Investigation of the porous structure of cellulosic substrates through confocal laser scanning microscopy.

At the most fundamental level, saccharification occurs when cell wall degrading enzymes (CWDEs) diffuse, bind to and react on readily accessible cellulose fibrils. Thus, the study of the diffusive behavior of solutes into and out of cellulosic substrates is important for understanding how biomass pore size distribution affects enzyme transport, binding, and catalysis. In this study, fluorescently labeled dextrans with molecular weights of 20, 70, and 150 kDa were used as probes to assess their diffusion into the porous structure of filter paper. Fluorescence microscopy with high numerical aperture objectives was used to generate high temporal and spatial resolution datasets of probe concentrations versus time. In addition, two diffusion models, including a simple transient diffusion and a pore grouping diffusion models, were developed. These models and the experimental datasets were used to investigate solute diffusion in macro- and micro-pores. Nonlinear least squares fitting of the datasets to the simple transient model yielded diffusion coefficient estimates that were inadequate for describing the initial fast diffusion and the later slow diffusion rates observed; on the other hand, nonlinear least squares fitting of the datasets to the pore grouping diffusion model yielded estimations of the micro-pore diffusion coefficient that described the inherently porous structure of plant-derived cellulose. In addition, modeling results show that on average 75% of the accessible pore volume is available for fast diffusion without any significant pore hindrance. The method developed can be applied to study the porous structure of plant-derived biomass and help assess the diffusion process for enzymes with known sizes.