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Jul 2015

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Procedure for Rhamnolipids Quantification Using Methylene-blue    

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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

  1. Borate Buffer Packs BupHTM (Thermo Fisher scientific, catalog number: 28384 )
  2. Centrifuge round bottom reaction tubes (30 ml) (Krackeler Scientific, COREX®, catalog number: 1-8445-30 )
  3. Greiner CELLSTAR® 96 well plates (clear polystyrene wells flat bottom) (Sigma-Aldrich, catalog number: M3687-60 EA )
  4. Microcentrifuge tube [2 ml, transparent polypropylene (PP) with Lid] (Sigma-Aldrich, Brand®, catalog number: Z628034-500 EA )
  5. Sterile culture tube (PP) (14 ml) (The Lab Depot Inc., catalog number: TLDT8235 )
  6. Centrifugal Filter (0.1 µm pore size, Non-Sterile) with Durapore® PVDF Membrane UFC40VV25 Ultrafree® (2 ml) (Merck Millipore Corporation, catalog number: UFC40VV25 )
  7. Aluminium foil
  8. Micropipettor (10 µl,100 µl and 1,000 µl) tips
  9. Pseudomonas aeruginosa (P. aeruginosa) PAO1 Wild-type (strain PAO0001) (http://www.pseudomonas.med.ecu.edu/)
  10. Chloroform (≥ 99% ) (Carl Roth GmbH & Co. KG, catalog number: 3313.1 )
  11. Ethyl acetate (> 99.5%) (Merck Millipore Corporation, EMSURE®, catalog number: 1096231000 )
  12. Hydrochloric acid (HCl) standard (1 N solution) (Sigma-Aldrich, Fluka analytical, catalog number: 318949-500 ml )
  13. HCl standard (0.2 N solution in water) (Sigma-Aldrich, Fluka analytical, catalog number: 343102-500 ml )
  14. Luria Bertani (LB) Broth (Lennox) Powder microbial growth medium (Sigma-Aldrich, catalog number: L3022-250 G )
  15. Methylene blue solution [1.4% (w/v) in 95% ethanol] (Sigma-Aldrich, catalog number: 1808-50 ml )
  16. 3-(N-morpholino) propanesulfonic acid (MOPS) (99.5%) (Sigma-Aldrich, catalog number: M1254-100 G )
  17. Rhamnolipids (90%, Standard) (Sigma-Aldrich, catalog number: R90 )
  18. Sodium hydroxide (NaOH) solution
  19. LB-MOPS broth (see Recipes)
  20. Borax buffer (50 mM) (see Recipes)
  21. Methylene blue aqueous solution (see Recipes)


  1. Micropipettor (100 µl-1,000 µl)
  2. Micropipettor (10 µl-100 µl)
  3. Autoclave
  4. Stopwatch
  5. Measuring Pipette (5 ml) (Jaytec Glass Limited, catalog number: WJ.485 )
  6. 96-well microplate spectrophotometer (e.g. Molecular Devices, model: SpectraMax M2 device )
  7. Vacuum concentrator (e.g. Savant SPEEDVAC SVC 200H: 200 x g)
  8. Tabletop centrifuge
  9. Vortex mixer


  1. P. aeruginosa PAO1 culture and rhamnolipids extraction
    1. P. aeruginosa PAO1 was grown with agitation (175 rpm) at 37 °C for 18 h in a sterile culture tube (14 ml) containing 5 ml LB-MOPS medium (initial A600 of the culture was between 0.020 and 0.025).
    2. After incubation, culture of P. aeruginosa in mid-stationary phase was centrifuged (3,200 x g, 24 °C, 5 min) to obtain supernatant and pellet. 4 ml of supernatant was filtered (0.1 µm filters, 20 x g) to remove cells. Then, pH of cell-free supernatant was adjusted to 2.3 ± 0.2 using 1 N HCl (≈ 80 µl).
    3. Rhamnolipids were extracted by mixing 4 ml of supernatant with 4 ml of ethyl acetate. The non-miscible mixture was vigorously vortexed (600 rpm) for 20 sec and phase separation was realized by centrifugation in a tabletop centrifuge at speed 100 x g for 1 min.
    4. The upper, rhamnolipid-containing organic/ethyl acetate phase was transferred to a new reaction tube (30 ml). Ethyl acetate phase should be carefully removed without taking any liquid from the top aqueous layer.
    5. Extraction procedure was repeated three times and ethyl acetate extracts were combined in the reaction tube and evaporated to dryness in speedvac centrifugal evaporator (generally evaporated in 30 min at 200 x g speed) and immediately quantified.

  2. Rhamnolipids quantification
    1. Formation of chloroform-methylene blue complex
      Dry rhamnolipid-extract was dissolved in 4 ml chloroform and mixed with 400 µl methylene blue solution (freshly prepared). Tubes were vigorously mixed by vortexing for 5 min, then incubated at ambient temperature for 15 min to allow for color formation.
      Note: The chloroform phase will develop a blue color, proportional to the concentration of rhamnolipids. Cover tubes with aluminum foil to minimize chloroform evaporation during incubation [Figure 1(d)].

      Figure 1. Expected results during chemical complexation reaction step. (a). Methylene blue complexation in chloroform containing 100 µg/ml of rhamnolipids (1) and chloroform without rhamnolipids (2) at T5 min. (b). Methylene blue complexation in chloroform containing 100 µg/ml of rhamnolipids (1) and chloroform without rhamnolipids (2) at T10 min. (c). Methylene blue complexation in chloroform containing 100 µg/ml of rhamnolipids (1) and chloroform without rhamnolipids (2) at T15 min. (d). Methylene blue aqueous solution (MB) and chloroform containing 100 µg/ml, 50 µg/ml, 25 µg/ml, 12.5 µg/ml and 0 µg/ml of rhamnolipids complexed with methylene blue. T represents incubation time for complexation.

    2. Measurements step
      One ml of the chloroform (lower) phase was transferred in a clear microcentrifuge tube (2 ml) and mixed by vortexing for 20 sec with 500 µl of HCl 0.2 N. Phase separation was then realized by centrifugation in a tabletop centrifuge at speed 100 x g for 1 min and the solution was left at room temperature for 10 min to optimize methylene blue extraction in HCl. Finally, 200 µl of the upper acidic phase, containing a portion of the complexed methylene blue, was transferred in a 96-well microplate and the absorbance was measured at 638 nm with SpectraMax M2 device against an HCl 0.2 N blank.
    3. Rhamnolipids calibration curve generation
      Rhamnolipids standard solution was prepared by dissolving 8 mg of rhamnolipids in 4 ml of chloroform. This solution is used to generate different concentration of rhamnolipids by dilution in chloroform (i.e. 12.5 µg/ml, 25 µg/ml, 50 µg/ml, 100 µg/ml and 200 µg/ml). For each concentration, formation of chloroform-methylene blue complex and measurements steps were carried out to generate calibration curve equation (Figure 2; Table 1).

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


  1. Incubation time for blue color development in chloroform phase should be equal for each tested sample.
  2. During chloroform phase transfer avoid touching the methylene blue solution with tips (Video 1).

    Video 1. Lower chloroform phase transfer and upper acidic phase transfer

  3. During upper acidic phase transfer, avoid pipetting chloroform which will attack/dissolve 96-well microplate. For that, prefer a clear microcentrifuge tube or transfer the chloroform/HCl solution into a clear tube for better visualization of the separated phase before pipetting the upper acidic phase. Check that all wells are free of chloroform before absorbance measurement.
  4. During all transfer phases use new tips for each sample.


  1. LB-MOPS broth
    Add 25 g of dry LB powder and 10 g of MOPS to 1,000 ml of deionized water
    Adjust pH to 7.2 ± 0.2 by adding NaOH solution
    Autoclave for sterilization
  2. Borax buffer solution
    Dissolve one borate buffer pack in 500 ml of deionized water (each pack makes 50 mM borate, pH 8.5)
    Store at 2-8 °C
  3. Methylene blue aqueous solution
    Add 200 µl of methylene blue solution to 4.8 ml of deionized water
    Adjust pH to 8.6 ± 0.2 by adding 15 µl of a 50 mM borax buffer
    Store at room temperature


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.


  1. Chayabutra, C., Wu, J. and Ju, L. K. (2001). Rhamnolipid production by Pseudomonas aeruginosa under denitrification: effects of limiting nutrients and carbon substrates. Biotechnol Bioeng 72(1): 25-33.
  2. Chandrasekaran, E. V. and BeMiller, J. N. (1980). In: Whistler, R. L. (ed). Methods in carbohydrate chemistry. Academic press, p 89.
  3. Helbert, J. R. and Brown, K. D. (1957). Color reaction of anthrone with monosaccharide mixtures and oligo- and polysaccharides containing hexuronic acids. Anal Chem 29:1464-1466.
  4. Pinzon, N. M. and Ju, L. K. (2009). Analysis of rhamnolipid biosurfactants by methylene blue complexation. Appl Microbiol Biotechnol 82(5): 975-981.
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Copyright: © 2016 The Authors; exclusive licensee Bio-protocol LLC.
How to cite: Rasamiravaka, T., Vandeputte, O. M. and Jaziri, M. E. (2016). Procedure for Rhamnolipids Quantification Using Methylene-blue. Bio-protocol 6(7): e1783. DOI: 10.21769/BioProtoc.1783.

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Anushree Ghosh
Washington University in St. Louis
Hi ,
It is a very informative and useful protocol.
I tried the given protocol but I need to do the analysis at low rhamnolipid concentration(0- 9mg/l) but all my absorbance is very similar to each other .
Also, when I try with higher concentration(>10mg/l) there is kind of jelly like formation.Is it supposed to be like that?
I modified the protocol a bit and let the rhamnolipid extract onto the chloroform layer for 10 hours.
Do you have any suggestions?

6/8/2018 10:15:20 AM Reply
shuchi liao
brown university

very good and detailed protocol, thanks!
but I have two issues:

1. I didn't get the "expected results" as shown in Figure 1, instead, when concentration is low(0-5ug/mL) : pink color, when concentration larger than 100ug/mL, dark blue which is out of the detection limit of my UV-Vis equipment.( see the figure below)
2. the maximum adsorption peak is not at 638nm, is near 650nm

I'll appreciate it very much if you can help me to figure out my issues!
5/30/2018 1:47:07 PM Reply
Tsiry Rasamiravaka
Laboratoire de Biotechnologie Végétale, Université Libre de Bruxelles, Belgium

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 use
the 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.



6/8/2018 4:42:44 AM

shuchi liao
brown university

Dear Tsiry,

Got it, Thank you so much!

Shuchi Liao

6/8/2018 6:29:55 AM

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