Quantifying diffusion in biofilms : from model hydrogels to living biofilms

Biofilms are primarily communities of microorganisms embedded in a complex exopolymer matrix. They are thought to play an important role as diffusive barriers in environmental systems and human health, resulting in increased resistance to disinfectants and antibiotics. Since mass transport in a biofilm is primarily due to molecular diffusion, it is critical to understand the main parameters influencing diffusive fluxes in a biofilm. In this thesis, a Pseudomonas fluorescens biofilm and two model hydrogels, (agarose and calcium alginate), were investigated. Both self-diffusion (Brownian motion) and mutual diffusion coefficients were quantified. Fluorescence correlation spectroscopy was used to measure the self-diffusion coefficients in a ca. 1 m3 confocal volume in the gels or biofilms, whereas a diffusion cell setup was employed for mutual diffusion measurements. In addition, microelectrode voltammetry was used to evaluate Donnan potential of the gels in order to determine its impact on diffusion.  For the agarose hydrogel, the combined observations of a decreasing self-diffusion coefficient coupled with increasing mutual diffusion as a function of a decreasing ionic strength have been attributed to the gel’s Donnan potential. Measurements of the Donnan effect (difference of -30 mV between ionic strengths of  10-4 and 10-1 M) and the corresponding accumulation of ions in the hydrogel (13x enhancement with respect to the bulk solution) indicated that electrostatic interactions can strongly influence the diffusive flux of cations, even in a weakly charged hydrogel, such as agarose. Somewhat surprisingly, for a more highly charged gel such as calcium alginate, varying ionic strength and pH resulted in only small changes to the diffusion of charged probes in the hydrogel. These results suggested that the direct effect of the cations on gel structure (due to an induced swelling or compression) was much more effective than the Donnan effect when influencing solute diffusion. Similarly, for a bacterial biofilm, self-diffusion coefficients were virtually constant across a range of examined ionic strengths (10-4-10-1 M) for both negatively and positively charged small solutes (Db/Dw≈85%) and nanoparticles (Db/Dw≈50%), suggesting that the obstruction effect of the biofilms again overwhelmed the charge effect.  The results of this work indicated that among the various major factors affecting diffusion in an oligotrophic environmental biofilm (steric exclusion, hydrophobic and electrostatic interactions), obstruction effects appeared to be the most important when attempting to understand the solute diffusion. While charge effects did not appear to be important to the self-diffusion of charged substrates in the alginate hydrogel or bacterial biofilm, they were key to understanding diffusion through another gel, with numerous biomedical and environmental applications, i.e. agarose. These results should be extremely useful when evaluating the bioavailability of the trace contaminants and nanoparticles in the environment.

Keyphrases: Agarose, Biofilm, Calcium alginate, Donnan Potential, Diffusion cell, Fluorescence correlation spectroscopy, Microelectrode voltammetry, Mutual diffusion, Self-diffusion