Effect of Biosurfactants on Remediation of Oil Contaminated Soil

Abstract

Crude oil exploration and exploitation to fulfill the fossil fuel demand of increasing world population is playing havoc with human and other life forms by contaminating soil in the vicinity of such sites. This work was aimed at evaluating potential of bioaugmentation with crude oil degrading bacteria, biostimulation and biosurfactant-enhanced bioavailability for bioremediation of crude oil contaminated soil and protects the environment. Crude oil degrading bacteria were isolated from oil contaminated soil and identified by partial sequencing of 16S rRNA gene. These strains were screened for crude oil degradation and production of rhamnolipids. Four of the strains, vis a vis Alcaligenes faecalis R8, Microbacterium oryzae R4, Ochrobactrum intermedium R2 and Pseudomonas aeruginosa R7, were found to be highly efficient for crude oil degradation. Theses strains also possessed alkane hydroxylase (alkB) gene. Three best rhamnolipid biosurfactant producing Pseudomonas aeruginosa strains i.e. R7, R21 and R25 were also selected. These strains produced different congeners of mono- and di-rhamnolipids as characterized by LC-MS. The produced rhamnolipids by selected bacterial strains were in good quality and quantity. These strains possessed rhamnolipids producing genes rhlA, rhlB and rhlC indicating the presence of rhl operon. A consortium of best oil degrading bacteria was inoculated in crude oil-contaminated soil in the vicinity of an oil field and supplemented with different combinations of rhamnolipids and nutrients. Maximum crude oil degradation (77.6%) was observed in the soil inoculated with hydrocarbon-degrading bacteria supplemented with rhamnolipids and nutrients. Moreover, addition of supplementary compounds enhanced bacterial survival as well as abundance and expression of alkane hydroxylase gene, alkB, in oil contaminated soil. A strong positive relationship (r = 0.94) observed between gene expression and crude oil reduction indicates that catabolic gene expression is essential for hydrocarbon mineralization. Two thermophilic bacterial strains, Geobacillus thermoglucosidasius RT-2 and Aeribacillus pallidus RT-10 efficiently utilized different hydrocarbons at 60°C. Addition of biosurfactants and nutrients resulted in enhanced degradation of hexadecane. Maximum hexadecane degradation (81%) occurred in the presence of rhamnolipids and nutrients. Both the strains were also quite efficient for crude oil degradation under thermophilic conditions. Results from the investigations collectively confirm that addition of rhamnolipids and nutrients enhance bacterial colonization and metabolic activity thus improving remediation of crude oil contaminated soil.