Dilute acid pretreatment (DAP) is commonly employed prior to enzymatic deconstruction of cellulose to increase overall sugar and subsequent ethanol yields from downstream bioconversion processes. Typically optimization of pretreatment is evaluated by determining hemicellulose removal, subsequent reactivity towards enzymatic deconstruction, and recoverable polysaccharide yields. In this study, the affect of DAP on the supramolecular and ultrastructure of lignocellulosic biomass was evaluated. A series of dilute acidic pretreatments, employing similar to 0.10-0.20 mol/m(3) H2SO4 at similar to 160-180 degrees C, for varying residence times were conducted on both Populus and switchgrass samples. The untreated and pretreated biomass samples were characterized by carbohydrate and lignin analysis, gel permeation chromatography (GPC) and C-13 cross polarization magic angle spinning (CPMAS) NMR spectroscopy. GPC analysis shows a reduction in the molecular weight of cellulose and change in its polydispersity index (PDI) with increasing residence time, indicating that pretreatment is actually degrading the cellulose chains. C-13 CPMAS and non-linear line-fitting of the C-4 region in the carbon spectrum of the isolated cellulose not only showed that the crystallinity index increases with residence time, but that the lateral fibril dimension (LFD) and lateral fibril aggregate dimension (LEAD) increase as well. (C) 2010 Elsevier Ltd. All rights reserved.