Assessing the Effect of Pretreatment Strategies on Biomass Deconstruction and Cellulose Accessibility for Clostridium thermocellum Compared to Free Enzymes

The reduced cost of consolidated bioprocessing (CBP) in converting lignocellulosic biomass to biofuels depends on the ability of a single organism to (1) produce ample enzymes with the ability to (2) deconstruct polysaccharides to metabolizable sugars which the same organism can then (3) ferment into a desired fuel with high yields. Clostridium thermocellum is a promising CBP organism based on its ability to hydrolyze cellulose and hemicellulose. However, to realize the economic potential of CBP, pretreatment will be needed to realize high sugar release yields. Although previous work has shown that the normalized sugar release rate of pure cellulose is consistent with that of pretreated substrates when hydrolyzed by cellulase cocktails, C. thermocellum digests pure cellulose at a faster rate than pretreated substrates. In addition, sugar release by C. thermocellum increases with pretreatment severity beyond that which is optimal for free enzyme systems. The difference in how free enzymes and C. thermocellum culture act on model and real substrates and their altered response to pretreatment severity has been hypothesized to result from the need for greater cellulose accessibility in non-ideal substrates for penetration by C. thermocellum's large enzyme complex, the cellulosome. In this study, dilute acid, hydrothermal, and enhanced lignin removal pretreatments with differing relative degrees of lignin and hemicellulose removal are coupled with downstream CBP to determine how the presence of lignin and hemicellulose impact deconstruction of Populus trichocarpa. In addition, cellulose accessibility is measured to determine its relationship to biomass conversion by C. thermocellum compared to free enzymes.