Abstract
Rhamnolipids produced by Pseudomonas aeruginosa (P. aeruginosa) represent a group of biosurfactants with various applications (e.g., bioremediation of oil spills, cosmetics, detergents and cleaners). The commonly used colorimetric methods for rhamnolipid quantification, including anthrone, phenol−sulfuric acid and orcinol based quantification (Helbert and Brown, 1957; Chandrasekaran and BeMiller, 1980), are laborious and operationally hazardous because of the strong acid/chemical emanation which can cause deterioration of instruments measurements (e.g., spectrophotometer). Therefore, the methylene-blue-based analysis appears as a promising alternative to safely quantify whole rhamnolipid molecules based on chemical complexation reaction (Pinzon and Ju, 2009). Indeed, methylene blue and rhamnolipids form a complex in a water-chloroform phase system. The rhamnolipids-methylene blue complex is partitioned into the chloroform phase which will develop a blue color that can be quantified at 638 nm to deduce rhamnolipids concentration. Here, we describe a variant of methylene-blue-based rhamnolipids quantification procedure that allows spectrophotometric quantification on standard 96-well plastic microplate contrarily to original methylene blue procedure that requires specific and expensive microplate due to chloroform chemical properties.
Keywords: Rhamnolipids, Pseudomonas aeruginosa, Methylene-blue, Complexation, Quantification
Materials and Reagents
Equipment
Procedure
Representative data
Figure 2. Expected calibration curve of different concentration of rhamnolipid (12.5 µg/ml, 25 µg/ml, 50 µg/ml, 100 µg/ml and 200 µg/ml). Absorbance of complexed methylene blue was measured at 638 nm with SpectraMax M2 device against an HCl (0.2 N) blank. Complexation and measurements were repeated three times and error bars represent the standard error of the mean. Table 1. Absorbance of complexed methylene blue measured at 638 nm with SpectraMax M2 device against an HCl (0.2 N) blank The applicability of methylene blue rhamnolipid method was previously verified by comparison of the analysis results with those obtained from the commonly used anthrone reaction technique (Pinzon and Ju, 2009). We show in Table 2 the main difference between both methods that highlighted the advantages of methylene blue rhamnolipid method. Table 2. Comparative table between methylene blue rhamnolipid and anthrone methods
Notes
Recipes
Acknowledgments
This protocol was adapted from the previously published studies (Pinzon and Ju, 2009). This work was supported by the project PIC-Madagascar 2009 and the postdoctoral fellowship program “ELAN 2015” of the ARES-CCD (Académie de Recherche et d’Enseignement Supérieur-Commission Coopération au Développement, Belgium). We declare no conflict of interest.
References
If you have any questions/comments about this protocol, you are highly recommended to post here. We will invite the authors of this protocol as well as some of its users to address your questions/comments. To make it easier for them to help you, you are encouraged to post your data including images for the troubleshooting.
Dear Liao,Thank you very much for your interest to this protocole, and thank you very much for testing the protocol at lower concentration. It's indeed interesting to notice that pink color.The maximum absorbance peak may vary according to the purety of the methylene blue used so in your case the max absorbance peak is 650nm. The pink color may be also due to the chloroform used (technical grade, analytic grade or HPLC grade) so usethe 0µg/ml as a blank.I suggest you to do the absorbance measurement at different Absorbance from 560nm to 660nm (with 20 nm steps), and for 100µg/ml to do a dilution before measurement.Then you can find the best absorbance that generate the best linear curve for you.RegardsTsiry
Dear Tsiry,Got it, Thank you so much!Best,Shuchi Liao