Journal of Hazardous Materials 276 (2014) 489498

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Journal of Hazardous Materials

journal homepage: www.elsevier.com/locate/jhazmat

Biosurfactant produced by novel Pseudomonas sp. WJ6 with

biodegradation of n-alkanes and polycyclic aromatic hydrocarbons
Wenjie Xia a,b,c, , Zhifeng Du b , Qingfeng Cui c , Hao Dong d , Fuyi Wang b, ,
Panqing He a , YongChun Tang a,
a
Power Environmental Energy Research Institute, Covina, CA 91722, USA
b
Key Laboratory of Analytical Chemistry for Living Biosystems, Institute of Chemistry, Chinese Academy of Science, Beijing 100190, PR China
c
Institute of Porous Flow & Fluid Mechanics, Chinese Academy of Sciences, Langfang 065007, PR China
d
State Key Laboratory of Heavy Oil Processing, China University of Petroleum, Beijing 102249, PR China

h i g h l i g h t s g r a p h i c a l a b s t r a c t

A novel Pseudomonas strain was iso- Long chain alkanes and polycyclic aromatic hydrocarbons have been threatening the global environment
lated from heavy oil contaminated because of toxicity and poor bioavailability. We isolated one novel Pseudomonas strain from heavy-oil con-
soil. taminated soil which could degrade long-chain alkanes and polycyclic aromatic hydrocarbons (PAHs) and
It can convert n-alkanes and poly- could produce the surfactin, fengycin and lichenysin, which are mostly recognized as common metabo-
cyclic aromatic hydrocarbons to lites produced by Bacillus sp. And the heavy oil could be easily and efciently removed from quartz sand
lipopeptides surfactant. by these lipopeptides. This paper showed that Pseudomonas is the excellent degrader of alkanes and PAHs
Lipopeptides surfactant have differ- and produce the well-known rhamnolipids and the easily neglected lipopeptides.
ent structures when using different
hydrocarbons.
Lipopetides surfactant showed great
performance on heavy oil sand wash-
ing.

a r t i c l e i n f o a b s t r a c t

Article history: Alkanes and polycyclic aromatic hydrocarbons (PAHs) have threatened the environment due to toxicity
Received 17 March 2014 and poor bioavailability. Interest in degradation of these hazardous materials by biosurfactant-producing
Received in revised form 25 April 2014 bacteria has been steadily increasing in recent years. In this work, a novel biosurfactant-producing Pseu-
Accepted 22 May 2014
domonas sp. WJ6 was isolated to degrade a wide range of n-alkanes and polycyclic aromatic hydrocarbons.
Available online 2 June 2014
Production of lipopeptide biosurfactant was observed in all biodegradable studies. These lipopeptides
were puried and identied by C18 RP-HPLC system and electrospray ionization-mass spectrometry.
Keywords:
Results of structural analysis showed that these lipopeptides generated from different hydrocarbons
n-Alkane
Aromatic hydrocarbons
were classied to be surfactin, fengycin and lichenysin. Heavy-oil sludge washing experiments demon-
Biodegradation strated that lipopeptides produced by Pseudomonas sp. WJ6 have 92.46% of heavy-oil washing efciency.
Lipopeptides The obtained results indicate that this novel bacterial strain and its lipopeptides have great potentials in
Pseudomonas the environmental remediation and petroleum recovery.
2014 Elsevier B.V. All rights reserved.

Corresponding authors at: Power Environmental Energy Research Institute, 738 Arrow Grand Circle, Covina, CA 91722, USA. Tel.: +1 626 250 4448/+86 10 62529069/
+1 626 858 5077; fax: +1 626 858 5077/+86 10 62529069/+1 626 858 5077.
E-mail addresses: wenjie.hsia@gmail.com, sjkr5201314@gmail.com (W. Xia), fuyi.wang@iccas.ac.cn (F. Wang), yongchun.tang@peeri.org (Y. Tang).

http://dx.doi.org/10.1016/j.jhazmat.2014.05.062
0304-3894/ 2014 Elsevier B.V. All rights reserved.
490 W. Xia et al. / Journal of Hazardous Materials 276 (2014) 489498
1. Introduction
Petroleum is the most important and predominant energy
resources and raw materials of chemical industry in modern soci-
ety. However, spills, leaks, and other releases of petroleum often
result in the contamination of soil and groundwater, especially
when associated with accidental spills on a large scale [1]. Main
sources of petroleum contamination are petrochemical industries,
oil eld installations, petroleum plants, liquid fuel distribution and
storage devices, transportation equipment for petroleum products,
airports and illegal drillings in pipe lines [2]. These pollutions have
signicant environmental impacts, and present substantial hazards
to human health and destroy the ecological balance which may take
years or even decades to recover.
Numerous researches have been done to diminish environ-
mental threads of petroleum and its risks to human health and
ecological system. Biodegradation of petroleum hydrocarbons by
microorganisms (such as bacteria, fungi, or yeast, and bacteria) has
been recognized as one predominant, effective and environment-
friendly way by which crude oil is eliminated from contaminated
sites [3]. Bacterial strains able to degrade petroleum hydrocarbons
are known to be ubiquitous in nature. For this reason, biodegrada-
tion by bacteria has been considered a potentially useful tool in the
cleansing of oil spills and the treatment of oil residues [4,5].
Petroleum is a complex mixture of hydrocarbons and related
compounds generally classied into four fractions: aliphatics (alka-
nes), aromatics, polars or resins and asphaltenes [6]. Alkanes can
constitute 50% to 95% of crude oil, depending on the oil source.
Alkanes of shorter chain length (C10C20) are more toxic, while
those of larger chain length (C2040) are hydrophobic solids dif-
cult to degrade [7,8]. Polycyclic aromatic hydrocarbons (PAHs)
are a widespread class of environmental chemical toxic pollutants,
and has several possible sources in the environment exist, such
as pyrolytic, diagenetic, and petroleum hydrocarbons [9]. PAHs
are aromatic compounds containing from two to eight conjugated
ring systems, and can have a range of substituents such as alkyl,
nitro, and amino groups in their structure. Nitrogen, sulfur, and
oxygen atoms can also be incorporated into their ring system
[10]. Crude oils contain 0.2 to 7% PAHs, with congurations rang-
ing from two to six rings [11]. And asphaltenes constituted 5%
to 20% of crude oil was detected with multiple PAH units con-
taining 25 or more rings inside asphaltene molecules and some
other heteroatom-containing (N, S and O) aromatic compounds
[12]. Therefore, alkanes and PAHs could be treated as typical and
predominant fraction of crude oil for biodegradation research.
Many literatures have reported the great abilities of bacteria
to utilize/degrade hydrocarbons with different carbon numbers
and structures [1317]. However, the general low bioavailability
of alkanes and PAHs, which are highly recalcitrant molecules that
can persist in the environment due to their hydrophobicity and
low water solubility, is a hindrance to microbial biodegradation
for achieving regulatory end-points. Consequently, there are much
more interests in isolating, identifying and understanding potential
mechanisms by which alkanes or PAHs degrading microorganisms
surmount the challenges imposed by these poorly available sub-
strates.
Production of biosurfactants by bacteria is considered an
important microbial strategy that inuences the bioavailability of
hydrophobic chemicals by changing the surface properties of bac-
terial cell or by dissolving and emulsifying these non-hydrophilic
hydrocarbons, and as well as releasing (or washing) the traped
hydrocarbons of porous medium in contaminated soil. Biosur-
factants are amphiphilic molecules consisting of hydrophobic
and hydrophilic moieties that tend to interact with interfaces of
various polarities and reduce the surface and interfacial tension of
solutions [18,19]. These surface active materials generally exhibit
low toxicity, high biodegradability and ecological acceptability,
which make biosurfactants as potential alternatives of chemically
synthesized surfactants in a variety of eld such as cosmetics,
food, pharmaceutics, agriculture, environmental remediation and
petroleum industry [20,21]. Many types of biosurfactants have
been synthesized from bacteria belonging to a wide variety of gen-
era, such as Pseudomonas, Bacillus, Acinetobacter and Mycobacterium
[22]. Lipopeptides are a group of biosurfactants with consisting of
a hydrophobic fatty acid moiety and a hydrophilic peptide moiety
in molecule. It has low critical micelle concentration (CMC), stable
emulsication properties, powerful surface activities and excellent
foaming characteristics, and shows stable physicalchemical prop-
erties at different temperatures and pH levels. Lipopeptides are
mostly synthesized by genus Bacillus with hydrophilic substrates
(e.g. molasses, glucose, source) as carbon source, and hardly
generated with hydrophobic hydrocarbons as sole carbon source,
such as alkanes and PAHs [23,24]. Pseudomonas species are always
considered to be ubiquitous hydrocarbon-degrader and have been
shown prominent ability of degrading alkanes and PAHs, and could
be isolated from diverse environments [14,23,25]. It is well known
that Pseudomonas sp. strains synthesize the glycolipid biosurfac-
tants (Rhamnolipids) when utilizing the hydrophobic substrates
[26]. However, it is rarely reported in literatures that Pseudomonas
sp. could convert alkanes or PAHs to lipopeptide surfactant.
The objective of this study is to isolate the promising
hydrocarbon-degrading and biosurfactant-producing bacterial
strain for bioremediation of petroleum-contaminated soil, and to
evaluate the abilities of biodegradation of hazardous hydrocar-
bons, and to analyze the characteristics and heavy oil-washing
potentials of produced biosurfactant. For this purpose, the heavy
oil-contaminated soil was collected from Xinjiang Oileld for
screening the high-performed bacterial strains. And we obtained
one novel Pseudomonas sp. WJ6 which show great abilities of
degrading the n-alkanes and PAHs compounds. And structural
diversity of lipopeptides biosurfactant produced during degra-
dation was analyzed. Additionally, characterization of surface
activities and heavy oil sand washing of these lipopeptides were
evaluated.
2. Materials and methods
2.1. Microorganisms
Heavy oil-contaminated soil was collected near one oilwell of
Xinjiang oileld (China). 20 g of soil sample was cultivated in 200 ml
mineral enrichment medium (MEM) amended with 10 g heavy oil
(from same oilwell, contains 44.46% of alkanes, 27.82% of aromat-
ics, 12.65% of polars or resins and 15.07 of asphaltenesa) as the sole
source of carbon and energy for 20 days at 37 C with 200 rpm agi-
tation under aerobic condition. Then 10 ml of culture supernatant
was collected and spread on LB agar plates for 3 days. Selection
of strains with ability to degrade hydrocarbons was conducted in
MEM amended with hydrocarbons (the mixtures of alkanes and
aromatic fractions separated from above crude oil according to the
method described previously [27], 1% w/v) for 7 days at 37 C with
200 rpm shaking. Strain WJ6 was selected due to its superior per-
formance of hydrocarbons emulsication and growth assessed at
multiple temperatures (450 C), pH (410), and salinities (015%
NaCl, w/v). The MEM contained (g/l): NaNO3 8.0, KH2 PO4 3.0,
Na2 HPO4 3.0, MgSO4 7H2 O 0.5, FeSO4 7H2 O 0.0028, NaMO O4 0.02,
yeast extract 0.5, pH 6.57.0; and 5 ml of a trace element solu-
tion containing (g/l): ZnSO4 7H2 O 0.3, CaCl2 4H2 O 0.2, CuSO4 5H2 O
0.2, MnSO4 H2 O 0.2 and vitamin C 0.1. The medium solution was
lter-sterilized through a 0.22 mm membrane lter (Millipore,
type GS).
The morphological, physiological and phylogenetic characteris-
tics of this isolate were analyzed according to Bergeys Manual of
 W. Xia et al. / Journal of Hazardous Materials 276 (2014) 489498
Systemic Bacteriology and the method of 16S rRNA sequencing as
described previously [21].
2.2. Biodegradation of n-alkanes and PAHs
Bacterial cell in 5 ml broth of LB culture (1010 CFU/ml) was
harvested by centrifugation at 5000 rpm for 10 min, washed in
triplicates with sterilized saline solution (0.9%, w/v) and inocu-
lated in 100 ml MEM (pH: 6.57.0) supplemented with liquid or
solid n-alkanes (n-dodecane, n-docosane, n-dotriacontane and n-
tetrocontane, >99% purity, sigma) and PAHs (uorene, naphthalene,
phenanthrene, pyrene, >99% purity, sigma), respectively. The nal
concentration of carbon source was 0.5% (w/v). One control con-
taining hydrocarbons with no cell was set up for determining
kinetics of growth and degradation. All cultures were incubated
for 20 days at 37 C with 180 rpm of shaking under aerobic con-
dition. Interval samples was collected every 2 days and analyzed
for cell growth based on CFU counts. The residual hydrocarbons
were extracted and measured by gas chromatograph (GC) accord-
ing to the methods described in literature [14]. All samples were
performed in triplicates and the results were expressed as mean
value and standard deviation.
2.3. Purication of biosurfactants
After 20 days incubation, the cell-free supernatant was obtained
by centrifugation at 10,000 rpm for 20 min at 4 C, and then was
acidied with 6 M HCl to pH 2.0 and stored at 4 C overnight.
The precipitates were harvested by centrifugation at 10,000 rpm
for 20 min at 4 C and lyophilized overnight. Biosurfactants were
extracted from the powder into the methanol under supersonic
and then dried at 40 C.
2.4. Thin-layer chromatography
Ten milliliter of puried biosurfactants in methanol was spotted
on a silica gel chromatography TLC plate (Silica gel 60, Merck, Darm-
stadt, Germany). The compounds were separated using a mobile
phase of chloroform/methanol/water in the ratio of 65:25:4 (v/v/v).
For the detection of peptides, the dry plates were sprayed with a
solution of 0.25% ninhydrin in acetone and kept at 105 C for 5 min.
For detection of lipids, the plates were treated with iodine [28].
2.5. Structural analysis of biosurfactants
The Electrospray ionization mass spectrometry (ESIMS) anal-
ysis was performed according to the method reported previously
[29] with modication. Negative mass spectra were obtained on a
Micromass Q-TOF mass spectrometer (Waters) coupled to a Dionex
HPLC system with use of a Gemini C18 column (2 50 mm, 100 A,
5 m, Phenomenex). The mass spectrometer was operated in the
data-dependent mode with a range of m/z 350 to 2000. The HPLC
mobile phases were A: H2 O containing 4.9% acetonitrile and 0.1%
triuoroacetate acid (TFA), and B: acetonitrile containing 4.9% H2 O
and 0.1% TFA. The biosurfactants were eluted with a gradient, start-
ing at 1% solvent B and holding for 3 min, then ramping to 80%
in 27 min, holding for 6 min, backing to 1% in 2 min and hold-
ing at 1% for 1 min. The eluents were directly infused into the
mass spectrometer through the ESI probe. The spray voltage of
the mass spectrometer was 3.30 kV and the cone voltage 35 V. The
desolvation temperature was 413 K and source temperature 353 K.
Nitrogen was used as both cone gas and desolvation gas with a ow
rate of 40 l/h and 400 l/h, respectively. The collision energy was set
to 10 V. Mass Lynx (ver. 4.0) software was used for analysis and post
processing.
491
Fig. 1. Phylogenetic relationship based on the 16S rDNA gene sequences between
strain WJ6 and species in the Pseudomonas as determined by the neighbor-joining
algorithm and evaluated by the maximum-likelihood and maximum parsimony
algorithms. Bootstrap values over 70% are shown at the nodes. Bar. 5 nucleotide
substitutions per 1000 nucleotides.
2.6. Fatty acid and amino acid analysis
Puried fractions from RP-HPLC were hydrolyzed with 6 M HCl
at 150 C for 3 h and the fatty acids (FAS) were extracted with ether
in triplicates and esteried with methanol at 100 C for 1 h, and
then analyzed by gas chromatograph mass spectrometer analysis
as previous described by Yang [30]. The aqueous fraction containing
the free amino acids was subjected to an automatic amino acid
analyzer (1100 series, Hewlett Packard, USA).
2.7. Surface tension and emulsication of puried lipopeptides
The surface tension was determined by a Tensiometer Kruss
K100 (Kruss GmbH, Hamburg, Germany) at room temperature,
according to the Du Nouys ring method [21], with the nal con-
centration of 50 mg/l. Emulsication activity of lipopeptides was
tested on kerosene according to the previous literature [21], with
concentration of 500 mg/l.
2.8. Sludge washing
Sands originated from the oileld were passed through a
0.15 mm sieve. 60 g of the sieved sands was mixed with 30 g heavy
oil (from Canada, viscosity = 13,210 mPa s, 16.8% of asphaltene),
vortexing at high speed until the mixture became muddy similar
to that of industrial oil sludge (tar sand), and then aging at 50 C for
3 days.
Sludge washing was carried out at 30 C as described previ-
ously with modication [31]. 4 g aliquot of sludge were mixed with
10 ml of biosurfactant solution (0.1 g/l) was shaken in a test tube at
100 rpm for 2 h, and then left for 1 h at 50 C to settle for the mixture
separated into three phases, e.g. solid, oil and water. The separa-
tion and weighting of oil layer and oil washing efciency (R) were
carried out according to methods described by Joseph et al. [32].
3. Results and discussion
3.1. Identication of microorganism
Strain WJ6 (deposited in the China General Microbiological
Culture Collection Center with the deposit number of CGMCC 4402)
was one of the 86 strains isolated from the heavy oil-contaminated
soil. The nearly-complete 16S rRNA gene sequence (1498 bp) of
strain WJ6 was obtained and phylogenetic analysis conducted to
determine that it belonged to Proteobacteria, showing the highest
16S rRNA sequence similarity of 99.358% with Pseudomonas
sp. (Fig. 1). The microorganism was gram-negative, facultative
492 W. Xia et al. / Journal of Hazardous Materials 276 (2014) 489498

anaerobic, oval to rod-shaped (0.30.5 m 0.51.0 m), and
motile with single agellum. Colonies (0.52 mm) growing on
LB agar for 2 days at 37 C were smooth, circular convex, wet
and yellowish-brown in color. The organism was able to grow at
550 C, pH 410 and 015% (w/v) NaCl, with the optimum growth
occurring at pH 6.57.5, 37 C and 02% (w/v) NaCl. It was positive
for catalase, urease, oxidase, aerobic nitrite reduction, anaerobic
nitrate reduction and denitrication, as well as for the hydrolysis of
starch and gelatin. Morphological, physiological and phylogenetic
properties indicated that strain WJ6 was a member of the genus
Pseudomonas, and the Genbank accession number is KF155141.
3.2. Biodegradation of n-alkanes and PAHs
Longer chain n-alkanes and PAHs in crude oil were generally
considered to be only slightly biodegradable due to their higher
hydrophobicity [8,33]. In this work, n-alkanes (C12, C22, C32 and
C40) and PAHs (uorene, naphthalene, phenanthrene, pyrene)
were tested as the sole carbon sources, the growth of strain WJ6
was observed in all cases. It grew obviously and rapidly with n-
alkanes (C12, C22, and C32), while it grew a bit slower when
utilizing C40 and polycyclic aromatic hydrocarbons, as shown in
Fig. 2. Comparisons with Pseudomonas strains reported by some
literatures indicated that strain WJ6 could use a broader range of
crude oil components as the sole carbon sources, including aliphatic
hydrocarbons, branched alkane, cyclane and aromatic hydrocar-
bons [14,25].
When utilizing the n-alkanes, a remarkable increase of CFU
counts was observed from 8 days to 12 days (Fig. 2ad). The
maximum CFU counts decreased with the chain-length increas-
ing. Stationary phase started on the 12th days for n-alkanes C12
and C22, while delayed by nearly 4 days for C32 and C40. Specif-
ically, n-dodecane (C12) was degraded by 46.65% in the 20-days
degradation. Comparatively, only 42.62%, 31.69% and 23.62% of C22,

Fig. 2. Degradation of different n-alkanes and polycyclic aromatic hydrocarbons as the sole carbon sources by Pseudomonas sp. WJ6. Opened square ( ) stands for residual
hydrocarbon in right vertical axis; Filled triangle ( ) stands for CFU count in the left vertical axis. (A)(D) represent the case of n-alkane C12, C22, C32 and C40; (E)(H)
represent the case of PAHs uorene, naphthalene, phenanthrene and pyrene, respectively. The values are mean standard deviations (n = 3).
 W. Xia et al. / Journal of Hazardous Materials 276 (2014) 489498
C32 and C40 were degraded, respectively (Fig. 2bd). The mecha-
nism of alkane biodegradation has been widely studied, and the
metabolic pathway, alkanehydroxylases enzymes and gene clus-
ters have been examined in details [34,35]. However, most studies
on the degradation pathways of alkanes have focused on short-
or medium-chain alkanes. Microorganisms degrading short-chain
alkanes (C2C4) have enzymes related to methane monooxyge-
nase [36], while strains degrading medium-chain alkanes (C5C11)
or long-chain alkane (>C12) frequently contain integral membrane
non-heme iron monooxygenase related to the well-characterized
Pseudomonas putida GPo1 Alkb alkane hydroxylase [37]. Some
strains contain alkane hydroxylating enzymes that belong to a fam-
ily of soluble cytochrome P-450s and that against C5C11 alkanes
[38]. In additions, several bacterial strains have been reported to
assimilate alkanes larger than C20 [25,39]. However the enzymes
responsible for the oxidation of long-chain alkanes are still poorly
characterized and only recently have started to be character-
ized.
PAHs are pollutants to environments because of their toxic and
carcinogenic potentials. Due to the high hydrophobicity of aromatic
rings, PAHs are much more resistant to biodegradation. However
degradation of PAHs by Pseudomonas sp. WJ6 was remarkable. As
shown in Fig. 2eh, uorene, naphthalene and phenanthrene were
obviously degraded, respectively, by 39.45%, 23.14% and 21.93% in
20 d. Compared to PAHs of two and three aromatic rings, pyrene
that contain four aromatic rings were much more resistant to
biodegradation with 19.50% of pyrene degraded. Degradation of
PAHs by microorganisms was extensively studied throughout the
world [40]. Pseudomonas strains have been reported to degrade n-
alkanes and PAHs [35]. Pseudomonas sp. F274 [41], Pseudomonas
sp. NCIB 9816-4 [42] and P. putida F1 [43] were isolated and
found to have the ability to degrade uorene, dibenzofuran and
Fig. 3. Thin-layer chromatography (TLC) of the biosurfactant obtained from Pseu-
domonas sp. WJ6 when utilizing different hydrocarbons. Lanes A and D for
n-dodecane, Lane B and E for uorene, Lane C and F for pyrene; Lanes A, B and C
were developed with a 0.1% ninhydrin solution to detect peptides, Erythrinus color
is positive. Lanes D, E and F were developed with iodine vapor to detect lipids,
lemony yellow is positive. (For interpretation of the references to color in this gure
legend, the reader is referred to the web version of this article.)
493
dibenzothiophene. Herein, this newly Pseudomonas sp. WJ6 can
degrade both alkanes and PAHs efciently. However, the metabolic
mechanism of the degradation of long-chain alkane and PAHs about
strain WJ6 remains uncertain. Further works about the degradation
mechanism will focus on the hydrophilic or hydrophobic charac-
teristic of cell surface, hydroxylase enzymes and gene cluster of
alkane or PAHs degradation.
3.3. TLC analysis of puried biosurfactant
In terms of the results of hydrocarbon degradation and cell
growth, the biosurfactants partially puried from the cultures of
biodegradation of hydrocarbons (n-dodecane, uorene and pyrene)
were selected and analyzed by thin layer chromatography (Fig. 3).
Three biosurfactants have an identical Rf value of 0.74 and showed
positive reactions with two spray reagents of ninhydrin and iodine,
indicating three biosurfctants maybe belongs to lipopeptide and
consisted of peptide and lipid moieties.
3.4. Structural analysis of biosurfactant
The partially puried biosurfactant produced by the WJ6 strain
with using hydrocarbons (n-dodecane, uorene, pyrene) were
subjected to the reverse-phase HPLC. With a linear gradient of ace-
tonitrile and water, three biosurfactants were resolved into several
fractions as shown in Fig. 4.
The separated HPLC fractions were then tested for surface activ-
ity by the drop-collapsing assay, the fractions that reduce the
surface tension were collected and the amino acid moieties were
analyzed and listed in Table 1.
HPLC fractions were also analyzed by electrospray ionization
mass spectrometry (ESI-MS) coupled to the HPLC system. As shown
in Fig. 5a, a series of singly-charged negative ions were observed for
the fraction 1 of the n-dodecane sample eluting at 10.46 min, hav-
ing a 14 Da difference in mass between the neighboring ion peaks.
Fig. 4. RP-HPLC chromatograms of partial biosurfactants produced by Pseudomonas
sp. WJ6 using (A) n-dodecane, (B) uorine and (C) pyrene as carbon source.
494 W. Xia et al. / Journal of Hazardous Materials 276 (2014) 489498

Fig. 5. Mass spectra of the HPLC fractions eluting at (a) 10.46, (b) 13.03, (c) 28.95, (d) 30.98 and (e) 33.23 min as shown in Fig. 4A of the biosurfactants produced by Pseudomonas
sp. WJ6 using n-dodecane as carbon source.

Based on the fatty acid (data not shown) and amino acid analysis,
the ve ions are assignable to the deprotonated homologs [M H]
([M H] at m/z 1447, 1461, 1475, 1491 and 1503) of the fengyin
A lipopeptides (Table 2) which compose of ten amino acids linked
to a C15, C16, C17, C18 or C19 hydroxy fatty acid chain, respec-
tively. Moreover, four minor ions peaks were observed at m/z 1469,
1497, 1513 and 1525 which correspond to the sodiated molecules
[M + Na] (Fig. 5a). These spectra are very similar to those reported
in the literature [4447], where protonated ions [M + H]+ at m/z
1435.6, 1449.9, 1463.7, 1477.8, 1491.7 and 1505.9 were observed
for fengycin homologs under positive MS mode.
The MS under negative mode showed deprotonated molecular
ions [M H] at m/z 1075, 1047, 1031, 1019 and 1005 with m/z
1031, 1047 and 1075 being the main ions for the HPLC fraction 2
eluting at 13.03 of n-dodecane sample (Fig. 5b). Yakimov et al. [48]
reported that the ve ion peaks have a 14 Da or 28 Da difference
in mass each another, except the ion at m/z 1031, possibly has one
double bond referring to the deprotonated molecular ions. Coupling
with the fatty acid (data not shown) and amino acid analysis, such
a distribution of molecular ions is in excellent agreement with that
of the lichenysin A lipopeptides homologs (Table 2), and the main
-hydroxy fatty acid residues in the lipophilic moiety of lichenysin

Table 1
The amino acid moieties of surface active fractions arising from RP-HPLC separation of biosurfactants produced by the WJ6 strain with using various hydrocarbons as carbon
source. The HPLC chromatograms are shown in Fig. 4.

Hydrocarbon Surface active fraction HPLC retention time (min) Amino acid moiety
n- Fraction 1 10.46 2 Glu, 1 Gln, 1 Orn, 2 Tyr, 1 Thr, 1 Ala, 1 Pro, 1Ile
Dodecane Fraction 2 13.03 1 Gln, 3Leu, 2 Val, 1 Asp, 1 IIe
Fraction 3 28.95 1 Glu, 3 Leu, 2 Val, 1 Asp
Fraction 4 30.98 1 Glu, 4 Leu, 1 Val, 1 Asp
Fraction 5 33.23 1 Glu, 3 Leu, 1 Val, 1 Asp, 1 IIe
Fluorene Fraction 6 7.49 2 Glu, 1 Gln, 1 Orn, 2 Tyr, 1 Thr, 1 Ala, 1 Pro, 1 Val
Fraction 7 10.13 1 Gln, 3Leu, 1 Val, 1 Asp, 1 IIe
Pyrene Fraction 8 8.44 2 Glu, 1 Gln, 1 Orn, 2 Tyr, 1 Thr, 1 Ala, 1 Pro, 1 Val
Fraction 9 13.91 1 Gln, 3Leu, 1 Val, 1 Asp, 1 IIe
 W. Xia et al. / Journal of Hazardous Materials 276 (2014) 489498 495

Table 2
Lipopeptides produced by Pseudomonas sp. WJ6 with three hydrocarbons as carbons source.

n-Dodecane Fluorene Pyrene

Surfactin Fraction 4: Surfactin A [Leu7]-C13,

[Leu7]-C14 No detected No detected

Fraction 3: Surfactin B [Val7]-C13,

[Val7]-C14, [Val7]-C15

Fraction 5: Surfactin D [Ile7]-C14,

[Ile7]-C15

Fengycin Fraction 1: Fengycin A [Ala6]-(C15, Fraction 6: Fengycin A [Ala6]-C14, Fraction 8: Fengycin A [Ala6]-C15.
C16, C17, C18, C19) [Ala6]-C15, [Ala6]-C19.

Lichenysin Fraction 2: Lichenysin A (C13,C14, C15, Fraction 7: Lichenysin A (C14, C15, C16, Fraction 9: Lichenysin A (C15, C16, C17
C16, C17), C15 has one double bond C18), C15 has one double bond C18), C15 has one double bond

A molecules are assignable to C13, C14, C15 (containing one double are assigned to be C14, C15 (containing a double bonds), C16 and
bond), C16 and C17, respectively. C18, respectively, similar to the results reported in the literature
For the n-dodecane sample, the negative MS mode analysis [49].
observed three deprotonated molecular ions [M H] at m/z 994, Two fractions, which showed surface active (Fig. 4c, Table 1)
1008 and 1022 for the fraction 3 eluting at 28.95 min (Fig. 5c), two arising from the HPLC separation of the pyrene sample were ana-
deprotonated molecular ions at m/z 1008 and 1022 for the fraction 4 logically analyzed by MS under negative mode. The results showed
eluting at 30.98 min (Fig. 5d), and two deprotonated molecular ions deprotonated molecular ions [M H] at m/z 1447 for the rst
at m/z 1022 and 1036 for the fraction 5 eluting at 33.23 min (Fig. 5e). fraction 8 eluting at 8.44 min (Fig. 7a). Combined with amino acid
These ion peaks have a 14 Da difference in mass each another, indi- analyses, the mass of this species is in excellent agreement with that
cating that these biosurfactants maybe belong to the homologs of of the fengycin A lipopeptides which composed of ten amino acids
lipopeptides. Pereira et al. [49] has described the surfactin mix- linked to a C15 -hydroxy fatty acid chain (Table 2) with similar
ture extract has protonated peaks with sodium adducts [M + Na]+ reported in the literature [40]. For the second fraction (fraction 9)
at m/z 1030.6, 1044.6 and 1058.7. Kowall et al. [28] described twelve eluting at 13.91 min, the negative MS showed deprotonated molec-
surfactins with molecule weight of 994, 1008, 1022 and 1036 Da, ular ions [M H] at m/z 1031, 1047, 1061 and 1075 (Fig. 7b). Taking
which contain different amino acids when same molecule weight the amino acid analysis result into account (Table 1), such a distri-
achieved. Therefore, in terms of the fatty acid (data not shown) bution of molecular ions is in excellent agreement with that the
and amino acid analysis, such a distribution of molecular ions of
fractions (fraction 3 to 5) is in excellent agreement with that of
the surfactin lipopeptides (Table 2). Fraction 3 was surfactin with
L-Val in position 7 of peptide ring linked with -hydroxy fatty
acid with chain lengths of 1315 carbon atoms; the fraction 4 con-
tains surfactins with L-Leu in position 7 of peptide ring linked with
-hydroxy fatty acid with chain lengths of 1314 carbon atoms.
Fraction 5 was surfactin with L-Ile in position 7 of peptide ring
linked with -hydroxy fatty acid with chain lengths of 1415 car-
bon atoms.
About the uorene sample, two fractions appeared to be drop-
collapsing active were collected and the corresponding analysis of
the amino acid showed in Table 1. The MS under negative mode
of MS showed deprotonated molecular ions [M H] at m/z 1433,
1447 and 1503 for the fraction 6 eluting at 7.49 min (Fig. 6a).
These results indicated that the distribution of molecular ions of
this fraction is in excellent agreement with that of the fengycin A
lipopeptides (Table 2), which composed of ten amino acids linked
to a C14, C15 or C19 -hydroxy fatty acid chain. The mass-spec
spectra reported are very similar to those obtained by Bie et al.
[45]. For the fraction 7 eluting at 10.13, four deprotonated molecu-
lar ions [M H] at m/z 1017, 1031, 1047 and 1075 were observed.
Combined with the amino acid analysis (Table 1), such a distribu-
tion of molecular ions is in excellent agreement with that of the
Fig. 6. Mass spectra of the HPLC fractions eluting at (a) 7.49 and (b) 10.13 min
lichenysin A lipopeptides (Table 2), and the main -hydroxy fatty
as shown in Fig. 4B of the biosurfactants produced by Pseudomonas sp. WJ6 using
acid residues in the lipophilic part of the lichenysin A homologs uorine as carbon source.
496 W. Xia et al. / Journal of Hazardous Materials 276 (2014) 489498
Fig. 7. Mass spectra of the HPLC fractions eluting at (a) 8.44 and (b) 13.91 min
as shown in Fig. 4C of the biosurfactants produced by Pseudomonas sp. WJ6 using
pyrene as carbon source.
homologs of the lichenysin A lipopeptides (Table 2), and the main
-hydroxy fatty acid residues in the lipophilic part of lichenysin A
molecules are C15 (containing a double bonds), C16, C17 and C18,
respectively.
Pseudomonas strains produce many types of bioactive com-
pounds which have great potential for biotechnological and
biomedical application [21,29,50]. Well-known group of com-
pounds includes glycolipid such as rhamnolipids [21] and
lipopeptides such as pseudophomins [51], viscosin [52], putisolvan
[53], and massetolide [54]. Bacteria of the genus Pseudomonas
showed prominent and overwhelming performance on the degra-
dation of alkanes and aromatic hydrocarbons than Bacillus strains.
However, few literatures have reported that Pseudomonas could
synthesize surfactin, fengycin and lichenysin, and these metabo-
lites show great structural diversities when utilizing different
n-alkanes and PAHs.
Surfactin was detected and had three isoforms with n-dodecane,
while no detection of surfactin in the case with using of uorene
and pyrene. However, fengycin and lichenysin were found in three
carbon source medium, as shown in Table 2.
As typical and representative members of lipopeptides, sur-
factin, fengycin and lichenysin are reputed to be the most powerful
microbial lipopeptides known so far [22]. Many types of surfactin,
fengycin and lichenysin were reported to be produced by Bacil-
lus genus from the hydrophilic carbon sources, such as molasses,
sucrose, yeast extracts and glucose [44,45,49,55]. Therefore, this
possibility and diversity of lipopeptides produced by Pseudomonas
sp. WJ6 maybe contributed to its characterization of hydrocarbon
degradation and some enzymes similar as existed in genus Bacil-
lus, and our further researches will focus on the mechanism of this
phenomenon.
3.5. Heavy oil washing efciency
There has been a growing interest in biosurfactant applications
in environmental remediation and heavy oil recovery because of
their lower toxicity, greater biodegradability and environmental
compatibility. The surface tension of three lipopeptides produced
Pseudomonas sp. WJ6 from different hydrocarbons (n-dodecane,
uorene and pyrene) were measured, and results showed that
Fig. 8. Oil washing efciency in response to washing time with lipopeptide
produced by Pseudomonas sp. WJ6 using n-dodecane as carbons source. The con-
centration of biosurfactant solution is 1000 g/l in 20 g/l KCl at pH = 9.86. (1): the
begin of sand washing, 0 min; (2): the end of the sand washing, 120 min.
they could decrease the surface tension of the distilled water from
71.82 mN/m to 28.93 mN/m, 32.08 mN/m and 31.24 mN/m, respec-
tively. Emulsication index of three lipopeptides against kerosene
as oil phase was 89.45%, 78.12% and 75.85%. Therefore, the lipopep-
tide from n-dodecane was chosen for heavy oil sludge washing.
Results of oil washing showed that the oil washing efciency
increased with washing time and approached an asymptote with
92.46% of the nal oil washing efciency at 120 min with low con-
centration of biosurfactant, and the treated sand at end point was
clearer than the original (Fig. 8). The outcomes of this work are
expected to provide a basis for developing environmentally friendly
and economically competitive approaches for heavy oily sludge
treatment.
4. Conclusions
Novel biosurfactant-producing Pseudomonas sp. WJ6 was able
to degrade the n-alkanes and PAHs. Biosurfactants produced during
degradation of different hydrocarbons were classied with differ-
ent isoforms and homologs of cyclic lipopeptides. Surfactin (A, B
and D), fengycin A and lichenysin A were detected with degrada-
tion of n-dodecane, while only fengycin A and lichenysin A were
detected with degradation of uorene and pyrene. These lipopep-
tides exhibited great performance of surface actives, emulsication
and heavy oil washing. Although the mechanism of degradation
and lipopeptide production need much more further studies, for-
tunately the results have demonstrated that this strain and its
biosurfactant have great potentials in the environmental remedia-
tion and petroleum industry.
Acknowledgments
We thank the 863 National High Technology Research and
Development Program of China (2013AA064402), and National
 W. Xia et al. / Journal of Hazardous Materials 276 (2014) 489498
Natural Science Foundation of China (Grant nos. 21020102039 and
21135006) for nancial support.
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