|ZHOU, SHANSHAN - University Of Kentucky|
|LI, HSIN-FEN - University Of Kentucky|
|GARLAPALLI, RAVINDER - University Of Kentucky|
|NOKES, SUE - University Of Kentucky|
|RANKIN, STEPHEN - University Of Kentucky|
|KNUTSON, BARBARA - University Of Kentucky|
Submitted to: Journal of Biotechnology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 11/8/2016
Publication Date: 1/10/2017
Citation: Zhou, S., Li, H., Garlapalli, R., Nokes, S.E., Flythe, M.D., Rankin, S.E., Knutson, B.L. 2017. Hydrolysis of model cellulose films by cellulosomes: Extension of quartz crystal microbalance techniques to multienzymatic complexes. Journal of Biotechnology. 241:42-49.
Interpretive Summary: Cellulose is a plant fiber, and one of the most abundant materials in the world. It is a major constituent renewable feedstocks collectively called "biomass". Biomass is inexpensive and renewable; thus, there is continued interest in using biomass as animal feed and converting it to high value products because (like biofuels and industrial chemicals). Biomass must be broken down to convert it to other products. Bacteria, like Clostridium thermocellum, and their enzymes can be used to break down cellulose and other fibers in biomass. The interaction of bacteria and their enzymes with fiber can be difficult to examine, but this study shows that a tool called a quartz crystal microbalance (QCM) can be used. A very thin film of cellulose was placed in the QCM and C. thermocellum or its enzymes were flowed over the film. The degree to which the bacterial cells and enzymes adhered to the cellulose film could be measured, as well as the decrease in mass as the enzymes broke down the cellulose. The QCM could also show when the enzymes were chemically inhibited. The impact of this research is to identify QCM as a powerful tool to study biomass degradation.
Technical Abstract: Clostridium thermocellum, a well-studied cellulolytic bacterium, produces highly active cellulases in the form of cellulosomes. The ability of the cellulose binding module within the cellulosome to adhere C. thermocellum cells to the cellulosic substrate is considered to contribute to its high cellulose degradation activity. Although the synergy of having cell-attached cellulosomes is widely accepted, the relative importance of cell-bound and cell-free cellulosomes on observed cellulose hydrolysis rates is unclear. In this study, a surface measurement technique, quartz crystal microbalance with dissipation (QCM-D), was used to examine the interactions between C. thermocellum and a model cellulose surface. To clearly differentiate the activity of cell-free cellulosome and cell-bound cellulosome, the distribution of cell-free cellulosome and cell-bound cellulosome in crude cell broth at different growth stages of C. thermocellum was quantified. For the C. thermocellum strain examined in this study (ATCC 27405) using cellobiose as carbon source in liquid fermentations, greater than 68% of the cellulosome in the crude cell broth existed unattached to the cell across multiple growthstages. The effect of substrate inhibition (0, 1, 3, 5, and 10 g/L cellobiose) on the hydrolysis rate of cellulose was measured using QCM and bulk hydrolysis measurements (Remazolbrilliant blue R dyed ß-glucan assay) for crude cell broth and cell-free cellulosome solutions. The initial hydrolysis rates of crude cell broth measured by QCM on uniform amorphous cellulose thin films were greater than that of cell-free cellulosome, but adsorbed “mass” of crude cell broth on the film was significantly greater than cell-free cellulosomes, potentially due to the differences in the corresponding masses of cell-bound and cell-free cellulosomes adhered to the substrate. Similar trends in hydrolysis inhibition by cellobiose for crude cell broth and cell-free cellulosomes suggest that models developed for the cell-free cellulosomes, which allow for more accurate interfacial adsorption analysis by QCM than their cell-attached counterparts, may provide insight into hydrolysis events in both systems.