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In the present study, a combined treatment strategy involving the addition of rhamnolipid, rhamnolipid-producing bacteria (Shewanella sp. BS4) and a native soil microbial community for the remediation of hydrocarbon-contaminated soil under pilot-scale conditions was adopted. The isolate BS4 (rhl+), demonstrating the highest emulsification activity and surface tension reduction efficiency, was identified based on 16 S rDNA sequencing as Shewanella sp. strain. Growth conditions for rhamnolipid production were optimized based on Central Composite Design (CCD) as 2.9% crude oil, a 54 × 106 CFU g−1 inoculation load of soil, a temperature of 30.5 °C, and a pH of 6.7. In situ bioremediation experiments, conducted using hydrocarbon-contaminated soil treated with the combination of rhamnolipid and rhamnolipid-producing bacteria, showed that the inoculated Shewanella sp. BS4, along with the indigenous soil microbial community, supported the highest hydrocarbon-degrading bacterial population and soil respiration activity, and this treatment resulted in 75.8% hydrocarbon removal efficiency, which was higher compared to contaminated soil devoid of any treatment.

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