AbstractEnzymatic hydrolysate of poplar wood was used in this study to produce biobutanol by a mutant M-18. Plackett-Burman and Box-Behnken designs were adopted in order to screen crucial factors from various nutrient factors affecting butanol production. These factors included reducing sugar content of hydrolysate, MgSO4•7H2O, yeast extract, K2HPO4, FeSO4•7H2O, CaCO3, and ammonium sulfate. The results demonstrated that a reduction in sugar content, K2HPO4, and CaCO3 were the most critical factors. Yeast extract was also found to have a significant effect on biobutanol production by performing an analysis of variance (ANOVA). Optimal variables were 44.53 g/L of reducing sugar concentration, 1.36 g/L of K2HPO4, and 4.65 g/L of CaCO3 according to the Box-Behnken design. A model was established and used to predict a maximum biobutanol production of 7.59 g/L. Optimal conditions of fermentation were determined by orthogonal tests. Three distinct factors with important effects on biobutanol production were explored. The pH was identified as having the most significant effect on biobutanol biosynthesis. Optimized fermentation conditions for biobutanol production were determined at an initial pH of 6.5, temperature 36 °C, and inoculum quantity 9%. Under these conditions, a maximum biobutanol production of 8.41±0.20 g/L was achieved in verification experiments in a 3 L fermentation tank.