experiments. The sheet graphite was exhaustively washed with ultrapure water, oven-dried at 90 °C for 48 h, and stored under nitrogen prior to use. Selected properties of sheet graphite are provided in SI Table S5. Vials containing the oxidant-treated DOM solution or water sample were amended with graphite and equilibrated on a rotary shaker in the dark at 23 ± 1 °C for 72 h. After the equilibration, solutions from each vial were analyzed for DOC. Graphite-to-solution ratios were adjusted to achieve 30−50% sorption of the unoxidized DOM to the graphite surface as measured by DOC. Control vials without graphite showed no measurable loss of DOM. The amount of sorbed DOM per unit mass of graphite (q) was calculated (eq 3):

−Δfn (1)

n

kDOM,oxidized

−2

where Δm is the change in sorbed mass density (ng·cm ), C is the mass sensitivity constant (17.7 ng·Hz−1·cm−2), Δf n is the measured frequency shift at the nth overtone (Hz), and n is the overtone number (3, 5, and 7). For model systems studied herein, normalization to three overtones showed overlapping Δf n/n values and small ΔD/Δf ratios, suggesting that eq 1 was applicable. Nevertheless, it should be noted that the sorbed DOM mass derived from eq 1 represents a wet mass because of the contribution of hydrodynamically coupled water molecules.51,52 Thus, increases in the apparent sorbed DOM mass may reflect either changes in the tendency of the DOM to sorb to the hydrophobic surface, or changes in the water content. Complementary techniques, as described below, were employed to distinguish these possibilities. QCM-D measurements typically commenced with flowing the DOM-free phosphate buffer until a stable frequency baseline was established (i.e., the averaged Δf n/n < 0.05 Hz· min−1). The oxidant-treated DOM solution or water sample was then delivered across the crystal surface by a KD Scientific syringe pump at a volumetric flow rate of 50 μL/min to maintain diffusion-controlled attachment rates in the flow chamber.52 Frequency shift data from the crystal overtones

q=

(C i − Ce)V D

(3)

where Ci is the initial DOC concentration of the DOM solution or water sample, Ce is the final DOC concentration, V is the sample volume, and D is the sheet graphite mass. To facilitate the comparison between graphite and QCM-D results (see SI section S4), the uptake of the oxidized DOM was normalized against that of the unoxidized DOM to determine the relative 5120

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Figure 1. Changes in the relative attachment efficiency (αR) of adenylate kinase (ADK), ribose-binding protein (RBP), lysozyme (LYZ), bovine serum albumin (BSA), and human serum albumin (HSA) upon chlorination, bromination, or ozonation as a function of the DOC-normalized oxidant dose. Error bars represent the range of duplicate measurements; where absent, bars fall within symbols. The bottom right panel illustrates the circular dichroism midpoint values (CD50) upon HOCl or HOBr treatment for ADK, RBP, and BSA reported in our previous work.40

maximum of αR exhibited approximately three- to four-fold increases at oxidant doses from 0.05 to 0.1 mmoloxidant/mmolC for O3 but migrated further toward 0.2 to 0.3 mmoloxidant/ mmolC for HOCl and HOBr. Additional techniques were applied to evaluate the trends observed via QCM-D. Comparison of the relative Bis-ANS binding capacity and αR revealed similar patterns of variations for BSA with respect to O3 dose (see SI Figure S2). Treatment of HSA with HOCl or O3 also resulted in similar patterns of αR, ΔmR, and qR (see SI Figure S3). These results suggest that changes measured by QCM-D primarily reflect changes in the sorption properties of proteins with oxidant dose, rather than changes in the water content, or other factors. The increase in the sorption tendency of proteins to the hydrophobic surface at low oxidant doses indicated by the increase in αR may result from the covalent modifications to the side chains of oxidizable amino acid residues. Although previous research has suggested that such modifications may lead to formation of aggregates via covalent cross-linkages between proteins (e.g., between carbonyl oxidation products and lysines),54,55 the QCM-D analysis relies upon noncovalent interactions between protein molecules and the hydrophobic surface of the sensor crystal. Earlier studies have also documented the oxidative modifications to amino acid side

extent of sorption to graphite qR (eq 4) and to the octadecanethiol-coated QCM-D sensor crystal ΔmR (eq 5) qDOM,oxidized qR = qDOM,unoxidized (4) ΔmR =

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ΔmDOM,oxidized ΔmDOM,unoxidized

(5)

RESULTS AND DISCUSSION Proteins. Figure 1 illustrates the dose-dependency of αR upon oxidation of model proteins. In all cases, changes in αR exhibited a biphasic response to increasing oxidant doses. For ADK, RBP, and LYZ, oxidation initiated up to five- to six-fold increases in αR at low oxidant doses from 0.01 to 0.02 mmoloxidant/mmolC, but αR declined rapidly at higher oxidant doses. Notably, O3 induced the most abrupt increases in αR at low doses as well as the most pronounced declines at high doses (see SI Figure S5 for higher resolution plots at low oxidant doses). Compared to HOCl, HOBr elicited steeper increases in αR at low doses and more rapid decreases with increasing doses. For BSA and HSA, variations in αR upon oxidation showed somewhat different patterns, in that the 5121

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Figure 2. Changes in the relative attachment efficiency (αR) of alginate (ALG), dextran (DEX), oleic acid (OA), and linoleic acid (LOA) upon chlorination, bromination, or ozonation as a function of the DOC-normalized oxidant dose. Error bars represent the range of duplicate measurements; where absent, bars fall within symbols.

range were in the order of O3 > HOBr > HOCl. The lower panels of Figure 2 illustrate the dose-dependency of αR upon oxidation of model unsaturated fatty acids. The oxidative treatments of fatty acids decreased αR, albeit to a lesser degree in comparison with polysaccharides. For both OA and LOA, HOCl and HOBr appeared to cause small but sustained decreases in αR up to oxidant doses of 0.05 mmoloxidant/mmolC (i.e., molar ratio of HOX:OA ≈ 1) and 0.1 mmoloxidant/mmolC (i.e., molar ratio of HOX:LOA ≈ 2), respectively, with minimal further changes at higher doses. In contrast, O3 induced continual and more significant decreases in αR. Similar trends were observed in ΔmR and qR after treatment of ALG and OA with HOCl or O3 (see SI Figure S3). Prior research has demonstrated that HOCl, HOBr, and O3 can all depolymerize polysaccharides into smaller, more polar fragments via specific or nonspecific oxidative modifications.67−69 Thus, the universal decreases in αR observed with ALG and DEX likely resulted from the formation of hydrophilic fragments enriched in oxygenated functional groups. Past studies have suggested that the initial reactions of HOCl and HOBr with unsaturated fatty acids form halohydrins via addition of HOCl and HOBr across double bonds,70−72 while O3 cleaves double bonds to form carbonyls and peroxides.73 Halohydrin carbonyl and peroxide products derived from either halogenation or ozonation reactions are believed to increase the polarity of parent fatty acids due to the incorporation of additional oxygen atoms into the product structures, which might explain the decreases in αR observed with OA and LOA. Humic and Fulvic Acids and Water Samples. The upper four panels of Figure 3 illustrate the dose-dependency of αR upon oxidation of model humic and fulvic acids. Treatment of humic and fulvic acids with HOCl and HOBr first increased αR by up to a factor of 2, followed by progressive decreases at higher oxidant doses. The maximum αR values required the lowest HOCl and HOBr doses for NLFA and SRFA (i.e., ∼0.2 mmoloxidant/mmolC), intermediate doses for LHA (i.e., ∼0.4

chains in proteins upon exposure to oxidants under both physiological and disinfection-relevant conditions.25,56−58 Chlorination and bromination can produce oxygenated residues,25 or more hydrophobic halogenated products, and, in the case of lysine, a nitrile.40 Ozonation forms predominantly oxygenated products.59,60 Oxidative modifications to amino acids disrupt the side chain interactions responsible for stabilizing protein secondary structure, triggering protein unfolding.26 The resulting exposure of the hydrophobic protein interior enhances the hydrophobic effect, leading to the formation of high molecular weight aggregates, although aggregates based upon covalent crosslinkages may also form.54,55,61−66 In our previous study, we had used circular dichroism (CD) to evaluate the loss of secondary structure in ADK, RBP, and BSA with increasing doses of HOCl and HOBr.40 The doses corresponding to 50% loss of secondary structure, the CD50, fell within the oxidant dose window prior to the occurrence of maximum αR (see the last panel in Figure 1). These results indicate that increasing tendency of sorption was likely accompanied by protein unfolding and aggregation, in line with exposure of the hydrophobic interior. At higher oxidant doses, the sorption tendency of proteins declined for all oxidants, pointing toward the covalent modifications of amino acid side chains to their hydrophilic forms. Previously, we applied sodium dodecyl sulfatepolyacrylamide gel electrophoresis to demonstrate the fragmentation of ADK, RBP, and BSA with increasing HOCl or HOBr doses.40 The QCM-D results indicate that, regardless of the size of the fragments, their sorption tendency decreased with oxidant exposure. Polysaccharides and Fatty Acids. The upper panels of Figure 2 illustrate the dose-dependency of αR upon oxidation of model polysaccharides. The oxidative treatments of polysaccharides decreased αR, with an initial rapid drop. For both ALG and DEX, declines in αR across the entire oxidant dose 5122

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Figure 3. Changes in the relative attachment efficiency (αR) of Leonardite humic acid (LHA), Nordic Lake fulvic acid (NLFA), Pahokee peat humic acid (PPHA), Suwannee River fulvic acid (SRFA), and water samples KY and CT upon chlorination, bromination, or ozonation as a function of the DOC-normalized oxidant dose. Error bars represent the range of duplicate measurements; where absent, bars fall within symbols.

mmoloxidant/mmolC), and the highest doses for PPHA (i.e., ∼0.5 mmoloxidant/mmolC). For all humic and fulvic acids, HOBr led to greater increases in αR than HOCl, particularly for the humic acids. O3 led to decreases in αR with increasing doses for both humic and fulvic acids. At low doses, HOCl and HOBr can react with phenolic moieties in humic and fulvic acids via electrophilic aromatic substitution reactions to form polyhalogenated products,74 which may increase their apparent hydrophobicity.75 The greater increase in the sorption tendency for HOBr treatment than for HOCl may result from HOBr being a more effective halogenating agent than HOCl.76,77 However, at high HOCl or HOBr doses, polyhalogenated phenolic products may undergo ring cleavage to form aliphatic, low molecular weight products.78 O3 preferentially reacts with phenolic moieties to form aliphatic aldehydes and carboxylic acids,79,80 reducing hydrophobicity. The lower two panels of Figure 3 illustrate the dosedependency of αR upon oxidation of KY and CT water samples. Treatment by HOCl and HOBr increased and decreased αR, with maxima near oxidant doses of 0.2 and 0.4 mmoloxidant/ mmolC for samples KY and CT, respectively. The increase in αR was greater for HOBr than HOCl. Treatment by O3, on the other hand, decreased αR across all doses. The similarity of the trends to those observed with humic and fulvic acids suggested

that the contribution from these constituents were more important than that from any protein, polysaccharide, or lipid constituents. Similar trends were observed in ΔmR and qR after treatment of LHA and sample KY with HOCl or O3 (see SI Figure S3). Implications. Results from two water samples suggest that the application of HOCl and HOBr increased the sorption tendency of DOM constituents to the hydrophobic surface at low doses, but the sorption tendency either returned to the level observed in the unoxidized samples, or in the case of the sample from the Kentucky utility, exhibited a decrease in the sorption tendency at the highest doses. The maximum increase in the sorption tendency was observed near doses commonly applied in practice (0.2−0.4 mmolHOCl/mmolC ≈ 1.2−2.4 mg chlorine/mgC). Thus, pretreatment with HOCl is anticipated to increase the sorption tendency to the hydrophobic surface, and thereby increase membrane fouling, but, as suggested by the results for sorption to sheet graphite, increase removal of DOM by activated carbon treatment. In contrast, the application of O3 uniformly decreased the sorption tendency to the hydrophobic surface, and therefore pretreatment with O3 would be expected to reduce membrane fouling. The similarity of the trends in αR with increasing oxidant doses between the water samples and humic and fulvic acids suggested that these constituents dominated the behavior for both water samples. 5123

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Although neither water sample could be categorized as pristine, neither was heavily impaired. Further research is needed to evaluate whether similar trends would be observed in highly impaired waters, such as municipal wastewater effluents within recycling plants, where a higher prevalence of proteins, polysaccharides, and lipids is expected. The fate of proteins at inflammatory sites has been a focus of biomedical research because their prevalence and high reactivity render them particularly important targets for the HOCl and HOBr produced by neutrophils and eosinophils, respectively. Formation of protein aggregates is associated with a range of inflammatory diseases (e.g., Alzheimer’s disease). Previous work has advocated that aggregate formation results from either noncovalent interactions or covalent cross-linkages between proteins.26,54,55,66 Our results indicate that low exposures to HOCl, HOBr, and even O3 increase the sorption tendency of proteins to the hydrophobic surface, likely by interactions between their hydrophobic cores exposed following protein unfolding. Accordingly, formation of covalent cross-linkages may not be necessary to drive protein aggregation. However, at sites of chronic inflammation, the prolonged exposure to oxidants is expected to eventually decrease such interactions. Recent research into the increasing cellular dysfunction associated with aging has implicated the formation of lipofuscin, an aggregate formed from interactions between oxidized lipids and proteins.26 Our results show that oxidants uniformly decreased the sorption tendency of lipids to the hydrophobic surface, concurring with the suggestion that lipofuscin forms from covalent cross-linkages between carbonyl products of lipid peroxidation and amines in protein side chains rather than noncovalent interactions.

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ASSOCIATED CONTENT

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AUTHOR INFORMATION

S Supporting Information *

Additional experimental details, tables, and figures as noted in the text. This material is available free of charge via the Internet at http://pubs.acs.org. Corresponding Author

*E-mail: [email protected]. Phone: 650-725-9298. Fax: 650-723-7058. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We thank Professor Paul R. Van Tassel (Yale University) for the use of QCM-D instruments. We are grateful to Dr. Stanley C. Howell (Yale University) for the supply of ADK and RBP, help with the microplate reader, as well as many useful discussions. We also thank Dr. Siamak Nejati (Yale University) for the XPS technical advice and data collection at Drexel University’s Centralized Research Facilities and Justin T. Jasper (University of California at Berkeley) for the DOC measurement. This work was partially funded by the Water Research Foundation (Project 4370).

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