Natural Organic Matter Alters Biofilm Tolerance to Silver Nanoparticles

Article pubs.acs.org/est

Natural Organic Matter Alters Biofilm Tolerance to Silver Nanoparticles and Dissolved Silver Stacy M. Wirth,† Gregory V. Lowry,†,‡ and Robert D. Tilton*,†,§ †

Center for the Environmental Implications of Nanotechnology, Carnegie Mellon University, Pittsburgh, Pennsylvania 15213, United States ‡ Department of Civil and Environmental Engineering, and §Department of Biomedical Engineering, Carnegie Mellon University, Pittsburgh, Pennsylvania 15213, United States S Supporting Information *

ABSTRACT: Motivated by the need to understand environmental risks posed by potentially biocidal engineered nanoparticles, the effects of silver nanoparticle (AgNP) exposure on viability in single species Pseudomonas fluorescens biofilms were determined via dye staining methods. AgNP dispersions, containing both particles and dissolved silver originating from the particles, negatively impacted biofilm viability in a dosedependent manner. No silver treatments (up to 100 ppm AgNPs) resulted in 100% biofilm viability loss, even though these same concentrations caused complete viability loss in planktonic culture, suggesting some biofilm tolerance to AgNP toxicity. Colloidally stable AgNP suspensions exhibited greater toxicity to biofilms than corresponding particle-free supernatants containing only dissolved silver released from the particles. This distinct nanoparticle-specific toxicity was not observed for less stable, highly aggregated particles, suggesting that biofilms were protected against nanoparticle aggregate toxicity. In both the stable and highly aggregated dispersions, dissolved silver made a significant contribution to overall toxicity. Therefore, despite increased colloidal stability when humic acid adsorbed to AgNPs, the presence of humic acid mitigated the toxicity of AgNP suspensions because it bound to silver ions in solution.

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INTRODUCTION The growing use of silver nanoparticles (AgNPs) in commercial and consumer products makes their release to waste streams and the environment inevitable. This has raised concern, since the broad spectrum biocidal properties that motivate AgNP use in consumer and medical applications could have detrimental consequences for environmental micro-organisms that are crucial to normal ecosystem function.1 Microbial toxicity of AgNPs and Ag+ is well documented for laboratory-grown planktonic bacterial cultures, where cells are suspended in nutrient-rich culture media.2−9 In most natural environments, however, surface attached biofilms, adherent bacterial communities surrounded by a self-produced matrix of extracellular polymeric substance (EPS), are the prevailing microbial community structure.10,11 Biofilms can be up to 1000 times more resistant to toxicants than their planktonic counterparts,12 and data obtained from planktonic cultures cannot be used to predict antibiotic effects in biofilms.10 Additionally, biofilms may be sinks for nanoparticles and their aggregates as they sediment in the environment.13,14 Thus, studies of nanoparticle effects on biofilms are necessary in order to predict potential environmental consequences of nanoparticle release, though few studies have addressed this topic.15−21 © 2012 American Chemical Society

Another critical feature when considering nanoparticles released into the environment is the presence of naturally occurring macromolecules that can adsorb to their surfaces. Humic acids, which comprise a major fraction of the ubiquitous natural organic matter (NOM) in most environments, are likely to interact with AgNPs. Increased colloidal stability following humic acid adsorption has been demonstrated for several nanoparticles,22−26 including silver.27,28 Therefore, the component of interest for environmental relevance is not simply the nanoparticle itself but the nanoparticle−macromolecule complex that forms in multicomponent environmental media. The adsorbed layer will affect colloidal stability and adherence to soil surfaces.29,30 This in turn will affect nanoparticle mobility in the environment, likely including transport in the biofilm matrix, and affect subsequent interactions with other environmental colloids, biomolecules, and cells, with probable implications for toxicity.31 Additionally, molecules that chelate silver ion may decrease its toxicity,32,33 so NOM may indirectly Received: Revised: Accepted: Published: 12687

April 17, 2012 October 19, 2012 October 30, 2012 October 30, 2012 dx.doi.org/10.1021/es301521p | Environ. Sci. Technol. 2012, 46, 12687−12696

Environmental Science & Technology

Article

ultracentrifuge OTB65B, Thermo Fisher Scientific, Waltham, MA) for 100 min at 115 000g and 4 °C, followed by supernatant removal, nanoparticle resuspension in ultrapure water by ultrasonication, and repeated six times, was performed for both particle types to remove unadsorbed PVP. After the sixth wash, nanoparticles were dispersed in ultrapure water to a 1000 ppm nominal concentration. LS AgNPs were dispersed by bath sonication for 1 min (Branson 1200, Branson Ultrasonics Corp., Danbury, CT) followed by probe ultrasonication at 2.9 W for 5 min. HS AgNPs were sonicated in the bath for 5 min and then probe ultrasonicated for 1 min. Ultrasonicating for longer times produced no further decrease in particle size, as measured by dynamic light scattering (Zetasizer Nano ZS, Malvern Instruments, Worcestershire, UK). Actual concentrations in these suspensions were measured by graphite furnace atomic absorption (GFAA) spectrometry (GBC 908AA and GF 3000, GBC Scientific Equipment, Melbourne, Australia) after dissolution in concentrated nitric acid. Due to losses during washing, actual concentrations were slightly lower than the 1000 ppm target concentration: 870 ppm for the LS AgNPs and 960 ppm for the HS AgNPs. Suspensions were serially diluted in minimal Davis media (MDM, described below) or MDM containing HA at 10 mg C/L (denoted MDM+HA) to obtain concentrations of 87, 8.7, and 0.87 ppm for LS AgNPs and 96, 9.6, and 0.96 ppm for HS AgNPs. All suspensions were agitated at room temperature in the dark on an orbital shaker at 160 rpm for 18 h, the length of toxicity experiments. After 18 h, a fraction of each suspension was ultracentrifuged (115 000g, 100 min, 4 °C) to obtain particle-free supernatants containing dissolved silver at concentrations corresponding to each AgNP concentration. The total silver concentration in the supernatants, consisting of free ionic silver, ionic silver complexed with HA if present, and any other dissolved silver species was measured by GFAA spectrometry after acidifying in 5% HNO3. To determine the contribution of HA-complexed silver ion to total silver in supernatants, filtrate silver concentrations were measured after HA-complexed silver was removed by Amicon Ultra-4 centrifugal filter units with 3 kDa molecular weight cutoffs (Millipore, Billerica, MA). UV absorbance of the filtrate confirmed that filtration removed more than 90% of the HA. AgNO3 solutions of known concentration were also mixed with 10 mg C/L HA in MDM for 18 h and filtered to determine the amount of Ag+ lost to HA complexation. Since substantial AgNP aggregation and sedimentation occurred during overnight mixing, the 87 and 96 ppm overnight-mixed suspensions were ultrasonicated at 2.9 W for 3 min (for LS AgNPs) or 1 min (for HS AgNPs) and then serially diluted in MDM or MDM+HA to 8.7 and 0.87 ppm (LS AgNPs) or 9.6 and 0.96 ppm (HS AgNPs), immediately prior to use in toxicity experiments. Hydrodynamic diameters and electrophoretic mobilities of these redispersed particles were measured using a Malvern Zetasizer Nano ZS. Humic acid adsorption to each type of AgNP was verified by solution depletion. Concentrated particle stocks were added to MDM+HA followed by orbital shaking in the dark for 18 h at 160 rpm and room temperature. Particles and adsorbed HA were removed by ultracentrifugation, and supernatant HA concentrations were estimated from absorbance at 254 nm. The adsorbed amount was calculated by closing the material balance between the initial HA amount and the amount remaining after particle removal.

affect AgNP toxicity by chelating toxic ions released from nanoparticles.34−36 The objectives of this work were to determine AgNP impacts on biofilms of the model bacterium Pseudomonas fluorescens ATCC 13525 and to determine whether humic acid modifies these effects. For comparison, toxicity toward planktonic P. fluorescens was also measured. Use of single species biofilms in well-defined media with controlled dosing of humic acid represents a step toward real environmental systems while preserving some compositional simplicity to isolate the effects of individual system constituents. Biofilm viability response to AgNP suspensions, as well as particle-free supernatants containing dissolved silver species from those suspensions, in the presence or absence of humic acid, was measured to determine the dose-dependent susceptibility of cells in this adherent growth mode. A potential mechanism for biofilm resistance to antimicrobials is hindered transport through the biofilm.37 Since nanoparticle transport in the biofilm will be affected by particle size and therefore aggregation state, two types of AgNPs with different colloidal stability were compared. The ability of silver treatments to cause biofilm detachment was also assessed. We show here that both particle stability and dissolved silver concentration influence AgNP toxicity to P. fluorescens biofilms.

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EXPERIMENTAL SECTION Preparation and Characterization of AgNP Suspensions. One type of AgNP (denoted LS for “low stability”) was purchased from Nanostructured and Amorphous Materials (Houston, TX) as a powder. These particles are stabilized by polyvinylpyrrolidone (PVP) and reported by the manufacturer to have an average primary particle diameter of 30−50 nm, based on transmission electron microscopy (TEM), 99.9% purity, and a specific surface area of 5−10 m2/g. The second particle type (denoted HS for “high stability”) was synthesized and characterized as previously described,9,38 are also PVP stabilized, and were obtained as a concentrated (>5 g/L) suspension that was never dried. These particles are spheroidal, with an average diameter of 18.6 ± 4 nm (sizes range from 10 to 30 nm) based on scanning electron microscopy (SEM) images. Particle stocks and the suspensions created from them were stored in the dark at room temperature (23 ± 2 °C). Pahokee peat reference humic acid (HA) was purchased from the International Humic Substances Society (IHSS, St. Paul, MN). Key properties of this HA, including elemental composition, are reported by the IHSS.39 A 100 mg/L stock was prepared in ultrapure water (Barnstead Nanopure, >18 MΩ cm resistivity), and the pH was adjusted with 0.1 M KOH to 7.1. The solution was magnetically stirred and heated to boiling, followed by overnight stirring at 23 ± 2 °C. The pH was again adjusted to 7.1, followed by sterile filtration through a 0.2 μm cellulose acetate filter. The final stock concentration was determined by measurement of absorbance at 254 nm and comparison to a calibration curve constructed by total organic carbon analysis (Model 1010 TOC Analyzer, OI Analytical). HA concentrations are therefore reported as milligrams of organic carbon per liter (mg C/L). This particular HA contains 56.84% (w/w) carbon. LS particles were dispersed to 500 ppm in ultrapure water by ultrasonication (Branson Sonifier 250, Branson Ultrasonics Corporation, Danbury, CT) for 5 min at 2.9 W. HS particles were diluted to 500 ppm in ultrapure water. A washing procedure, where the suspension was ultracentrifuged (Sorvall 12688

dx.doi.org/10.1021/es301521p | Environ. Sci. Technol. 2012, 46, 12687−12696


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400 μL of ultrapure water to remove unbound dye. Bound dye was resolubilized in 200 μL of 95 vol % ethanol for 15 min, 100 μL was transferred to an empty well, and crystal violet absorbance was measured at 585 nm with a microplate reader (SpectraMax M2, Molecular Devices, Sunnyvale, CA). The percentage difference between these absorbance values and the average value for biofilms at time zero, before any silver treatment, indicated the effect of the added silver on the net biofilm growth or detachment during 18 h silver exposure. Since crystal violet binds both to cells and to components of the extracellular matrix, this assay does not discriminate cellular biomass from EPS.43 Biofilm viability was assessed using the LIVE/DEAD BacLight bacterial viability kit (Invitrogen Corp., Carlsbad, CA), containing propidium iodide (PI) and SYTO9 fluorescent dyes. These dyes differentiate between cells with intact or damaged cell membranes. This distinction often correlates with viability, and we use that association here. More details about these dyes, including a calibration curve for planktonic P. fluorescens, can be found in the Supporting Information (Figure S1). The manufacturer’s protocol was modified so measurements could be made directly in the wells in which biofilms had been grown and exposed to nanoparticles. Briefly, the liquid was pipetted from each well, and each biofilm was gently washed three times to remove nonadherent cells and stained with the BacLight mixture, prepared according to the manufacturer’s instructions. After 15 min incubation in the dark, free stain was removed, wells were refilled with ultrapure water, and each stain’s fluorescence intensity was measured in nine separate points of each well using the microplate reader. To determine the dye fluorescence intensity values corresponding to 100% cell death, biofilms killed by 1 h incubation in 70% isopropanol were also stained. Silver did not interfere with staining (Supporting Information, Figures S2−S4). The ratio of SYTO9 to PI intensity determined for the 70% isopropanol killed biofilms was nonzero, despite the fact that these biofilms were completely killed (as verified by agar plating). This nonzero intensity ratio was subtracted from the ratios in the other wells, including the silver-free biofilm control wells. The percentage difference in this adjusted ratio for each well compared to the silver-free control, termed the change in viability, was calculated. The silver-free control was either MDM or MDM+HA. This analysis does not rely on the planktonic calibration curve but rather on two biofilm reference states: silver-free control and isopropanol killed. By this method, a treatment that is no different from the silver-free control would give a 0% change, while a treatment giving the same response as the 70% isopropanol treatment would give a −100% change. To determine potential effects of cells growing at the air− water interface and incomplete removal of nonadherent cells, a subset of wells had the air−water interface aspirated and wells rinsed during media switches, prior to silver treatment and before crystal violet or BacLight analysis. No statistical difference (P > 0.05) in biomass amounts or viability was observed for these biofilms and those treated as described above. All statistical analyses were performed with unpaired twotailed t tests. A P value less than 0.05 was used to indicate a statistically significant difference at the 95% confidence level.

Bacterial Strain and Culture Conditions. The bacterial strain was Pseudomonas fluorescens ATCC 13525. All cell culture and AgNP exposure work was performed in MDM containing 0.7 g/L K2HPO4, 0.2 g/L KH2PO4, 1 g/L (NH4)2SO4, 0.5 g/L sodium citrate, 0.1 g/L MgSO4·7H2O, and 1 g/L glucose as the carbon source at a pH of 7.2 ± 0.1. This media was chosen for its comparatively low ionic strength (∼50 mM) and its use in previous microbial exposure studies where minimal nanoparticle aggregation was desired.40 The media composition does not represent any specific ecological niche but represents a model system in which to assess effects of critical variables, namely, AgNP and/or dissolved silver concentration, colloidal stability, and presence of HA, on biofilms. For each experiment, a starting culture was prepared by inoculating autoclavesterilized MDM with a P. fluorescens frozen stock (−80 °C) and growing aerobically overnight at room temperature with 160 rpm orbital mixing. AgNP Toxicity to Established Biofilms. Since our goals at this stage were to determine the effects of humic acid on toxicity and to observe the possible contribution of dissolved silver to growth inhibition or viability loss, we used nanoparticle concentrations where one might expect to find a short-term toxic effect (1−100 ppm). Concentrations of AgNPs released from wastewater treatment plants to the environment, the most likely mode of entry, are predicted to be much lower, with some estimates reaching parts per million levels.41,42 However, other modes of entry (i.e., accidental release from a production facility) cannot be ruled out and could give locally higher concentrations. Biofilms were grown in sterile, nontreated 96well flat-bottom polystyrene microplates (Costar) using established methods.43,44 Briefly, P. fluorescens cultures in log phase growth were diluted in MDM to an OD600 of 0.01 (1 × 107 CFU/mL), and 200 μL of this culture was added to wells of 6 rows of a 96-well microplate. The remaining wells were blanks containing sterile MDM. During biofilm growth and AgNP exposure, plates were kept in the dark at 23 ± 2 °C with 160 rpm orbital shaking. Following a 5 h adhesion period, the liquid in the wells (containing nonadherent cells) was removed and replaced with 200 μL of sterile MDM. Media was replaced again after an additional 16 h to replenish depleted nutrients. After an additional 6 h (27 h after inoculation), the amount of adherent biomass in a subset of wells was measured by the crystal violet assay, as described below. Liquid was gently pipetted from the remaining wells and replaced by 200 μL of 0.87, 8.7, or 87 ppm LS AgNPs or 0.96, 9.6, or 96 ppm HS AgNPs in MDM or MDM+HA, or the corresponding particlefree supernatants. In some cases, AgNO3 solutions in MDM or MDM+HA were added to the biofilms. The time at which silver in any form was added to established biofilms was defined as time zero. At time zero, control wells received sterile MDM or MDM+HA containing no silver. Each treatment was repeated in a minimum of six wells per experiment; the overall experiment was repeated at least four times (at least 24 wells per treatment). For each treatment, half of the biofilm wells were analyzed for viability and the other half for total biomass. Following 18 h silver exposure, total biomass adhered to the wells, that is, total biofilm, was assessed by the crystal violet staining method of O’Toole and Kolter.45 Briefly, the liquid in each well was gently removed by pipet, followed by rinsing three times with 25 mM NaCl to remove nonadherent cells and drying for 30 min. Each well was then stained with 200 μL of a 1 wt % crystal violet (Sigma Chemical Company, St. Louis, MO) solution for 15 min, followed by washing five times with 12689



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Figure 1. Biofilm viability results for LS (A) and HS AgNPs (B). Percentage difference in viability from silver-free control treatments indicates dosedependent loss of P. fluorescens biofilm viability following treatment with silver nanoparticles (green triangles or red squares) and their corresponding particle-free supernatants (blue circles or diamonds). For all treatments, 100 ppm decreased viability significantly more than the corresponding 10 ppm treatment (P < 0.05). Some treatments had no significant difference between 1 and 10 ppm (LS supernatant, LS+HA supernatant, HS supernatant, HS+HA particles), while the others had significantly greater viability loss at 10 ppm. Biofilms receiving treatments containing HA (open symbols) had significantly (P < 0.05) greater viability than biofilms receiving the corresponding treatments without HA (closed symbols) at all concentrations. Data points are the average from at least four separate experiments, with at least four replicate wells per experiment. Error bars represent 95% confidence intervals. Lines are to guide the eye.

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RESULTS AND DISCUSSION Particle Characterization. AgNP size distributions determined by dynamic light scattering (DLS) are included in Figure S5 of the Supporting Information. LS AgNP suspensions dispersed in pure water showed a bimodal intensity-weighted hydrodynamic diameter distribution, with the main peak at 200 nm and a small peak near 27 nm. The main peak in the number weighted distribution in water was near 25 nm, likely corresponding to primary particles. The difference between intensity- and number-weighted distributions indicated that the initial suspension contained a fraction of aggregated particles that could not be fully dispersed. When LS AgNPs dispersed in ultrapure water were transferred to MDM or MDM+HA, the main peak of the intensity weighted hydrodynamic diameter distribution shifted from 200 nm to about 400 nm, indicating substantial aggregation. The number-weighted distribution peak shifted to about 300 nm, indicating aggregation of primary particles. In both MDM and MDM+HA, LS particles sedimented completely within a few hours of ultrasonication, while in ultrapure water, some particles remained suspended for days. Other evidence for substantial LS particle aggregation is provided by UV−vis absorbance spectra (Supporting Information, Figure S6). These display the expected localized surface plasmon resonance (LSPR) absorbance peak at 398 nm for discrete LS AgNPs when initially dispersed in water, but it disappears completely in spectra from LS particles in MDM or MDM+HA due to LS AgNP aggregation and sedimentation. After redispersal just prior to addition to biofilms, LS AgNP suspensions contain dispersed aggregates with diameters greater than 70 nm (Supporting Information, Figure S5E), which also sedimented entirely in several hours. Thus, experiments using LS AgNPs assessed the effects of aggregating and sedimenting silver nanoparticles, rather than well-dispersed primary nanoparticles. The HS AgNPs maintained their stability when transferred from water to MDM or MDM+HA, as indicated by stable DLS size distributions (Supporting Information, Figure S5). The intensity-weighted hydrodynamic diameter distribution for these particles was also bimodal, with a main peak at 100 nm and a smaller peak at 13 nm. The number-weighted

hydrodynamic diameter distribution has a peak at an apparent diameter of 9 nm, likely corresponding to primary particles. This exact value is unrealistic, given the size distribution in SEM images, and it reflects uncertainty due to the imprecise inversion of the autocorrelation function in the presence of both primary and aggregated particles. The important observation is that, in contrast to the LS AgNPs, after 24 h in MDM or MDM+HA (Supporting Information, Figure S5D, F), some HS AgNPs remain suspended and these suspended particles are mostly unaggregated. However, HS AgNP sedimentation did occur, though to a smaller degree than for LS particles. After 18 h, the LSPR peak at 408 nm in the absorbance spectra (Supporting Information, Figure S7) decreased by 71% for the HS suspension in MDM, indicating some particle loss to aggregation and sedimentation but by only 33% for the particles in MDM+HA, indicating a stabilizing role of HA. HA adsorbed to both AgNP types, but to differing extents. The amount of HA adsorbed to the HS AgNPs was 1.78 mg C/ g particles, nearly 20 times greater than the amount adsorbed to the LS AgNPs, 0.098 ± 0.016 mg C/g particles. The origins of this difference in HA adsorption affinity are not yet known, and it must be borne in mind that both particle types already have PVP on their surfaces. After HA adsorption, electrophoretic mobility became more negative for both particle types. For LS particles, electrophoretic mobility changed from −2.75 ± 0.18 to −3.4 ± 0.17 (μm cm)/(V s), and for HS particles, the change was from −1.48 ± 0.18 to −2.50 ± 0.26 (μm cm)/(V s). This is consistent with the anionic character of HA.28 Particle Dissolution. Dissolved silver concentrations in suspension supernatants after 18 h are shown in Figure S8 of the Supporting Information. Dissolved silver was about 2.5 times more concentrated for the smaller and more stable HS AgNPs than for the LS AgNPs. This is consistent with reports that AgNP dissolution (kinetics and equilibrium solubility) is surface area dependent.46−49 For example, the Ostwald− Freundlich equation (Supporting Information, eq S1), which relates particle solubility to size, predicts that HS AgNPs will have an equilibrium solubility 1.7 times greater than that for the LS AgNPs. This is close to the observed value of 2.5. Also, of interest is the effect of HA on dissolved silver. HA might 12690



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promote particle dissolution by chelating dissolved silver and increasing the driving force for dissolution,50 although HA adsorption to the particle could also block surface sites and hinder dissolution, as previously observed for quantum dots51 and nanosilver.52 Here, for particles dispersed in MDM, HA had no significant effect on total dissolved silver concentrations in the suspension supernatants for either LS or HS AgNPs. When supernatants from suspensions in MDM+HA were filtered to remove HA, dissolved silver concentrations decreased significantly, indicating that much of the silver was actually complexed with HA. Filtration had no effect on dissolved silver concentrations in supernatants without HA, indicating that ultracentrifugation had removed all the particles and that filtration did not remove dissolved silver from solution. Silver complexation by HA has implications for toxicity, since the bioavailability of HA-complexed silver likely differs from that of the free silver, as discussed below. Effects on Viability in Established Biofilms. Pre-existing biofilms are difficult to eradicate with conventional antibiotics10,53 and could potentially also show tolerance to biocidal nanoparticles. Viability results for established biofilms treated with LS (Figure 1A) or HS AgNPs (Figure 1B) or particle-free supernatants from those suspensions indicate a dose-dependent toxicity. In the absence of HA, both particle types caused significant (P < 0.05) viability loss at all tested concentrations. However, 100% viability loss did not occur even at the highest concentrations tested (87 ppm LS AgNPs or 96 ppm HS AgNPs), suggesting some biofilm tolerance to AgNPs. For comparison, P. fluorescens planktonic cultures treated with 87 ppm LS AgNPs or 96 ppm HS AgNPs in MDM for 18 h showed 100% viability loss based on viable plate counts. Biofilms also showed tolerance to dissolved silver treatments, since particle-free supernatants brought about 100% viability loss in planktonic cultures at concentrations that did not give 100% biofilm viability loss. (For details see the Supporting Information, Table S1). To confirm incomplete viability loss in biofilms, AgNP exposed biofilms were scraped from wells and streaked onto nutrient agar. Some colony growth from scraped biofilm material was observed in all cases, even when 10 μL drops of liquid from above the biofilm (which contained high concentrations of planktonic cells in silver-free control experiments) showed no growth on nutrient agar. These results support the conclusion that P. fluorescens biofilms are more tolerant than planktonic cultures to AgNPs. Effect of Humic Acid on Viability. The percent change in viability between biofilms treated with only MDM+HA and biofilms receiving silver treatments in the presence of HA was determined. HA significantly (P < 0.05) decreased viability loss for both particle types at every AgNP concentration (Figure 1). Other studies have suggested that adsorption of large macromolecules such as humic acid to nanoparticles could prevent direct contact with cells, decreasing toxicity.28 Nevertheless, Figure 1 also shows that particle-free supernatants from suspensions in MDM+HA were less toxic than the corresponding HA-free supernatants for all AgNP concentrations. Thus, the toxicity-mitigating effect of HA was not limited to the particles. We hypothesized that bioavailable silver ion concentration was decreased by complexation to anionic HA. Filtering HA from HA-containing AgNO3 solutions decreased the total silver measured by GFAA (Figure 2). This confirms that HA binds to Ag+. Prior work has shown that HA complexation decreases Ag+ toxicity to fathead minnows and

Figure 2. Silver concentrations in AgNO3 solutions prepared in either MDM (closed symbols) or MDM with 10 mg C/L HA (open symbols). Unfiltered samples yielded identical results with or without HA (black squares). Filtration (red circles) removed HA-complexed silver and had no effect on the silver concentration in HA-free samples (top red line). Filtration decreased silver concentrations in samples that originally contained HA (lower red curve) by a constant 56 ± 11%. Error bars represent one standard deviation, based on duplicate measurements.

Daphnia magna,54 consistent with the decreased toxicity toward biofilm P. fluorescens. Figure 3 shows viability data for biofilms exposed to particlefree dissolved silver, including precisely prepared AgNO3

Figure 3. Viability change produced by particle-free solutions as a function of free silver concentration (after filtration) shows that solutions obtained from LS suspension supernatants (circles), HS suspension supernatants (diamonds), and AgNO3 solutions (squares) produced similar effects. Loss of viability when HA was present (open symbols) was similar to the HA-free conditions (closed symbols) when the free silver concentration was similar. Error bars represent 95% confidence intervals based on at least three replicate wells. Data is shown only for those supernatant experiments where free silver concentration was measured.

solutions and AgNP suspension supernatants, where free silver concentration was measured. Here, “free silver” is defined as dissolved silver that is able to pass through a 3 kDa filter and is therefore not bound to HA, though the presence of soluble complexes between Ag+ and other media components (i.e., PO43−, SO42−, C6H5O73−) is likely.55,56 Figure 3 shows similar biofilm viability loss for solutions with similar free silver concentrations, regardless of the silver source (AgNO3 or AgNP supernatants) or the presence of HA. This supports the hypothesis that HA decreased toxicity by removing bioavailable silver ion. No additional mitigating effects from HA were observed. Since dissolved silver contributes to the overall 12691



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toxicity of particle-containing AgNP suspensions, loss of bioavailable silver ion to HA complexation in those suspensions would also result in decreased toxicity. Particle-Specific Effects on Viability. AgNP suspensions contain both AgNPs and dissolved silver, and the contribution of each to overall toxicity to bacteria is a subject of debate. Some studies have suggested that dissolved Ag+ is the main toxic agent, with AgNPs serving mainly as a source of Ag+.32,33,55,57 Other studies have shown a distinct role for the nanoparticles themselves in AgNP suspension toxicity, although the particle-specific toxicity mechanism remains unclear.5,49,58,59 Here, we define “particle-specific” as any effect on the biofilm that was not evident when dissolved silver was introduced by itself. This definition does not necessarily indicate a different biochemical pathway for particles and ions. (For example, AgNPs could adsorb to cells and deliver a high localized Ag+ flux to the cell wall. The biochemical toxicity mechanism could be the same, but the particle would play a prominent role. We do not distinguish such effects here.) In the following, all concentrations refer to the AgNP concentration in the suspension that was either used directly or ultracentrifuged to generate the corresponding supernatant. Figure 1 shows that, in the absence of HA, similar viability loss occurred for LS particle suspensions and their corresponding particle-free supernatants (particles were significantly more toxic than the supernatant at 100 ppm only; P = 0.03). A greater deviation between particle suspensions and their supernatants existed for the HS particles, particularly at 9.6 ppm (−74.6% and −50.4% viability loss for particles and supernatant, respectively; P = 10−5). The 9.6 ppm HS AgNP suspension toxicity could not be attributed exclusively to the dissolved silver in the suspension. These results indicate a minimal particle-specific toxic effect for the highly aggregated LS particles, whereas more colloidally stable HS particles did contribute a particle-specific toxic effect. To more thoroughly scrutinize the possibility of a particlespecific toxic effect, biofilm viability was plotted in Figure 4 as a

function of free silver concentration for exposure experiments with HS AgNPs, LS AgNPs, or particle-free solutions. (Data from Figure 3 has been averaged for clarity in Figure 4.) Only data from experiments where both viability and free silver concentration were measured in the same well is included in this plot. Figure 4 indicates constant low toxicity at low free silver concentrations. One-way ANOVA shows no statistical difference between the mean viabilities for all tested treatments at free silver concentrations less than 0.02 ppm (excluding the outlier at 0.08 ppm) The overall average change in viability at these low concentrations is −20.8 ± 14.0%. At intermediate concentrations (0.02 to ∼0.1 ppm), dose-dependent viability loss is evident. In this regime, HS AgNPs were significantly (P < 0.05) more toxic than either LS AgNPs or dissolved silver (no particles) with the same free silver concentration. LS AgNPs, on the other hand, had similar toxicity to the free silver at all concentrations. This supports the conclusion that HS AgNPs have a particle-specific toxic effect. HS particles caused higher viability loss than LS particles even though the LS particles were completely sedimented onto the biofilm soon after addition, giving locally higher concentrations on the biofilm surface, whereas a significant fraction (>25% after 18 h) of HS particles remained suspended above the biofilm. This emphasizes the importance of colloidal stability in determining AgNP toxicity to biofilms. EPS could act as a barrier to antimicrobial transport into biofilms and play a role in the extraordinary antimicrobial tolerance of biofilms.37 Transport hindrance in hydrogels becomes significant and increasingly severe for macromolecules (e.g., proteins) that are several nanometers or larger.60 The relatively large sizes of AgNPs and especially their aggregates suggest that transport hindrance could play a role in biofilm tolerance. Hindered nanoparticle diffusion in biofilms has been demonstrated by Peulen and Wilkinson,19 who observed negligible diffusion of carboxylated polymer nanoparticles with diameters greater than 50 nm in laboratory grown P. fluorescens biofilms with a reported thickness less than 30 μm. The presence of EPS in biofilms of the current study was evident in the diffuse light scattering in SYTO9 stained biofilms viewed with epifluorescence microscopy (Supporting Information, Figures S9 and S10). A microscopy method (details in the Supporting Information) similar to that of Bakke and Olsson61 showed that the biofilms studied here had an average thickness of 18 ± 10 μm at time zero, suggesting that a size-dependent transport hindrance is feasible here. Additionally, Stojak and coworkers62 showed a size-dependent response of Legionella pneumophila biofilm morphology to gold nanoparticles. Effects seen with 4 or 18 nm nanoparticles were absent for 50 nm particles. The lack of a nanoparticle-specific effect for the highly aggregated LS AgNPs on biofilms in the current study suggests that EPS hindered AgNP aggregate penetration into the biofilms, while the much smaller silver ion was able to diffuse through the EPS and exert toxicity. Nevertheless, comparison of dissolved silver toxicity in biofilm and planktonic cultures indicates that even the dissolved silver toxicity to biofilms was limited, since concentrations causing 100% planktonic culture viability loss in MDM did not cause total biofilm viability loss. A possible explanation for this is ionic silver reaction with EPS components.63,64 We suggest that the more stable HS particles may be able to diffuse into the biofilm matrix and gain closer access to cells. In this case, increased toxicity compared to dissolved silver might result from direct interactions between AgNPs and cells or from particle dissolution from AgNP

Figure 4. Loss of viability as a function of free silver concentration from experiments where free silver concentrations were measured. Particle-free toxicity (blue diamonds) is represented by average values from supernatants and AgNO3 solutions with similar free silver concentrations. LS AgNPs (green triangles) had similar toxicity to particle-free silver solutions (blue diamonds) at similar free silver concentrations. HS AgNPs (red squares) without HA (closed symbols) and with HA (open symbols) were more toxic than both the free silver and LS AgNPs at intermediate free silver concentrations (0.03−0.1 ppm). Points marked with a black × had significantly greater loss of viability (P < 0.05) than the corresponding particle-free supernatants. Error bars represent 95% confidence intervals. 12692



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sources within the biofilm matrix delivering locally high Ag+ concentrations. This could allow toxic interactions with cells to occur without interference from ionic silver reaction with the biofilm EPS. This hypothesis remains to be tested. Figure 4 also shows that HS AgNPs exerted particle-specific toxicity even in the presence of HA. This is in contrast to previous studies with planktonic cultures that demonstrated that NOM coatings on a variety of nanoparticle types,31,65,66 including silver,28 mitigated particle-specific toxicity, likely by creating a physical barrier to cell−particle contact. The difference likely results from the different modes of bacterial growth, planktonic versus biofilm, and the different types of original particle coatings. For instance, Fabrega and coworkers28 used citrate-capped AgNPs that lack the PVP steric stabilizing layers and could likely make more direct contact with cell walls in the absence of HA. Adsorbing HA would create a physical barrier to contact where none previously existed, decreasing toxicity. In the current study, the macromolecular PVP layer on the particles and the biofilm EPS likely hindered direct nanoparticle−cell wall contact, even in the absence of HA. Although it remains to be tested by future research, we hypothesize that the particle-specific toxic effects observed in this biofilm study are more likely due to silver ion delivery in close proximity to cells within the matrix, with a corresponding avoidance of Ag+ scavenging by EPS, rather than direct bacterial contact. Effect of AgNPs on Biomass Amounts. Biofilm killing and biofilm removal are distinct phenomena. Agents that promote one do not necessarily affect the other.67 Change in the adherent biomass amounts, determined by crystal violet assay, occurred during biofilm exposure to AgNPs or corresponding particle-free supernatants (Figure 5). Silverfree MDM- or MDM+HA-treated controls both had overall growth (including suspended cells and cells adhered to the wells), based on optical density measurements (Supporting Information, Figure S11). MDM caused no change in the adherent biomass amount while MDM+HA caused adherent biomass loss. Biofilm shedding following changes in media composition, such as occurred here with HA addition to biofilms grown in MDM without HA, has been previously observed.68 Both AgNPs and their supernatants at low concentrations (8.7 ppm for LS or 0.96 ppm for HS AgNPs) in the presence of HA stimulated an increase in adherent biomass. Thus, low concentration silver not only prevented the shedding observed with HA alone but promoted biofilm biomass production. Biofilm stimulation was greater for LS AgNPs and supernatants when HA was present. HS AgNPs had no stimulatory effect without HA. For both particle types with HA present, stimulation was more pronounced for the particles than for their supernatants, suggesting a particle-specific component to this effect. The observed stimulation could result from increased cell growth and/or EPS production, which have both been observed as a stress response for biofilms treated with low concentration antibiotics.69,70 A comparison of the overall cell growth (planktonic + adherent cells) in the control and silver-treated wells shows that overall growth was not enhanced by silver treatments that showed increased biofilm amounts (Supporting Information, Figure S11). Biomass stimulation by silver was therefore specific to the adherent cells. The increased adherent biomass at low silver concentrations perhaps could explain the constant low viability loss at low free silver concentrations shown in Figure 4. Potentially, cells lost to

Figure 5. Change in adherent biomass based on crystal violet assay after 18 h exposure of established biofilms grown in MDM to LS AgNPs (A) or HS AgNPs (B). Results are shown for particles (green and red) and supernatants (blue) without HA (closed bars) and with HA (open bars). No significant change in biomass occurred for control biofilms incubated with MDM for 18 h (solid black line), although control biofilms incubated with MDM+HA (dotted black line) lost adherent biomass. Asterisks (*) indicate a significant change from the time 0 biomass amount. Plus signs (+) indicate significant difference from the corresponding control. Shaded regions represent 95% confidence intervals for control biofilms. Error bars represent 95% confidence intervals from a minimum of nine total biofilms, from at least three independent experiments.

AgNP toxicity provided a nutrient source for biofilm growth, maintaining relatively constant overall biofilm viability with the cell growth rate equaling the death rate. Since environmental exposures are expected to be low in most instances, this finding suggests that AgNP toxicity to environmental biofilms will be low and biofilm growth may even be stimulated. At higher silver concentrations, cell growth could not keep up with cell death, leading to the dose−response region of Figure 4. Similar stimulatory effects at low doses have been observed for biofilms treated with carbon nanotubes18 and planktonic bacteria treated with AgNPs.57 Both particle types (and associated supernatants) caused significant biofilm removal at high concentrations (87 ppm LS AgNPs or 96 ppm HS AgNPs), though removal was always significantly less than 100%. For LS AgNPs and their supernatants with HA, biomass loss at 87 ppm was no greater than that observed for HA alone. This could indicate that, at these conditions, silver did not contribute to biofilm loss, although we cannot distinguish between the contribution of silver and HA to biomass removal. All other high concentration (87 or 96 ppm) silver treatments resulted in significantly greater adherent biomass loss than the corresponding silver-free controls. At these high concentrations, no significant difference in biomass loss was observed between AgNPs and their supernatants. Biomass loss appears to result from dissolved 12693



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Carnegie Mellon University and Malvern Instruments for use of light scattering equipment.

silver, with the greater biomass decrease caused by HS AgNPs resulting from the approximately 2.5 times greater dissolved silver concentrations in those suspensions compared to LS AgNPs (Supporting Information, Figure S8). Environmental Implications. The toxicity of AgNP suspensions toward P. fluorescens biofilms involved combined effects of dissolved silver and silver nanoparticles. Particlespecific toxicity becomes important for colloidally stable particles that may be capable of penetrating the protective biofilm EPS and delivering toxic silver ion directly to adherent cells. Therefore, engineered or incidental modifications to AgNPs that increase colloidal stability may increase their potential hazard to environmental biofilms, even if this leads to less accumulation of the particles in biofilms by sedimentation. This potential hazard would depend on the surface modifying agent. Here, loss of bioavailable free silver as a result of humic acid complexation decreased overall acute biofilm toxicity, despite the colloidal stabilizing effect of humic acid on the AgNPs. This decreased toxicity is consistent with previous planktonic studies.28,31,65,66 Nevertheless, unlike previous planktonic culture studies that suggested that NOM adsorption would mitigate particle-specific toxicity to environmental bacteria, this study indicates that silver ion toxicity toward biofilms is mitigated while nanoparticle-specific toxicity is maintained for colloidally stable silver nanoparticles. NOM adsorption could enhance accumulation of stable nanoparticles within biofilms, as opposed to aggregate sedimentation on the surface, and affect chronic environmental toxicity through continuous ion release within the biofilms.

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ASSOCIATED CONTENT

S Supporting Information *

BacLight details, particle size distributions and absorbance spectra for AgNPs suspended in various media, AgNP dissolution data, Ostwald−Freundlich relation, AgNP toxicity to planktonic bacteria, epifluorescence microscopy details, and overall growth (by OD600) during AgNP exposure. This material is available free of charge via the Internet at http:// pubs.acs.org.

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REFERENCES

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AUTHOR INFORMATION

Corresponding Author

*Phone: (412) 268-1159; e-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This material is based upon work supported by the National Science Foundation (NSF) and the Environmental Protection Agency (EPA) under NSF Cooperative Agreement EF0830093, Center for the Environmental Implications of NanoTechnology (CEINT). Any opinions, findings, conclusions, or recommendations expressed in this material are those of the author(s) and do not necessarily reflect the views of the NSF or the EPA. This work has not been subjected to EPA review and no official endorsement should be inferred. S.M.W. is funded by a National Science Foundation Graduate Research Fellowship. The authors thank Clément Levard in the Department of Geological & Environmental Sciences at Stanford University for synthesizing the HS AgNPs and the PPG Industries Colloids, Polymers and Surfaces Laboratory at 12694



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Natural Organic Matter Alters Biofilm Tolerance to Silver Nanoparticles

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