(447c) Novel Inverse Isotope Effect for Cellulase Hydrolysis of Deuterated Switchgrass

Authors: 
Bhagia, S., University of Tennessee, Knoxville
Meng, X., University of Tennessee-Knoxville
Dunlap, J., University of Tennessee
Evans, B. R., Center for Structural Molecular Biology, Oak Ridge National Laboratory
Bali, G., BioEnergy Science Center, Oak Ridge National Laboratory
Chen, J., Oak Ridge National Laboratory
Reeves, K. S., Oak Ridge National Laboratory
Davison, B. H., BioEnergy Science Center and Oak Ridge National Laboratory
Ragauskas, A. J., University of Tennessee
Neutron scattering enables the study of structural and dynamic properties of lignocellulosic biomass at multiple length scales in a non-destructive manner. High levels of deuterium substitution are desired to enable contrast variation techniques which reduce background. However, changes in physical and chemical properties during growth and adaptation to D2O solutions need to be identified in model plants used for neutron scattering. In this study, enzymatic hydrolysis of bacterial cellulose, and cellulose and holocellulose isolated from switchgrass showed the expected kinetic isotope effect, with deuteration lowering glucose yields by 17, 18 and 4% of theoretical yield, respectively. However, the opposite trend was found for deuterated switchgrass, in which glucose yield was 5% higher than that obtained with protiated switchgrass at lower enzyme loading. These data indicated that alterations to lignin might be responsible for this novel inverse isotope effect. Lignin content of deuterated switchgrass was about 2% higher than that of protiated switchgrass, while NMR spectroscopy indicated no significant structural differences in lignin, and Simons’ staining found comparable cellulose surface area. However, extensive CFM and TEM imaging showed that deuterated switchgrass had abnormal lignin distribution in some of its cell walls and many of them were collapsed possibly due to reduced rigidity, which would render them easier to deconstruct by cellulases. The changes in morphology resembled those of drought stressed plants, consistent with abiotic stress due to growth in deuterated media. These protio/deutero investigations clearly illustrate the key role of component distribution in the multilamellar cell wall architecture on biomass recalcitrance.