Biochemical and molecular characterization of potent bacterial biosurfactant producers isolated from hydrocarbon contaminated environments

Samples from geographically diverse habitats with extensive exposure to hydrocarbons or extreme environments were examined for the presence of biosurfactant producing microorganisms. Fifty isolated colonies obtained after four cycles of enrichment of the collected samples, were first subjected to qualitative screening comprising of different methods as oil displacement method, blood hemolysis, CTAB agar method followed by secondary quantitative screening using emulsification activity, emulsification index and measurement of surface tension by the DuNouy ring method. Screening results showed fourteen potent biosurfactant producers with reduction in the surface tension values of the minimal salt medium (MSM) from 60.23±2.04 mN/m (uninoculated control) to 28-33 mN/m. Biochemical and molecular characterization by 16S rRNA sequencing of these fourteen isolates identified them as belonging to the genera Pseudomonas, Achromobacter, Lysinibacillus, Bacillus, Citrobacter and Ochrobactrum. Among these, Achromobacter sp. (PS1) showed significant surface tension value of 33mN/m with excellent emulsion activity of 98.2 EU/ml and high stable emulsion index (EI24) of 70.59 % using diesel, retaining 90% of emulsion stability even after 7 days. Characterization by TLC and GC-MS of the extracted biosurfactants produced by these fourteen isolates of different genera respectively revealed the production of different types of biosurfactants with Achromobacter sp. (PS1) producing a rhamnolipid type of biosurfactant. This study indicates that stressed environments and hydrocarbon contaminated sites can serve as potential sources of diverse biosurfactant producing microorganisms.