The Heterogeneous Diffusion of Polystyrene Nanoparticles and the Effect on the Expression of Quorum-Sensing Genes and EPS Production as a Function of Particle Charge and Biofilm Age.

Biofilms are abundantly present in both natural and engineered environmental systems and will likely influence broader particle fate and transport phenomena. While some developed models describe the interactions between nanoparticles and biofilms, studies are only beginning to uncover the complexity of nanoparticle diffusion patterns. With the knowledge of the nanoparticle potential to influence bacterial processes, more systematic studies are needed to uncover the dynamics of bacteria-nanoparticle interactions. This study explored specific microbial responses to nanoparticles and the heterogeneity of nanoparticle diffusion. Pseudomonas aeruginosa biofilms (cultivated for 48 and 96 hours, representing early and late stages of development) were exposed to charged (aminated and carboxylated) polystyrene nanoparticles. With a combination of advanced fluorescence microscopy and real time quantitative PCR, we characterized the diffusion of polystyrene nanoparticles in P. aeruginosa biofilms and evaluated how biofilms respond to the presence of nanoparticles in terms of the expression of key EPS production-associated genes (pelA and rpsL) and quorum-sensing associated (lasR) genes. Our findings show that nanoparticle diffusion coefficients are independent of the particle surface charge only in mature biofilms and that the presence of nanoparticles influences bacterial gene expression. Independent of the particle's charge polystyrene nanoparticles down-regulated pelA in mature biofilms. By contrast, charge-specific responses were identified in lasR and rpsL gene expression. The targeted genes expression analysis and heterogeneous diffusion models demonstrate that particle charge influences nanoparticle mobility and provides significant insight into the intrinsic structural heterogeneity of P. aeruginosa biofilms. These findings suggest that biofilm maturity and particle charge are essential factors to consider when evaluating the transport of nanoparticles within a biofilm matrix.

Heterogeneous Diffusion of Polystyrene Nanoparticles through an Alginate Matrix: The Role of Cross-linking and Particle Size.

Most bacteria in natural and engineered environments grow and exist in biofilms. Recent investigations have shown that nanoparticles (NPs) interact with environmental biofilms, but these interactions are still not well characterized. Extracellular polymeric substances (EPS) are polymers secreted by bacteria to establish the functional and structural integrity of biofilms, and EPS porosity is a major contributor to NP access to and diffusion in biofilms. We used a synergistic combination of total internal reflection fluorescence microscopy and image correlation spectroscopy to monitor and map diffusion of fluorescent NPs in alginate yielding a detailed picture of the heterogeneous structure and connectivity of pores within a model EPS polymer. Using different sizes (20, 100, and 200 nm) of carboxylated polystyrene NPs, we examined how NP diffusive behaviors change as a result of calcium-induced cross-linking of the alginate matrix. This study reveals that cross-linking decreases NP diffusion coefficients and pore accessibility in an NP size-dependent manner and that NP movement through alginate matrices is anisotropic and heterogeneous. These results on heterogeneous and size-dependent movement within biofilms have important implications for future studies and simulations of NP-biofilm interactions.