Swelling by Hydrochloric Acid Partially Retains Cellulose-I Type Allomorphic Ultrastructure but Enhances Susceptibility toward Cellulase Hydrolysis Such as Highly Amorphous Cellulose

Abstract

Enzymatic conversion of cellulosic biomass into fermentable sugars such as glucose is a slow and catalytically inefficient process, largely due to limited accessibility of cellulose. Cellulose crystallinity can be reduced to increase substrate accessibility toward cellulolytic enzymes by either swelling or dissolving biomass in chemicals such as concentrated acids (e.g., phosphoric acid or H3PO4, sulfuric acid or H2SO4) or ionic liquids. Phosphoric acid swollen cellulose (PASC) is one such highly digestible form of regenerated amorphous cellulose (RAC), enriched along with some cellulose-II allomorph that can be readily produced in the lab and is, therefore, the most widely reported form of amorphous cellulose in the literature. However, concentrated hydrochloric acid (HCl) can also be used to produce RAC completely free of inorganic esters, which is structurally more representative of native cellulosic substrates and, therefore, is a useful alternative model substrate for cellulolytic enzyme assays. Here, unlike previous reports, we found that concentrated HCl swells cellulose into a gel-like state at temperatures close to freezing (4 °C), while only partially hydrolyzing and dissolving a small fraction of cellulose as large cellodextrins. Raman spectroscopy analysis suggests that cellulose-I transitions from its native allomorphic state to an intermediate swollen, gel-like state that retains some "memory" of the original starting structure, which facilitates its transition back into a cellulose-I-like state upon full solvent removal and lyophilization. Surprisingly, the cellulose regenerated from this HCl-treated cellulose gel-like state resulted in an amorphous PASC-like substrate before drying with comparable enzymatic digestibility.