Applied Microbiology and Biotechnology

, Volume 82, Issue 5, pp 975–981 | Cite as

Analysis of rhamnolipid biosurfactants by methylene blue complexation

Methods

Abstract

Rhamnolipids, produced by Pseudomonas aeruginosa, represent an important group of biosurfactants having various industrial, environmental, and medical applications. Current methods for rhamnolipid quantification involve the use of strong hazardous acids/chemicals, indirect measurement of the concentration of sugar moiety, or require the availability of expensive equipment (HPLC-MS). A safer, easier method that measures the whole rhamnolipid molecules would significantly enhance strain selection, metabolic engineering, and process development for economical rhamnolipid production. A semi-quantitative method was reported earlier to differentiate between the rhamnolipid-producing and non-producing strains using agar plates containing methylene blue and cetyl trimethylammonium bromide (CTAB). In this study, a rapid and simple method for rhamnolipid analysis was developed by systematically investigating the complexation of rhamnolipids and methylene blue, with and without the presence of CTAB. The method relies on measuring the absorbance (at 638 nm) of the rhamnolipid−methylene blue complex that partitions into the chloroform phase. With P. aeruginosa fermentation samples, the applicability of this method was verified by comparison of the analysis results with those obtained from the commonly used anthrone reaction technique.

Keywords

Biosurfactant Pseudomonas aeruginosa Rhamnolipid Methylene blue Analysis 

Notes

Acknowledgments

The study was supported by the U.S. Department of Transportation, Office of the Secretary, Grant No. DTOS59-07-G-00050. The authors also thank Dr. Tsung Min Kuo (USDA-ARS-NCAUR, Peoria, IL) for providing the P. aeruginosa strain isolated from the soil samples at a biodiesel plant, and Ms. Jennifer Lilly and Mr. Aaron Cook for their assistance in carrying out the experimental procedures.

References

  1. Benincasa M (2007) Rhamnolipid produced from agroindustrial wastes enhances hydrocarbon biodegradation in contaminated soil. Curr. Microbiol 54(6):445–449CrossRefGoogle Scholar
  2. Chandrasekaran EV, BeMiller JN (1980) Constituent analysis of glycosaminoglycans. In: Whistler RL, BeMiller JN (eds) Methods in carbohydrate chemistry, vol 8. Academic Press, New York, pp 89–96Google Scholar
  3. Chayabutra C, Ju L-K (2000) Degradation of n-hexadecane and its metabolites by Pseudomonas aeruginosa under microaerobic and anaerobic denitrifying conditions. Appl Environ Microbiol 66(2):493–498CrossRefGoogle Scholar
  4. Chayabutra C, Ju L-K (2001) Polyhydroxyalkanoic acids and rhamnolipids are synthesized sequentially in hexadecane fermentation by Pseudomonas aeruginosa ATCC 10145. Biotechnol Progr 17(3):419–423CrossRefGoogle Scholar
  5. Chayabutra C, Wu J, Ju L-K (2001) Rhamnolipid production by Pseudomonas aeruginosa under denitrification: effects of limiting nutrients and carbon substrates. Biotechnol Bioeng 72(1):25–33CrossRefGoogle Scholar
  6. Desai JD, Banat IM (1997) Microbial production of surfactants and their commercial potential. Microbiol Mol Biol Rev 61(1):47–64Google Scholar
  7. Eaton AD, Clesceri LS, Greenberg AE (1995) Standard methods for the examination of water and wastewater. American Public Health Association, Washington, DCGoogle Scholar
  8. Gunther NWIV, Nunez A, Fett W, Solaiman DKY (2005) Production of rhamnolipids by Pseudomonas chlororaphis, a nonpathogenic bacterium. Appl Environ Microbiol 71(5):2288–2293CrossRefGoogle Scholar
  9. Heyd M, Kohnert A, Tan TH, Nusser M, Kirschhoefer F, Brenner-Weiss G, Franzreb M, Berensmeier S (2008) Development and trends of biosurfactant analysis and purification using rhamnolipids as an example. Analytical and Bioanalytical Chemistry 391(5):1579–1590CrossRefGoogle Scholar
  10. Hodge JE, Hofreiter BT (1962) Determination of reducing sugars and carbo- hydrates. In: Whistler RL, Wolfrom ML (eds) Methods in carbohydrate chemistry, vol 1. Academic, New York, pp 380–394Google Scholar
  11. Jurado E, Fernandez-Serrano M, Nunez-Olea J, Luzon G, Lechuga M (2006) Simplified spectrophotometric method using methylene blue for determining anionic surfactants: Applications to the study of primary biodegradation in aerobic screening tests. Chemosphere 65(2):278–285CrossRefGoogle Scholar
  12. Koga M, Yamamichi Y, Nomoto Y, Irie M, Tanimura T, Yoshinaga T (1999) Rapid determination of anionic surfactants by improved spectrophotometric method using methylene blue. Anal Sci 15(6):563–568CrossRefGoogle Scholar
  13. Mercade ME, Manresa MA (1994) The use of agroindustrial byproducts for biosurfactant production. J Am Oil Chem Soc 71(1):61–4CrossRefGoogle Scholar
  14. Siegmund I, Wagner F (1991) New method for detecting rhamnolipids excreted by Pseudomonas species during growth on mineral agar. Biotechnol Tech 5(4):265–8CrossRefGoogle Scholar
  15. Soberon-Chavez G, Lepine F, Deziel E (2005) Production of rhamnolipids by Pseudomonas aeruginosa. Appl Microbiol Biotechnol 68(6):718–725CrossRefGoogle Scholar
  16. Turney ME, Cannell DW (1965) Alkaline methylene blue method for determination of anionic surfactants and for amine oxides in detergents. J Am Oil Chem Soc 42(6):544–6CrossRefGoogle Scholar
  17. Wang Q, Fang X, Bai B, Liang X, Shuler PJ, Goddard WA, Tang Y (2007) Engineering bacteria for production of rhamnolipid as an agent for enhanced oil recovery. Biotechnol Bioeng 98(4):842–853CrossRefGoogle Scholar
  18. Wild M, Caro AD, Hernandez AL, Miller RM, Soberon-Chavez G (1997) Selection and partial characterization of a Pseudomonas aeruginosa mono-rhamnolipid deficient mutant. FEMS Microbiol Lett 153(2):279–285CrossRefGoogle Scholar
  19. Zhang Q (2007) Collection of Trichoderma reesei cellulase by foaming. The University of Akron, Akron, OHGoogle Scholar
  20. Zhu L, Zhang M (2008) Effect of rhamnolipids on the uptake of PAHs by ryegrass. Environ Pollut (Amsterdam, Netherlands) 156(1):46–52Google Scholar

Copyright information

© Springer-Verlag 2009

Authors and Affiliations

  1. 1.Department of Chemical and Biomolecular EngineeringThe University of AkronAkronUSA

Personalised recommendations