Skip to main content

Advertisement

SpringerLink
Log in

High-performance liquid chromatography purification of biosurfactant isoforms produced by a marine bacterium

  • Original Paper
  • Published:
Analytical and Bioanalytical Chemistry Aims and scope Submit manuscript

Abstract

A marine Bacillus strain produced biosurfactant during its growth in a defined glucose-containing medium. An efficient method for separation and purification of biosurfactant isoforms was developed and optimized in high-performance liquid chromatography (HPLC) by manipulating solvent gradient program and flow rates. Starting with an initial run time of 60 min, the final optimized method had a significantly reduced run time of 20 min. By using this method, all the surface-active isoforms (fractions A–D) were eluted within 12 min of elution with much shortened retention time of each component. The purity levels of the isoforms were enhanced using the optimized method as evident from their lower CMC values. Among the four surface-active fractions, antimicrobial action was solely displayed by HPLC fraction A. FTIR analysis revealed all the HPLC fractions to be lipopeptide in nature and MALDI-ToF mass spectral analysis showed that these belonged to the fengycin family containing C15, C16, and C17 fengycins.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5

Similar content being viewed by others

References

  1. Mukherjee S, Das P, Sen R (2006) Towards commercial production of microbial surfactants. Trends Biotechnol 24:509–515

    Article  CAS  Google Scholar 

  2. Das P, Mukherjee S, Sen R (2008) Genetic regulation of the biosynthesis of microbial surfactant: an overview. Biotechnol Genet Eng Rev 25:165–186

    CAS  Google Scholar 

  3. Rodrigues L, Banat IM, Teixeria J, Oliveira R (2006) Biosurfactant: potential application in medicine. J Antimicrob Chemother 57:609–618

    Article  CAS  Google Scholar 

  4. Desai JD, Banat IM (1997) Microbial production of surfactants and their commercial potential. Microbiol Mol Biol Rev 61:47–64

    CAS  Google Scholar 

  5. Das P, Mukherjee S, Sen R (2008) Antimicrobial potential of a lipopeptide biosurfactant derived from a marine Bacillus circulans. J Appl Microbiol 104:1675–1684

    Article  CAS  Google Scholar 

  6. Cameotra SS, Makkar RS (2004) Recent applications of biosurfactant as biological and immunological molecules. Curr Opin Microbiol 7:262–266

    Article  CAS  Google Scholar 

  7. Maneerat S, Phetrong K (2007) Isolation of biosurfactant-producing marine bacteria and characteristics of selected biosurfactant. Songklanakarin J Sci Technol 29:781–791

    Google Scholar 

  8. Vater J, Kablitz WC, Franke P, Mehta N, Cameotra SS (2002) Matrix-assisted laser desorption ionization- time of flight mass spectrometry of lipopeptide biosurfactant in whole cells and culture filtrates of Bacillus subtilis C-1 isolated from petroleum sludge. Appl Environ Microbiol 68:6210–6219

    Article  CAS  Google Scholar 

  9. Romera D, Ad V, Rakotoaly RH, Dufour SE, Veening JW, Arrebola E, Cazorla FM, Kuipers OP, Paquot M, Garcia AP (2007) The iturin and fengycin family of lipopeptide are key factor in antagonism of Bacillus subtilis towards Podosphaera fusca. Mol Plant Microbe Interact 20:430–440

    Article  Google Scholar 

  10. Sun L, Lu Z, Bie X, Lu F, Yang S (2006) Isolation and characterization of a co-producer of fengycins and surfactins, endophytic Bacillus amyloliquefaceins ES-2, from Scutellaria baicalensis georgi. World J Microbiol Biotechnol 22:1259–1266

    Article  CAS  Google Scholar 

  11. Deleu M, Paquot M, Nylander T (2008) Effect of fengycin, a lipopeptide produced by Bacillus subtilis, on model biomembranes. Biophys J 94:2667–2679

    Article  CAS  Google Scholar 

  12. Mukherjee S, Das P, Sivapathasekaran C, Sen R (2009) Antimicrobial biosurfactant from marine Bacillus circulans: extracellular synthesis and purification. Lett Appl Microbiol 48:281–288

    Article  CAS  Google Scholar 

  13. Sen R, Swaminathan T (2005) Characterization of concentration and purification parameters and operating conditions for the small-scale recovery of surfactin. Process Biochem 40:2953–2958

    Article  CAS  Google Scholar 

  14. Lin SC, Chen YC, Lin YM (1998) General approach for the development of high-performance liquid chromatography methods for biosurfactant analysis and purification. J Chromatogr A 825:149–159

    Article  CAS  Google Scholar 

  15. Thaniyavarn J, Roongsawang N, Kameyama T, Haruki M, Imanaka T, Morikawa M, Kanaya S (2003) Production and characterization of biosurfactant from Bacillus licheniformis F2.2. Biosci Biotechnol Biochem 67:1239–2003

    Article  CAS  Google Scholar 

  16. McInerney MJ, Javaheri M, Nagle DP Jr (1990) Properties of biosurfactant produced by Bacillus licheniformis strain JF-2. J Ind Microbiol 5:95–102

    Article  CAS  Google Scholar 

  17. Das K, Mukherjee AK (2005) Correlation between diverse cyclic lipopeptides production and regulation of growth and substrate utilization by B.subtilis strains in a particular habitat. FEMS Microbiol Ecol 54:479–489

    Article  Google Scholar 

  18. Pueyo MT Jr, CB RAMC, Pd M (2009) Lipopeptides produced by a soil Bacillus megaterium strain. Microb Ecol 57:367–378

    Article  CAS  Google Scholar 

  19. Leenders F, Stein TH, Kablitz B, Franke P, Vater J (1999) Rapid typing of Bacillus subtilis strains by their secondary metabolites using matrix-assisted laser desorption/ionization mass spectrometry of intact of cells. Rapid Commun Mass Spec 13:943–949

    Article  CAS  Google Scholar 

  20. Pabel CT, Vater J, Wilde C, Franke P, Hofemeister J, Adler B, Bringmann G, Hacker J, Hentschel (2003) Antimicrobial activities and matrix-assisted laser desorption/ionization mass spectrometry of Bacillus isolates from the marine sponge aplysina aerophoba. Marine Biotechnol 5:424–434

    Article  CAS  Google Scholar 

  21. Fernandes PAV, Arruda IRD, Santos A, AAd A, Maior AMS, Ximenes EA (2007) Antimicrobial activity of surfactants produced by Bacillus subtilis R14 against multidrug resistant bacteria. Braz J Microbiol 38:704–709

    Google Scholar 

  22. Kim PI, Bai I, Bai D, Chae H, Chung S, Kim Y, Park R, Chi YT (2004) Purification and characterization of a lipopeptide produced by Bacillus thuringiensis CMB26. J Appl Microbiol 97:942–949

    Article  CAS  Google Scholar 

Download references

Acknowledgments

RS and CS acknowledge the Department of Biotechnology (DBT), the Government of India for the project grant (BT/PR-6827/AAQ/03/263/2005) in marine biotechnology. SM acknowledges CSIR, New Delhi for the Senior Research Fellowship. RS acknowledges Department of Biotechnology, IIT Kharagpur. Authors acknowledge Palashpriya Das for taking pains in strain isolation and for her all-round support throughout this work.

Author information

Authors and Affiliations

  1. Department of Biotechnology, Indian Institute of Technology, Kharagpur, West Bengal, 721302, India

    C. Sivapathasekaran, Soumen Mukherjee, Ramapati Samanta & Ramkrishna Sen

Authors
  1. C. Sivapathasekaran
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Soumen Mukherjee
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Ramapati Samanta
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Ramkrishna Sen
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Ramkrishna Sen.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Sivapathasekaran, C., Mukherjee, S., Samanta, R. et al. High-performance liquid chromatography purification of biosurfactant isoforms produced by a marine bacterium. Anal Bioanal Chem 395, 845–854 (2009). https://doi.org/10.1007/s00216-009-3023-2

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s00216-009-3023-2

Keywords

Search

Navigation