(750d) Bacterial Biofilm Susceptibility to Silver Nanoparticle Dispersions: Impacts of Colloidal Stability, Humic Acid, and Dissolved Silver

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.