Production and characterization of a glycolipid biosurfactant from Bacillus megaterium using economically cheaper sources

  • R. Thavasi
  • S. JayalakshmiEmail author
  • T. Balasubramanian
  • Ibrahim M. Banat
Original Paper


Criteria selected for screening of biosurfactant production by Bacillus megaterium were hemolytic assay, bacterial cell hydrophobicity and the drop-collapse test. The data on hemolytic activity, bacterial cell adherence with crude oil and the drop-collapse test confirmed the biosurfactant-producing ability of the strain. Accordingly, the strain was cultured at different temperatures, pH values, salinity and substrate (crude oil) concentration in mineral salt medium to establish the optimum culture conditions, and it was shown that 38°C, 2.0% of substrate concentration, pH 8.0 and 30‰ of salt concentration were optimal for maximum growth and biosurfactant production. Laboratory scale biosurfactant production in a fermentor was done with crude oil and cheaper carbon sources like waste motor lubricant oil and peanut oil cake, and the highest biosurfactant production was found with peanut oil cake. Characterization of partially purified biosurfactant inferred that it was a glycolipid with emulsification potential of waste motor lubricant oil, crude oil, peanut oil, diesel, kerosene, naphthalene, anthracene and xylene.


Biosurfactants Emulsification Biodegradation Crude oil Waste lubricant oil Peanut oil 



The authors thank the authorities of Annamalai University for providing the facilities and Department of Ocean Development and Council of Scientific and Industrial Research Govt. of India for their financial support to carryout the work in a successful way.


  1. Arima K, Kakinuma A, Tamura G (1968) Surfactin, a crystalline peptide surfactant produced by Bacillus subtilis: isolation, characterization and its inhibition of fibrin clot formation. Biochem Biophys Res Commun 31:488–494CrossRefGoogle Scholar
  2. Banat IM (1993) The isolation of a thermophilic biosurfactant producing Bacillus sp. Biotechnol Lett 15:591–594CrossRefGoogle Scholar
  3. Banat IM, Makkar RS, Cameotra SS (2000) Potential commercial applications of microbial surfactants. Appl Microbiol Biotechnol 53:495–508CrossRefGoogle Scholar
  4. Benincasa M, Contiero J, Manresa MA et al (2002) Rhamnolipid production by Pseudomonas aeruginosa LBI growing on soapstock as the sole carbon source. J Food Eng 54:283–288CrossRefGoogle Scholar
  5. Bernheimer AW, Avigad LS (1970) Nature and properties of a cytological agent produced by Bacillus subtilis. J Gen Microbiol 61:361–369Google Scholar
  6. Besson F, Michel G, (1992) Biosynthesis of iturin and surfactin by Bacillus subtilis. Evidence for amino acid activating enzymes. Biotechnol Let 14:1013–1018CrossRefGoogle Scholar
  7. Betts RP, Bankers P, Banks JG (1989) Rapid enumeration of viable microorganisms by staining and direct microscopy. Lett Appl Microbiol 9:199–202CrossRefGoogle Scholar
  8. Bodour AA, Maier RM (1998) Application of a modified dropcollapse technique for surfactant quantification and screening of biosurfactant-producing microorganisms. J Microbiol Methods 32:273–280CrossRefGoogle Scholar
  9. Bredholt H, Bruheim P, Potocky M et al (2002) Hydrophobicity development, alkane oxidation and crude-oil emulsification in a Rhodococcus species. Can J Microbiol 48:295–304CrossRefGoogle Scholar
  10. Buchanan RE, Gibbons NE, Cowan ST et al (1974) Bergey’s manual of determinative bacteriology. Williams and Wilkinns Co, BaltimoreGoogle Scholar
  11. Carrillo P, Mardaraz C, Pitta-Alvarez S et al (1996) Isolation and selection of biosurfactant-producing bacteria. World J Microbiol Biotechnol 12:82–84CrossRefGoogle Scholar
  12. Cooper DG, Goldenberg BG (1987) Surface-active agents from two Bacillus species. Appl Environ Microbiol 53:224–229Google Scholar
  13. Desai JD, Banat IM (1997) Microbial production of surfactants and their commercial potential. Microbiol Mol Biol Rev 61:47–64Google Scholar
  14. Deshpande M, Daniels L (1995) Evaluation of sophorolipid biosurfactant production by Candida bombicola using animal fat. Bioresour Technol 54:143–150CrossRefGoogle Scholar
  15. Deziel E, Paquette G, Villemur R et al (1999) Biosurfactant production by a soil Pseudomonas strains growing on poly aromatic hydrocarbons. Appl Environ Microbiol 62:1908–1912Google Scholar
  16. Dubois M, Gilles KA, Hamilton JK et al (1956) Colorimetric method for determination of sugars and related substances. Anal Chem 28:350–356CrossRefGoogle Scholar
  17. Fernandez-Linares L, Acquaviva M, Bertrand J-C et al (1996) Effect of sodium chloride concentration on growth and degradation of eicosane by marine halotolerent bacterium Marinobacter hydrocarbonoclastieus. Appl Microbiol 19:113–121Google Scholar
  18. Folch JM, Lees M, Stanly HS (1956) A simple method for the isolation and quantification of total lipids from animal tissues. J Biol Chem 226:497–509Google Scholar
  19. Fox SL, Bala GA (2000) Production of surfactant from Bacillus subtilis ATCC 21332 using potato substrates. Bioresour Technol 75:235–240CrossRefGoogle Scholar
  20. Goldman S, Shabtai Y, Rubinovitz C, Rosenberg E et al (1982) Emulsan in Acinetobacter calcoaceticus RAG-I: distribution of cell-free and cell associated cross-reacting materials. Appl Environ Microbiol 44:165–170Google Scholar
  21. Jain DK, Collins-Thompson DL, Lee H et al (1991) A drop-collapsing test for screening surfactant-producing microorganisms. J Microbiol Methods 13:271–279CrossRefGoogle Scholar
  22. Johnson M, Boese-Marrazzo D (1980) Production and properties of heat stable extracellular hemolysin from Pseudomonas aeruginosa. Infect Immun 29:1028–1033Google Scholar
  23. Juwarkar A, Khirsagar DG (1991) Emulsification and oil degradation by marine bacteria. Indian J Mar Sci 20:78–79Google Scholar
  24. Li Z-Y, Lang S, Wagner F et al (1984) Formation and identification of interfacial-active glycolipids from resting microbial cells. Appl Environ Microbiol 48:610–617Google Scholar
  25. Makkar RS, Cameotra SS (1999) Biosurfactant production by microorganisms on unconventional carbon sources-a review. J Surf Det 2:237–241CrossRefGoogle Scholar
  26. Marahiel M, Denders W, Krause M et al (1977) Biological role of gramicidin S in spore function. Studies on gramicidinc-S negative mutants of Bacillus brevis 9999. Eur J Microbiol 99:49–52Google Scholar
  27. Mercade ME, Manresa MA (1994) The use of agroindustrial byproducts for biosurfactant production. J Am Oil Chem Soc 71:61–64CrossRefGoogle Scholar
  28. Mercade ME, Manresa MA, Robert M et al (1993) Olive oil mill effluent (OOME). New substrate for biosurfactant production. Bioresour Technol 43:1–6CrossRefGoogle Scholar
  29. Moran A, Alejandra M, Martinez F et al (2002) Quantification of surfactin in culture supernatant by hemolytic activity. Biotechnol Lett 24:177–180CrossRefGoogle Scholar
  30. Mukherjee AK, Das K (2005) Correlation between diverse cyclic lipopeptides production and regulation of growth and substrate utilization by Bacillus subtilis strains in a particular habitat. FEMS Microbiol Ecol 54:479–489CrossRefGoogle Scholar
  31. Mulligan CN, Cooper DG, Neufeld RJ (1984) Selection of microbes producing biosurfactants in media without hydrocarbons. J Ferment Technol 62:311–314Google Scholar
  32. Neu TR, Poralla K (1990) Emulsifying agent from bacteria isolated during screening for cells with hydrophobic surfaces. Appl Microbiol Biotechnol 32:521–525Google Scholar
  33. Nitschke M, Pastore GM (2004) Biosurfactant production by Bacillus subtilis using cassava processing effluent. Appl Biochem Biotechnol 112:163–172CrossRefGoogle Scholar
  34. Rahman KSM, Rahman TJ, McClean S et al (2002) Rhamnolipid biosurfactant production by strains of Pseudomonas aeruginosa using low-cost raw materials. Biotechnol Prog 18:1277–1281CrossRefGoogle Scholar
  35. Rodrigues LR, Teixeira JA, van der Mei HC, Oliveira R (2006) Isolation and partial characterization of a biosurfactant produced by Streptococcus thermophilus A. Colloids Surf B Biointerfaces 53:105–112CrossRefGoogle Scholar
  36. Rosenberg E, Zuckerberg A, Rubinovitz C et al (1979) Emulsifier of Arthrobacter RAG-I: isolation and emulsifying properties. Appl Environ Microbiol 37:402–408Google Scholar
  37. Rosenberg M, Gutnick DL, Rosenberg E (1980) Adherence of bacteria to hydrocarbons: a simple method for measuring cell-surface hydrophobicity. FEMS Microbiol Lett 9:29–33CrossRefGoogle Scholar
  38. Sandrin C, Peypoux F, Michel G (1990) Coproduction of surfactin and iturin A lipopeptides with surfactant and antifungal properties by Bacillus subtilis. Biotechnol Appl Biochem 12:370–375Google Scholar
  39. Sheppard JD, Mulligan CN (1987) The production of surfactin by Bacillus subtilis grown on peat hydrolysate. Appl Microbiol Biotechnol 27:110–116CrossRefGoogle Scholar
  40. Tahzibi A, Kamal F, Assadi MM (2004) Improved production of rhamnolipids by a Pseudomonas aeruginosa mutant. Iran Biomed J 8:25–31Google Scholar
  41. Thavasi R, Jayalakshmi S (2003) Bioremediation potential of hydrocarbonoclastic bacteria in Cuddalore harbour waters (India). Res J Chem Environ 7:17–22Google Scholar
  42. Tuleva BK, Ivanov RG, Christova NE (2001) Biosurfactant production by an new Pseudomonas putida strain. Z Naturforsch 57:356–360Google Scholar
  43. Yonebayashi H, Yoshida S, Ono K et al (2000) Screening of microorganisms for microbial enhanced oil recovery process. Sekiyu Gakkaishi 43:59–69Google Scholar
  44. Youssef NH, Duncan KE, Nagle DP et al (2004) Comparison of methods to detect biosurfactant production by diverse microorganisms. J Microbiol Methods 56:339–347CrossRefGoogle Scholar
  45. Youssef N, Simpson DR, Duncan KE et al (2007) In situ biosurfactant production by Bacillus strains injected into a limestone petroleum reservoir. Appl Environ Microbiol 73:1239–1247CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media B.V. 2007

Authors and Affiliations

  • R. Thavasi
    • 1
  • S. Jayalakshmi
    • 1
    Email author
  • T. Balasubramanian
    • 1
  • Ibrahim M. Banat
    • 2
  1. 1.CAS in Marine BiologyAnnamalai UniversityParangipettaiIndia
  2. 2.School of Biomedical SciencesUniversity of UlsterColeraineNorthern Ireland, UK

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