AbstractThis work reports on the exploitation of Beta vulgaris for biosurfactant production by Bacillus licheniformis STK 01 and its optimization using statistical modeling of response surface methodology (RSM). Three variables were investigated: agro-waste concentration, pH, and temperature. The response and contour plots of the RSM showed perfect interaction among the variables, with the highest surface tension reduction of the culture medium to 30 mN/m observed at 42 °C, a pH of 8, and a substrate concentration of 4% (w/v). The biosurfactant produced demonstrated a high tendency for hydrocarbon emulsification. Furthermore, by numerical optimization techniques, the optimum conditions were found to be as follows: a pH of 6.72, an agro-waste concentration of 4% (w/v), and a temperature of 44.5 °C. The experiment conducted to validate the optimum conditions obtained showed a biosurfactant with remarkable surface activity, lowering the surface tension of the broth to 30 mN/m, when the organism was grown on B. vulgaris, and to 23.5 mN/m, when grown in glucose medium – the later representing one of the highest surface tension reductions ever reported for a biosurfactant. This study revealed, among others, that the exclusive utilization of cheap solid agro-waste without supplementation with a refined nutrient source is feasible and could ensure the economic sustainability of biosurfactant production.