Response surface methodology (Box-Behnken) to improve a liquid media formulation to produce biosurfactant and phenanthrene removal by Pseudomonas putida

A culture medium formulation was established using a Box-Behnken experimental design aimed at enhancing biosurfactant production and phenanthrene removal by Pseudomonas putida CB-100. The independent variables selected (+, 0, −, levels) were: glucose (9.1, 13.6, and 18.2 g/l), NH4Cl (0.5, 0.75, and 1.0 g/l), and yeast extract (0.005, 0.0075, and 0.01 g/l), with 200 mg/l phenanthrene at 37°C, shaken at 150 rpm, at pH 7, for 5 days. Analyses of results by one-way analysis of variance showed that biosurfactant production was improved by the glucose–ammonium chloride interaction at high values (p < 0.004). Phenanthrene removal was enhanced by the ammonium chloride-yeast extract interaction at low and high values, respectively (p < 0.02). The highest phenanthrene removal (82.4%) was observed in formulation 11, which had a C:N ratio of 5:1 and a C:P ratio of 10:1. In addition, 47.5 mN/m surface tension, 20% emulsion capacity, and 23.5 mg/l biosurfactant production were obtained. With this medium, we followed the kinetic growth of P. putida for 118 h, the culture conditions were the same as those used in the experimental design. At 46 h, we obtained 57.8% phenanthrene removal and 27 mg/l biosurfactant production. The critical micelle concentration of the biosurfactant was 430 mg/l. The biosurfactant produced by P. putida was characterized as a rhamnolipid type.