Revisiting size-exclusion chromatography for measuring structural changes in raw and pretreated mixed hardwoods and switchgrass.

The study of the biomass porous structure and its role in defining the accessibility of cell-wall-degrading enzymes (CWDEs) to the substrate is very important for understanding the cellulase-cellulose reaction system. Specific pore volume and specific surface area are two important measures of accessibility and a variety of methods have been used to make these measurements. For this study a size exclusion chromatography system was developed to measure specific pore volume and specific surface areas for raw and pretreated mixed-hardwood and switchgrass. Polyethylene glycol (PEG) probes of known molecular diameter (1.8-13 nm) were allowed to diffuse into the pore structure of the various biomass substrate packed in the column and subsequently eluted to generate high resolution concentration measurements with excellent reproducibility. Replicate measurements of probe concentrations from this system consistently yielded coefficient of variance of less than 1.5%. Our results showed that particle size reduction had a smaller influence on the specific pore volume distribution of raw mixed-hardwoods, whereas for switchgrass the larger particles yielded a significantly lower estimate for the pore volume distribution compared to the smaller particles. Our results also clearly showed that our bi-phasic pretreatment yielded the largest increase in pore volume accessibility for mixed-hardwoods relative to switchgrass. From these results a pore size change mechanism was proposed that could explain the influence of size reduction and pretreatment on pore volume measurements.