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Enzymatic hydrolysis of cellulose provides a renewable source of monosaccharides for production of variety of biochemicals and biopolymers. Unfortunately, the enzymatic hydrolysis of cellulose is often incomplete, and the reasons are not fully understood. We have monitored enzymatic hydrolysis in terms of molecular density, ordering and autofluorescence of cellulose structures in real time using simultaneous CARS, SHG and MPEF microscopy with the aim of contributing to the understanding and optimization of the enzymatic hydrolysis of cellulose. Three cellulose-rich substrates with different supramolecular structures, pulp fibre, acid-treated pulp fibre and Avicel, were studied at microscopic level. The microscopy studies revealed that before enzymatic hydrolysis Avicel had the greatest carbon-hydrogen density, while pulp fibre and acid-treated fibre had similar density. Monitoring of the substrates during enzymatic hydrolysis revealed the double exponential SHG decay for pulp fibre and acid-treated fibre indicating two phases of the process. Acid-treated fibre was hydrolysed most rapidly and the hydrolysis of pulp fibre was spatially non-uniform leading to fractioning of the particles, while the hydrolysis of Avicel was more than an order of magnitude slower than that of both fibres.

Visualization of structural changes in cellulosic substrates during enzymatic hydrolysis using multimodal nonlinear microscopy