Journal of Applied Microbiology ISSN 1364-5072

ORIGINAL ARTICLE

Isolation and characterization of a novel biosurfactant

produced by hydrocarbon-degrading bacterium Alcanivorax
dieselolei B-5
N. Qiao1,2 and Z. Shao1
1 Key Laboratory of Marine Biogenetic Resources, The Third Institute of Oceanography, State Oceanic Administration, Xiamen, China
2 School of Life Science, Xiamen University, Xiamen, China

Keywords
Alcanivorax dieselolei, biosurfactant, linear
lipoamino, lipopeptide, proline lipids.
Correspondence
Zongze Shao, The Third Institute of
Oceanography, State Oceanic Administration,
Daxue Road 178, Xiamen 361005, Fujian,
China. E-mail: shaozz@163.com
2009 0487: received 14 March 2009, revised
27 July 2009 and accepted 30 July 2009
doi:10.1111/j.1365-2672.2009.04513.x
Abstract
Aims: Our goal was to identify a novel biosurfactant produced by a marine
oil-degrading bacterium.
Methods and Results: Biosurfactants were produced by Alcanivorax dieselolei
strain B-5T growing with diesel oil as the sole carbon and energy source. Cul-
ture supernatant was first extracted with chloroform methanol (1 : 1, v v),
then further purified step by step with a normal phase silica gel column, a
Sephadex LH20 gel column and a preparative thin layer plate. The main com-
ponent was determined to be a lipopeptide; it was chemically characterized
with nuclear magnetic resonance, liquid chromatography-quadrupole ion-trap
mass spectrometry, amino acid analysis and GCMS and was found to be a
mixture of proline lipids. The monomers of the proline lipids were composed
of a proline residue and a fatty acid (C14:0, C16:0 or C18:0). The critical micelle
concentration of the mixed proline lipids was determined to be 40 mg l)1.
Moreover, activity variations in ranges of pH, temperature and salinity were
also detected and showed reasonable stability.
Conclusions: Alcanivorax dieselolei B-5 produced a novel linear lipoamino
biosurfactant, characterized as a proline lipid.
Significance and Impact of the Study: A proline lipid was characterized for the
first time as a bacterial biosurfactant. This product has potential in both
environmental and industrial applications.

Although biosurfactants have common amphipathic

Introduction
Biosurfactants comprise a diverse group of surface-active
chemical compounds that are produced by a variety of
micro-organisms. These amphipathic molecules usually
have a hydrophilic and a hydrophobic domain, tend to
accumulate at interfaces, form micelles, lower the surface
tension and thereby enhance the solubility of poorly solu-
ble compounds in water (Rosenberg 1993; Schippers et al.
2000). Because of their low toxicity and high efficacy, bio-
surfactants are potential candidates for novel industrial
and environmental applications such as enhanced oil
recovery (Banat 1995; Banat et al. 2000) and speedy bio-
remediation of oil-contaminated sites (Kumara et al.
2006).
characteristics, their primary structures show wide-
ranging diversity. Glycolipids are the most intensely stud-
ied biosurfactants, and the main representative of this
class is rhamnolipid, produced by Pseudomonas bacteria
(Zhang and Miller 1994; Providenti et al. 1995; Deziel
et al. 1996; Sim et al. 1997; Perfumo et al. 2006). Lipo-
peptides, another important group, are usually produced
by species of Bacillus, Lactobacillus, Streptococcus and
Pseudomonas bacteria. Surfactin is a member of the lipo-
peptide group (Arima et al. 1968; Kowall et al. 1998; Liu
et al. 2008). Lipopeptides produced by Gram-negative
strain Pseudomonas have been reported as agents for the
biocontrol of phytopathogenic fungi (Stanghellini and
Miller 1997; Nielsen et al. 1999; Henriksen et al. 2000;

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Journal compilation 2009 The Society for Applied Microbiology, Journal of Applied Microbiology 108 (2010) 12071216 1207
A lipopeptide from strain B-5
Thrane et al. 2000), or as phytotoxins (Hutchison and
Gross 1997; Kuiper et al. 2004). Lipopeptides produced
by Gram-positive Bacillus, Lactobacillus and Streptococcus
strains, play a key role in the attachment of bacteria to
surfaces (Neu 1996; Busscher et al. 1997; Velraeds et al.
2000; Meylheuc et al. 2001; Mireles et al. 2001).
Bacteria of the Alcanivorax genus have been confirmed
as important marine alkane degraders. The cosmopolitan
Alcanivorax borkumensis strain SK2 produces glucose
lipids, one of which consists of four 3-hydroxydecanoic
acids linked together by ester bonds and coupled glyco-
sidically to the C-1 of glucose (Abraham et al. 1998).
Strain B-5, isolated from oil-contaminated surface water
of the Bohai Sea of China and identified as a new species
of Alcanivorax (Liu and Shao 2005), is the second in its
genus that has been reported to produce a biosurfactant,
reducing the surface tension of its culture from 65
to 298328 mN m)1. In this article, biosurfactants in
the culture supernatant of strain B-5 were purified and
characterized.
Materials and methods
Bacterium and medium
Alcanivorax dieselolei B-5T was used throughout this work
and for biosurfactant extraction. It was cultivated in
mineral salt medium (MSM) [(NH4)2SO4, 10 g l)1; KCl,
11 g l)1; NaCl, 11 g l)1; FeSO47H2O, 2810)4 g l)1;
KH2PO4, 34 g l)1; K2HPO43H2O, 44 g l)1; MgSO4,
05 g l)1; yeast extract, 05 g l)1; and trace element solu-
tion, 05 ml l)1 in distilled water] with 2% (v v) diesel oil
as the sole carbon source. The trace element solution (fil-
ter-sterilized) contained the following: ZnSO4, 029 g l)1;
CaCl2, 024 g l)1; CuSO4, 025 g l)1; and MnSO4,
017 g l)1. All chemicals were of analytical grade unless
specified. The n-hexadecane was 98% pure and purchased
from Fluka (Buchs, Switzerland). Bacteria were cultured
in a 5-l Chemostat (200 rev min)1) at 28C for 5 days.
Surface activity tests
Three methods (De nouy ring, oil spreading test and
biofilm on parafilm test) were used to detect surfactant
activity. Each test was performed in triplicate. Surface
tension was measured using a digital tensiometer
equipped with a De nouy ring (McInerney et al. 1990).
Pure water and ethanol were used as standards. The value
of surface tension was the average of three readings for
the same measurement.
For the oil spreading test, 50 ml of distilled water was
added to a large Petri dish (25 cm diameter) followed by
the addition of 20 ll of crude oil to the surface of the
1208 Journal compilation 2009 The Society for Applied Microbiology, Journal of Applied Microbiology 108 (2010) 12071216
N. Qiao and Z. Shao
water. The 10 ll of culture or samples was then added to
the oil surface. The diameter of the clear zone on the oil
surface was measured after 30 s, as previously described
(Morikawa et al. 2000). The negative control with
distilled water had no clear zone at all. The minimum
active dose (MAD) of biosurfactant was defined as the
amount necessary to obtain a detectable clear zone on an
oil layer (Maneerat et al. 2006).
The biofilm on parafilm test (with a slight modifica-
tion) was used to determine the surface tension of
purified surfactant (Kuiper et al. 2004). More specifically,
the purified surfactants were dissolved in water and then
were put as 25 -ll droplets on the hydrophobic side of
the parafilm. The diameters of the droplets were exam-
ined; the larger the diameter, the higher the surface activ-
ity. Methylene blue was added to stain the droplets for
photographic purposes, which had no influence on the
shape of the droplets. Sterile water was used as a surfac-
tant-absent control.
Emulsification activity was measured according to a
previously described method (Cooper and Goldenberg
1987), with a slight modification. Five millilitres of
n-hexadecane was added to 5 ml culture supernatant or
biosurfactant extract (05%, w v) and then vortexed at
high speed for 2 min. The mixture was then allowed to
stand still for 10 min prior to measurement. Emulsification
activity was defined as the height of the emulsion layer
divided by the total height and expressed as percentage.
To detect the physical chemistry characteristics of the
proline lipid, the effects of salinity, pH and temperature
treatments were examined using the oil spreading test, as
previously described (Morikawa et al. 2000). In the salin-
ity test, the effects of NaCl, CaCl2 and MgCl2 were tested
with the purified proline lipid dissolved in potassium
phosphate buffer (PBS, 50 mmol l)1, pH 70) (02%,
w v). In the temperature test, a mixture of lipid and
water was treated for 1 h at temperatures ranging from
20 to 100C, and for 15 min at 121C. In the pH test, the
lipid solution was first adjusted from pH 3 to pH 10 with
HCl (100 mmol l)1) or NaOH (100 mmol l)1), before
measuring the surface activity. Each experiment was
repeated three times.
Biosurfactant extraction and purification
Three litres of culture broth was used to extract and
purify the biosurfactant. The culture was first centrifuged
for 25 min at 11 000 rev min)1 at 4C. A hydrophobic
layer floating on the surface was scraped out and washed
with three volumes of hexane to remove alkanes. The
crude material was extracted with chloroform methanol
(1 : 1). The solvent was then removed by rotary evapora-
tion at 40C under reduced pressure.
2009 The Authors
N. Qiao and Z. Shao
Next, the crude extract was loaded onto a silica gel
column (Peng et al. 2007). The solvents used for the
adsorption silica gel column chromatography of the
surfactant were gradually increased in polarity: hexane,
chloroform methanol (95 : 5, v v), chloroform methanol
water (65 : 15 : 2, v v v) and methanol. The chloroform
methanol water (65 : 15 : 2, v v v) eluate was demon-
strated to have the highest surface activity, and the
compounds in this eluate were further separated by a
Sephadex LH20 gel column. The velocity of flow was 12 s
per drop, and methanol was the only flow solvent.
Four reagents were used to test the category of the sur-
factant: anthrone sulfuric acid reagent (for glycolipids);
02% ninhydrin reagent (for lipopeptides); cobalt chlo-
ride acetone reagent (for phospholipids); and bromocre-
sol green (for lipid-organic acids).
After passage through the Sephadex LH20 gel column,
45 eluates were collected; samples 2432 exhibited a high
surface activity and a positive reaction to ninhydrin
reagent. Thus, the nine samples were pooled together for
the following purification.
First, the nine samples were concentrated to 1 ml, then
loaded on the preparative layer plates (1 mm; Merck) and
run with chloroform methanol water (65 : 15 : 2, v v v)
solvent. The silica containing the separated lipopeptide-
positive spot was scraped from the glass plate and
extracted with chloroform methanol (1 : 1, v v). Other
TLC spreading systems (to confirm the purity of
the extracted compound) included solvent system II
(chloroform methanol, 10 : 1, v v), system III (chloro-
form methanol water, 85 : 15 : 2, v v v) and system IV
(chloroform methanol water, 65 : 25 : 4, v v v).
Characterization of the biosurfactant: LCQ-MS; NMR;
AAS; GCMS
The purified biosurfactant was characterized by liquid
chromatography-quadrupole ion-trap mass spectrometry
(LCQ-MS), amino acid analysis (AAS), gas chromatogra-
phymass spectrometry (GCMS) and nuclear magnetic
resonance (NMR) to identify its molecular weight, hydro-
philic group, hydrophobic group and whole chemical
structure, respectively.
MS characterization and detection of the biosurfactant
was carried out using an LCQ quadrupole ion-trap mass
spectrometer (Finnigan MAT, San Jose, CA, USA) with
electrospray ionization (ESI). Standard solutions and
samples (dissolved in methanol) were infused into the
mass spectrometer at a flow rate of 10 ll min)1. In the
ESI source, the nitrogen sheath and auxiliary gas flows
were maintained at 50 and 5, respectively; these refer to
arbitrary values set by the software. The heated capillary
temperature was 275C, and the spray voltage was set to
2009 The Authors
Journal compilation 2009 The Society for Applied Microbiology, Journal of Applied Microbiology 108 (2010) 12071216
A lipopeptide from strain B-5
5000 V. The positive ion mode was used and scans were
run over the 1002000 m z range.
The purified surfactant was hydrolysed with 6 mol l)1
HCl at 110C for 24 h. The resulting solution was then
extracted with ether at least three times (Maneerat et al.
2006). Composition analysis of the hydrolytes was
achieved with GCMS and AAS methods. The solvent
phase and the aqueous phase, which contained fatty acids
and amino acids, respectively, were separated and
collected. The solvent phase containing fatty acids was
subjected to methanolysation for GCMS analysis (Peng
et al. 2008). The 1-ll sample (from the solvent phase)
was injected into a Shimadzu GCMS (QP2010), which
was equipped with a RTX-5MS capillary column
(30 m 02 mm). The carrier gas was helium, the flow
rate was 15 ml min)1, and the operating temperature of
the injector was 260C. The temperature gradient was set
from 60 to 260C at a rate of 5C min)1, with an isother-
mal period of 10 min at the end. The electron impact ion
source was maintained at 200C. Electron impact mass
spectra were recorded at 70 keV. The mass spectrum of
each fatty acid methyl ester was matched to the NIST
database.
The aqueous fraction containing the amino acids was
subjected to AAS (GL Biochem, Shanghai, China), using
phenyl isothiocyanate (PTA) to generate the derived
amino acids of PTA-amino acid (PTA-AA). PTA-AA
standard solutions and samples (2 ll) were subjected to
HPLC, using a Venusii-AA analysis column (46 mm
250 mm, 5 lm). An acetonitrile water gradient was used;
the gradient started with 80% acetonitrile for 3 min and
was raised to 100% acetonitrile for 35 min, after which it
was kept at that concentration for 2 min. The HPLC flow
rate was 06 ml min)1. NMR spectra were recorded on a
Bruker 400 MHz NMR spectrometer at 30C, using
samples in deuterated chloroform (CDCl3).
Antimicrobial assay
Antimicrobial activity was measured as described by
Suppasil et al. (2006). Two strains of Gram-positive
bacteria (Bacillus subtilis CMCC 63501; Staphylococcus
aureus CMCC 26003), a Gram-negative bacterium
(Escherichia coli CMCC 44103), a yeast (Candida albicans
AS 2.538) and a fungus (Rhizoctonia solani CBS627.77)
were used in this assay. The activity tests were used with
the TLC bio-autography method (Merck GF254, 06 mm)
(Galindo-Cuspinera and Rankin 2005).
Amino acid supplementation test
To test for branched chains of amino acids in the compo-
sition of the hydrophobic moiety of the lipopeptide,
1209
A lipopeptide from strain B-5 N. Qiao and Z. Shao

1 g l)1 l-alanine, l-valine, l-leucine and l-isoleucine were on the surface of the culture supernatant. The hydropho-
added separately to MSM medium with 2% (v v) n-hex- bic layer contained surface-active compounds that could
adecane as the sole carbon and energy source. These lower waters surface tension to 328 mN m)1, while the
amino acids can be used as precursors of branched-chain supernatant and cells lost their surface activity and only
fatty acids (Kaneda 1966). One litre of culture was loaded reduced surface tension to 5965 mN m)1.
in a 2-l flask and cultured at 180 rev min)1 at 28C for The biosurfactant in the hydrophobic layer was
5 days; a control was set up without added amino acids. extracted, run on a TLC plate and detected using several
The biosurfactant from each treatment was purified and colour reaction reagents as described in M&M. The
subjected to fatty acid analysis as described earlier. highest surface-activity compound was observed in the
chloroform methanol water (65 : 15 : 2, v v v) eluate,
from which a spot (Rf = 055) on the TLC plate was
Results
found to react positively to ninhydrin reagent. Glycoli-
pids, phospholipids and lipid-organic acids were also
Biosurfactant production
found in other eluates, but in much smaller quantities.
We found that A. dieselolei B-5 produced biosurfactants Subsequently, preparation of the purified lipopeptide was
only in the presence of hydrocarbons, but not with other conducted using column and preparative TLC.
carbon sources such as glucose, glycerol and citric acid.
In another report, hydrocarbons also significantly induced
Structural characterization of the purified lipopeptide
biosurfactant production in hydrocarbon-degrading bacte-
ria (Parkinson 1985). Only one amino acid was found from the aqueous phase
After 5 days of incubation with n-hexadecane as the of the acid hydrolyte of the purified lipopeptide, and it
sole carbon source in MSM medium, the surface tension was identified as proline. When the derived PTA-AA
of the B-5 culture reached 298328 mN m)1. After products were subjected to HPLC analysis, there was only
centrifugation, a hydrophobic layer formed and floated one peak at 20441 min (Fig. 1a), which matched with

(a) mV
90
23913

33805

80
24976

28295

70
25978

28738

31022

60
15213

50
20234
16284

19527

40
14438

18828
17983
5518

30
6722

26888
23419

20
34364
26585

31630

10
0
3 6 9 12 15 18 21 24 27 30 33 min

(b) mV
72
64
20441

56
48
Figure 1 Amino acid analysis of the hydro-
40
philic moiety of the lipopeptide produced by
32 strain B-5. (a) HPLC profile of 17 kinds of
24 PTA-AA run within 38 min. The peak at 202-
34 min was PTA-proline. (b) PTA-AA profile of
16
33880
19121

hydrophilic hydrolytes of strain B-5 run under

13588

8 the same conditions as in (a). The only peak

0 occurred at 20441 min and matched to
PTA-proline. PTA-AA, phenyl isothiocyanate-
8 4 8 12 16 20 24 28 32 36 min amino acid.

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1210 Journal compilation 2009 The Society for Applied Microbiology, Journal of Applied Microbiology 108 (2010) 12071216
N. Qiao and Z. Shao
the PTA-proline peak (20234 min) in the standard
PTA-AA sample (Fig. 1b).
Three kinds of fatty acids were found in the lipid phase
of the acid hydrolyte using GCMS. They were C14:0,
C16:0 and C18:0 (with distributions of 4976%, 4738% and
286%, respectively); all of them were normal fatty acids,
and no branched-chain fatty acids or 3-OH fatty acids
were detected.
Analysis of the intact molecules with LCQ-MS revealed
three molecular ion peaks with molecular masses [M+H]
of 327, 355 and 383, respectively (Fig. 2). There was a
difference of 28 between each of the molecular masses,
which exactly matches the mass of two CH2 groups
(14 2). Evidently, the varying molecular masses of 327,
355 and 383 correspond to the fatty acids C14:0, C16:0 and
C18:0, which occur in different monomers. Based on the
results of the AAS, GCMS and LCQ-MS, the molecular ion
with mass 355 is deduced as: C16:0 proline  H2 O H
256  43 115  13  18  01 1  01 354  56 (Fig. 3).
The proposed structure was further confirmed by 1H-
NMR (Fig. 4). In 1H-NMRd spectra, we detected the pro-
line functional group as P1, 38 ppm; P1, 365 ppm;
P2 P3, 20 ppm; P3, 23 ppm; P4, 44 ppm and the fatty
acid functional group as CH3, 087 ppm; (CH2)n,
12146 ppm; COCH2, 222 ppm; COCH2CH2,
167 ppm.
Characteristics of the proline lipids
The critical micelle concentration (CMC) of the proline
lipids was determined to be 40 mg l)1 approximately. The
MAD of the proline lipid mixture was measured with the
oil displacement test and compared to synthetic deter-
gents. The mixture showed higher activity than all the
tested synthetic detergents. As shown in Table 1, the
MAD of the proline lipids was 0125 lg, which was lower
than those of Tween 20, Tween 80, Triton X-100 and
sodium dodecyl sulfate (SDS), respectively.
2823
100
Relative abundance
80
60
40
3278
20 2653
2093
0
200
Figure 2 Molecular mass spectra of the lipopeptide produced by strain B-5. An LCQ quadruple ion-trap mass spectrometer utilizing electrospray
ionization was used for MS analysis of the purified compound. The positive ion mode was used, scans were run over the 1002000 m z range,
and results displayed anticipant signals at molecular masses [M+H] of 327, 355 and 383. Samples of the compound dissolved in methanol were
injected for MS analysis.
2009 The Authors
Journal compilation 2009 The Society for Applied Microbiology, Journal of Applied Microbiology 108 (2010) 12071216
A lipopeptide from strain B-5
O
N
COOH
Figure 3 Deduced molecular structure of the lipopeptide produced
by strain B-5. The molecular structure of the monomer with a molecular
mass of 355 was drawn using Chemdraw software version 8.0,
based on PTA-AA and MS results. The structure was proposed to
be a proline and a C16:0 fatty acid, linked by an acylamide bond.
PTA-AA, phenyl isothiocyanate-amino acid.
The effects of salinity and salts were examined using
the oil spreading test, by observing the variations in the
diameter of oil drops. Without NaCl, the diameter was
30 cm. At 39% salinity, NaCl enhanced the surface
activity (d = 3436 cm), while at 12% salinity the
diameter was 31 cm, and at 15% salinity a significant
reduction occurred (d = 25 cm). The effect of MgCl2 was
tested from 20 to 100 mmol l)1 and resulted in the larg-
est diameter, 3239 cm at 50 mmol l)1. However, CaCl2
above 3 mmol l)1 lowered the surface activity; the diame-
ters were 3132 cm at concentrations from 0 to
3 mmol l)1, but the diameter fell sharply to 2 cm when
the concentration was raised to 4 mmol l)1 (Fig. 5).
In the heat stability test, the surfactant activity showed
no obvious changes (d = 3032 cm) after 1 h of treat-
ment at 40100C, while there was a slight reduction in
diameter after 15 min of treatment at 121C (d =
26 cm). At a pH of 310, the diameter varied from 21
to 32 cm, with the largest diameter occurring at pH 4
(Fig. 5).
The emulsification test showed that the mixture of pro-
line lipids had strong emulsification activity, 75 3%
with n-hexadecane. Moreover, the emulsion was stable at
room temperature for 48 h. However, the proline lipids
did not exhibit any antimicrobial activity against any of
the tested strains, at concentrations of 10150 mg l)1.
3550
3832 5631 5925 7548 8624 9765 1036212028 13332 14816 1631017048 18603 19194
400 600 800 1000 1200 1400 1600 1800 2000
m/z
1211
A lipopeptide from strain B-5 N. Qiao and Z. Shao

(a)

Oil-spreading diameter (cm)

35
3
25
2
15
COCH2CH2 1
(CH2)n
05
0
2 4 6 8 10
(b) pH

Oil-spreading diameter (cm)

4
35
3
CH3 25
2
15
1
05
0
20 40 60 80 100 121
T (C)
P4 (c)
P1
P1 4
COCH2 Oil-spreading diameter (cm) 35
P2/P3 3
P3
25
2
15
1
5 4 3 2 1 ppm 05
0
0 1 3 5 7 9 12 15
Figure 4 1H nuclear magnetic resonance spectrum of the lipopeptide NaCl (%)
(d)
produced by strain B-5. The chemical groups represented by each
Oil-spreading diameter (cm)

peak are given in the figure. 45

4
35
3
25
Table 1 Comparison of minimum active dose (MAD) by oil displace-
2
ment test 15
1
Compounds MAD (lg)* 05
0
Proline lipid 0125 005 0 50 90 120 160
Tween 20 03 007 (e)
MgCl2 (mmol l1)
Oil-spreading diameter (cm)

Tween 80 015 005 35

Triton X-100 03 008 3
SDS 60 012 25

* standard deviation (SD) from triplicate experiments. 2

15
1
Effects of amino acid amendment
05
When alanine, valine, leucine and isoleucine were added 0
0 1 2 3 4 5 6
to MSM medium as the precursors of branched-chain
CaCl2 (mmol l1)
fatty acids, some changes occurred in the chemical com-
position and surface activity. Compared to the control to Figure 5 Effects of physical and chemical factors on the surface
which no amino acids were added, the fatty acid C14:0 activity of the lipopeptide. (ae) Treatments of pH, temperature, NaCl,
disappeared in all of the samples treated with amino CaCl2 and MgCl2, respectively. The activity was examined with the oil
acids, and the proportion of C16:0 fatty acid increased spreading test method, in triplicate.

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1212 Journal compilation 2009 The Society for Applied Microbiology, Journal of Applied Microbiology 108 (2010) 12071216
N. Qiao and Z. Shao
according to the length of the fatty acid chain of
the amino acids (alanine < valine < leucine isoleucine).
Moreover, the proportion of C18:0 fatty acid decreased
significantly in the presence of isoleucine. Correspond-
ingly, the surface activities detected with the parafilm and
oil spreading methods increased depending on the amino
acid added: alanine < valine < leucine isoleucine. How-
ever, the surface tension of the culture broth did not
change depending on the amino acid added and showed
only slight variations.
Discussion
More and more biosurfactants are being used for envi-
ronmental and industrial purposes (Javaheri et al. 1985;
White et al. 1985; Shepherd et al. 1995; Desai and Banat
1997; Ron and Rosenberg 2001, 2002; Arutchelvi et al.
2008; Franzetti et al. 2008). Lipopeptides such as surfac-
tin, which is produced by B. subtilis, are among the most
important biosurfactants as they are mostly cyclic and
usually more efficient than glycolipids (Bernheimer and
Avigad 1970; Cooper and Zajic 1980).
In this report, a proline lipid was found to be pro-
duced by A. dieselolei B-5. This strain is the first-reported
lipopeptide producer in the genus Alcanivorax. Bacteria of
this genus are the most prevalent oil degraders in marine
environments. The first bacterium of this genus to be
discovered was A. borkumensis SK2, which was identified
as a biosurfactant producer producing a mixture of
glucose lipids (Abraham et al. 1998). In a previous study,
we also tried to search for glucose lipids in B-5 cultures,
but did not obtain any such compounds (Liu and Shao
2005). In this report, however, we identified the hydro-
phobic layer floating on the culture of B-5, from which
proline lipids were found as the main surface-active com-
ponent. Moreover, these lipids are different from other
linear lipopeptides previously reported.
Most lipopeptide biosurfactants have been noted for
their antibiotic activity (Morikawa et al. 1993; Sorensen
et al. 2001; Batrakov et al. 2003). One kind of linear
lipopeptide biosurfactant is N-acylglutamine, which is pro-
duced by various bacteria including Myroides odoratus, and
was suggested as an elicitor of plant volatiles (Spiteller et al.
2000). Another kind of linear lipopeptide biosurfactant is
ornithine lipid, which has no significant antimicrobial
activity but has strong emulsification ability. It exists widely
in outer (mainly) and inner cell membranes (Wilkinson
et al. 1982; Laneelle et al. 1990), with the exception of the
strain Myroides sp. SM1, which produces ornithine lipid in
culture supernatant (Maneerat et al. 2006).
In contrast, the proline lipids of strain B-5 had no anti-
microbial activity, but showed strong emulsification abil-
ity. Moreover, the lipid is not membrane-bound, but is
2009 The Authors
Journal compilation 2009 The Society for Applied Microbiology, Journal of Applied Microbiology 108 (2010) 12071216
A lipopeptide from strain B-5
found in the hydrophobic layer of culture broth. Thus,
the biosurfactant produced by B-5 would be environmen-
tally friendly and synergetic to hydrocarbon degradation,
as it is not toxic to other bacteria and is released freely
from the producing cells.
Biosurfactants usually have lower CMC values and
stronger emulsification ability than synthesized surfactants
(Parkinson 1985); for example, the CMC value of rhamno-
lipids was 50 mg l)1 and that of surfactin was 45 mg l)1
(Whang et al. 2008). The proline lipids of B-5 showed a
CMC value of 40 mg l)1.
In addition, the B-5 proline lipid was still active in the
presence of high concentrations of NaCl, MgCl2 and
CaCl2. Sodium, calcium and magnesium salts are gener-
ally present in seawater and are also frequently found in
industrial water, but an excess of salts could eliminate
surface activity (Kim et al. 1997). Thus, biosurfactants
that tolerate higher salinity are very propitious for
bioremediation of petroleum pollution in seawater and
hydrocarbon pollution in industrial water. Salts such as
NaCl, MgCl2 and CaCl2 were consistently found to
activate biosurfactant activity with other strains isolated
from seawater or petroleum reservoirs (Yakimov et al.
1995). Moreover, the proline lipid of B-5 was structurally
steady and retained stable activity within a wide range of
temperatures and pH. It is known that high temperature
could reduce negative ion surfactant adsorption and
increase positive ion surfactant adsorption to granules
(Franzetti et al. 2008), while low temperature could
enhance the surface tension of liquids and interfere with
the effects of surfactant activity (Hoffmann et al. 2001);
extreme pH could alter the structure and activity of surf-
actants and reduce solubility in water (Zhu et al. 1993;
Acharya et al. 2004). Therefore, the proline lipid, which
has high surface activity at extreme pH and temperature
levels, serves an excellent candidate for application to
extreme environments.
It was reported that, as precursors of branched-chain
fatty acids, aliphatic amino acids could alter the ratio of
iso to normal even-numbered fatty acids and the ratio of
anteiso to iso odd-numbered fatty acids (Zhu et al. 2005).
When alanine, valine, leucine and isoleucine were added
to the culture broth separately, the fatty acid composition
of the lipopeptide changed. It has been found that
biosurfactant activity depends on the ratios of both iso to
normal even-numbered fatty acids and anteiso to iso
odd-numbered fatty acids (Youssef et al. 2005).
In the case of B-5, the fatty acid composition of the
proline lipids did change in response to the addition of
aliphatic amino acids but only in the proportion of
normal fatty acids, not in the occurrence of branched-
chain fatty acids. As in our previous reports (Peng et al.
2007, 2008), 3-OH fatty acid was not found in proline
1213
A lipopeptide from strain B-5
lipids, although it is believed to be very important in both
glycolipid and lipopeptide biosurfactants (Maier and
Soberon-Chavez 2000).
In conclusion, a linear lipopeptide biosurfactant was
produced by A. dieselolei B-5. It is unique in its chemical
composition and can lower water surface tension to 298
328 mN m)1; it has a CMC value of 40 mg l)1, and its
activity remains stable throughout the wide ranges of pH
and temperature. The proline lipid is believed to have
potential for further development because of its low
toxicity, high efficiency with a low CMC value and
outstanding tolerance for extreme pH and temperature.
These predominant characteristics make it an excellent
candidate for enhanced oil recovery and various other
applications, more specifically, in the treatment of
oil-containing water, microbial enhanced oil recovery and
the foodstuff industry; it might be a competitive candi-
date for applications in extreme environments.
Acknowledgements
The authors acknowledge Q.L. Lai., T.H. Zhong and L.J.
Yu for their collaboration during the GCMS, NMR and
LCQ-MS analyses, respectively. This report was supported
by the National Natural Science Foundation of China
(30670051), the Science Foundation of the Fujian
Province of China, and National Infrastructure of Natural
Resources for Science and Technology Program of China
(no. 2005DKA21209).
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