Structural and physico-chemical characterization of a dirhamnolipid biosurfactant purified from Pseudomonas aeruginosa: application of crude biosurfactant in enhanced oil recovery

The present study describes the structural characterization and biotechnological application of a dirhamnolipid biosurfactant produced by Pseudomonas aeruginosa strain NBTU-01 isolated from a petroleum oil-contaminated soil sample. Characterization of partially purified biosurfactant by LC-MS/MS analysis indicated predominant production (78%) of dirhamnolipids Rha-Rha-C10-C10 and Rha-RhaC12-C10 with a minor production of monorhamnolipids (22%). NMR analysis of the purified major biosurfactant produced by P. aeruginosa strain NBTU-01 identified it as dirhamnolipid and its deduced structure was L-rhamnosyl-L-rhamnosyl-b-hydroxydecanoyl-b-hydroxydecanoate. The LC-MS/MS analysis of intracellular proteins of P. aeruginosa strain NBTU-01 identified key enzymes and other proteins associated with regulation and biosynthesis of rhamnolipids. Critical micelle concentration of purified dirhamnolipid biosurfactant was determined at 72 +/- 2.25 mg l(-1) and it reduced the surface tension of water from 72.2 to 29.5 mN m(-1). The crude rhamnolipid biosurfactant effectively emulsified crude petroleum-oil, diesel, kerosene and coconut oil, and removed 70 +/- 3.5% crude petroleum oil from a saturated sand pack column. Heating the crude rhamnolipid biosurfactant at 100 degrees C for 5 h did not affect oil recovery from the sand pack column. Moreover, rhamnolipid biosurfactant showed stability at pH values between 6.0 and 10.0. The crystallization and melting temperature of purified dirhamnolipid biosurfactant was found to be 99 and 134 degrees C, respectively, suggesting it can withstand thermal denaturation and is suitable for application in high temperature wells for tertiary oil recovery.