Statistical optimization for the production of lichenase by a newly isolated Bacillus licheniformis UEB CF in solid state fermentation using pea pomace as a novel solid support

Abstract

A newly isolated Bacterium strain UEB CF was selected from southern Tunisia, exhibiting the highest clear zone on agar plates containing barley flour by staining with Congo red. On the basis of 16S rDNA sequence analysis, this strain was identified as Bacillus licheniformis. Its capacity to produce lichenase was assessed under solid-state fermentation (SSF) using pea pomace as an abundant agro-industrial substrate. Cultivation conditions of B. licheniformis UEB CF for lichenase production were optimized using Doehlert design. The impact on enzyme production of four independent variables, namely cultivation time, moisture-to-substrate ratio (L/S), inoculum volume and culture temperature were evaluated by response surface methodology (RSM). A second-order polynomial model was determined by the multiple regression analysis of the experimental data. A statistical analysis showed a satisfactory adjustment of the quadratic model with the experimental data. Contour plots were generated to assess the change in the response surface and to understand the relationship between the enzyme production yield and the culture conditions. A ridge analysis revealed that the optimum values of the tested variables for maximizing enzyme production were as follows cultivation time, 84h; L/S ratio 4ml/gs; inoculum volume, 0.8ml (8×107CFU/ml) and culture temperature, 44°C. The experiment carried out under these conditions led to a lichenase production yield of 280U/g dry substrate, which was in agreement with the predicted value of 293±49U/gds by the model.