Clean Fractionation Pretreatment Reduces Enzyme Loadings for Biomass Saccharification and Reveals the Mechanism of Free and Cellulosomal Enzyme Synergy

Enzymatic depolymerization of polysaccharides is often a key step in the production of fuels and chemicals from lignocellulosic biomass. Historically, model cellulose substrates have been utilized to reveal insights into enzymatic saccharification mechanisms. However, translating findings from model substrates to realistic biomass substrates is critical for evaluating enzyme performance. Here, we employ a commercial fungal enzyme cocktail, purified cellulosomes, and combinations of these two enzymatic systems to investigate saccharification mechanisms on corn stover deconstructed either via clean fractionation (CF) or deacetylated dilute sulfuric acid pretreatments. CF is an organosolv pretreatment method utilizing water, MIBK, and either acetone or ethanol with catalytic amounts of sulfuric acid to fractionate biomass components. The insoluble cellulose-enriched fraction (CEF) from CF contains mainly cellulose, with minor amounts of residual hemicellulose and lignin. Enzymatic digestions at both low and high solid loadings demonstrate that CF reduces the amount of enzyme required to depolymerize polysaccharides relative to deacetylated dilute acid-pretreated corn stover. Transmission and scanning electron microscopy of the digested biomass provides evidence for the different mechanisms of enzymatic deconstruction between free and cellulosomal enzyme systems and reveals the basis for the synergistic relationship between the two enzyme paradigms on a process-relevant substrate. These results also demonstrate that the presence of lignin is more detrimental to cellulosome action than to free fungal cellulases. As enzyme costs are a major driver for biorefineries, this study provides key inputs for evaluation of CF as a pretreatment method and synergistic mixed enzyme systems as a saccharification strategy for biomass conversion.