Bacterial biofilms are heterogeneous reaction systems wherein limiting substrates, essential nutrients, cellular antagonists, and cellular by-products are exchanged between the surrounding fluid and the site of the reaction (i.e., the cell). In numerous circumstances, the performance of biofilm processes can be limited by both internal and external mass transfer rates (e.g., antibiotic treatment of a biofilm infection, biocide challenge to eliminate biofilm formation in heat exchange systems, biofilm reactor treatment of wastewaters or waste gases, and in situ hazardous waste decontamination). Recent advances in characterizing biofilm architecture have led to the need to reformulate concepts of the mass transport mechanism in biofilms.
For about 25 years, estimates of substrate consumption for biofilms of known thickness have been based on the steady-state solution of equation 14,
where C is the solute concentration, Rc is all reactions transforming solute C, t is the stoichiometric coefficient for the transformation, x is the coordinate of molecular transport, and 3eff is the effective diffusivity of solute in the biofilm. These various models are based on the assumption that transport of reactants (i.e., substrates) into the biofilm and export of products is by molecular diffusion alone and that bulk transport (convection) does not occur within the biofilm. These models also do not account for (1) any heterogeneities in the biofilm gel structure, (2) any locally dependent bacterial concentrations, or (3) any nonlinearity in diffusion coefficients.
Traditionally, either 3eff is estimated as the solute diffusivity in pure water, reduced by an arbitrary fraction, or 3eff is determined for the particular biofilm system in question from diffusion experiments. For small ions and uncharged small organic compounds, both Libicki et al. (78) and Siegrist and Gujer (79) indicate that transport in thin microbial layers is indeed by molecular diffusion. Both reports indicate that the decrease in observed diffusivity values relative to those in pure water is a function of the mi-crobial aggregate cell-to-polymer composition. Siegrist and Gujer (79) provided the first report to insinuate that molecular diffusion may not be the only mass transport process acting within a biofilm and that convective mass transport may predominate in the upper layers of thick, filamentous biofilms.
Results of Drummond (80) and Bryers and Drummond (81) suggest these dense alginate clusters are themselves porous, with a wide distribution of pore sizes ranging from angstroms to tenths of micrometers. Their results have shown the transport of larger solutes (i.e., macromolecules) through the hydrated biofilm polysaccharide gel matrix is controlled by transport mechanisms far more complex than molecular diffusion. Overall effective diffusion coefficients for various molecules, increasing in molecular weight and differing in molecular size and stereochemistry, were determined using half-cell diffusion chambers. Transient diffusion studies were carried out at constant temperature and pH using bacterial biofilms of increasing thickness. Half-cell diffusion results were considered erroneous due to the "perforated" nature of the biofilms.
The existence of water channels in these biofilms was documented and found to significantly affected mass transfer coefficient estimates obtained from half-cell studies. Consequently, a local microtransport technique was developed based on the fluorescence recovery after photobleach-ing (FRAP) technique of Poitevin and Wahl (82). FRAP is a local, instantaneous, and noninvasive method that can be repeatedly carried out over a wide expanse of biofilm. Corrections of the FRAP "tracer" diffusion coefficients must be made in order to estimate "Fickian" diffusivities. Diffusion coefficients determined for large macromolecule solutes (dextrans, proteins, DNA fragments) moving inbio-films did not correlate simply with increasing molecular weight except within the homologous chemical groups. Transport of macromolecules through the biofilm was affected not only by molecular weight but by the size or length of the molecule in solution. Using a computer-
actuated laser and motorized microscope stage, FRAP analysis across a large section of biofilm illustrates that local mass transfer coefficients can vary significantly in a biofilm over very short distances (Fig. 7).
Thus, research reported by Drummond (80) and Bryers and Drummond (81) and independently corroborated by Stoodley et al. (84) has shown biofilms are not architecturally spatially continuous. Rather, biofilms are structurally nonuniform, consisting of dense microbial clusters surrounded by tortuous water channels that perforate the biofilm matrix. Thus, theory and experiments addressing overall mass transport within a real biofilm system must account for both convective transport and molecular diffusion.
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