Applied Microbiology and Biotechnology

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

Analysis of rhamnolipid biosurfactants by methylene blue complexation



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.


Biosurfactant Pseudomonas aeruginosa Rhamnolipid Methylene blue Analysis 



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.


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Copyright information

© Springer-Verlag 2009

Authors and Affiliations

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

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