Understanding biomass pore structural changes during enzymatic hydrolysis is critical for elucidating the intrinsic hydrolysis reaction mechanisms and interactions between cell wall degrading enzymes (CWDEs) and biomass. Mixed hardwoods (MHW) and switchgrass (SG) were pretreated using biphasic carbon dioxide (CO2) and water (H2O) at 200 bar and 210°C. Resulting samples were hydrolyzed by CWDE mixtures, and the reducing sugar yield was measured by high performance liquid chromatography at different hydrolysis time points. Hydrolyzed biomass was treated with protease to remove bound CWDEs, and the treated biomass accessible pore volume was measured using a size-exclusion chromatography system. The influence of biomass drying was evaluated by measuring the pore size distribution of dried biomass samples by rehydrating and repacking them into the chromatography system. Results showed a 30% initial decline of specific pore volume for both pretreated MHW and SG after the first 2 h of hydrolysis, which correlated well with hydrolysis rates that decreased by 26.3% and 36.3% for pretreated MHW and SG, respectively, 2 h after the reaction started. Temporal pore volume measurements obtained during enzymatic hydrolysis clearly showed that CWDEs had reduced accessibility to the substrates over the course of the reaction. Also, particle size analyses of the substrates after reaction with CWDEs revealed a shift in the particle size distribution toward smaller particles. This is another way that CWDEs modify the biomass structure. Finally, results from drying experiments showed that drying could universally decrease the accessible porous volume of pretreated and hydrolyzed biomass by up to 80%, which suggested that drying could cause the irreversible collapse of pores.
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