(599m) Quartz Crystal Microbalance Investigation of Cellulose Hydrolysis By Clostridium Thermocellum on Model Cellulose Films

The high cost of enzymatic hydrolysis of polymeric cellulose to soluble sugars is a key bottleneck in the production of lignocellulosic-based biofuels and biochemicals at an industrial scale. Approaches to optimize the hydrolysis conditions through an understanding of the complex interactions between cellulase enzymes/enzyme mixtures, lignocellulosic feedstock, feedstock pretreatment, and reaction conditions are required to make the depolymerization more efficient. Quartz crystal microbalance with dissipation (QCM-D), which allows for monitoring in situ and in real time the binding and catalytic activity of cellulases on model cellulose substrates, has been successfully used to study the hydrolysis of cellulose films using commercial fungal cellulase mixtures. An alternative to mixtures of free cellulase enzymes is the use of the cellulase system of whole cells, such as complexed cellulases of C. thermocellum, also known as cellulosome.  In addition to having a highly active cellulase system capable of degrading cellulose into soluble sugars, C. thermocellum further utilizes the sugars to produce ethanol.  This makes it a potential microorganism for consolidated processing (CBP).

In this work, the interaction and hydrolytic activity of C.thermocellum on amorphous cellulose surfaces is measured using QCM. The ability to extend the use of QCM to whole cell-based cellulases is supported by AFM and confocal microscopy of the thin films before and after processing.  To clearly differentiate the activity free cellulosome and cell-bound cellulosome, the distribution of free cellulosome and cell-bound cellulosome in crude cell broth at different growth stages of C.thermocellum was quantified. For this strain, approximately 70% of the cellulosome in the crude cell broth is shown to exist unattached to the cell. The hydrolysis activity of free cellulosome and crude cell broth are measured by QCM on uniform amorphous (LiCl/DMAc dissolved) cellulose films at 50ºC.  Cellobiose, a known inhibitor of cellulases, has the same inhibition pattern on crude cell broth and free cellulosome over the concentration range of 0-10g/L, as monitored by QCM. Further analysis of cellobiose inhibition of the cellulase activity of C. thermocellum is supported by interfacial models of cellulase activity and comparison to traditional cellulase assays.