Optimization of Biosurfactant Production by Bacillus licheniformis Isolated from Oil Contaminated Egyptian Soil

OPTIMAL conditions for obtaining elevated biosurfactant production by a local strain of Bacillus licheniformis strain No. 5 were investigated. Modified minimal salt medium, pH 7.0, containing crude oil (1 %), urea (2 g/l), KH2PO4 + K2HPO4 (2+2 g/l), MgSO4 (0.2 g/l), yeast extract (1 g/l), and trace elements solution (0.1 %) was found to be the most suitable for growth and emulsifying activity by this bacterium. High biosurfactant production was obtained after incubation for 7 days at 30°C. By providing the previous conditions, the emulsion index (E24%) was increased 3–fold as compared to that obtained via growth in the original minimal salt medium. In bioreactor batch culture, an agitation speed of 300 rpm attained the highest microbial growth (1.9 g cell dry weight/l) and an E24= 50.90 % after 7 days of incubation. In fed-batch culture, the pulsed addition of crude oil during the first 2 – 3 days of incubation enhanced the emulsification activity by 1.3–fold. The greatest E24 was obtained using black grain oil (89.09 %), followed by that obtained against diesel oil (87.27 %). The highest stability of emulsion index was recorded on diesel oil, which remained stable for 10 days (E240 = 81.81 %). The biosurfactant showed an almost stable surface activity profile over a wide range of pH values (from 6 to 12). The maximum emulsification activity was obtained at pH 8. The reduction in E24 after exposure of the biosurfactant to 121oC for 15 min against diesel oil and toluene was 43.7 % and 28.6 %, respectively. Chemical analyses of the purified biosurfactant showed that it is a lipoprotein. Significant emulsification activity was detected towards different aliphatic and aromatic hydrocarbons and vegetable oils. The purified biosurfactant contained 41.7% C, 7.4% H and 5.8 % N and was comprised of 36.2% proteins, 12.3% lipids and 5.6 % carbohydrates.