Bovine Serum Albumin Adsorption to Iron-Oxide Coated Sands Can

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Bovine Serum Albumin Adsorption to Iron-Oxide Coated Sands Can Change Microsphere Deposition Mechanisms Raymond M. Flynn,*,† Xinyao Yang,†,§ Thilo Hofmann,*,‡ and Frank von der Kammer‡ †

School of Planning, Architecture & Civil Engineering, Queen’s University Belfast, David Keir Building, Stranmillis Road, Belfast BT9 5AG N. Ireland, U.K. ‡ Department of Environmental Geosciences, University of Vienna, Althanstrasse 14, 1090 Vienna, Austria S Supporting Information *

ABSTRACT: Particulate colloids often occur together with proteins in sewageimpacted water. Using Bovine Serum Albumin (BSA) as a surrogate for protein in sewage, column experiments investigating the capacity of iron-oxide coated sands to remove latex microspheres from water revealed that microsphere attenuation mechanisms depended on antecedent BSA coverage. Dual pulse experiment (DPE) results suggested that where all BSA was adsorbed, subsequent multiple pore volume microsphere breakthrough curves reflected progressively reduced colloid deposition rates with increasing adsorbed BSA content. Modeling colloid responses suggested adsorption of 1 μg BSA generated the same response as blockage by between 7.1 × 108 and 2.3 × 109 deposited microspheres. By contrast, microsphere responses in DPEs where BSA coverage of the deposition sites approached/reached saturation revealed the coated sand maintained a finite capacity to attenuate microspheres, even when incapable of further BSA adsorption. Subsequent microsphere breakthrough curves demonstrated the matrix’s colloid attenuation capacity progressively increased with continued microsphere deposition. Experimental findings suggested BSA adsorption on the sand surface approaching/reaching saturation generated attractive deposition sites for colloids, which became progressively more attractive with further colloid deposition (filter ripening). Results demonstrate that adsorption of a single type of protein may either enhance or inhibit colloid mobility in saturated porous media.

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INTRODUCTION Sewage constitutes one of the most widely distributed waste products associated with human activity. Numerous studies have demonstrated how contact with it can impact public health and how appropriate means of treatment are necessary to minimize possibilities of waterborne disease outbreaks.1−3 Release of incompletely treated sewage and associated wastewater to the wider environment may adversely impact the quality of water destined for human consumption; this may include releases from improperly functioning septic tanks and leaking sewers.4−6 Potential pollutants of concern in sewage and wastewater include nutrients, organic matter and pathogenic micro-organisms.7 Micro-organisms (bacteria, viruses, and protozoa), in particular, display considerable potential to impact human health since high levels are often released to water, yet very small numbers may be capable of causing infection.8 The presence of micro-organisms associated with sewage have been noted in groundwater samples collected in a wide range of hydrogeological settings; these include karst,9 sandstone,10 and sand and gravel aquifers.11 Artificial tracer tests completed by Ryan et al,12 in a sand and gravel aquifer locally impacted by septic tank contamination showed that colloids (particles with at least one dimension less than 1 μm, including many microorganisms13,14) were more mobile in those parts of the aquifer impacted by sewage. The authors hypothesized that adsorbed © 2012 American Chemical Society

organic matter present in the sewage hindered colloid attachment to fixed surfaces in the aquifer. Laboratory studies investigating the influence of organic matter on the mobility of colloids have often resulted in conflicting findings. Studies by Johnson and Logan15 noted that organic matter could enhance micro organism transport through porous media; by contrast, Marschall et al.16 observed that some organic compounds may hinder it. Organic matter (OM) content in water is routinely expressed by the operationally defined terms total organic carbon (TOC) and dissolved organic carbon (DOC). However, the huge array of organic compounds that may be present in water renders the utility of these parameters for measuring their influence on colloid mobility questionable; many may have no effect. Recent research by Harvey et al.17 noted that a minor proportion of the wide variety of compounds making up DOC in a sewagecontaminated plume had a disproportionate effect on enhancing bacterial mobility, further demonstrating that the nature of the organic matter present in an aqueous system will determine its influence on colloid mobility. Received: Revised: Accepted: Published: 2583

September 3, 2011 January 11, 2012 January 23, 2012 January 23, 2012 dx.doi.org/10.1021/es202048c | Environ. Sci. Technol. 2012, 46, 2583−2591

Environmental Science & Technology

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column experiments were the same as those employed by Yang et al.,23 (Details provided in Supporting Information, S1) . Fluoresbrite yellow-green fluorescent stained polystyrene latex microspheres (microspheres) with carboxylic functional groups and a nominal diameter of 0.2 μm (Polysciences Inc., Eppelheim, Germany) were used as the particulate colloid tracer. Microsphere suspension preparation followed the procedure described by Yang et al.,23 that is, a microsphere reservoir with a concentration of 10.4 ppm (2.5 × 109 colloids mL−1) was ultrasonically dispersed following mixing of a 5% (v/v) microsphere suspension with tracer-free flush solution. Bovine serum albumin (BSA) (Acros Organics,Geel, Belgium) acted as the protein tracer in all experiments. The compound has been widely used as a model form of organic matter to represent the behavior of proteins in wastewater effluent.28−30 BSA has a molecular weight of 66.4 KDa and an isoelectric point of approximately pH 4.7.31 Although some debate exists concerning the best model to describe BSA, there is a consensus that surface properties vary across the molecule giving rise to charge heterogeneity,32 along with hydrophobic and hydrophilic regions.33 Studies investigating BSA adsorption onto metal oxides have shown the process to be surface charge dependent20 and a function of pH.21 Moreover, investigations by Rezwan et al.22 have demonstrated that BSA adsorption to aluminum oxide under near neutral conditions (pH 7.2) and low ionic strength (10 mM) could reverse their surface charge. The same authors note that the configuration of BSA permits it to adsorb in two different modes, which make it either more or less attractive for adsorption of negatively charged substances. At high surface coverage the adsorption mode was less negative. BSA source concentrations employed in the current experimental series ranged from 6.6 mg/L (1 × 10−7 M) to 266 mg/L (4 × 10−6 M). These concentrations aimed to correspond to protein levels of typical relatively undiluted wastewaters reported in Raunkjær et al.,34 and when it has undergone (forty-fold) dilution with protein free water. Double Pulse Column Experiments. Prior to each experiment 75 pore volumes (PV) of tracer-free flushing solution were passed through the column apparatus to ensure chemical and hydrodynamic stability. Following stabilization, the first multiple PV tracer pulse in a double pulse experiment (DPE), was passed through the column matrix. In all but one series of DPEs (DPE-0), BSA was injected as the first tracer pulse. The first pulse was followed by a multiple pore volume flush of tracer-free solution, before the injection of a second 13 PV pulse of 10.4 ppm microsphere dispersion, succeeded by a final flush with tracer-free solution. BSA pulses, injected at identical concentrations, but for variable durations, aimed to investigate the effects of adsorption of progressively greater masses of protein to the column matrix, on microsphere breakthrough. Injection of different BSA concentrations aimed to examine their possible effects of contrasting BSA adsorption mechanisms on microsphere breakthrough. Three groups of DPEs were carried out: • DPE-0. Experiments where microspheres were injected into the column matrix in both the first and second tracer pulse, that is, no BSA was injected. These experiments provided an experimental control by which the impact of BSA on microsphere response could be compared. • DPE-1, DPE-2, and DPE-3. Experiments where masses of BSA injected were significantly lower than the BSA adsorption capacity of the sand, that is, three PV of

Studies of fundamental processes controlling micro-organism transport and attenuation through porous media are complicated by biological processes. These may include motility, growth/ inactivation, temporal changes in micro-organism surface properties and high levels of natural variability in a population.18 Abiotic colloids provide a useful surrogate for understanding the physical and chemical factors influencing micro organism fate and transport, without the need to consider biological processes; this has included batch studies investigating the role of organic matter on colloid stability. Saleh et al.19 quantified the influence of a range of organic substances on the stability of suspended colloidal carbon nanotubes and observed that the protein bovine serum albumin (BSA) proved highly effective in stabilizing colloidal suspensions by adsorbing to nanotube surfaces. Comparable colloid responses to the presence of BSA have been observed in other batch studies employing metal oxide suspensions.20−22 The impact of OM on colloid migration through saturated porous media affected by sewage pollution has been demonstrated by Harvey et al.17 using static column systems. However, published examples of quantification of coverage of colloid deposition sites by particular types of OM in dynamic settings remain scarce. Studies by Yang et al.23 employed easily detectable abiotic colloidal microsphere tracers, having comparable sizes to some micro-organisms, to investigate the influence of humic acids on colloid migration through saturated porous media. In their study, multiple pulse dynamic column experiments highlighted how microsphere deposition onto iron oxide coated sand proved relatively insensitive to changes in pH and ionic strength (IS) in the absence of organic matter. Conversely, the introduction of OM, in the form of Suwanee River Humic Acid, resulted in significantly heightened sensitivity of the colloid deposition to process to both pH and IS.24 This further demonstrated the potential importance of adsorbed organic matter in natural environments in influencing particulate colloid mobility, as also noted by Kumpulainen et al.25 Kiely26 provided an overview of the organic composition of sewage and noted that protein constitutes an important organic matter type in wastewater effluent. The qualitative influence of protein on microorganism mobility in saturated porous media has been more recently highlighted. Research by Lutterodt et al.27 noted that protein expression in bacterial cells played an important role in their adsorption to saturated quartz sand. However, to the best of the authors’ knowledge, no studies have been published to date investigating the effects of individual protein types on particulate colloid adsorption in saturated porous media. This study aims to investigate and quantify the impact of a single model protein on the mobility of colloidal microspheres (microspheres) in a saturated porous medium. Dual pulse dynamic column experiments (DPEs), completed by injecting different concentrations of the protein for different durations, followed by a pulse of microspheres, allowed the impact of adsorbed protein on colloid mobility to be investigated. A numerical model was employed to simulate microsphere response in a protein free system. Comparison of extrapolated model output to microsphere breakthrough in DPEs, following protein injection, provided a means of equating the impact of the protein adsorbed to the column matrix to an equivalent number of microspheres, necessary to generate the same response.

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MATERIALS AND METHODS Materials. With the exception of the organic matter (protein) tracer, all materials and apparatus employed in 2584

dx.doi.org/10.1021/es202048c | Environ. Sci. Technol. 2012, 46, 2583−2591


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microsphere BTC, and the subsequent gradual increase in concentration. Where increases in concentration did not correspond, following the microsphere BTC inflection point, simulations matched the inflection point alone. The latter approach permitted direct comparison of nonideal responses, generated in the presence of BSA, with those anticipated in a BSA-free system. Although the scope of the current work did not aim to reproduce BSA responses, where they were observed in the column effluent, the ADE equation (eq 1) provided a means of comparing BSA BTCs to a nonreactive tracer response. This was achieved by superimposing the rising and falling limbs of a BSA BTC onto an ADE simulation; the simulation employed transport terms (v and D) determined by fitting to experimental nitrate responses in DPE-0.

6.6 mg/L BSA (DPE-1), 3 PV of 13.3 mg/L BSA (DPE-2), and 12 PV of 6.6 mg/L BSA (DPE-3). These experiments aimed to assess the impact of the adsorption of small quantities of BSA (at levels significantly below deposition site saturation) on the breakthrough of a microsphere pulse. • DPE-4, DPE-5, and DPE-6. Experiments where masses of BSA adsorbed approached/reached the maximum deposition site adsorption capacity of the sand, that is, 8 PV of 66.4 mg/L BSA (DPE-4), 8 PV of 265.6 mg/L BSA (DPE-5), and 32 PV of 265.6 mg/L BSA (DPE-6). Injection of microspheres, following BSA adsorption, aimed to investigate the influence of high levels of BSA deposition site saturation on microsphere fate and transport. To ensure consistency, all column experiments were completed in triplicate. Zeta Potential and Particle Size Measurements. Measuring colloid electrophoretic mobility and size provided an insight into the surface charge and the potential of the microspheres to aggregate; this was completed in the presence of BSA at concentrations corresponding to source levels employed in column experiments. Using Smoluchowski’s theory, measured mobility values were converted into zeta potential. Dynamic light scattering (DLS) measurements, performed simultaneously, provided information on microsphere particle size using the cumulative fit of the autocorrelation function. Numerical Modeling. Transport and attenuation of nonreactive substances passing through saturated porous media under steady-state flow conditions can be described using the advective-dispersive equation (ADE).35 For a one-dimensional system, the change in concentration (C) at a given distance (z), as a function of time (t) because of the effects of advective velocity (v), and dispersion(D) may be simulated with the following: ∂C ∂ 2C ∂C =D 2 −v ∂t ∂z ∂z

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RESULTS AND DISCUSSION BSA-Free Microsphere Breakthrough Curves. Figure 1a summarizes the results of microsphere breakthrough curves generated for DPE-0 along with the ADE model fit to the nitrate responses observed; responses remained consistent in triplicate experiments. The BTCs reveal a sharp rise in relative concentration to an inflection point at 7% ± 0.5%. After this point a more gradual rise to 9% occurred, resulting from the injection of 13 pore volumes of microspheres. Shortly following the injection of tracer-free flush water, a rapid decline in concentration occurs, with little tailing, from a maximum relative concentration to close to zero. Microsphere concentrations remained close to zero until the breakthrough of a second colloid pulse when a rapid rise in concentration occurred up to an inflection point corresponding closely to the maximum encountered at the end of the first colloid pulse. After this point microsphere levels continued to rise at a comparable rate to those observed in the first pulse, before dropping back toward zero with the onset of the effects of flushing. Microspheres responses observed in column effluent reflect high levels of attenuation and show that the iron-oxide coated sand has a high capacity to retain the colloids. On the other hand, the gradual increase in colloid concentration observed in column effluent, accompanying the sustained injection of microspheres, reflects a gradual decline in the sand’s attenuation capacity. The rapid decline in concentration and insignificant tailing, similar to that observed with a nonreactive tracer, suggests that this process is irreversible over the time frame of the experiments. Similarly, the close correspondence between the concentration observed in the inflection point of the rising limb of the second microsphere pulse BTC and the maximum noted at the end of the first not only reflects the capacity of the column matrix to irreversibly adsorb microspheres, but also that the deposition of colloids limits the rate at which subsequent colloid deposition can occur. Kretzschmar et al.37 observed comparable phenomena in column experiments employing weathered granite saprolite and attributed these responses to colloidal blocking; the same process is believed to have operated in DPE-0. BSA Responses. No BSA was detected in column effluent in experiments where less than 100 μg was injected. The absence of BSA in column effluent in DPE-1 through DPE-3 demonstrates that the column matrix can completely attenuate the protein and suggests that the sand retains the capacity to adsorb further BSA. In experiments where greater masses of BSA were injected breakthrough curves had a consistent form. BTCs for DPE-4

(1)

Where reaction occurs, appropriate terms may be added to account for deposition/adsorption and release, onto and from stationary matrix surfaces. The random sequential adsorption (RSA) model of Johnson and Elimelech36 was employed to simulate microsphere experimental response. The model provides a means of simulating colloidal blocking in porous media. Details of the model are provided in Supporting Information (Section S2). Model calibration of microsphere responses was completed by fitting the particle transfer coefficient (K) to the rising limb of the (first) microsphere BTC. The subsequent, more gradual, rise in microsphere concentration, observed as breakthrough continued, was fitted by adjusting the maximum surface coverage (θmax). The RSA model provided a means of simulating the microsphere breakthrough curves (BTCs), generated in the absence of BSA. These simulations provided a means of quantifying how many microspheres were necessary to generate the rise in concentration observed. Matching extrapolated model output to microsphere BTCs, following BSA injection, permitted calculation of the equivalent number of microspheres in a BSAfree experiment, necessary to generate the same response. Simulations, where low masses of BSA were injected/ adsorbed, aimed to match both the inflection point on the 2585



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Figure 1. DPE breakthrough curves for (A) DPE-0, where no BSA was injected. In (B) DPE-4, (C) DPE-5, and (D) DPE-6, BSA was detected in the column effluent (error within ±2.5%). Hollow circles: Microsphere relative concentration. Gray triangles: BSA relative concentration. Black circles in A: Nitrate relative concentration. Solid gray line: Conservative tracer simulation, simulated with velocity: 18 m/day, Dispersion: 1.62 m2/day. Experimental data from Yang.38

volumes needed to be injected in experiments employing 66.4 mg/L BSA. By contrast, in experiments employing 265.6 mg/L BSA injection of only 0.35 ± 0.05 extra pore volumes achieved the same increase in relative concentration; this reflects the more rapid rise in BSA levels in those experiments employing higher concentrations. On the other hand, calculation of the equivalent mass necessary to generate the same increase in relative concentration reveals that up to twice as much BSA must be injected in experiments employing 265.6 mg/L (DPE-5 and DPE-6). In the latter part of each BTC’s rising limb the rise in BSA levels, toward unity, occurred more gradually at lower concentration. Nonetheless between 25% and 50% more BSA needed to be injected at higher concentrations to achieve the increase in relative concentration to 80%. Moreover, despite the injection of multiple pore volumes, relative concentrations had not reached unity after the injection of eight pore volumes of 265.6 mg/L BSA; BSA concentrations in column effluent eventually reached unity following injection of 25 pore volumes

through DPE-6 demonstrate significant differences compared to responses expected for a nonreactive substance, as simulated using transport parameters determined from nitrate response and eq 1 (Figure 1a). Figure 1b−d presents the results of DPE4 through DPE-6 and compares BSA behavior with simulated nonreactive tracer responses. Comparison of the simulated and experimental responses shows that initially no BSA was detected, despite the injection of multiple pore volumes of the protein. This was then followed by a rapid rise in relative concentration to an inflection point, after which concentrations increased, gradually approaching unity. The point where a rapid rise in relative concentration occurred on the BSA BTC depends on the source concentration. In those experiments employing 66.4 mg/L BSA, the increase to C/C0 = 0.2 occurred after approximately 5.5 pore volumes of BSA had been injected. In those experiments employing 265.6 mg/L BSA the rise occurred following injection of approximately 2.8 pore volumes. In order to generate a rise in relative concentration to C/C0 = 0.5, a further 0.7 ± 0.05 pore 2586



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Figure 2. Translated (shifted) experimental microsphere breakthrough data (circles) and RSA model simulated responses (black line) for DPE 0 through DPE 3. Gray circles in DPE-0 show the microsphere response observed following the injection of microspheres in the first experimental pulse, instead of BSA. These data provided calibration parameters for the RSA model output. (RSA parameters: maximum surface coverage 0.1 ± 0.01, transfer rate coefficient 0.034 ± 0.002). White circles represent the microsphere response in the second pulse of all DPEs. Shifting the (second pulse) microsphere BTC along x-axis to correspond to RSA model output permitted calculation of the number of microspheres necessary to generate the same response as that generated by the preceding pulse of BSA in each DPE.

at which the inflection point occurs on the microsphere BTC is higher than that generated in the first pulse of DPE-0 (Figure 2). Moreover, the level at which this occurs increased with the mass of BSA adsorbed in the preceding pulse. Approximately 50 μg of BSA generates a 1% rise in the inflection point on the microsphere BTC, independently of whether 6.6 mg/L or 13.3 mg/L BSA were used. In a similar manner, the gradual rise in relative concentration observed in the microsphere BTC, following the inflection point, occurs at a comparable rate to that observed in DPE-0, again irrespective of the concentration of the BSA injected in the previous pulse. In DPE1 through DPE-3, the difference in microsphere BTC observed, compared to the first microsphere BTC of DPE-0, reflects the ability of the protein to enhance the microsphere’s mobility in a manner akin to colloidal blocking. This occurs due to adsorbed BSA hindering microsphere deposition. The proportional relationship between the height of the inflection point on the BTC and the mass of BSA adsorbed in DPE1 through DPE-3 further underscores the influence of the protein in reducing the matrix’s capacity to attenuate microspheres in experiments when all BSA injected was retained by the porous medium.

of 265.6 mg/L BSA (DPE-6). Following the onset of the effects of flushing, a rapid decline in BSA concentration occurred with slight to no tailing, relative to the simulated response, before concentrations returned to zero. The greater mass of BSA required to reach a given inflection point in experiments using 266 mg/L BSA (DPE-5 and DPE-6), compared to those using 66 mg/L (DPE4), suggests that at higher concentrations BSA may be adsorbed on the matrix surface in a more compact configuration resulting in lower coverage of adsorption sites per unit mass adsorbed. Relative concentrations of unity observed in column effluent in DPE-6 reflect BSA deposition site saturation of the column matrix. Microsphere Responses with Low BSA Coverage. DPE experimental microsphere BTC results varied depending on BSA masses injected into the column. (Supporting Information Figure S3 presents experimental breakthrough curves DPE-1DPE-3.) Overall, microsphere BTCs generated following the injection of BSA in DPE-1 through DPE-3 display comparable responses to those generated in the absence of the protein (DPE-0), with one crucial difference; the relative concentration 2587



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RSA Modeling Results. Application of an RSA model, calibrated using the first pulse of the DPE-0 microsphere BTC (to estimate K and θmax), permitted the number of colloids required to generate an increase in relative concentration due to blocking to be determined. Figure 2a presents the results of DPE-0 where the second microsphere BTC has been merged with the first. The RSA model output corresponds well with the experimental data and shows that the model can effectively simulate the blocking process under BSA-free conditions. Matching extrapolated RSA model output to microsphere BTCs for DPE-1 through to DPE-3, using the same calibration parameters (K and θmax) as DPE-0, reveals that the model provides a good fit to experimental data where small masses of BSA (less than 100 μg) have been injected (Figure 2b-2d). The model adequately simulates the gradual rise in microsphere concentration from the point where the shifted microsphere BTC inflection point intersects the model output. This point needed to be shifted further along the axis with increasing mass of adsorbed BSA, reflecting the increasing influence of adsorbed BSA on the column matrix’s microsphere adsorption capacity (Figure 3).

This suggested that the RSA model could be used to determine the number of microspheres that need to be deposited to generate the same response as the adsorbed pulse of BSA. Shifting the microsphere BTC along the x-axis, to match the gradual rise following the BTC inflection point, permitted calculation of the equivalent number of pore volumes of microspheres that must be injected to have the same effect as the BSA (deposited in DPE’s first pulse). Integration of the RSA model-generated curve indicated what proportion of the supplemental number of pore volumes injected would be recovered in the column effluent. As a corollary to this, subtraction of this proportion from the total number of supplemental pore volumes of colloids injected provided the amount of extra microspheres that would need to be deposited onto the column matrix to generate the microsphere BTC inflection point observed in a BSA-free system. Over the whole experimental period, dividing the extra number of colloids required to correspond to an experimental response generated by an adsorbed mass of BSA, provided a global estimate of the number of colloid deposition sites that could be covered by a unit mass of BSA. Table 1 summarizes the number of additional pore volumes of microspheres that would need to be injected under BSA-free conditions to generate the same response as DPE-1 through DPE-6. Calculations using RSA model-derived data indicated that in DPE-1 through DPE-3 the deposition of more BSA would require the injection and deposition of progressively greater numbers of microspheres onto the column matrix to generate an equivalent response in a BSA-free system. However, this appears to be independent of concentration injected when less than 100 μg of BSA was adsorbed to the column matrix. Microsphere Responses with High BSA Coverage. Microsphere responses observed in DPE-4 through DPE-6, where BSA was detected in the column effluent (Figure 1b−d), contrast with those noted where lower masses of BSA were injected. (Table S3 in Supporting Information summarizes all BTCs with representative parameters). Inflection points in microsphere BTCs occur at significantly higher levels than those observed in DPE-0, or in DPE-1 through DPE-3. This is consistent with observations made in experiments with lower BSA coverage, i.e. greater adsorbed mass of BSA gives rise to greater coverage of colloid deposition sites on the column matrix, as reflected in higher microsphere concentrations in column effluent. However, microsphere responses following inflection on the BTC rising limbs in DPE-4 through DPE-6 contrast sharply with those observed in DPE-0 through DPE-3. A decline in relative concentration of between 0.5% and 1% per pore volume occurred with sustained microsphere injection, that is, inflection points are progressively lower with greater BSA coverage.

Figure 3. Plot of rise in microsphere BTC inflection point with mass of BSA adsorbed in preceding pulse for DPE-0 through DPE-3 (margin of experimental error within area of data points).

The good correspondence between experimental and model data using the same model parameters as employed in a BSAfree system (DPE-0) indicates that the microsphere blocking rate, after BSA adsorption, does not differ from that which would be observed if microspheres alone would be injected. Furthermore, the consistency in response observed with increasing mass of BSA adsorbed, irrespective of source concentration (6.6 mg/L or 13.3 mg/L) suggests that the mass of protein deposited had the same effect on subsequent microsphere responses, irrespective of concentration employed.

Table 1. Number of Pore Volumes (PV) of Microspheres Necessary to Generate the Same Microsphere BTC Inflection Point as BSA in DPE 1 through DPE 6 experiment

BSA adsorbed- μg (as % of mass injected)

DPE1 DPE2 DPE3 DPE4 DPE5 DPE6

21.3 (100%) 42.5 (100%) 85.0 (100%) 317 (57%) 476 (21%) 467 (5.5%)

no. of PV of microspheres to inject to reach equivalent inflection point 5.9 27.2 87.1 226.8 179.8 131.8

± ± ± ± ± ±

2 6 1 7 4 1

microspheres to be adsorbeda 1.52 6.69 1.99 4.24 3.62 2.86

± ± ± ± ± ±

0.01 0.16 0.00 0.12 0.05 0.01

× × × × × ×

1010 1010 1011 1011 1011 1011

equivalent no. of microspheres/mg BSA adsorbed 7.14 1.57 2.34 1.34 7.60 6.12

± ± ± ± ± ±

0.03 0.04 0.00 0.04 0.10 0.02

× × × × × ×

108 109 109 109 108 108

a

Calculated by subtracting equivalent pore volumes of microspheres that must be injected from the simulated number of pore volumes recovered in column effluent (as determined from the RSA model), multiplied by colloid source concentration. 2588



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Figure 4. Translated experimental microsphere breakthrough data (circles) and RSA model simulated responses (black line) for DPE 4 through DPE 6. RSA model calibration parameters determined from the first pulse BSA-free DPE 0 (gray circles) (maximum surface coverage 0.1 ± 0.01, transfer rate coefficient 0.034 ± 0.002). Shifting the microsphere BTC along x-axis to correspond to RSA model output permitted calculation of the number of microspheres necessary to generate the same inflection point as that generated by the preceding pulse of BSA in each DPE.

The decline in microsphere concentration following inflection reflects the unexpected role played by BSA in both enhancing colloid mobility, as seen with higher inflection points on microsphere BTCs, and inhibiting it, as shown in declining microsphere concentrations in column effluent with sustained colloid injection. Moreover, RSA model cannot reproduce the microsphere responses observed in DPE-4 through DPE-6. However, matching BTC inflection points to extrapolated RSA model output provides an indication of the number of colloids that would be necessary to reach the same concentration under BSA-free conditions (Figure 4); this permitted comparison with findings from DPE-1 through DPE-3. Mass balance calculations, coupled with RSA model output, suggest that with greater BSA coverage a smaller number of microspheres must be deposited to reach the inflection point in a subsequent microsphere BTC. It has already been noted that comparison of conservative tracer simulations with BSA responses reveals that the later part

of the BSA breakthrough curves takes considerably longer to reach unity than that of a nonreactive substance. As DPE-4 through DPE − 6 results demonstrate, BSA maximum concentrations closer to unity generate lower inflection points in the subsequent microsphere breakthrough curve. Moreover, DPE-6’s results show that the column matrix retains an ability to attenuate microspheres, even when all BSA deposition sites are covered; this suggests an alternative microsphere attenuation mechanism operates as BSA deposition sites approach saturation. Zeta Potential and Size Measurements. Zeta potential measurements show microsphere potential to be lowest in the absence of BSA (−68 mV ± 1 mV) but gradually increased to −42 mV ± 1 mV in the presence of 266 mg/L BSA. This increase occurred at an approximately constant rate of 10 mV for each 10-fold increase in BSA concentration. In contrast to studies by Patil et al.20 and Rezwan et al.22 these findings suggest that the presence of BSA slightly reduces electrostatic repulsive forces between particulate colloids (microspheres). 2589



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where progressively greater BSA adsorbed to the column matrix, generating more attractive colloid deposition surfaces. Further studies promise to shed light on the fundamental processes driving these phenomena. The BTCs generated in this study demonstrate that BSA can influence the fate and transport of colloidal particles passing through saturated porous media. The changes in attenuation process observed, with increasing deposition site coverage show that deposited organic matter may not only influence the rate at which colloids are attenuated, but also the mechanism. The results observed demonstrate that attenuation processes operating in porous media, in the presence of variable concentrations of a single form organic matter, can enhance or inhibit colloid transport. The phenomena observed may help reconcile the conflicting responses observed in studies investigating particulate colloid/organic matter interactions in porous media.

Despite the rise in zeta potential, size data suggest no significant difference in particle diameter between solutions, which consistently remained between 215 and 240 nm. (see Supporting Information S2). The consistency in particle size determined for different BSA concentrations suggest that aggregation did not occur and that repulsive forces operating between particles maintained a monodisperse microsphere-BSA system at the range of BSA concentrations employed. The zeta potential and size data are consistent with microsphere responses in experiments completed at low BSA coverage (DPE-1 through DPE-3), where BSA deposition has an effect equivalent to blocking in a colloid free system, i.e. the deposited protein renders the column matrix more repulsive to suspended microspheres. BSA Reconfiguration. The sustained decline in microsphere inflection point, observed with increasing BSA coverage in DPE-4 through DPE-6 suggests that as adsorbed BSA approached saturation, the protein deposited onto the column matrix in an alternative manner; this permitted microspheres to deposit onto it. Furthermore, the sustained decline in microsphere concentration after the BTC inflection point suggests that microsphere deposition onto the protein-covered surface formed an additional type of deposition site. This became progressively more attractive to microspheres that subsequently passed through the matrix, resulting in enhanced deposition rates; this is reflected by the decline in relative concentration in the column effluent as microsphere injection continued (filter ripening33). Zeta potential data suggest that the attractive depositional conditions experienced by microspheres passing through the BSA coated sand in DPE-4 through DPE-6 arise not because of BSA-microsphere interactions alone, but are due to the influence of BSA being deposited on the column matrix in such a way as to encourage microsphere deposition. Despite the change in depositional conditions in the column matrix arising from BSA deposition, comparison with nonreactive tracer simulations, revealing little tailing in either microsphere or BSA breakthrough curves, suggests that deposition of both materials is an irreversible process under the experimental conditions imposed. The mechanism responsible for the ripening phenomena observed in colloid breakthrough curves, associated with higher coverage of the column matrix by BSA (DPE-4 through DPE-6), is suspected to be due to a change in the configuration of BSA molecules deposited on the matrix at conditions approaching/ reaching saturation. Rezwan et al.22 noted similar phenomena when investigating BSA adsorption onto aluminum oxide and attributed a change in the adsorption regime to a lack of space on the adsorbing metal oxide surfaces. This resulted in BSA depositing in a more compact configuration with a less negatively charged domain of the molecule facing away from the adsorbing surface. This proved more favorable to further deposition by negatively charged substances. A comparable process is suspected to have occurred in this study. BSA depositing onto the matrix, when conditions approached saturation, took on a configuration that promoted adsorption of negatively charged microspheres (in contrast to the BSA configuration at lower site coverage, which promoted repulsion). Deposition of colloids onto the compactly adsorbed protein generated a new surface that further enhanced colloidal deposition rates leading to the declining concentrations observed with time in the colloid breakthrough curve. This is consistent with the decrease in microsphere inflection point in DPE-4 through DPE-6,

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ASSOCIATED CONTENT

S Supporting Information *

Experimental materials and apparatus description, numerical modeling, zeta potential and size data for microspheres in the presence of BSA, and summary breakthrough curve parameters for DPE-0 to DPE-6 and breakthrough curves for DPE-0 through DPE-3. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected] (R.F.); [email protected] (T.H.). Tel.: +44(0)2890974044 (R.F.); +43(1)427753380 (T.H.). Fax: +43(1)42779533 (T.H.). Present Address §

Provincial Key Laboratory of Agricultural Environmental Engineering, Sichuan Agricultural University, Huimin Road 211, Chengdu 611130, China

Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS Xinyao Yang’s research was supported by a Queen’s University Belfast Scholarship. REFERENCES

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Bovine Serum Albumin Adsorption to Iron-Oxide Coated Sands Can

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