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Procedure for Rhamnolipids Quantification Using Methylene-blue

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Jul 2015



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 (鼠李糖脂), Pseudomonas aeruginosa (铜绿假单胞菌), Methylene-blue (亚甲基蓝), Complexation (络合), Quantification (量化)

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.


由绿脓杆菌(铜绿假单胞菌)产生的鼠李糖脂代表一组具有各种应用的生物表面活性剂(例如生物修复的溢油,化妆品,洗涤剂和清洁剂)。常用的鼠李糖脂定量的比色方法,包括蒽酮,苯酚 - 硫酸和基于苔藓醇的定量(Helbert和Brown,1957; Chandrasekaran和BeMiller,1980)是费力和操作上危险的,因为强酸/化学散发仪器测量的恶化(例如分光光度计)。因此,基于亚甲蓝的分析显示为基于化学络合反应安全定量整个鼠李糖脂分子的有希望的替代物(Pinzon和Ju,2009)。事实上,亚甲基蓝和鼠李糖脂在水 - 氯仿相系统中形成复合物。将鼠李糖脂 - 亚甲基蓝复合物分配到氯仿相中,其将形成蓝色,其可以在638nm下定量以推断鼠李糖脂浓度。在这里,我们描述基于亚甲基蓝的鼠李糖脂定量程序的变体,允许在标准96孔塑料微量滴定板相反的原始亚甲蓝程序,由于氯仿化学属性需要特定和昂贵的微孔板分光光度定量。

关键字:鼠李糖脂, 铜绿假单胞菌, 亚甲基蓝, 络合, 量化


  1. 硼酸盐缓冲液BupH TM (Thermo Fisher scientific,目录号:28384)
  2. 离心圆底反应管(30ml)(Krackeler Scientific,COREX ,目录号:1-8445-30)
  3. 96孔板(透明的聚苯乙烯孔平底)(Sigma-Aldrich,目录号:M3687-60EA)。
  4. 微量离心管[2ml,带有盖的透明聚丙烯(PP)](Sigma-Aldrich,Brand ,目录号:Z628034-500EA)
  5. 无菌培养管(PP)(14ml)(Lab Depot Inc.,目录号:TLDT8235)
  6. 使用Durapore PVDF膜UFC40VV25 Ultrafree(2ml)(Merck Millipore Corporation,目录号:UFC40VV25)的离心过滤器(0.1μm孔径,非无菌)
  7. 铝箔
  8. 微量移液器(10μl,100μl和1,000μl)提示
  9. 绿脓杆菌(绿脓杆菌)PAO1野生型(菌株PAO0001)(http://www.pseudomonas.med.ecu.edu/
  10. 氯仿(≥99%)(Carl Roth GmbH& Co.KG,目录号:3313.1)
  11. 乙酸乙酯(> 99.5%)(Merck Millipore Corporation,EMSURE ,目录号:1096231000)
  12. 盐酸(HCl)标准(1N溶液)(Sigma-Aldrich,Fluka analytical,目录号:318949-500ml)
  13. HCl标准品(0.2N水溶液)(Sigma-Aldrich,Fluka analytical,目录号:343102-500ml)
  14. Luria Bertani(LB)肉汤(Lennox)粉末微生物生长培养基(Sigma-Aldrich,目录号:L3022-250G)
  15. 亚甲基蓝溶液[在95%乙醇中的1.4%(w/v)](Sigma-Aldrich,目录号:1808-50ml)
  16. 3-(N-吗啉代)丙磺酸(MOPS)(99.5%)(Sigma-Aldrich,目录号:M1254-100G)
  17. 鼠李糖脂(90%,标准)(Sigma-Aldrich,目录号:R90)
  18. 氢氧化钠(NaOH)溶液
  19. LB-MOPS肉汤(见配方)
  20. 硼砂缓冲液(50mM)(参见配方)
  21. 亚甲蓝水溶液(见配方)


  1. 微量移液器(100μl-1000μl)
  2. 微量移液器(10μl-100μl)
  3. 高压灭菌器
  4. 秒表
  5. 测量移液管(5ml)(Jaytec Glass Limited,目录号:WJ.485)
  6. 96孔微板分光光度计(例如Molecular Devices,型号:SpectraMax M2装置)
  7. 真空浓缩器( Savant SPEEDVAC SVC 200H:200 x g )
  8. 台式离心机
  9. 涡流搅拌器


  1. p。铜绿假单胞菌 PAO1培养和鼠李糖脂提取
    1. p。铜绿假单胞菌PAO1在37℃下搅拌(175rpm)生长18小时 在含有5ml LB-MOPS培养基的无菌培养管(14ml)中 (培养物的初始A 600在0.020和0.025之间)。
    2. 后 ?孵育,培养P 。 离心(3200×g,24℃,5分钟),得到上清液和沉淀。 4 ?过滤上清液(0.1μm过滤器,20×g/g)以除去 细胞。然后,使用,将无细胞上清液的pH调节至2.3±0.2 ?1N HCl(≈80μl)。
    3. 通过混合4ml提取鼠李糖脂 的上清液与4ml乙酸乙酯。不可混溶的混合物 剧烈涡旋(600rpm)20秒,并进行相分离 通过在台式离心机中以100×g /分钟的速度离心来实现 ?1分钟。
    4. 上层含有鼠李糖脂的有机/乙酸乙酯 将相转移到新的反应管(30ml)中。乙酸乙酯 相应小心地移除,而不从顶部取任何液体 ?水层。
    5. 提取程序重复三次 和乙酸乙酯萃取液在反应管中合并 在speedvac离心蒸发器(一般 在200×g/min速度下在30分钟内蒸发)并立即定量。

  2. 鼠李糖脂定量
    1. 氯仿 - 亚甲蓝复合物的形成
      干 将鼠李糖脂提取物溶解在4ml氯仿中并与400混合 μl亚甲基蓝溶液(新鲜制备)。剧烈振动 通过涡旋混合5分钟,然后在环境温度下温育 15分钟以允许颜色形成 注意:氯仿相会 ?发展蓝色,与浓度成正比 鼠李糖脂。覆盖管铝箔,以减少氯仿 孵化期间的蒸发[图1(d)]。

      图1.预期 结果在化学络合反应步骤。 (a)。亚甲基蓝 在含有100μg/ml鼠李糖脂(1)和氯仿的氯仿中的络合作用 氯仿,没有鼠李糖脂(2)在T 5分钟。 (b)。亚甲基蓝 在含有100μg/ml鼠李糖脂(1)和氯仿的氯仿中的络合作用 氯仿,没有鼠李糖脂(2)在T 10分钟。 (C)。亚甲基蓝 在含有100μg/ml鼠李糖脂(1)和氯仿的氯仿中的络合作用 氯仿,没有鼠李糖脂(2)在T 15分钟。 (d)。亚甲基蓝 水溶液(MB)和含有100μg/ml,50μg/ml,25μg/ml的氯仿 μg/ml,12.5μg/ml和0μg/ml的与亚甲基复合的鼠李糖脂 蓝色。 T代表络合的孵育时间
    2. 测量步骤
      将1ml氯仿(下)相转移到澄清的 微量离心管(2ml),并通过用500涡旋20秒混合 μl的HCl 0.2N。然后通过离心进行相分离 ?台式离心机以速度100×g离心1分钟,并且溶液是 在室温下放置10分钟以优化亚甲基蓝 在HCl中萃取。最后,200μl的上部酸性相,含有 ?一部分络合的亚甲蓝,转移到96孔中 ?并使用SpectraMax M2在638nm测量吸光度 ?装置对抗HCl 0.2N空白
    3. 鼠李糖脂校准曲线生成
      鼠李糖脂标准溶液通过将8mg的 鼠李糖脂在4ml氯仿中的溶液。此解决方案用于生成 通过在氯仿中稀释(即12.5μg/ml,25μg/ml,50μg/ml,100μg/ml和200μg/ml)的不同浓度的鼠李糖脂。每个 浓度,氯仿 - 亚甲蓝复合物的形成 进行测量步骤以产生校准曲线 方程(图2;表1)。


图2.不同浓度的鼠李糖脂(12.5μg/ml,25μg/ml,50μg/ml,100μg/ml和200μg/ml)的预期校准曲线。复合亚甲基蓝的吸光度在638nm下用SpectraMax M2装置针对HCl(0.2N)空白测量。复合和测量重复三次,误差条表示平均值的标准误差
表1.在638nm下用SpectraMax M2装置对HCl(0.2N)空白测量的络合亚甲基蓝的吸光度



  1. 氯仿相中蓝色显色的孵育时间对于每个测试样品应当相等
  2. 在氯仿相转移期间避免接触亚甲基蓝溶液(见视频1)。

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  3. 在上部酸性相转移期间避免移取氯仿,其将攻击/溶解96孔微板。为此,优选清洁的微量离心管或将氯仿/HCl溶液转移到透明管中以在移取上部酸性相之前更好地观察分离的相。在测量吸光度之前,检查所有孔都没有氯仿。
  4. 在所有转移阶段,为每个样本使用新提示。


  1. LB-MOPS肉汤
    将25g干LB粉末和10g MOPS加入到1,000ml去离子水中
    通过加入NaOH溶液将pH调节至7.2±0.2 高压灭菌器
  2. 硼砂缓冲液
  3. 亚甲蓝水溶液
    通过加入15μl50mM硼砂缓冲液将pH调节至8.6±0.2 在室温下贮存


该协议改编自以前发表的研究(Pinzon和Ju,2009)。这项工作得到了PIC-Madagascar项目2009年和ARES-CCD(比利时的Acédémiede Recherche et d'EnseignementSupérieur-CommissionCoopérationauDéveloppement)的博士后研究金方案"ELAN 2015"的支持。我们声明没有利益冲突。


  1. Chayabutra,C.,Wu,J.and Ju,L.K。(2001)。 反硝化后由绿脓假单胞菌生产鼠李糖脂:限制营养物和碳的影响底物。 Biotechnol Bioeng 72(1):25-33
  2. Chandrasekaran,E.V.and BeMiller,J.N。(1980)。 In:Whistler,R.L。(ed)。碳水化合物化学方法。学术出版社,第89页
  3. Helbert,J.R。和Brown,K.D。(1957)。 蒽酮与单糖混合物和含有己糖醛酸的寡糖和多糖的显色反应。 em> Anal Chem 29:1464-1466。
  4. Pinzon,N.M。和Ju,L.K。(2009)。 通过亚甲基蓝络合作用分析鼠李糖脂生物表面活性剂。 Appl Microbiol Biotechnol < em> 82(5):975-981。
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Copyright: © 2016 The Authors; exclusive licensee Bio-protocol LLC.
引用: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.



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