Characterization Of Rhamnolipids (2009)

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Characterization of rhamnolipids produced by a Pseudomonas aeruginosa mutant strain grown on waste oils Zulfiqar A. Raza ab; Zafar M. Khalid a; Ibrahim M. Banat c a National Institute for Biotechnology and Genetic Engineering, Faisalabad, Pakistan b Department of Textile Engineering, University of Faisalabad, Faisalabad, Pakistan c School of Biomedical Sciences, Faculty of Life and Health Sciences, University of Ulster Coleraine, Northern Ireland, UK Online Publication Date: 01 November 2009

To cite this Article Raza, Zulfiqar A., Khalid, Zafar M. and Banat, Ibrahim M.(2009)'Characterization of rhamnolipids produced by a

Pseudomonas aeruginosa mutant strain grown on waste oils',Journal of Environmental Science and Health, Part A,44:13,1367 — 1373 To link to this Article: DOI: 10.1080/10934520903217138 URL: http://dx.doi.org/10.1080/10934520903217138

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Journal of Environmental Science and Health Part A (2009) 44, 1367–1373 C Taylor & Francis Group, LLC Copyright
ISSN: 1093-4529 (Print); 1532-4117 (Online) DOI: 10.1080/10934520903217138

Characterization of rhamnolipids produced by a Pseudomonas aeruginosa mutant strain grown on waste oils ZULFIQAR A. RAZA1,2 , ZAFAR M. KHALID1 and IBRAHIM M. BANAT3 1

National Institute for Biotechnology and Genetic Engineering, Faisalabad, Pakistan Department of Textile Engineering, University of Faisalabad, Faisalabad, Pakistan 3 School of Biomedical Sciences, Faculty of Life and Health Sciences, University of Ulster Coleraine, Northern Ireland, UK

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Pseudomonas aeruginosa EBN-8 mutant rhamnolipids produced on waste oils were investigated using normal-phase thin layer chromatography and fast atom bombardment mass spectrometry. Negative ion mode mass spectra yielded [M – H]− ions and their fragment ions, which gave some indications on the sequence of rhamnolipid biosynthesis. Five rhamnolipid homologs [viz. RC10 C10 (m/z 503), RC12 C10 or RC10 C12 (531), RRC10 C8 or RRC8 C10 (621), RRC10 C10 (649) and RRC12 C10 or RRC10 C12 (677)] were detected in four rhamnolipid combinations under the different carbon sources. The prevalence of rhamnolipids was confirmed by Fourier transform infrared and one-dimensional proton nuclear magnetic resonance. We also observed some correlations between the tensioactive characteristics and structural chemistry of the rhamnolipid surfactants. Keywords: Biosurfactant, mass spectrometry, NMR, Pseudomonas aeruginosa, rhamnolipid.

Introduction Glycolipids are the most common microbial surfactants reported in literature. They contain carbohydrate moieties linked to long-chain aliphatic acids or hydroxyl aliphatic acids. The most effective glycolipids with reference to the surface-active properties are trehalose lipids of Rhodococcus erythropolis,[1] sophorose lipids of Candida bombicola[2] and rhamnose lipids of Pseudomonas species.[3,4] Rhamnolipid has been identified and characterized as a prominent extra-cellular metabolite of Pseudomonas aeruginosa.[5] They are thought to play a role in both virulence and survival; however their exact function remains unclear. The rhamnolipid molecules contain one or two β-hydroxy fatty acids of various chain length (C8 -C22 ) esters linked to a mono-rhamnose (R) or di-rhamnose (RR) moiety and are produced as complex mixture by Pseudomonas sp.[6] The four most predominant studied rhamnolipid homologs of P. aeruginosa are RRC10 C10 , RC10 C10 , RRC10 and RC10 , obtained when using different hydrophilic and hydrophobic substrates.[7]

Address correspondence to Dr. Zulfiqar Ali Raza, Department of Textile Engineering, University of Faisalabad, Faisalabad, Pakistan. E-mail: [email protected] Received March 31, 2009.

The hydroxyl group of one of the fatty acids is involved in glycosidic linkage with the reducing end of rhamnose disaccharides, whereas the hydroxyl group of the second acid is involved in ester formation. The properties of rhamnolipid mixtures depend on the type and proportion of the constituent rhamnolipid homologs which vary according to the bacterial strain, culture conditions, process strategy and medium composition, particularly the carbon source.[8,9] Individually, rhamnolipids are non-toxic. The main component, rhamnose, is a sugar usually used as a food additive. The breakdown products of rhamnolipids are of little toxicological concern.[10] Rhamnolipids are biodegradable and have potential ability to replace synthetic surfactants.[11] Versatility in the biosurfactant structures imparts in them several assorted features including surface activity, aqueous dispersion of hydrocarbons and growth stimulation of hydrocarbons degrading microbes.[4,12−16] In the present investigation, purification and chemical characterization of rhamnolipids produced on waste oils was carried out using different analytical techniques. The heterogeneity in the number of rhamnose rings and in the composition of β-hydroxy fatty acid chains has been determined for the major as well as minor components present in the rhamnolipid mixtures. The surface-active and emulsification properties of aqueous solutions of the purified rhamnolipids have also been investigated to detect any correlations to the chemical composition of the rhamnolipids, under consideration.

1368 Materials and methods Materials

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The cell-free culture broths (CFCBs) of P. aeruginosa EBN8 mutant strain, grown on the minimal salts media containing either canola or soybean waste frying oils (WFOs), or either canola or soybean oil refinery wastes (ORWs) (obtained from a local vegetable oil refinery) as carbon substrates, were used as separate rhamnolipids’ sources. Silica gel (60 F254 )-coated aluminum sheets (20 × 20 cm), silica gel (230–400 mesh) and sulfuric acid (Merck); acetic acid, ethanol and α-naphthol (BDH); chloroform (Fisher); L-rhamnose (Hopkin & Williams); orcinol (Fluka), and methanol (Riedel-de Haen); all the chemicals were used without any pre-treatment. Cultivation conditions The EBN-8 mutant strain has previously been reported to produce biosurfactants using the abovementioned carbon sources in the iron-limited minimal salts media, under the shake flask conditions (37◦ C, pH 7 and 100 rpm) using a 1% v/v inoculum size.[17,18]

Raza et al. Fourier transform infrared (FT-IR) and one dimensional proton nuclear magnetic resonance (1 D 1 H NMR) analyses, up to 12 spots of the chloroform-dissolved rhamnolipids extract were applied on a TLC plate. For the recovery of the separated products, the portions of the none-sprayed plates were scratched off corresponding at the relevant expected points. The scrapings of horizontally aligned spots of same R f values were separately collected, and the rhamnolipids were re-extracted with two 8 mL volumes of chloroform:methanol (1:2, v/v) mixture. The solvent fraction was centrifuged at 8000 rpm for 10 min to remove the silica gel residue, solvent collected, micro-filtered and air dried.

Mass spectrometry A matrix of total rhamnolipid products was prepared in glycerol and its chemical analysis was performed on a double focusing mass spectrometer (JMS-HX110, JEOL, Tokyo) in negative ion mode, using a gun voltage of 1 kV, accelerating voltage of 10 kV and emission current of 10 mA. Xenon gas was used as a primary source of ionization under the pressure of 10−5 to 10−7 Torr. The scanning mass range was from 0 to 700 Da.

Preparative column chromatography Rhamnolipids were purified using column chromatography. Pooled crude biosurfactants’ extract was freeze-dried as described by Raza et al.[17] A column (30 × 4 cm) was prepared with 60 g activated silica gel slurry in hexane. One gram crude biosurfactants extract was dissolved in 5 mL of chloroform, micro-filtered (pore size: 3 µm) and loaded on the silica gel column. The loaded column was washed with hexane (approx. 200 mL) to remove any phospholipids from the product, then with chloroform (approx. 300 mL) to eliminate neutral lipids and finally with chloroform:methanol mobile phase applied in a sequence: 1:1 v/v (200 mL) and 1:2 v/v (200 mL) to elute rhamnolipids.[19] The flow rate was 1 mL min−1 . Separate biosurfactant fractions (20 mL size) were collected, freeze-dried and weighed; amongst them the total rhamnolipid fractions were assayed. Thin layer chromatography (TLC) Each of the purified rhamnolipid fractions was separately solubilized in chloroform (0.3 gL−1 ) and 10 µL of this solution were applied in triplicates on a silica gelcoated aluminum sheet. The thin layer chromatograms were developed in the chloroform/methanol/acetic acid (65/25/4, v/v) system and visualized using Molish reagent: α-naphthol (15% in ethanol)/sulfuric acid/ethanol/water (10.5/6.5/40.5/4.0, v/v).[20] The retardation factors (R f ) of the rhamnose lipid purple spots were determined. To have at least 25 mg of a purified rhamnolipid fraction, to perform

FT-IR and NMR analyses The IR spectra of TLC-purified rhamnolipids isolated from the EBN-8 mutant culture, cultivated on different carbon sources, were recorded on an FT-IR spectrometer (8201 PC, Shimadzu, Germany) in the spectral region 4000– 400 cm−1 at a resolution of 2 cm−1 , using a 0.23 mm KBr liquid cell. For 1 D 1 H NMR analysis, TLC-purified rhamnolipid product was re-dissolved in deuterated chloroform (CDCl3 ) and analyzed with an NMR machine (Avance 400 MHz, Bruker, Germany).

Physicochemical properties Equilibrated surface tension and interfacial tension (IFT) of the rhamnolipids solutions (0.1% w/v), prepared in 0.1 M NaHCO3 vs. hexadecane, were measured by using ¨ digital tensiometer (K10T, Kruss, ¨ a de Nouy Hamburg, Germany). Surface tension vs. dilutions curves (not shown) were plotted by measuring the surface tension of rhamnolipid dilutions in the range of 200–10 mg L−1 . The critical micelle concentration (CMC) was determined from the break point of the curve at which the surface tension of the solution abruptly increased. The emulsification index (E24 ) value of the rhamnolipids solution was determined using the method described by Cooper and Goldenberg.[21] All the experiments were carried out in triplicates. The data reported are averages of three analyses and typical variations in the results were less than 5%.

Characterization of rhamnolipids of P. aeruginosa

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Table 1. Different biosurfactants’ fractions within 1 g (portion) of crude biosurfactants produced by P. aeruginosa EBN-8 mutant grown on different carbon sources and extracted by column chromatography. Values are expressed in g/g or as specified otherwise.

Carbon source

Phospholipids (Hexane phase)

Neutral lipids (CHCl3 phase)

1st RLs fr (CHCl3 :CH3 OH, 1:1, v/v phase)

2nd RLs fr (CHCl3 :CH3 OH, 1:2, v/v phase)

RL’s part (1st+ 2nd frs) (%)

Canola WFO Soybean WFO Canola ORW Soybean ORW

0.22 ± 0.01 0.23 ± 0.01 0.20 ± 0.01 0.22 ± 0.01

0.22 ± 0.01 0.18 ± 0.01 0.29 ± 0.01 0.28 ± 0.01

0.22 ± 0.01 0.25 ± 0.01 0.20 ± 0.01 0.21 ± 0.01

0.34 ± 0.02 0.34 ± 0.02 0.31 ± 0.02 0.29 ± 0.01

56.00 59.00 51.00 50.00

RLs = rhamnolipids, fr = fraction, WFO = waste frying oil, ORW = oil refinery waste.

Results

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Quantitative analysis of biosurfactants produced P. aeruginosa cultures have been reported to produce higher rhamnolipid concentrations when grown on waterimmiscible substrates (hydrocarbons and vegetable oils) than when growing on water-soluble carbon sources (glucose, glycerol, ethanol and manitol).[22] The present study confirmed the ability of industrial wastes of vegetable origin to be used as carbon sources for rhamnolipids production reported earlier.[17,18] The renewable waste materials are preferable carbon sources for biosurfactant production for the added advantage of cost reduction and sustainability for industrial output.[23,24] A comparison of different biosurfactant component fractions, collected during column chromatography of crude biosurfactants is shown in Table 1. Total rhamnolipid yields were highest using soybean WFO as carbon source (59.0% w/w) of the crude extract and slightly less rhamnolipid yields 50.0–56.0% w/w were obtained using the other waste oils (Table 1). The average incorporation of phospholipids and neutral lipids in the crude extracts were 22 and 24%, respectively.

Severance and identification of rhamnolipid homologs Most previous studies on rhamnolipids production[19,20,25] using P. aeruginosa strains grown on different substrates and under various fermentation conditions have reported two prime rhamnolipids of RC10 C10 (C26 H48 O9 ; Mol. mass = 504 amu) and RRC10 C10 (C32 H58 O13 ; Mol. mass = 650 amu). Two supplementary hydrophilic rhamnolipid compounds containing one β-hydroxydecanoic acid unit (RRC10 and RC10 ) have also been occasionally reported.[5] In our case, the TLC extracts showed mixtures of different rhamnolipid homologs in two main groups a monorhamnolipids (R f = 0.73) and di-rhamnolipids (R f = 0.52). Di-rhamnolipids were the predominant species over mono-rhamnolipids with all the carbon sources examined, however only the di-rhamnolipids were detected with canola WFO (Table 2).

The mass spectrometric analysis of the rhamnolipid structures (Fig. 1) showed several types of rhamnolipid homologs with their molecular masses ranging from m/z 504 to 678. The proton abstraction of rhamnolipid molecules yielded [M – H]− anions at m/z 503 (from RC10 C10 ), 531 (RC12 C10 or RC10 C12 ), 621 (RRC10 C8 or RRC8 C10 ), 649 (RRC10 C10 ) and 677 (RRC12 C10 or RRC10 C12 ). Different carbon sources led to different combinations of rhamnolipid species in different proportions (Table 2). Soybean WFO induced the widest diversity in its associated rhamnolipids mixture in the range of m/z 503–677. The mass spectra confirmed the presence of two major classes of rhamnolipids, i.e. mono- and di-rhamnolipids with the latter being the predominant in all the samples analyzed. Several researchers reported the predominance of mono-rhamnolipid RC10 C10 ,[20,26,27] while others reported di-rhamnolipid RRC10 C10 as the main component of the rhamnolipid mixtures.[9,28,29] Our crude extract had RRC10 C10 as the predominant component with all the carbon sources tested, and the rhamnolipids with C8 or C12 fatty acid chain were present as minor components (Table 2). For the identification of characteristic functional groups, the FT-IR spectra of rhamnolipids were recorded in the spectral region of 4000–400 cm−1 (IR spectra are not shown). Several C-H stretching bands of -CH2 - and -CH3 groups were observed in the region 3000–2700 cm−1 . The carbonyl stretching peak was observed at 1635 cm−1 , which is characteristic of ester compounds. The ester carbonyl group was also confirmed from the peak at 1013 cm−1 , which corresponds to C-O stretching vibration. The lack of characteristic bands for organic acids that usually appear at 3500–2700 cm−1 , 1635–1650 cm−1 and 1013–1018 cm−1 indicates the presence of an ester compound. The prevalence of rhamnolipids in the glycolipid biosurfactants was confirmed by the 1 D 1 H NMR analysis. The NMR spectroscopy is based on transitions in atoms with a magnetic moment under the influence of applied external magnetic field. The data of 1 H shifts of the absorption frequencies are shown in Table 3. The NMR results indicate that the purified biosurfactant comprises two principal rhamnolipid homologs, i.e. RC10 C10 and RRC10 C10 .

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Fig. 1. Representative negative mode mass spectra of P. aeruginosa EBN-8 mutant showing rhamnolipids produced on soybean waste frying oil (upper) and soybean oil refinery waste (lower) as carbon sources.

Physicochemical properties of rhamnolipids Rhamnolipid mixtures are viscous honey color materials, highly soluble in chloroform, methanol, ethyl acetate, diethyl ether, alkaline aqueous media including ammonium bicarbonate solution, slowly soluble in water in pH range of 6.5–7.5, insoluble in hexane and petroleum ether, and precipitate at pH ≤ 2.[30] The most frequently used criteria for biosurfactant’s performance evaluation have been the surface and interfacial tensions, and the CMC. Once the rhamnolipids are excreted into the nutrients medium

up to the level of CMC, they reduce the surface tension of the medium to ≤30 mN m−1 . Below the CMC, all biosurfactant hydrophilic monomer head groups accumulate at the air/water interface, whereas above the CMC, the hydrophobic interactions between the amphiphiles of biosurfactant molecules may contribute to create micro-emulsions in which micellization takes place.[31] The CMC values of 25–45 mg L−1 of purified rhamnolipids produced on either substrate (Table 4) were consistent with the values (27–54 mg L−1 ) reported by MillerMaier and Bodour.[32] The lowest CMC of 25 mg L−1 was

Table 2. Negative mode mass spectrometry results of P. aeruginosa EBN-8 mutant rhamnolipids (RLs) produced using different carbon sources. C-source Canola WFO Soybean WFO

Canola ORW

Soybean ORW

RL structure

Pseudomolecular ion (m/z)

RA

Ion fragment (m/z)

RRC10 C10 RC10 C10 RC12 C10 /RC10 C12 RRC10 C8 /RRC8 C10 RRC10 C10 RRC12 C10 /RRC10 C12 RC10 C10 RRC10 C8 /RRC8 C10 RRC10 C10 RRC12 C10 /RRC10 C12 RC10 C10 RRC10 C8 /RRC8 C10 RRC10 C10 RRC12 C10 /RRC10 C12

649 503 531 621 649 677 503 621 649 677 503 621 649 677

40 25 7 8 60 9 10 6 20 4 18 8 50 9

479, 367, 313, 169 333, 169, 163 333, 311, 169, 163 479, 311, 169 479, 311, 169, 163 479, 311, 169, 163 333, 169, 133 169 479, 169 479, 169 333, 169, 133 451, 169 479, 169 479, 169

RA = Relative abundance, WFO = waste frying oil, ORW = oil refinery waste.

Characterization of rhamnolipids of P. aeruginosa Table 3. 1 D 1 H NMR chemical shifts (in ppm) of principal rhamnolipids produced by EBN-8 mutant. Moiety Rhamnose(s)

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Hydroxy fatty acid

Proton location 

C-1  C-2  C-3  C-4  C-5 -CH3 (ring)  C-1  C-2  C-3  C-4  C-5 -CH3 (ring) C-1 C-2 C-3 -(CH2 )5 -CH3 -CH2 -COO-COO-CH2 -O-C-H

RC10 C10

RRC10 C10

4.69 3.63 3.60 3.47 3.59 1.23–1.47 — — — — — — 4.14 2.33 1.63 1.23–1.47 0.88 2.33 4.92 4.19

4.71 4.14 3.51 3.46 3.49 1.24–1.47 4.73 3.73 3.49 3.32 3.62 1.23–1.31 4.19 2.33 1.58 1.23–1.31 0.85 2.31 4.78 4.69

observed with the solution of purified rhamnolipids mixture, containing higher concentrations of the less mobile (di-rhamnolipid) homologs of RRC10 C8 /RRC8 C10 , RRC10 C10 and RRC12 C10 /RRC10 C12 produced with soybean WFO as carbon source, while its corresponding CFCB exhibited the CMC of 42 mg L−1 . A similar trend was observed with each of the carbon sources that lower amounts of purified rhamnolipids are required to reach the CMC values as compared to that of crude biosurfactants in their respective CFCBs. A significant difference of 48 mg L−1 was observed between the CMC values of purified rhamnolipids produced with soybean ORW and that of its respective CFCB (Table 4).

Discussion The predominance of mono or di-rhamnolipid is most likely a result of carbon sources and/or due to the specific cultivation conditions. The rhamnolipids produced by EBN-8 mutant differed both quantitatively and qualitatively with the carbon source. This suggests that the production of a desirable rhamnolipid homolog can be directed simply by controlling the carbon source. Conversely, Mata-Sandoval et al.[33] found that P. aeruginosa UG2 could produce rhamnolipid mixtures of the similar compositions in spite of using different carbon sources while Deziel et al.[34] reported that rhamnolipids produced by P. aeruginosa 57RP strain differed both in quantity and pro-

1371 portion depending on the carbon source. Syldatk et al.[5] proposed that environmental conditions and fermentation approach can also affect the distribution of rhamnolipid homologs. The analysis of mass spectra of different rhamnolipids led to a sub-conclusion that some fragments are common to all rhamnolipid structures, for example, [RRC10 C10 – H]− ion of m/z 649 fragmented to form two major fragment − ions of m/z 479 (RRC− 10 ) and 169 (C10 ) (Fig. 1). The mass spectrum peak at m/z 479 was detected in two of the rhamnolipid congeners (RC10 C10 and RRC12 C10 /RRC10 C12 ), which represents the rupture of ester link between two alkylic chains of rhamnolipids; and the m/z 169 ion peak represents the fatty acid moiety with the loss of additional hydrogen. The other characteristic peak in mass spectrum appeared at m/z 311, which corresponds to the rupture of the link in the rhamnose-alkylic chain of di-rhamnolipids. Other common peaks were detected at m/z 163 and 333 (RC− 10 ) in the mass spectra for two mono-rhamnolipids (RC10 C10 and RC12 C10 /RC10 C12 ), which correspond to the rupture of the rhamnose-alkylic chain and the ester links, respectively. The proportions of different constituents of rhamnolipid mixtures were obtained from the relative intensities of their [M – H]− ions. According to Deziel et al.[34] most of the ions above m/z 447 are rhamnolipid pseudomolecular [M – H]− ions and the most ions between m/z 163 and 503 are fragment ions produced by cleavage. Interestingly, twenty eight distinct rhamnolipid homologs have been reported that differ in fatty acid chain composition as well as in a number of rhamnose moieties. Nevertheless, these variants have always been found as minor components of the rhamnolipid mixtures. The variations observed in the surface-active properties of different rhamnolipid mixtures, obtained by using different carbon sources, are mostly due to the different profiles of rhamnolipid homologs produced. Mata-Sandoval et al.[33] suggested that larger fatty acid chains confer higher hydrophobicity to the rhamnolipid molecules, and so they start micellization at lower concentrations than the species richer in shorter chain fatty acids. Haba et al.[29] suggested that lower contribution of the hydrophobic dirhamnolipid to the rhamnolipids mixture could raise the CMC higher than that of a rhamnolipids mixture richer in mono-rhamnolipid contents. The mono-rhamnolipids have higher proportion of fatty acids, which makes them more hydrophobic while an extra rhamnose ring confers more hydrophilicity to di-rhamnolipid and additional homologous fatty acid chain can increase its hydrophobicity. The E24 values of the purified rhamnolipid mixtures were higher than that of the respective CFCBs containing the crude biosurfactants. A general correlation was also observed between the rhamnolipids’ composition and its E24 . The E24 values of the mixed component rhamnolipids solutions, containing higher relative abundance of less mobile homologs such as di-rhamnolipids were higher (as with

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Table 4. Surface-active characteristics of biosurfactant containing P. aeruginosa EBN-8 cell-free culture broth (CFCB) produced on different carbon sources in comparison to purified rhamnolipids (RLs) solution (0.1% w/v) prepared in 0.1 M NaHCO3 . CFCB with crude biosurfactants

Purified RLs solution

C-source

ST (mN m−1 )

IFT (mN m−1 )

E24 (%)

CMC (mg L−1 )

ST (m Nm−1 )

IFT (mN m−1 )

E24 (%)

Pure RLs CMC (mg L−1 )

Canola WFO Soybean WFO Canola ORW Soybean ORW

32.0 ± 1.0 30.3 ± 0.9 28.5 ± 0.7 29.3 ± 0.5

>1 >1 0.70 ± 0.03 0.90 ± 0.04

62 ± 4.2 68 ± 4.6 68 ± 0.4 65 ± 0.4

45 ± 2 42 ± 2 56 ± 3 75 ± 4

29.0 ± 0.7 28.5 ± 0.7 30.0 ± 0.8 28.9 ± 0.6

0.9 ± 0.1 1.2 ± 0.1 1.0 ± 0.1 1.0 ± 0.1

70 ± 3 75 ± 4 68 ± 3 73 ± 4

35 ± 3 25 ± 2 45 ± 4 27 ± 2

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Results represent averages with ± S. E. from three independent experiments. ST = surface tension, IFT = interfacial tension, E24 = emulsification index, CMC = critical micelle concentration, WFO = waste frying oil, ORW = oil refinery waste.

soybean WFO) than the rhamnolipids solutions containing their lesser contribution (as with canola ORW) (Table 4). Such patterns could be due to four possible reasons: (i) different rhamnolipid mixtures produce different micellar arrangements depending upon the input of diverse rhamnolipid species, (ii) in addition to rhamnolipids, other types of biosurfactants including phospholipids and/or neutral lipids may also involve in micellization, (iii) the presence and contribution of coagulates and traces of residual growth substrates, and the complex formation between biosurfactants and the cationic species of the media, and (iv) the presence of impurities such as non-metabolized coextracted fatty acids from the culture media.

Conclusion In conclusion, the structure of a biosurfactant is particularly important in conferring surface-active properties and molecular aggregates formation. Carbon source selection for production directly affects the type of rhamnolipid homolog product. Future cost effective production depends on finding improved biosynthetic strategies and downstream processing needed to obtain the level of product purity of rhamnolipid desirable for particular applications. Crude rhamnolipids containing broths seem suitable for many environmental remediation applications however a host of interesting features of purified rhamnolipids has a lot of potential in many biomedical applications.[35] Moreover investigating the effects of carbon sources on the mixed rhamnolipid species produced, and the distinct roles and characteristics of each homolog produced are of great importance to be better elucidated, both in individual and mutual properties.

Acknowledgments The authors acknowledge the Director, International Center for Chemical and Biological Sciences, University of Karachi for the mass spectrometry and 1 H NMR facili-

ties and the Higher Education Commission, Islamabad for the research funds.

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