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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
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 Journal of Environmental Science and Health Part A (2009) 44, 1367–1373
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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
2
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
Downloaded By: [Raza, Zulfiqar Ali] At: 18:47 5 October 2009

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 The hydroxyl group of one of the fatty acids is involved

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 Rhodococ-
cus erythropolis,[1] sophorose lipids of Candida bombicola[2]
and rhamnose lipids of Pseudomonas species.[3,4] Rhamno-
lipid 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 sur-
vival; 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 hydropho-
bic substrates.[7]
Address correspondence to Dr. Zulfiqar Ali Raza, Department
of Textile Engineering, University of Faisalabad, Faisalabad,
Pakistan. E-mail: zarazapk@yahoo.com
Received March 31, 2009.
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 rham-
nolipid 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 com-
ponent, rhamnose, is a sugar usually used as a food additive.
The breakdown products of rhamnolipids are of little toxi-
cological 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, aque-
ous 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 deter-
mined for the major as well as minor components present
in the rhamnolipid mixtures. The surface-active and emul-
sification properties of aqueous solutions of the purified
rhamnolipids have also been investigated to detect any cor-
relations to the chemical composition of the rhamnolipids,
under consideration.
 1368
Materials and methods
Materials
The cell-free culture broths (CFCBs) of P. aeruginosa EBN-
8 mutant strain, grown on the minimal salts media contain-
ing either canola or soybean waste frying oils (WFOs), or
either canola or soybean oil refinery wastes (ORWs) (ob-
tained from a local vegetable oil refinery) as carbon sub-
strates, were used as separate rhamnolipids’ sources. Silica
gel (60 F254 )-coated aluminum sheets (20 × 20 cm), sil-
ica 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
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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]
Preparative column chromatography
Rhamnolipids were purified using column chromatogra-
phy. 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 phos-
pholipids from the product, then with chloroform (approx.
300 mL) to eliminate neutral lipids and finally with chloro-
form: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 frac-
tions (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 sepa-
rately solubilized in chloroform (0.3 gL−1 ) and 10 µL of
this solution were applied in triplicates on a silica gel-
coated 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
Raza et al.
Fourier transform infrared (FT-IR) and one dimensional
proton nuclear magnetic resonance (1 D 1 H NMR) anal-
yses, up to 12 spots of the chloroform-dissolved rhamno-
lipids 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 ex-
pected points. The scrapings of horizontally aligned spots
of same R f values were separately collected, and the rham-
nolipids were re-extracted with two 8 mL volumes of chlo-
roform: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.
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 rham-
nolipid 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
a de Noüy digital tensiometer (K10T, Krüss, Hamburg,
Germany). Surface tension vs. dilutions curves (not shown)
were plotted by measuring the surface tension of rhamno-
lipid dilutions in the range of 200–10 mg L−1 . The criti-
cal 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 re-
ported are averages of three analyses and typical variations
in the results were less than 5%.
 Characterization of rhamnolipids of P. aeruginosa
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.
Phospholipids Neutral lipids
Carbon source (Hexane phase) (CHCl3 phase)
Canola WFO 0.22 ± 0.01 0.22 ± 0.01
Soybean WFO 0.23 ± 0.01 0.18 ± 0.01
Canola ORW 0.20 ± 0.01 0.29 ± 0.01
Soybean ORW 0.22 ± 0.01 0.28 ± 0.01
RLs = rhamnolipids, fr = fraction, WFO = waste frying oil, ORW = oil refinery waste.
Results
Quantitative analysis of biosurfactants produced
P. aeruginosa cultures have been reported to produce
higher rhamnolipid concentrations when grown on water-
Downloaded By: [Raza, Zulfiqar Ali] At: 18:47 5 October 2009
immiscible substrates (hydrocarbons and vegetable oils)
than when growing on water-soluble carbon sources (glu-
cose, 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 rhamno-
lipid 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 differ-
ent rhamnolipid homologs in two main groups a mono-
rhamnolipids (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 exam-
ined, however only the di-rhamnolipids were detected with
canola WFO (Table 2).
1369
1st RLs fr 2nd RLs fr RL’s part
(CHCl3 :CH3 OH, (CHCl3 :CH3 OH, (1st+ 2nd frs)
1:1, v/v phase) 1:2, v/v phase) (%)
0.22 ± 0.01 0.34 ± 0.02 56.00
0.25 ± 0.01 0.34 ± 0.02 59.00
0.20 ± 0.01 0.31 ± 0.02 51.00
0.21 ± 0.01 0.29 ± 0.01 50.00
The mass spectrometric analysis of the rhamnolipid
structures (Fig. 1) showed several types of rhamnolipid ho-
mologs 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 ). Dif-
ferent carbon sources led to different combinations of
rhamnolipid species in different proportions (Table 2).
Soybean WFO induced the widest diversity in its associ-
ated 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 sam-
ples analyzed. Several researchers reported the predomi-
nance of mono-rhamnolipid RC10 C10 ,[20,26,27] while others
reported di-rhamnolipid RRC10 C10 as the main compo-
nent 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 preva-
lence of rhamnolipids in the glycolipid biosurfactants was
confirmed by the 1 D 1 H NMR analysis. The NMR spec-
troscopy 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 pu-
rified biosurfactant comprises two principal rhamnolipid
homologs, i.e. RC10 C10 and RRC10 C10 .
 1370 Raza et al.
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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, di-
ethyl 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 crite-
ria 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 ten-
sion of the medium to ≤30 mN m−1 . Below the CMC,
all biosurfactant hydrophilic monomer head groups ac-
cumulate at the air/water interface, whereas above the
CMC, the hydrophobic interactions between the am-
phiphiles of biosurfactant molecules may contribute to cre-
ate micro-emulsions in which micellization takes place.[31]
The CMC values of 25–45 mg L−1 of purified rhamno-
lipids produced on either substrate (Table 4) were consis-
tent with the values (27–54 mg L−1 ) reported by Miller-
Maier 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.

Pseudomolecular
C-source RL structure ion (m/z) RA Ion fragment (m/z)

Canola WFO RRC10 C10 649 40 479, 367, 313, 169

Soybean WFO RC10 C10 503 25 333, 169, 163
RC12 C10 /RC10 C12 531 7 333, 311, 169, 163
RRC10 C8 /RRC8 C10 621 8 479, 311, 169
RRC10 C10 649 60 479, 311, 169, 163
RRC12 C10 /RRC10 C12 677 9 479, 311, 169, 163
Canola ORW RC10 C10 503 10 333, 169, 133
RRC10 C8 /RRC8 C10 621 6 169
RRC10 C10 649 20 479, 169
RRC12 C10 /RRC10 C12 677 4 479, 169
Soybean ORW RC10 C10 503 18 333, 169, 133
RRC10 C8 /RRC8 C10 621 8 451, 169
RRC10 C10 649 50 479, 169
RRC12 C10 /RRC10 C12 677 9 479, 169
RA = Relative abundance, WFO = waste frying oil, ORW = oil refinery waste.
 Characterization of rhamnolipids of P. aeruginosa 1371

Table 3. 1 D 1 H NMR chemical shifts (in ppm) of principal portion depending on the carbon source. Syldatk et al.[5]
rhamnolipids produced by EBN-8 mutant. proposed that environmental conditions and fermentation
approach can also affect the distribution of rhamnolipid
Moiety Proton location RC10 C10 RRC10 C10
homologs.

Rhamnose(s) C-1 4.69 4.71 The analysis of mass spectra of different rhamnolipids

C-2 3.63 4.14 led to a sub-conclusion that some fragments are common to
C-3

3.60 3.51 all rhamnolipid structures, for example, [RRC10 C10 – H]−

C-4 3.47 3.46 ion of m/z 649 fragmented to form two major fragment
ions of m/z 479 (RRC− −

C-5 3.59 3.49 10 ) and 169 (C10 ) (Fig. 1). The mass
-CH3 (ring) 1.23–1.47 1.24–1.47 spectrum peak at m/z 479 was detected in two of the rham-

C-1 — 4.73 nolipid congeners (RC10 C10 and RRC12 C10 /RRC10 C12 ),

C-2 — 3.73 which represents the rupture of ester link between two

C-3 — 3.49 alkylic chains of rhamnolipids; and the m/z 169 ion peak

C-4 — 3.32 represents the fatty acid moiety with the loss of addi-

C-5 — 3.62 tional hydrogen. The other characteristic peak in mass
-CH3 (ring) — 1.23–1.31 spectrum appeared at m/z 311, which corresponds to
Hydroxy fatty acid C-1 4.14 4.19 the rupture of the link in the rhamnose-alkylic chain of
C-2 2.33 2.33
di-rhamnolipids.
C-3 1.63 1.58
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-(CH2 )5 - 1.23–1.47 1.23–1.31

-CH3 0.88 0.85
-CH2 -COO- 2.33 2.31
-COO-CH2 - 4.92 4.78
-O-C-H 4.19 4.69
observed with the solution of purified rhamnolipids mix-
ture, containing higher concentrations of the less mo-
bile (di-rhamnolipid) homologs of RRC10 C8 /RRC8 C10 ,
RRC10 C10 and RRC12 C10 /RRC10 C12 produced with soy-
bean 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 spe-
cific cultivation conditions. The rhamnolipids produced by
EBN-8 mutant differed both quantitatively and qualita-
tively with the carbon source. This suggests that the pro-
duction of a desirable rhamnolipid homolog can be di-
rected simply by controlling the carbon source. Conversely,
Mata-Sandoval et al.[33] found that P. aeruginosa UG2
could produce rhamnolipid mixtures of the similar com-
positions 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-
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 in-
tensities of their [M – H]− ions. According to Deziel et al.[34]
most of the ions above m/z 447 are rhamnolipid pseudo-
molecular [M – H]− ions and the most ions between m/z
163 and 503 are fragment ions produced by cleavage. Inter-
estingly, 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 compo-
nents of the rhamnolipid mixtures.
The variations observed in the surface-active properties
of different rhamnolipid mixtures, obtained by using dif-
ferent carbon sources, are mostly due to the different pro-
files 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 di-
rhamnolipid 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 homolo-
gous 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 ob-
served between the rhamnolipids’ composition and its E24 .
The E24 values of the mixed component rhamnolipids solu-
tions, containing higher relative abundance of less mobile
homologs such as di-rhamnolipids were higher (as with
 1372 Raza et al.
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

Pure RLs
ST IFT E24 CMC ST IFT E24 CMC
C-source (mN m−1 ) (mN m−1 ) (%) (mg L−1 ) (m Nm−1 ) (mN m−1 ) (%) (mg L−1 )

Canola WFO 32.0 ± 1.0 >1 62 ± 4.2 45 ± 2 29.0 ± 0.7 0.9 ± 0.1 70 ± 3 35 ± 3
Soybean WFO 30.3 ± 0.9 >1 68 ± 4.6 42 ± 2 28.5 ± 0.7 1.2 ± 0.1 75 ± 4 25 ± 2
Canola ORW 28.5 ± 0.7 0.70 ± 0.03 68 ± 0.4 56 ± 3 30.0 ± 0.8 1.0 ± 0.1 68 ± 3 45 ± 4
Soybean ORW 29.3 ± 0.5 0.90 ± 0.04 65 ± 0.4 75 ± 4 28.9 ± 0.6 1.0 ± 0.1 73 ± 4 27 ± 2
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 ties and the Higher Education Commission, Islamabad for
their lesser contribution (as with canola ORW) (Table 4). the research funds.
Such patterns could be due to four possible reasons: (i)
Downloaded By: [Raza, Zulfiqar Ali] At: 18:47 5 October 2009

different rhamnolipid mixtures produce different micellar

arrangements depending upon the input of diverse rham-
nolipid species, (ii) in addition to rhamnolipids, other types
of biosurfactants including phospholipids and/or neutral
lipids may also involve in micellization, (iii) the pres-
ence and contribution of coagulates and traces of resid-
ual 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 co-
extracted fatty acids from the culture media.
Conclusion
In conclusion, the structure of a biosurfactant is particu-
larly important in conferring surface-active properties and
molecular aggregates formation. Carbon source selection
for production directly affects the type of rhamnolipid ho-
molog product. Future cost effective production depends
on finding improved biosynthetic strategies and down-
stream 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] More-
over 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 im-
portance to be better elucidated, both in individual and
mutual properties.
Acknowledgments
The authors acknowledge the Director, International Cen-
ter for Chemical and Biological Sciences, University of
Karachi for the mass spectrometry and 1 H NMR facili-
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