A wide variety of biosurfactant-producing microorganisms were isolated from hydrocarbon-contaminated soil and were screened for biosurfactant production using conventional methods including oil spreading test, emulsification index, emulsification activity and CTAB agar test. Among the isolated bacteria, A2 isolate, a Gram negative bacterium was selected for further studies based on its highest activity and was identified by 16S rDNA sequencing as Pseudomonas aeruginosa. The presence of specific genes responsible for the biosynthesis of mono-rhamnolipid (rhlB) and dirhamnolipid (rhlC) were detected. Optimization of different cultural conditions (carbon source, carbon concentration, nitrogen source, nitrogen concentration, pH, incubation time, and inoculum concentration) were performed to achieve maximum production of biosurfactant. Production of biosurfactant was estimated in terms of oil spreading test, emulsification index, emulsification activity and biomass as 15 cm, 60 %, 1.831 ± 0.025 and 2.851 ± 0.043 g/l respectively. The obtained results demonstrated that the maximum rhamnolipid production (5.42 ± 0.475 g/l) happened using olive oil at a concentration of 2% as carbon source, 2 g/l of urea as nitrogen source, inoculum size of 3 %, pH: 7, and 6 days incubation period at 30°C. The analysis of the extracted biosurfactant by TLC, FTIR spectra and GC-MS analysis confirmed that the biosurfactant nature was rhamnolipid. The rhamnolipid could decrease the surface tension of water to 28.49 mN/m and exhibited good stabilities at high temperatures (up to autoclaving at 121°C), salinities (up to 10 % NaCl), and pH values (up to pH: 10 except 4 and 2 pH).

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