Article pubs.acs.org/est
Transport of Industrial PVP-Stabilized Silver Nanoparticles in Saturated Quartz Sand Coated with Pseudomonas aeruginosa PAO1 Biofilm of Variable Age Michael R. Mitzel†,‡ and Nathalie Tufenkji*,† †
Department of Chemical Engineering and ‡Department of Natural Resource Sciences, McGill University, Montreal, Quebec H3A 0C5, Canada S Supporting Information *
ABSTRACT: Understanding the environmental fate and transport of engineered nanoparticles (ENPs) is of paramount importance for the formation and validation of regulatory guidelines regarding these new and increasingly prevalent materials. The present study assessed the transport of an industrial formulation of poly(vinylpyrrolidone)-stabilized silver nanoparticle (PVP-nAg) in columns packed with watersaturated quartz sand and the same sand coated with Pseudomonas aeruginosa PAO1 biofilm of variable age (i.e., growth period). Physicochemical characterization studies indicate that the PVP-nAg is stable in suspension and exhibits little change in size or electrophoretic mobility with changing ionic strength (IS) in either NaNO3 or Ca(NO3)2. The collector surface had a relatively homogeneous biofilm coating, as determined by CLSM, and a near uniform distribution of biomass and biofilm thickness following column equilibration. Transport experiments in clean sand revealed changes in the particle deposition behavior only at and above 10 mM IS Ca(NO3)2 and showed no discernible change in PVP-nAg transport behavior in the presence of 1 to 100 mM NaNO3. Transport experiments in P. aeruginosa-coated sand indicated significantly reduced retention of PVP-nAg at low IS compared to clean sand, irrespective of biofilm age. Nanoparticle retention was also generally reduced in the biofilm-coated sand at the higher IS, but to a lesser extent. The decreased retention of PVP-nAg in biofilm-coated sand compared to clean sand is likely due to repulsive electrosteric forces between the PVP coatings and extracellular polymeric substances (EPS) of the biofilm. Additionally, the slope of the rising portion of the PVP-nAg breakthrough curve was noticeably steeper in biofilm conditions than in clean sand. More mature biofilm coating also resulted in earlier breakthrough of PVP-nAg compared to younger biofilm coatings, or to the clean sand, which may be an indication of the effect of repulsive surface forces combined with selective pore size exclusion from the pores of denser, more developed biofilm. These results, when considered with other literature, indicate the importance in considering the flow dynamics, pore network and structure, the effective particle size, and particle permeability with regard to the biofilm matrix when considering the possible influence of biofilms on ENP transport.
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INTRODUCTION
Although bare or weakly stabilized nAg are not expected to transport large distances in the natural subsurface, mainly because of aggregation or dissolution,14−17 it remains unclear what the transport behavior of more stabilized nAg (e.g., using poly(vinylpyrrolidone); PVP) may be.14,18,19 Considering that industrial formulations may increase the stability (and likely the environmental mobility) of these potentially deleterious ENPs, investigations of the transport potential of industrially sourced nAg and their interactions with environmentally relevant surfaces will help elucidate factors contributing to ENP environmental risk.20,21
Nanomaterials can be found in a range of manufactured goods, and the commercial applications involving novel engineered nanoparticles (ENPs) are ever increasing.1−3 Silver nanoparticles (nAg), largely because of their antimicrobial properties, have recently found their way into a wide variety of common consumer items (e.g., toothpaste, clothing, household cleaners, and surfaces of various items).4−7 However, there remain concerns regarding the impact of nAg on environmental and human health.8−12 The potential environmental risks associated with ENPs will largely depend on their behavior and fate following release into natural environmental systems;12,13 in particular, antimicrobial ENPs, such as nAg, may have deleterious effects on fragile subsurface microbial communities that are important for biogeochemical cycling. © 2014 American Chemical Society
Received: Revised: Accepted: Published: 2715
October 14, 2013 February 4, 2014 February 5, 2014 February 19, 2014 dx.doi.org/10.1021/es404598v | Environ. Sci. Technol. 2014, 48, 2715−2723
Environmental Science & Technology
Article
been treated as a uniform layer coating the grain surfaces (Figure 1B), allowing the application of CFT to the interpretation of ENP transport behavior in biofilm-laden systems. However, some methods used to grow biofilms result in a collector that may be more accurately depicted by Figure 1C, consisting of a patchy, or inhomogeneous, biofilm coating. One reason for the inhomogeneity of coating reported in previous studies24,28 is that a biofilm grown in the packed column may experience gradients in nutrient and oxygen concentrations over the column length. In addition, when biofilm is grown inside the column, it may grow across the void spaces of the grains. Biofilm which spans the spaces between the collector grains can lead to another mechanism of retention; namely, physical trapping, which is not accounted for in CFT (Figure 1C) and, as a result, may confound our understanding of the nature of collector−particle interactions. The formation of a biofilm, defined as a multilayer coating of cells embedded in a biopolymer matrix, allows the bacteria to maximize passive transport processes (i.e., diffusion) by forming micropores or channels for liquid to penetrate within the accumulating biomass.30 The result is that a larger density of cells can survive through passive processes where they would otherwise require active transport processes to remove waste or sequester nutrients. Sauer et al.31 observed that the biofilm formed by a monoculture of P. aeruginosa PAO1 displayed distinct phenotypes during development, which they characterized in five stages (earliest time observed): (i) reversible attachment (>0 min), (ii) irreversible attachment (2 h), (iii) maturation-1 (3 days), (iv) maturation-2 (6 days), and (v) dispersal (9−12 days). Hence, in the present study, 24 and 72 h were selected as incubation points to represent a young or newly forming P. aeruginosa biofilm (24 h) and a maturing biofilm (72 h). A longer incubation period of 96 h was also included, as this is a growth time comparable to that used in other studies examining ENP or colloid transport in biofilmcoated granular porous media.22,24−26,28 In this paper, we systematically examined the transport behavior of an industrial preparation of PVP-stabilized nAg in laboratory-scale columns packed with quartz sand. To understand the interaction of this ENP with biofilms that are present on the surface of collector grains in aquatic environments, we conducted experiments using P. aeruginosa PAO1 biofilm of varying age (24, 72, and 96 h incubation). Moreover, we developed a method for producing a biofilm-coated collector surface which aims to (i) minimize the influence of biofilm webbing in the pore space within the experimental design, such that we can directly assess the interactions between the nAg particles and the biofilm-coated grains; (ii) be performed with the assurance of sterility, easily and more frequently, without large amounts of growth medium or multiple column apparatuses, (iii) coat the entire surface of the granular material to allow the assessment of ENP transport and deposition upon approach of an ENP to biofilm at the collector surface.
Transport of ENPs in water-saturated granular environments, such as those representative of groundwater aquifers, river banks (bank filtration), or deep bed (rapid sand) filters, has been commonly studied with a packed chromatography column. These experiments are generally conducted under relatively pristine conditions that do not represent the complexity or heterogeneity of natural and engineered aquatic environments.16 For instance, biofilms are ubiquitous in aquatic environments, yet there is limited information on the influence of these microbial structures on the transport and fate of ENPs in granular aquatic matrixes. The transport behavior of ENPs and other colloids in biofilm-laden granular environments has been examined using columns packed with glass beads or quartz sand upon which is grown a biofilm of Escherichia coli,22−24 Pseudomonas spp.,25−28 or Bacillus spp.28 Several different approaches have been used to grow the biofilm on the collector (grain) surfaces, where the majority of studies have used the experimental column as a growth chamber, using either clean or preinoculated sand to develop a biofilm through nutrient supply and incubation within the column for a period of time ranging from 24 h, 3 to 5 days, or even multiple weeks at room temperature or 35 ± 2 °C.22−28 Despite these methodological differences, nearly every study reports increased or unchanged retention of ENPs (e.g., quantum dots,27 zerovalent iron,24 nZnO,22 nC60,26 and nAg28) or other colloids (e.g., latex27 or bacteria25) in the presence of biofilm. Pore clogging and resultant physical straining has been highlighted as a key mechanism controlling retention of ENPs in biofilm-laden systems and, thus, may be a concern when data is interpreted using classical colloid filtration theory (CFT).22,24 Moreover, the presence of relatively sparse biofilm on the collector may lead to a more heterogeneous collector surface and thus complicate the interpretation of column transport experiments. Yao et al.29 presented a classic description of CFT in which they described three fundamental particle transport mechanisms: interception, sedimentation, and dif f usion (Figure 1A). In recent studies,22−28 the presence of a biofilm grown on collector surfaces of a granular matrix has, at least in theory,
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MATERIALS AND METHODS Preparation and Characterization of PVP-nAg Suspensions. PVP-nAg was obtained from NanoValid as a 4% (w/w) stock (pH 6.5 ± 0.5) suspended in deionized water (DI). PVP-nAg was generated through a proprietary synthesis procedure by Collorobia Italia and is currently used as an antimicrobial coating for tiles (e.g., granite, quartz, or porcelain). Suspensions of PVP-nAg were prepared at various ionic strengths (IS) in both sodium nitrate (NaNO3) and
Figure 1. Schematic of transport mechanisms, as defined in Yao et al.:29 i. interception, ii. sedimentation, iii. dif f usion. (A) Particle transport in clean granular matrix. (B) Theoretical schematic of particle transport behavior applied to a collector grain with a uniform biofilm coating. (C) A theoretical schematic of the key retention mechanisms in a biofilm-coated granular matrix with the commonly used approach to grow the biofilm in the packed column, which may lead to biofilm “webbing” that spans the pore spaces between collector grains and imparts a fourth mechanism: iv. physical trapping. 2716
dx.doi.org/10.1021/es404598v | Environ. Sci. Technol. 2014, 48, 2715−2723
Environmental Science & Technology
Article
incubator (100 rpm) and grown for 24 h at 35 ± 2 °C. The shaking speed selected was fast enough for the liquid to move while minimizing any visually detectable movement of the sand grains. For the longer growth conditions, the bottles were removed from the incubator every 20−24 h, the media was decanted, and 12−14 mL of fresh, sterile LB was added to top the bottle to 15 mL. The bottle was returned to the incubator and this process repeated every 24 h. Sand batches coated with LB, but not inoculated, were prepared following the same procedure as the biofilm-coated sand and considered as a concurrent negative control with each inoculated batch of sand. LB-coated sand was used in column experiments under selected conditions and analyzed using confocal microscopy. In every investigated case, there was no detectable difference between the LB-coated and the clean sand (i.e., confocal images, and biomass estimates) and the data collected for this control condition are not included herein. Characterization of the Collector Surface. Confocal laser scanning microscopy (CLSM) was also performed using aliquots of sand grains randomly collected from the top and bottom of selected columns, namely, those injected with an inert tracer. Sand grains were stained using FilmTracer FM 143 Green Biofilm Cell Stain and FilmTracer SYPRO Ruby Biofilm Matrix Stain (Molecular Probes, Invitrogen) following the guidelines provided by the manufacturer (Supporting Information) and imaged using a Zeiss LSM 510 META equipped with argon laser (458 and 488 nm) set to two channels (Ch1: BP530−600; Ch2: BP 560−615). Clean and LB-coated conditions were investigated as negative controls and there was no discernible difference observed between these conditions. For both the clean and LB-coated sand, CLSM images produced no fluorescent signals (completely black images) and are not included herein. There were no detectable differences between the top and bottom sand grains with respect to CLSM imaging, and therefore the representative images in the Supporting Information were selected from the top sections of the 10 mM NaNO3 condition. Average biofilm thicknesses were calculated from all experimental conditions using a procedure described in Supporting Information. An estimate of the total biomass retained in each quarter section of the biofilm-coated sand columns was determined from the combusted mass using a method described previously (following the tracer experiments).27 Streaming potential analysis (Electrokinetic Analyzer, Anton Paar) was also used to characterize the electrokinetic properties of the clean and biofilm-coated sand surfaces in 1 mM of NaNO3 and Ca(NO3)2 following a method described previously.27 Column Transport Experiments. Transport experiments were performed in triplicate using glass columns (16 mm inner diameter, GE Life Sciences). To ensure uniform packing, clean and biofilm-coated sand was wet packed to a length of 8 cm into glass columns using gentle vibration. The packed-bed porosity of clean sand, determined by the displacement method, was 0.37. Clean sand columns were conditioned with particle-free background electrolyte for 10 pore volumes (PVs) using a method described previously.35 Biofilm-coated sand was packed in a similar method to that of the clean sand, with slight modifications (Supporting Information). Biofilmcoated sand columns were similarly equilibrated for a minimum of 10 PVs. During this time, the effluent of the column was monitored using a UV−visible spectrophotometer (1-cm flowthrough cell, Agilent 8453) at 450, 500, and 600 nm
calcium nitrate (Ca(NO3)2) (trace metal analysis grade, SigmaAldrich). NaNO3 and Ca(NO3)2 were selected as the electrolytes for these experiments to prevent possible precipitation of ionic silver in the PVP-nAg suspension due to the presence of chloride. NaNO3 and Ca(NO3)2 were prepared in filtered (0.22 μm cellulose acetate filter, Fisher) DI at IS of 1, 10, or 100 mM. 3-(N-Morpholino)propanesulfonic acid (MOPS buffer; Fisher) was added to each electrolyte solution to a final concentration of 0.1 mM and the pH of the solution adjusted to 7.03 ± 0.02 using NaOH or HNO3. For use in the column experiments, PVP-nAg stock was mixed by inversion, diluted to a 20 mg/L suspension in 50 mL NaNO3 or Ca(NO3)2 solution, and vortexed for 30 s at maximum speed for 15 min before injection into the column. The hydrodynamic diameters of the PVP-nAg were characterized using dynamic light scattering (DLS; ZetaSizer Nano ZS, Malvern) and nanoparticle tracking analysis (NTA; NanoSight LM10, NanoSight). Laser Doppler velocimetry in conjunction with phase analysis light scattering (Zetasizer Nano ZS) was used to measure electrophoretic mobility (EPM) concurrent with the column experiments. All EPM measurements were performed at 25 °C, with an applied electrical field (E) of 4.9 ± 0.1 V/m. Additionally, transmission electron microscope (TEM) images of PVP-nAg were obtained using a Philips CM200 TEM (Advanced Microscopy Techniques Corp.) and were prepared by adding a small droplet (20 μL) of 20 mg/L PVP-nAg suspended in DI onto a carbon/Formvarcoated copper grid with mean size determined from 68 individual particles across 14 images following procedures described previously.32 Dissolution of PVP-nAg was assessed using inductively coupled plasma atomic emission spectroscopy (ICP-AES; Thermo Jarrell Ash, Trace Scan) at selected IS in both NaNO3 and Ca(NO3)2 over the time frame of the transport experiments by filtering 1 mL of 20 mg/L PVP-nAg suspended in 1 and 100 mM IS NaNO3 or Ca(NO3)2 using a 3kD Amicon Ultracentrifuge filter (
