ISOLATION AND ANTIMICROBIAL ACTIVITY OF RHAMNOLIPID (BIOSURFACTANT) FROM OIL-CONTAMINATED SOIL SAMPLE USING HUMIC-ACID SALTS-VITAMIN AGAR

Abstract

In this study, actinomycetes were isolated from the soil sample contaminated with petroleum oil in Naval Dockyard near Visakhapatnam. The isolate was identified as Streptomyces coelicoflavus comprising 16S rDNA sequence. Ten isolates of soil actinomycetes were initially screened in humic acid-salts vitamin agar plates. Soil organic matter is generally rich in humic acid and fluvic acid. Humic acid is an important ingredient for the extraction of biosurfactant producing actinomycetes. In the present study, humic acid substances used for screening of biosurfactant producing Actinomycetes were extracted by using soil organic matter. Humic substances were extracted by treating this organic soil with alkalis like dilute sodium hydroxide. The ten isolates were further grown in Kim’s medium, containing olive oil as the sole source of carbon, for extracellular biosurfactant activity by oil spreading method. Out of these ten, only three isolates namely, NDYS-1, NDYS-3, NDYS-4, showed promising biosurfactant activity compared to the standard, sodium laryl sulphate. These three promising isolates were further screened for their biosurfactant activity by using Para film–M method, Emulsification index and Phenol H2SO4. Out of these three, only NDYS-4 showed maximum biosurfactant activity for the above tests. The result showed that the extracellular surface active agent produced by the isolate NDYS-4 was Rhamnolipid in nature, and was further confirmed by orcinol assay taking L-rhamnose as standard. Then crude extract obtained from this isolate was used in contact bioautography for antibacterial activity. It was observed that as the antibacterial activity increased, the biosurfactant activity also increased and vice versa.