Spatiotemporal dynamics of cellulose during enzymatic hydrolysis studied by infrared spectromicroscopy

Cellulose, a sustainable source of fermentable glucose and nanomaterials, exists as highly-ordered, solvent-inaccessible fibrils held by intra- and intermolecular hydrogen bonds. Cellulose hydrolysis rates and extents are limited by the availability and accessibility of productive cellulase binding sites at the water-cellulose interface. There is a need to understand how spatial heterogeneity of celluloses impacts hydrolysis kinetics. We report a real-time, in situ infrared spectromicroscopy study of enzymatic cellulose hydrolysis in buffer with micrometer-scale spatial mapping. Algal cellulose depletion by a purified cellobiohydrolase Cel7A was tracked as time-resolved decreases in the absorption peak intensities of the glycosidic bond (1161 cm-1), C2-O2 (1112 cm-1), C3-O3 (1059 cm-1), and C6-O6 rotamers (1034 cm-1, 1013 cm-1, and 997 cm-1). Depletion kinetics varied spatially, with peak intensities decreasing to zero in some areas and plateauing in others. Hydration impacted cellulose ordering where C6-O6 and C3-O3 peaks were sharp, narrow, and centered at higher frequencies when hydrated, but broader, less well-defined and centered at lower frequencies when dried. Temporal trends of the Lateral Order Index (LOI), Total Crystallinity Index (TCI), and Hydrogen Bonding Index (HBI) indicated that hydrolysis by Cel7A preferentially removed less extensively hydrogen bonded cellulose, without significantly affecting the overall crystallinity of highly ordered cellulose. Spatial and temporal resolved mapping of infrared spectra of cellulose during enzymatic hydrolysis towards developing sustainable bioconversion processes to materials, fuels, and chemicals.