Insight to factors limiting enzymatic hydrolysis of cellulose-rich substrates

The slow release of monomeric sugars during enzymatic hydrolysis is the major obstacle to large-scale implementation of biorefineries due to high cost of enzymes. Enzymatic hydrolysis of lignocellulosic biomass is often incomplete and, therefore, it is of great importance to understand the limitations of the process. Among the limitations of enzymatic hydrolysis, structural properties of lignocellulose have an effect of enzymatic hydrolysis efficiency. In the current work, we used a combination of approaches to visualize and quantify the spatial polymer distribution in cellulosic substrates (derived from spruce pulp, cotton and Avicel, including preparation of nanocrystalline cellulose) and monitoring of interactions between cellulose degrading enzymes and the substrate.

The structural properties of the substrates during an incrementing time of hydrolysis were analyzed by solid-state cross-polarization magic angle spinning carbon-13 nuclear magnetic resonance (CP/MAS ¹³C-NMR), Coherent Anti-Stokes Raman Scattering (CARS) and Second Harmonic Generation (SHG) microscopy. We have analysed the characteristics of the complex supramolecular structure of cellulose on the nanometre scale in terms of the spatial distribution of fibrils and fibril aggregates, the accessible surface area and the crystallinity during enzymatic hydrolysis. Influence of the porosity of the substrates and the hydrolysability was also investigated. A strong correlation was found between the average pore size of the starting material and the enzymatic conversion yield. The degree of crystallinity was maintained during enzymatic hydrolysis of the cellulosic substrates.