Extraction and antimicrobial activity of rhamnolipid biosurfactant produced by Pseudomonas aeruginosa UKMP14T

Aims: Rhamnolipids are seeking utmost attention as a new class of biosurfactants having promising potential in diverse fields as they offer a wide range of advantages over chemically synthesised surfactants. However, the high extraction costs make large scale production face difficulty. In present study, hydrocarbon degrading bacteria Pseudomonas aeruginosa UKMP14T was exploited for its biosurfactant producing ability including a comparative study between different extraction procedures for its recovery. In addition to this, the recovered biosurfactant was explored for its potential application as an antimicrobial agent. Methodology and results: The production of rhamnolipid biosurfactant was confirmed through various detection methods which are drop-collapse test, oil spreading assay, emulsification index, cetyltrimethylammonium bromide (CTAB) assay and hemolytic assay. The test strain P. aeruginosa UKMP14T showed positive results for all the detection assays. Following this, shake flask cultivation was carried out for several time intervals (1, 3, 5, 7 and 9 days) to discover the optimum time for rhamnolipid biosurfactant production. The results were evaluated by quantifying the rhamnolipid yield using Anthrone method and maximum yield was obtained on day 7. Then, three commonly employed rhamnolipid biosurfactant extraction methods (acid precipitation, solvent extraction and zinc sulphate precipitation) were incorporated for the extraction of rhamnolipid biosurfactant. Among these methods, organic solvent extraction (using methanol, chloroform and acetone in 1:1:1 ratio) gave the highest yield (7.37 +/- 0.81 g/L) of biosurfactant, followed by zinc sulphate precipitation (5.83 +/- 0.02 g/L), whereas acid precipitation gave the lowest yield (2.8 +/- 0.12 g/L) and required longer time (30 days). Finally, the antimicrobial activity of several concentrations of rhamnolipid was tested using modified microdilution method and highest antibacterial activity (in the form of percent reduction in growth) of 95.05% and 91.89% was recorded for Escherichia coli ATCC 10536 and Staphylococcus aureus ATCC 11632, respectively, at 100 mu g/mL concentration of rhamnolipid biosurfactant. Conclusion, significance and impact of study: The ability of P. aeruginosa UKMP14T in producing rhamnolipid biosurfactant was confirmed. Despite the higher yield obtained by organic solvent extraction method, the recovery technique (involving the separation of solvent system) caused some loss in product. In addition, the transfer and storage of rhamnolipid was challenging using solvent extraction in comparison to acid precipitation and zinc sulphate precipitation. On the other hand, recovery using acid precipitation suffered from lowest yield of rhamnolipid. Therefore, zinc sulphate precipitation is prioritised over the other two methods. Furthermore, the antimicrobial potential of rhamnolipid biosurfactant was tested successfully for as low as 10 mu g/mL concentration against E. coli ATCC 10536 and S. aureus ATCC 11632. Therefore, the recovery cost of a high value product like rhamnolipid can be reduced by incorporating the results of this study in the downstream processing and promote rhamnolipid biosurfactant as a potential antimicrobial agent.