Forty-two morphologically different fungal strains were isolated from different soil samples and agricultural wastes and screened for β-glucosidase activity under solid-state fermentation. Eight species were chosen as the most active β-glucosidase producers and were subjected to primary morphological identification. β-Glucosidase was highly produced by Aspergillus terreus, which showed the highest activity, and was subjected to full identification using scanning electron microscopy and molecular identification. Initial screening of different variables affecting β-glucosidase production was performed using Plackett-Burman design and the variables with statistically significant effects were identified. The optimal levels of the most significant variables with positive effect and the effect of their mutual interactions on β-glucosidase production were determined using Box-Behnken design. Fifteen variables including temperature, pH, incubation time, inoculum size, moisture content, substrate concentration, NaNO3, KH2PO4, MgSO4 · 7H2O, KCl, CaCl2, yeast extract, FeSO4 · 7H2O, Tween 80, and (NH4)2SO4 were screened in 20 experimental runs. Among the 15 variables, NaNO3, KH2PO4 and Tween 80 were found as the most significant factors with positive effect on β-glucosidase production. The Box-Behnken design was used for further optimization of these selected factors for better β-glucosidase production. The maximum β-glucosidase production was 4457.162 U g(-1).

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