Monday, April 30, 2012
Napoleon Ballroom C-D, 3rd fl (Sheraton New Orleans)
The basis for the recalcitrance of crystalline cellulose microfibrils that make up a large portion of lignocellulosic biomass remains unanswered. This study looks into the structure of cellulose microfibrils using atomic force microscopy (AFM), relating the microstructure of cellulose with cellulase accessibility, measured as the extent of hydrolysis and binding by a purified cellobiohydrolase. In our previous work, we examined never-dried cellulose in buffer by AFM. However, the cellulose microfibrils required grafting to 5-(4,6-Dichlorotriazinyl)aminofluorescein (DTAF) in order to immobilize it onto the glass imaging substrate. In the present study, methyltrimethoxysilane is used to create a hydrophobic surface on the glass that successfully adheres unlabeled cellulose via hydrophobic interactions. Our cellulose, harvested from Gluconacetobacter xylinus, is washed thoroughly with separate alkali washing and acid-chlorite bleaching to remove all protein from the culturing step. The cellulose microfibrils are dispersed by ultrasonication, which has been observed to impact microstructure and length of the cellulose microfibrils. We present results demonstrating changes in cellulose microstructure due to sonication, and consequent impact on the kinetics of hydrolysis and binding by fluorescently-labeled Trichoderma reesei Cel7A. Surface topography and chemical properties of immobilized microfibrils studied by tapping mode AFM using both regular (silicon) and chemically-functionalized AFM tips will be presented.