Int. J. Pharm. Investigation, 2021;11(2):204-207 204 International Journal of Pharmaceutical Investigation, Vol 11, Issue 2, Apr-Jun, 2021 Original Article Compositional Characterization of Biosurfactant Produced from Pseudomonas aeruginosa ENO14-MH271625 and its Application in Crude Oil Bioremediation Ekramul Haque 1, *, Mohd Aamir Bin Riyaz 2 , Sriram Shankar 1 , Saqib Hassan 1 1 Department of Microbiology, School of Life Sciences, Pondicherry University, Puducherry, INDIA. 2 Department of Chemistry, School of Physical, Chemical and Applied Sciences, Pondicherry University, Puducherry, INDIA. ABSTRACT Objectives: This study aimed to compositionally characterize the biosurfactant and its application in crude oil bioremediation. Methods: Production of biosurfactant was carried out in Bushnell Hass Broth (BHB), supplemented with 1% glucose. Compositional characterizations were performed by 1 H-NMR and ESI-MS analysis. The biodegradation of crude oil was carried out by using a 0.1% (g/v) of purifed biosurfactant (ENO14BS). The experiment has three sets namely Set A, Set B and Set C. The experiments are as follows: Set A contained 50 ml sterilized BHB medium with bacterial cells (5% v/v, O.D. 600 ~ 1.0), 2% (v/v) of crude oil and ENO14BS (0.1 % w/v). Set B contained 50 ml of sterilized BHB medium with bacterial cells (5% v/v, O.D. 600 nm ~ 1.0) and 2% (v/v) of crude oil. Set C (abiotic control) contained 50 ml of sterilized BHB with 2% (v/v) of crude oil only. This experiment was performed for 7 days. Results: Structural elucidation by NMR and Electrospray Ionization-Mass Spectroscopy (ESI- MS) showed the presence of six uncommon rhamnolipid homologs, m/z (mass to charge ratio) = 358.98 [M+H] + , Rha-C 12:2 ; m/z = 391.28 [M+H] + , Rha–C 6 –C 6 ; m/z = 427.38 [M+H] + , Rha–C 17:3 ; m/z = 447.32 [M+H] + , Rha–C 8 - C 8 ; m/z = 507.22 [M+H]+, Rha-Rha-C 12:1 ; m/z = 648.17 [M+H] + , Rha-Rha- C 10 -C 10:1 . The application of biosurfactant (ENO14BS) in biodegradation of crude oil was performed at a laboratory scale. The biosurfactant (0.1% w/v) amended microcosm showed up to 73% crude oil degradation (31% higher than with culture alone) in four days. Conclusion: We conclude that ENO14BS biosurfactant produced by Pseudomonas aeruginosa ENO14 using glucose in this study has shown its potential for use in the crude oil bioremediation process. Keywords: Rhamnolipid, NMR, ESI-MS, Crude oil, Bioremediation. Correspondence Mr. Ekramul Haque Senior Research Fellow (SRF), Department of Microbiology, School of Life Sciences, Pondicherry University, Puducherry-605014, INDIA. Email: hekramul37@gmail.com DOI: 10.5530/ijpi.2021.2.36 This is an open access article distributed under the terms of the Creative Commons Attribution-NonCommercial-ShareAlike 4.0 License, which allows others to remix, tweak, and build upon the work non-commercially, as long as the author is credited and the new creations are licensed under the identical terms. INTRODUCTION Te Petroleum industry is considered one of the central and fastest growing industrial sectors. Petroleum and its by-products consist of a complex composition of hydrocarbons and environmental concerns such as oil spills and leakages cause serious havoc to the natural ecosystem and biosphere due to their mutagenic, immunotoxic, carcinogenic and neurotoxic properties. 1,2 Restoration of such condition is carried out naturally through micro-organisms which can degrade and clean up the toxic pollutants present at petroleum contaminated sites and also for enhanced oil recovery from reservoirs. 3-6 Such bioremediation strategies for environmental restoration are favoured over conventional physiochemical strategies due to their cost efective and eco-friendly characteristics. 7,8 Moreover, Microbial degradation is considered as the ultimate natural process for restoring natural conditions in petroleum contaminated areas. 9 For instance, the notable role of indigenous microbes has been documented in bioremediation/biodegradation mechanisms. 10,11 Furthermore, hydrocarbon degradation involves various groups of organisms such as fungi, algae and bacteria. 12,13,2 Tey are ubiquitous ranging from soil, freshwater and marine habitats. 4 However, bacteria possess an array of bioactive agents that play a signifcant role in degrading petroleum as well as in microbial enhanced oil recovery (MEOR) process by producing biosurfactants. Also, such bioactive compounds have signifcant commercial importance. 14-17 Moreover, Varjani and Upasani, 6 have reported the applicability of rhamnolipid biosurfactant produced from an efcient hydrocarbon degrading Pseudomonas sp. in MEOR process. Furthermore, various studies have highlighted the importance of bioremediation by biodegradation as the most infuential and efective strategy for eliminating hydrocarbon contaminants from the environment. 18,9 In this study, we have compositionally characterized the biosurfactant and used it for crude oil bioremediation on laboratory scale. MATERIALS AND METHODS Bacterial strain and cultivation Biosurfactant producing P. aeruginosa ENO14 was isolated from crude oil contaminated seawater of Ennore, Tamilnadu, India. Culture condition of this isolate has been described earlier. 19 Production and purifcation of Biosurfactant Production, recovery and purifcation of biosurfactant was carried out as per the protocol described by Haque et al. 20 Te purifed biosurfactant (ENO14BS) was used for further compositional characterization and crude oil bioremediation. Nuclear magnetic resonance (NMR) spectroscopy analysis NMR spectra ( 1 H NMR) of the ENO14BS solution were measured at room temperature using a Bruker DRX Advance 400 MHz spectrometer. Twenty-fve milligrams of purifed ENO14BS sample (dissolved in 1 ml of 99.8% CDCl 3 ) was used for the analysis. 21