Arabian Journal for Science and Engineering

, Volume 42, Issue 6, pp 2349–2359 | Cite as

Isolation and Molecular Characterization of Biosurfactant-Producing Bacterial Diversity of Fimkassar Oil Field, Pakistan

  • Afshan Hina Naeem
  • Sana Mumtaz
  • Abdul Haleem
  • Muneer Ahmed Qazi
  • Zulfiqar Ali Malik
  • Javid Iqbal Dasti
  • Safia Ahmed
Research Article - Biological Sciences


Oil-contaminated sites potentially harbor biosurfactant-producing microorganisms. The present study relates to isolation of biosurfactant-producing bacteria from Fimkassar oil field, Chakwal, Pakistan, and their molecular screening for two important genes viz. srfA and rhlB, responsible for production of surfactin and rhamnolipid biosurfactants, respectively. In total, 38 out of 79 different bacterial isolates showing growth on crude-oil-containing media were screened for biosurfactant production. Evidently, 34.2% (\(n=13\)) of the isolates were found to inherit srfA gene, while 15.8% (\(n=6\)) of the isolates contained rhlB gene. Subsequently, 16S ribosomal RNA sequence homology studies confirmed the gene-positive isolates to be the species of genera Bacillus, Brevundimonas, Alcaligenes, Pseudomonas, Serratia, Proteus and Stenotrophomonas. The unusual presence of genes in these microorganisms indicates the possibility of horizontal gene transfer and retention or presence of gene orthologs. Although all the gene-positive isolates showed biosurfactant production under submerged fermentative conditions, maximum production in terms of biosurfactant activities (i.e., \(E_{24} = 59.5\pm 4.0\%\); \(\hbox {SFT} = 27.2\pm 1.1\, \hbox {mN/m}\); \(\hbox {ODA} = 3.5\,\pm \,0.2\, \hbox {cm}\)) was revealed by Bacillus subtilis strain SWW1. Surfactin nature of biosurfactant produced was confirmed by thin-layer chromatography and Fourier transform infrared spectroscopy.


Biosurfactant Rhamnolipid Emulsification index Surface tension reduction srfA gene rhlB gene 


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The authors are highly grateful to Higher Education Commission (HEC), Government of Pakistan, for funding this study under HEC-Project No. 1366, entitled “Production and characterization of biosurfactants produced by indigenous microorganisms”. The authors have no conflict of interest to declare.


  1. 1.
    Chen, M.L.; Penfold, J.; Thomas, R.K.; Smyth, T.J.P.; Perfumo, A.; Marchant, R.; Banat, I.M.; Stevenson, P.; Parry, A.; Tucker, I.; Grillo, I.: Mixing behavior of the biosurfactant rhamnolipid with a conventional anionic surfactant, sodium dodecyl benzene sulfonate. Langmuir 26, 17958–17968 (2010)CrossRefGoogle Scholar
  2. 2.
    Maier, R.M.: Biosurfactants: evolution and diversity in bacteria. Adv. Appl. Microbiol. 52, 101–121 (2003)CrossRefGoogle Scholar
  3. 3.
    Daniels, R.; Vanderleyden, J.; Michiels, J.: Quorum sensing and swarming migration in bacteria. FEMS Microbiol. Rev. 28, 261–289 (2004)CrossRefGoogle Scholar
  4. 4.
    Das, P.; Mukherjee, S.; Sen, R.: Genetic regulations of the biosynthesis of microbial surfactants: an overview. Biotechnol. Genet. Eng. Rev. 25, 165–186 (2008)CrossRefGoogle Scholar
  5. 5.
    Vedaraman, N.; Venkatesh, N.: Production of surfactin by Bacillus subtilis MTCC 2423 from waste frying oils. Braz. J. Chem. Eng. 28, 175–180 (2011)CrossRefGoogle Scholar
  6. 6.
    Řezanka, T.; Siristova, L.; Sigler, K.: Rhamnolipid-producing thermophilic bacteria of species Thermus and Meiothermus. Extremophiles 15, 697–709 (2011)CrossRefGoogle Scholar
  7. 7.
    Rooney, A.P.; Price, N.P.; Ray, K.J.; Kuo, T.M.: Isolation and characterization of rhamnolipid-producing bacterial strains from a biodiesel facility. FEMS Microbiol. Lett. 295, 82–87 (2009)CrossRefGoogle Scholar
  8. 8.
    Özdemir, G.; Malayoglu, U.: Wetting characteristics of aqueous rhamnolipids solutions. Colloids Surf. B Biointerfaces 39, 1–7 (2004)CrossRefGoogle Scholar
  9. 9.
    Müller, M.M.; Hausmann, R.: Regulatory and metabolic network of rhamnolipid biosynthesis: traditional and advanced engineering towards biotechnological production. Appl. Microbiol. Biotechnol. 91, 251–264 (2011)CrossRefGoogle Scholar
  10. 10.
    Peypoux, F.; Bonmatin, J.M.; Wallach, J.: Recent trends in the biochemistry of surfactin. Appl. Microbiol. Biotechnol. 51, 553–563 (1999)CrossRefGoogle Scholar
  11. 11.
    Steller, S.; Sokoll, A.; Wilde, C.; Bernhard, F.; Franke, P.; Vater, J.: Initiation of surfactin biosynthesis and the role of the SrfD-thioesterase protein. Biochemistry 43, 11331–11343 (2004)CrossRefGoogle Scholar
  12. 12.
    Hamoen, L.W.; Venema, G.; Kuipers, O.P.: Controlling competence in Bacillus subtilis: shared use of regulators. Microbiology 149, 9–17 (2003)Google Scholar
  13. 13.
    Hsieh, F.C.; Li, M.C.; Lin, T.C.; Kao, S.S.: Rapid detection and characterization of surfactin-producing Bacillus subtilis and closely related species based on PCR. Curr. Microbiol. 49, 186–191 (2004)Google Scholar
  14. 14.
    Lesuisse, E.; SCHANCK, K.; Colson, C.: Purification and preliminary characterization of the extracellular lipase of Bacillus subtilis 168, an extremely basic pH-tolerant enzyme. Eur. J. Biochem. 216, 155–160 (1993)Google Scholar
  15. 15.
    Holt, J.G.K.; Sneath, N.R.; Staley, P.H.; Williams, J.T.; Stanley, T. (eds.): Bergey’s Manual of Determinative Bacteriology, 9th edn. Lippincott Williams & Wilkins, Baltimore (1994)Google Scholar
  16. 16.
    Bodour, A.A.; Drees, K.P.; Maier, R.M.: Distribution of biosurfactant-producing bacteria in undisturbed and contaminated arid southwestern soils. Appl. Environ. Microbiol. 69, 3280–3287 (2003)CrossRefGoogle Scholar
  17. 17.
    Tamura, K.; Nei, M.; Kumar, S.: Prospects for inferring very large phylogenies by using the neighbor-joining method. Proc. Natl. Acad. Sci. USA 101, 11030–11035 (2004)CrossRefGoogle Scholar
  18. 18.
    Rzhetsky, A.; Nei, M.: A simple method for estimating and testing minimum-evolution trees. Mol. Biol. Evol. 9, 945–967 (1992)Google Scholar
  19. 19.
    Nei, M.; Kumar, S.: Molecular Evolution and Phylogenetics. Oxford University Press, Oxford (2000)Google Scholar
  20. 20.
    Saitou, N.; Nei, M.: The neighbor-joining method: a new method for reconstructing phylogenetic trees. Mol. Biol. Evol. 4, 406–425 (1987)Google Scholar
  21. 21.
    Kumar, S.; Stecher, G.; Tamura, K.: MEGA7: Molecular Evolutionary Genetics Analysis Version 7.0 for Bigger Datasets. Mol. Biol. Evol. 33(7), 1870–1874 (2016)Google Scholar
  22. 22.
    Morikawa, M.; Ito, M.; Imanaka, T.: Isolation of a new surfactin producer Bacillus pumilus A-1, and cloning and nucleotide sequence of the regulator gene, psf-1. J. Ferment. Bioeng. 74, 255–261 (1992)CrossRefGoogle Scholar
  23. 23.
    Felsenstein, J.: Confidence limits on phylogenies: an approach using the bootstrap. Evolution 39(4), 783–791 (1985)Google Scholar
  24. 24.
    Grangemard, I.; Wallach, J.; Maget-Dana, R.; Peypoux, F.: Lichenysin. Appl. Biochem. Biotechnol. 90, 199–210 (2001)CrossRefGoogle Scholar
  25. 25.
    Tuleva, B.K.; Ivanov, G.R.; Christova, N.E.: Biosurfactant production by a new Pseudomonas putida strain. Z. Naturforsch. C 57, 356–360 (2002)CrossRefGoogle Scholar
  26. 26.
    Jinfeng, L.; Lijun, M.; Bozhong, M.; Rulin, L.; Fangtian, N.; Jiaxi, Z.: The field pilot of microbial enhanced oil recovery in a high temperature petroleum reservoir. J. Pet. Sci. Eng. 48, 265–271 (2005)CrossRefGoogle Scholar
  27. 27.
    Joshi, S.; Bharucha, C.; Desai, A.J.: Production of biosurfactant and antifungal compound by fermented food isolate Bacillus subtilis20B. Bioresour. Technol. 99, 4603–4608 (2008)Google Scholar
  28. 28.
    Lorenz, M.G.; Wackernagel, W.: Bacterial gene transfer by natural genetic transformation in the environment. Microbiol. Rev. 58, 563–602 (1994)Google Scholar
  29. 29.
    Logsdon, J.M.; Faguy, D.M.: Evolutionary genomics: Thermotoga heats up lateral gene transfer. Curr. Biol. 9, 747–751 (1999)CrossRefGoogle Scholar
  30. 30.
    Nelson, K.E.; Clayton, R.A.; Gill, S.R.; Gwinn, M.L.; Dodson, R.J.; Haft, D.H.; Hickey, E.K.; Peterson, J.D.; Nelson, W.C.; Ketchum, K.A.; McDonald, L.: Evidence for lateral gene transfer between Archaea and bacteria from genome sequence of Thermotoga maritima. Nature 399, 323–329 (1999)Google Scholar
  31. 31.
    Williamson, N.R.; Fineran, P.C.; Ogawa, W.; Woodley, L.R.; Salmond, G.P.: Integrated regulation involving quorum sensing, a two-component system, a GGDEF/EAL domain protein and a post-transcriptional regulator controls swarming and RhlA-dependent surfactant biosynthesis in Serratia. Environ. Microbiol. 10, 1202–1217 (2008)CrossRefGoogle Scholar
  32. 32.
    Hatt, J.K.; Rather, P.N.: Characterization of a novel gene, wosA, regulating FlhDC expression in Proteus mirabilis. J. Bacteriol. 19, 1946–1955 (2008)Google Scholar
  33. 33.
    Menkhaus, M.; Ullrich, C.; Kluge, B.; Vater, J.; Vollenbroich, D.; Kamp, R.M.: Structural and functional organization of the surfactin synthetase multienzyme system. J. Biol. Chem. 268, 7678–7684 (1993)Google Scholar
  34. 34.
    Perez, K.J.; dos Santos Viana, J.; Lopes, F.C.; Pereira, J.Q.; dos Santos, D.M.; Oliveira, J.S.; Velho, R.V.; Crispim, S.M.; Nicoli, J.R.; Brandelli, A.; Nardi, R.M.: Bacillus spp. isolated from puba as a source of biosurfactants and antimicrobial lipopeptides. Front. Microbiol. 8, 1–14 (2017)CrossRefGoogle Scholar
  35. 35.
    Conti, E.; Stachelhaus, T.; Marahiel, M.A.; Brick, P.: Structural basis for the activation of phenylalanine in the non-ribosomal biosynthesis of gramicidin S. EMBO J. 16, 4174–4183 (1997)CrossRefGoogle Scholar
  36. 36.
    Peypoux, F.; Bonmatin, J.M.; Wallach, J.: Recent trends in the biochemistry of surfactin. Appl. Microbiol. Biotechnol. 51, 553–563 (1999)CrossRefGoogle Scholar
  37. 37.
    Qazi, M.A.; Malik, Z.A.; Qureshi, G.D.; Hameed, A.; Ahmed, S.: Yeast extract as the most preferable substrate for optimized biosurfactant production by rhlB gene positive Pseudomonas putida SOL-10 isolate. J. Bioremediat. Biodegrad 4, 1–10 (2013)Google Scholar
  38. 38.
    Mukherjee, A.K.; Das, K.: Correlation between diverse cyclic lipopeptides production and regulation of growth and substrate utilization by Bacillus subtilis strains in a particular habitat. FEMS Microbiol. Ecol. 54, 479–489 (2005)Google Scholar
  39. 39.
    Cooper, D.G.; Macdonald, C.R.; Duff, S.J.B.; Kosaric, N.: Enhanced production of surfactin from Bacillus subtilis by continuous product removal and metal cation additions. Appl. Environ. Microbiol. 42, 408–412 (1981)Google Scholar
  40. 40.
    Joshi, S.; Bharucha, C.; Jha, S.; Yadav, S.; Nerurkar, A.; Desai, A.J.: Biosurfactant production using molasses and whey under thermophilic conditions. Bioresour. Technol. 99, 195–199 (2008)CrossRefGoogle Scholar

Copyright information

© King Fahd University of Petroleum & Minerals 2017

Authors and Affiliations

  1. 1.Department of MicrobiologyQuaid-i-Azam UniversityIslamabadPakistan
  2. 2.Institute of Water Resource Engineering and ManagementMehran University of Engineering and TechnologyJamshoroPakistan
  3. 3.Department of Microbiology, Faculty of Natural ScienceShah Abdul Latif UniversityKhairpurPakistan

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