Breakdown of Cell Wall Nanostructure in Dilute Acid Pretreated Biomass Sai Venkatesh Pingali,* ,† Volker S. Urban,* ,† William T. Heller, † Joseph McGaughey, ‡ Hugh O’Neill, † Marcus Foston, § Dean A. Myles, † Arthur Ragauskas, § and Barbara R. Evans* ,‡ Center for Structural Molecular Biology and Molecular Bioscience and Biotechnology Group, Chemical Sciences Division, Oak Ridge National Laboratory, Oak Ridge, Tennessee 37831, and Institute of Paper Science and Technology, School of Chemistry and Biochemistry, Georgia Institute of Technology, Atlanta, Georgia 30332 Received April 26, 2010; Revised Manuscript Received July 8, 2010 The generation of bioethanol from lignocellulosic biomass holds great promise for renewable and clean energy production. A better understanding of the complex mechanisms of lignocellulose breakdown during various pretreatment methods is needed to realize this potential in a cost and energy efficient way. Here we use small- angle neutron scattering (SANS) to characterize morphological changes in switchgrass lignocellulose across molecular to submicrometer length scales resulting from the industrially relevant dilute acid pretreatment method. Our results demonstrate that dilute acid pretreatment increases the cross-sectional radius of the crystalline cellulose fibril. This change is accompanied by removal of hemicellulose and the formation of R g ∼ 135 Å lignin aggregates. The structural signature of smooth cell wall surfaces is observed at length scales larger than 1000 Å, and it remains remarkably invariable during pretreatment. This study elucidates the interplay of the different biomolecular components in the breakdown process of switchgrass by dilute acid pretreatment. The results are important for the development of efficient strategies of biomass to biofuel conversion. Introduction Lignocellulosic biomass produced by terrestrial plants has the potential to be an abundant, renewable feedstock for the production of ethanol and other transportation fuels. 1,2 Of the many types of plants that have been examined as potential feedstocks for production of ethanol and other fuels, herbaceous crops, particularly grasses, offer a number of advantages. These include fast growth, established agricultural cultivation, and potential for dual-purpose production, providing both grain for food and straw (stalks) for biofuel conversion. Switchgrass (Panicum Virgatum), a native North American prairie grass, is being developed as the main herbaceous crop for biofuel production. Switchgrass offers several advantages, including high yields, perennial growth, production of seeds, and adapt- ability to poor soils. 3,4 All lignocellulosic biomass is largely composed of three component biopolymers: cellulose, a linear polymer of -1,4- linked glucose chains assembled into partially crystalline fibers; hemicellulose, a heterogeneous branched polymer of pentose and hexose sugars; and lignin, which is composed of extensively cross-linked methoxy-substituted phenyl propane units. Cel- lulose, which forms the main structural component of the plant cell walls, is an attractive source of glucose for fermentative ethanol production, but must be first depolymerized by enzy- matic or chemical hydrolysis. In lignocellulosic biomass, enzymatic access to the cellulose fibers is impeded by hemi- cellulose and lignin layers. Hydrolysis is further impeded by the crystalline, fibrous structure of cellulose. 5 As a result, efficient production of fermentable sugars from lignocellulosic biomass requires deconstruction of the plant cell walls by mechanical and chemical pretreatment. Typically, biomass pretreatment includes size reduction by chipping and grinding, followed by chemical swelling with alkali or acid, 1,5 schematically illustrated as pathway a of Figure 1. 6,7 The most effective pretreatments increase the gross material * To whom correspondence should be addressed. E-mail: pingalis@ ornl.gov; urbanvs@ornl.gov; evansb@ornl.gov. † Center for Structural Molecular Biology, Oak Ridge National Laboratory. ‡ Molecular Bioscience and Biotechnology Group, Oak Ridge National Laboratory. § Georgia Institute of Technology. Figure 1. Schematic of (a) dilute acid pretreatment and (b) component- extraction processes. Biomacromolecules XXXX, xxx, 000 A 10.1021/bm100455h  XXXX American Chemical Society