Rhamnolipid production by a novel thermophilic hydrocarbon-degrading Pseudomonas aeruginosa AP02-1

  • Amedea Perfumo
  • Ibrahim M. Banat
  • Francesco Canganella
  • Roger Marchant
Applied Microbial and Cell Physiology


Thermophilic bacterial cultures were isolated from a hot spring environment on hydrocarbon containing mineral salts media. One strain identified as Pseudomonas aeruginosa AP02-1 was tested for the ability to utilize a range of hydrocarbons both n-alkanes and polycyclic aromatic hydrocarbons as sole carbon source. Strain AP02-1 had an optimum growth temperature of 45°C and degraded 99% of crude oil 1% (v/v) and diesel oil 2% (v/v) when added to a basal mineral medium within 7 days of incubation. Surface activity measurements indicated that biosurfactants, mainly glycolipid in nature, were produced during the microbial growth on hydrocarbons as well as on both water-soluble and insoluble substrates. Mass spectrometry analysis showed different types of rhamnolipid production depending on the carbon substrate and culture conditions. Grown on glycerol, P. aeruginosa AP02-1 produced a mixture of ten rhamnolipid homologues, of which Rha-Rha-C10-C10 and Rha-C10-C10 were predominant. Rhamnolipid-containing culture broths reduced the surface tension to ≈28 mN and gave stable emulsions with a number of hydrocarbons and remained effective after sterilization. Microscopic observations of the emulsions suggested that hydrophobic cells acted as emulsion-stabilizing agents.



This work was partially supported by the CEC EU Structural Funds, Building Sustainable Prosperity, Measure 5.1 ‘Sustainable Management of the Environment and Promotion of the Natural and Built Heritage (BSP7473), Environment and Heritage Service, N. Ireland. We also like to thank Dr. A.R. Taddei of the Centre for Electron Microscopy at University of Tuscia, Italy, for EM negative stain analyses.


  1. Al-Tahhan RA, Sandrin TR, Bodour AA, Maier RM (2000) Rhamnolipid-induced removal of lipopolysaccharide from Pseudomonas aeruginosa: effect on cell surface properties and interaction with hydrophobic substrates. Appl Environ Microbiol 66:3262–3268CrossRefPubMedGoogle Scholar
  2. Arino S, Marchal R, Vandecasteele J-P (1996) Identification and production of a rhamnolipidic biosurfactant by a Pseudomonas species. Appl Microbiol Biotechnol 45:162–168CrossRefGoogle Scholar
  3. Balch WE, Fox GE, Magrum LJ, Woese CR, Wolfe RS (1979) Methanogens: reevaluation of a unique biological group. Microbiol Rev 43:260–296PubMedGoogle Scholar
  4. Banat IM (1995a) Biosurfactants production and possible uses in microbial enhanced oil recovery and oil pollution remediation: a review. Bioresour Technol 51:1–12CrossRefGoogle Scholar
  5. Banat IM (1995b) Characterization of biosurfactants and their use in pollution removal—state of the art (review). Acta Biotechnol 15:251–267CrossRefGoogle Scholar
  6. Banat IM, Samarah N, Murad M, Horne R, Banerjee S (1991) Biosurfactant production and use in oil tank clean-up. W J Microb Biotechnol 7:80–88CrossRefGoogle Scholar
  7. Banat IM, Makkar RS, Cameotra SS (2000) Potential commercial applications of microbial surfactants. Appl Microbiol Biotechnol 53:495–508CrossRefPubMedGoogle Scholar
  8. Banat IM, Marchant R, Rahman TJ (2004) Geobacillus debilis sp. nov., a novel obligately thermophilic bacterium isolated from a cool soil environment, and reassignment of Bacillus pallidus to Geobacillus pallidus comb.nov. Int J Syst Evol Microbiol 54:2197–2201CrossRefPubMedGoogle Scholar
  9. Beal R, Betts WB (2000) Role of rhamnolipid biosurfactants in the uptake and mineralization of hexadecane in Pseudomonas aeruginosa. J Appl Microbiol 89:158–168CrossRefPubMedGoogle Scholar
  10. Benincasa M, Abalos A, Oliveira I, Manresa A (2004) Chemical structure, surface properties and biological activities of the biosurfactant produced by Pseudomonas aeruginosa LBI from soapstock. Antonie van Leeuwenhoek 85:1–8CrossRefPubMedGoogle Scholar
  11. Bodour AA, Drees KP, Maier RM (2003) Distribution of biosurfactant-producing bacteria in undisturbed and contaminated arid southwestern soils. Appl Environ Microbiol 69:3280–3287CrossRefPubMedGoogle Scholar
  12. Bond PL, Smirga SP, Banfield JF (2000) Phylogeny of microorganisms populating a thick, subaerial predominantly lithotrophic biofilm at extreme acid mine drainage site. Appl Environ Microbiol 66:3842–3849CrossRefPubMedGoogle Scholar
  13. Bouchez-Naïtali M, Rakatozafy H, Marchal R, Leveau J-Y, Vandecasteele J-P (1999) Diversity of bacterial strains degrading hexadecane in relation to the mode of substrate uptake. J Appl Microbiol 86:421–428CrossRefPubMedGoogle Scholar
  14. Cameotra SS, Makkar RS (1998) Synthesis of biosurfactants in extreme conditions. Appl Microbiol Biotechnol 50:520–529CrossRefPubMedGoogle Scholar
  15. Cooper DJ, Goldenberg BG (1987) Surface active agents from two Bacillus species. Appl Environ Microbiol 53:224–229Google Scholar
  16. Desai JD, Banat IM (1997) Microbial production of surfactants and their commercial potential. Microbiol Mol Biol Rev 61:47–64PubMedGoogle Scholar
  17. Déziel E, Paquette G, Villemur R, Lépine F, Bisaillon J-G (1996) Biosurfactant production by a soil Pseudomonas strain growing on polycyclic aromatic hydrocarbons. Appl Environ Microbiol 62:1908–1912Google Scholar
  18. Déziel E, Lépine F, Dennie D, Boismenu D, Mamer OA, Villemur R (1999) Liquid chromatography/mass spectrometry analysis of mixtures of rhamnolipids produced by Pseudomonas aeruginosa strain 57RP grown on mannitol or naphthalene. Biochim Biophys Acta 1440:244–252PubMedGoogle Scholar
  19. Déziel E, Lépine F, Milot S, Villemur R (2000) Mass spectrometry monitoring of rhamnolipids from a growing culture of Pseudomonas aeruginosa strain 57RP. Biochim Biophys Acta 1485:145–152PubMedGoogle Scholar
  20. Dèziel E, Comeau Y, Villemur R (2001) Initiation of biofilm formation by Pseudomonas aeruginosa 57RP correlates with emergence of hyperpiliated and highly adherent phenotypic variants deficients in swimming, swarming, and twitching motilities. J Bacteriol 183:1195–1204CrossRefPubMedGoogle Scholar
  21. Dorobantu LS, Yeung AKC, Foght JM, Gray MR (2004) Stabilization of oil–water emulsions by hydrophobic bacteria. Appl Environ Microbiol 70:6333–6336CrossRefPubMedGoogle Scholar
  22. Dua M, Singh A, Sethunathan N, Johri AK (2002) Biotechnology and bioremediation: successes and limitations. Appl Microbiol Biotechnol 59:143–152CrossRefPubMedGoogle Scholar
  23. King EO, Ward MK, Raney DE (1954) Two simple media for the demonstration of pyocyanin and fluorescein. J Lab Clin Med 44:301–302PubMedGoogle Scholar
  24. Korda A, Santas P, Tenente A, Santas R (1997) Petroleum hydrocarbon bioremediation: sampling and analytical techniques, in situ treatments and commercial microorganisms currently used. Appl Microbiol Biotechnol 48:677–686CrossRefPubMedGoogle Scholar
  25. Kosaric N, Gray NCC, Cairns WL (1983) Microbial emulsifiers and de-emulsifiers. In: Rehm HJ, Reed G (eds) Biotechnology, vol 3. Verlag Chemie, Weinheim, pp 575–592Google Scholar
  26. Lang S, Wullbrandt D (1999) Rhamnose lipids-biosynthesis, microbial production and application potential. Appl Microbiol Biotechnol 51:22–32CrossRefPubMedGoogle Scholar
  27. Le Borgne S, Quintero R (2003) Biotechnological processes for the refining of petroleum. Fuel Process Technol 81:155–169CrossRefGoogle Scholar
  28. Maier RM, Soberón-Chávez G (2000) Pseudomonas aeruginosa rhamnolipids: biosynthesis and potential applications. Appl Microbiol Biotechnol 54:625–633CrossRefPubMedGoogle Scholar
  29. Makkar RS, Cameotra SS (2002) An update on the use of unconventional substrates for biosurfactant production and their new applications. Appl Microbiol Biotechnol 58:428–434CrossRefPubMedGoogle Scholar
  30. Marchant R, Banat IM, Rahman TJ, Berzano M (2002) The frequency and characteristics of highly thermophilic bacteria in cool soil environments. Environ Microbiol 4:595–602CrossRefPubMedGoogle Scholar
  31. Morikawa M, Hirata Y, Imanaka T (2000) A study on the structure–function relationship of lipopeptide biosurfactants. Biochim Biophys Acta 1488:211–218PubMedGoogle Scholar
  32. Mulligan CN (2005) Environmental applications for biosurfactants. Environ Pollut 133:183–198CrossRefPubMedGoogle Scholar
  33. Niehaus F, Bertoldo C, Kähler M, Antranikian G (1999) Extremophiles as source of novel enzymes for industrial application. Appl Microbiol Biotechnol 51:711–729CrossRefPubMedGoogle Scholar
  34. Noordman WH, Janssen DB (2002) Rhamnolipid stimulates uptake of hydrophobic compounds by Pseudomonas aeruginosa. Appl Environ Microbiol 68:4502–4508CrossRefPubMedGoogle Scholar
  35. Prabhu Y, Phale PS (2003) Biodegradation of phenanthrene by Pseudomonas sp. strain PP2: novel metabolic pathway, role of biosurfactant and cell surface hydrophobicity in hydrocarbon assimilation. Appl Microbiol Biotechnol 61:342–351PubMedGoogle Scholar
  36. Rahman KSM, Rahman TJ, McClean S, Marchant R, Banat IM (2002) Rhamnolipid biosurfactant production by strains of Pseudomonas aeruginosa using low-cost raw materials. Biotechnol Prog 18:1277–1281CrossRefPubMedGoogle Scholar
  37. Rashid MH, Kornberg A (2000) Inorganic polyphosphate is needed for swimming, swarming, and twitching motilities of Pseudomonas aeruginosa. Proc Natl Acad Sci U S A 97:4885–4890CrossRefPubMedGoogle Scholar
  38. Ron EZ, Rosenberg E (2002) Biosurfactants and oil bioremediation. Curr Opin Biotechnol 13:249–252CrossRefPubMedGoogle Scholar
  39. Rosenberg M, Gutnick D, Rosenberg E (1980) Adherence of bacteria to hydrocarbons: a simple method for measuring cell-surface hydrophobicity. FEMS Microbiol Lett 9:29–33CrossRefGoogle Scholar
  40. Siegmund I, Wagner F (1991) New method for detecting rhamnolipids excreted by Pseudomonas species during growth on mineral agar. Biotechnol Tech 5:265–268CrossRefGoogle Scholar
  41. Trevors JT, Thomson DLC, Lee H, Jain DK (1991) A drop-collapsing test for screening surfactants producing microorganisms. J Microbiol Methods 13:271–279CrossRefGoogle Scholar
  42. Van Delden C, Iglewski BH (1998) Cell-to-cell signalling and Pseudomonas aeruginosa infections. Emerg Infect Dis 4:551–560PubMedCrossRefGoogle Scholar
  43. Van Hamme JD, Singh A, Ward OP (2003) Recent advances in petroleum microbiology. Microbiol Mol Biol Rev 67:503–549CrossRefPubMedGoogle Scholar
  44. Zhang Y, Miller RM (1992) Enhanced octadecane dispersion and biodegradation by a Pseudomonas rhamnolipid surfactant (biosurfactant). Appl Environ Microbiol 58:3276–3282PubMedGoogle Scholar
  45. Zhang Y, Miller RM (1994) Effect of a Pseudomonas rhamnolipid biosurfactant on cell hydrophobicity and biodegradation of octadecane. Appl Environ Microbiol 60:2101–2106PubMedGoogle Scholar
  46. Zhang Y, Miller RM (1995) Effect of rhamnolipid (biosurfactant) structure on solubilization and biodegradation of n-alkanes. Appl Environ Microbiol 61:2247–2251Google Scholar
  47. Zhang Y, Maier WJ, Miller RM (1997) Effect of rhamnolipids on the dissolution, bioavailability and biodegradation of phenanthrene. Environ Sci Technol 31:2211–2217CrossRefGoogle Scholar

Copyright information

© Springer-Verlag 2005

Authors and Affiliations

  • Amedea Perfumo
    • 1
    • 2
  • Ibrahim M. Banat
    • 2
  • Francesco Canganella
    • 1
  • Roger Marchant
    • 2
  1. 1.Department of Agrobiology and AgrochemistryUniversity of TusciaViterboItaly
  2. 2.Microbial Biotechnology Group, School of Biomedical SciencesUniversity of UlsterColeraineUK

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