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Background  Enzymatic hydrolysis of lignocellulosic biomass (mainly plant cell walls) is a critical process for biofuel production. This process is greatly hindered by the natural complexity of plant cell walls and limited accessibility of surface cellulose by enzymes. Little is known about the plant cell wall structural and molecular level component changes after pretreatments, especially on the outer surface. Therefore, a more profound understanding of surface cellulose distributions before and after pretreatments at single-molecule level is in great need. In this study, we determined the structural changes, specifically on crystalline cellulose, of natural, dilute sulfuric acid pretreated and delignified cell wall surfaces of poplar, switchgrass, and corn stover using single molecular atomic force microscopy (AFM) recognition imaging.    Results  The AFM tip was first functionalized by a family 3 carbohydrate-binding module (CBM3a) ( Clostridium thermocellum  Scaffoldin) which specifically recognizes crystalline cellulose by selectively binding to it. The surface structural changes were studied at single molecule level based on the recognition area percentage (RAP) of exposed crystalline cellulose over the imaged cell wall surface. Our results show that the cell wall surface crystalline cellulose coverage increased from 17-20% to 18-40% after dilute acid pretreatment at 135°C under different acid concentrations and reached to 40-70% after delignification. Pretreated with 0.5% sulfuric acid, the crystalline cellulose surface distributions of 23% on poplar, 28% on switchgrass and, 38% on corn stover were determined as an optimized result. Corn stover cell walls also show less recalcitrance due to more effective pretreatments and delignification compared to poplar and switchgrass.    Conclusions  The dilute acid pretreatment can effectively increase the cellulose accessibility on plant cell wall surfaces. The optimal acid concentration was determined to be 0.5% acid at 135°C, especially for corn stover. This study provides a better understanding of surface structural changes after pretreatment such as lignin relocation, re-precipitation, and crystalline cellulose distribution, and can lead to potential improvements of biomass pretreatment.

Mapping out the structural changes of natural and pretreated plant cell wall surfaces by atomic force microscopy single molecular recognition imaging