Technical Abstract: Ethanol production from grass is desirable due to the large amount of biomass it produces. However, a pretreatment is necessary before fermentation to increase ethanol yield. Tifton 85 bermudagrass was treated with a newly designed pressurized batch hot water reactor. Multiple temperatures, pressures, and reaction times were evaluated, and reducing sugars liberated during enzymatic hydrolysis were determined. Pressure had a negligible effect on digestibility of the grass, and a reaction temperature of 230°C for two minutes was the most effective in releasing reducing sugars. Fermentations were conducted with untreated grass and with grass treated for two minutes at 200°C or 230°C to confirm that the increase in reducing sugar concentration resulted in an increased ethanol yield. Following hydrolysis with 2 filter paper units (FPU) of a mixed cellulase enzyme cocktail per gram dry weight of grass, fermentations were performed with engineered Escherichia coli strain LY01. Grass treated at 230°C produced 14.7 g/L of ethanol, which was significantly higher than 200°C treated grass (11.0 g/L) and untreated grass (9.0 g/L). Ferulic and para-coumaric acids were also released during the fermentations. Based on these results, pressurized batch hot water reactor pretreatment is effective in increasing ethanol yield of grass in fermentation.