The objective of this study was to optimize the physico-enzymatic pretreatment of P. roxburghii fallen foliage (needles) to produce reducing sugars through response surface methodology (RSM) with central composite face centered design (CCD). Under this, five parameters, i.e., concentration of laccase, cellulose and xylanase, steam explosion pressure and incubation period, at three levels with twenty six runs were taken into account. Cellulase, xylanase and laccase enzymes with activity 4.563, 38.32 and 0.05 IU/mL, respectively, were produced from locally isolated microbial strains. The analysis of variance (ANOVA) was applied for the validation of the predicted model at 95% of confidence level. This model predicted 334 mg/g release of reducing sugars on treating P. roxburghii fallen foliage with 1.18 mL of cellulose, 0.31 mL of xylanase and 0.01 mL of laccase, 14.39 psi steam explosion pressure and 24 h of incubation time. The experimental results obtained were in good agreement to predicted values, making it a reliable optimized model for five factors in combination to predict reducing sugar yield for ethanol production for bio-fuel industry.
