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The role of particle size in carbohydrate fractionation upon pretreatment and glucan yields upon enzymatic hydrolysis was investigated at two different temperatures, to examine the possibility of pretreating under milder conditions smaller particles, in order to satisfy pilot-scale operational constraints. Maize stover was knife-milled through 1-mm and 0.5-mm screens and pretreated by soaking in aqueous ammonia pretreatment at 60 or 110°C for 6 h. Pretreated solids were analyzed for composition and a material balance calculated for glucan, xylan, and lignin. At 60°C, milling resulted in greater delignification compared to unmilled biomass. Delignification was more uniform at 110°C. Pretreated solids were washed and cellulase hydrolysis carried out at 10% w/w solids loading, with low and high enzyme loadings. Liquid samples were drawn and concentration data developed through HPLC to calculate 48-h glucan and xylan hydrolytic yields. The differences in hydrolytic yield between milled and unmilled treatments were found to vary with pretreatment temperature and enzyme loading. The results show that while particle size impacts carbohydrate recovery and hydrolytic yield, it is less important in bioprocessing than pretreatment temperature and enzyme loading, possibly owing to the particles' morphology rather than the size.