277827 Bacterial Biofilm Susceptibility to Silver Nanoparticle Dispersions: Impacts of Colloidal Stability, Humic Acid, and Dissolved Silver

Thursday, November 1, 2012: 4:20 PM
412 (Convention Center )
Stacy M. Wirth, Department of Chemical Engineering, Carnegie Mellon University, Pittsburgh, PA, Gregory V. Lowry, Department of Civil and Environmental Engineering, Carnegie Mellon University, Pittsburgh, PA and Robert D. Tilton, Department of Chemical Engineering and Department of Biomedical Engineering, Carnegie Mellon University, Pittsburgh, PA

This work is motivated by the need to understand environmental risks posed by potentially biocidal engineered nanoparticles. Silver nanoparticles (AgNPs) are of particular concern since they are increasingly used in commercial and consumer products and their release to waste streams and the environment is inevitable.  The broad spectrum biocidal properties that motivate AgNP use in consumer and medical applications could have detrimental consequences for environmental microorganisms that are crucial to normal ecosystem function.  In order to understand the environmental risks of engineered AgNPs, an understanding of how they interact with environmental bacteria, which typically exist as surface attached biofilms rather than the more frequently studied planktonic, or suspended, cultures, is necessary.  This study therefore addressed AgNP toxicity towards single species Pseudomonas fluorescens biofilms using dye staining methods.  Both silver nanoparticles and dissolved silver negatively impacted biofilm viability in a dose-dependent manner.  However, no silver treatments (up to the very high concentration of 100 ppm AgNPs) resulted in 100% biofilm viability loss, even though these same concentrations caused complete viability loss in planktonic cultures, suggesting some biofilm tolerance to AgNP toxicity. Exposure of AgNPs to environmental conditions will result in various processes, such as aggregation, dissolution and adsorption of naturally occurring macromolecules, which could impact nanoparticle toxicity towards biofilms.  In this study, addition of the macromolecule humic acid (HA), which comprises a major fraction of the natural organic matter that is ubiquitous in most environments, to AgNP dispersions was found to decrease the dispersion toxicity.  The decreased toxicity was found not to result from HA adsorption to particle surfaces but rather from HA complexation of dissolved silver introduced as part of the AgNP dispersions.  Therefore, dissolved silver plays at least a partial role in the overall AgNP dispersion toxicity towards biofilms. The effects of aggregation were assessed by comparing the toxicities of two types of AgNPs with different colloidal stabilities. The more stable dispersions exhibited a distinct nanoparticle-specific toxicity that was not observed for less stable, highly aggregated particles.  This lack of particle-specific toxicity from highly aggregated particles suggests that the biofilm is somehow protected from nanoparticle aggregate toxicity.  One potential explanation is that the biofilm extracellular polymeric substance presented a barrier to aggregate transport into the biofilm while the small, stable AgNPs were able to diffuse through the biofilm matrix and gain closer access to cells. Changes in the amount of biofilm during silver exposure were measured using the crystal violet assay.  High concentration silver treatments resulted in loss of adherent biomass, though a significant amount of biofilm remained in all cases.  However, low concentration silver treatments actually stimulated biofilm growth, with greater amounts of adherent biomass resulting from these low concentration silver treatments than from silver-free controls. The enhanced growth was specific to adherent cells. Overall, this study indicates that both silver nanoparticles themselves and dissolved silver originating from the nanoparticles contribute to overall AgNP dispersion toxicity towards bacterial biofilms.  Environmental interactions which impact the colloidal stability of AgNPs or the bioavailability of dissolved silver will greatly impact toxicity towards biofilms.


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