Microbial Production of Biosurfactants from Some El-Korma Governorate Microbial Isolates for Bioremediation of Crude Oil Spills in the Different Environments

Biosurfactants are surface –active amphipathic molecules produced by a plethora of microorganisms. The aim of the present study was production of biosurfactants from some bacterial isolates for bioremediation of crude oil spills. Twenty five bacterial isolates were isolated from twenty crude oil contaminated soils located in repairing cars stations in Khorma governorate,Taif, Kingdom of Saudi Arabia (KSA). Ten pure bacterial isolates were screened qualitatively and quantitatively for the ability to surface active compounds (SACs) biosynthesis in liquid culture media containing crude oil as sole carbon and energy source by blood agar lysis and oil spread techniques. The most potent bacterial isolate was selected and displayed highest reduction of surface tension by using surface tension measurement. The selected bacterial isolate was identified as Bacillus subtilis-KG82-KSA based on the morphological, physiological, biochemical characteristics and 16S rRNA gene sequencing analysis. Maximum SACs biosynthesis by B. subtilisKG82-KSA strain respectively under study could be obtained using crude oil in concentrations of 0.5 (mg/100ml); initial pH values,7; sodium chloride concentrations, 1 (%);carbon sources, sucrose; best carbon source concentrations,2.5 (mg/100ml);nitrogen sources, ammonium ferrous sulphate ; best nitrogen concentrations, 0.133 (mg/100ml), vitamin sources, thiamine HCl; best vitamin concentrations, 200 (ppm) under shaking conditions of 100 rpm, incubation temperature and incubation period are at 40oC for 7 days. The vegetable oils proved to increase SACs biosynthesis by B. subtilis-KG82-KSA bacterial strain. The bacterial strain showed high salt tolerance and their successful biosynthesis of SAC in a vast pH and temperature domain after only seven days and reduce surface tension. SAC could be biosynthesized from B. subtilis-KG82-KSA which could be extracted by chloroform: ethanol (2:1v/v) solvent and purified by using TLC and Silica gel chromatography. The SAC were identified as Iturin based on UV, IR and HPLC analysis techniques. Detection the qualitative and quantitative of amino acids were performed by amino acids analyzer, H-Nuclear Magnetic Resonance (NMR) analysis, fatty acid analysis and sugar pattern. These results indicated that biosurfactant iturin has a strong potential to be applied as a remediation agent for the clean –up of oil spills in sea and soil positive effects on the bioremediation of crude oil spills.