Process Biochemistry 49 (2014) 16991707

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Process Biochemistry
journal homepage: www.elsevier.com/locate/procbio

Identication and biochemical characteristics of lipopeptides from

Bacillus mojavensis A21
Hanen Ben Ayed a , Noomen Hmidet a, , Max Bchet b , Marlne Chollet b ,
Gabrielle Chataign b , Valrie Leclre b , Philippe Jacques b , Moncef Nasri a
a
b

Laboratoire de Gnie Enzymatique et de Microbiologie, Universit de Sfax, Ecole Nationale dIngnieurs de Sfax, B.P. 1173-3038 Sfax, Tunisia
ProBioGEM EA1026, PolytechLille/IUTA, Universit Lille-Nord de France, F-59655 Villeneuve dAscq, France

a r t i c l e

i n f o

Article history:
Received 25 March 2014
Received in revised form 30 June 2014
Accepted 3 July 2014
Available online 11 July 2014
Keywords:
NRPS
PCR
MALDI-TOF-MS
Fengycin
Surfactin
Pumilacidin

a b s t r a c t
This study reports the potential of a marine bacterium, Bacillus mojavensis A21, to produce lipopeptide
biosurfactants. The crude lipopeptide mixture was found to be very effective in reducing surface tension to 31 mN m1 . PCR experiments using degenerate primers revealed the presence of nonribosomal
peptide synthetases genes implied in the biosyntheses of fengycin and surfactin. Matrix-Assisted Laser
Desorption Ionization Time-of-Flight Mass Spectrometry (MALDI-TOF-MS) performed on whole cells of
B. mojavensis A21 conrmed the presence of lipopeptides identied as members of surfactin and fengycin
families. Further, a detailed analysis performed by MALDI-TOF-TOF revealed the presence of pumilacidin
compounds. The crude lipopeptide mixture was tested for its inhibitory activity against Gram-positive
and Gram-negative bacteria, and fungal strains. It was found to display signicant antimicrobial activity.
Strain A21 lipopeptide mixture was insensitive to proteolytic enzymes, stable between pH 3.0 and 11.0,
and resistant to high temperature. Production of lipopeptides is a characteristic of several Bacillus species,
but to our knowledge this is the rst report involving identication of pumilacidin, surfactin and fengycin
isoforms in a B. mojavensis strain.
2014 Elsevier Ltd. All rights reserved.

1. Introduction
The genus Bacillus is known to produce a broad spectrum of biologically active molecules with great potential
for medical and biotechnological applications. Among these
molecules, biosurfactants have received great attention in different elds, including phytosanitary sector, medicine, cosmetics,
food and feed additives, bioremediation, etc., [1]. Structurally,
biosurfactants are amphiphilic molecules and comprise various
different chemical structures, such as glycolipids, lipopeptides,
polysaccharideprotein complexes, phospholipids, fatty acids and
neutral lipids [2]. Compared to chemical surfactants, biosurfactants have several advantages, including low toxicity, high
biodegradability under natural conditions, ecological acceptability
and effectiveness at extreme temperatures and pH values [3].
Lipopeptides are among the most commonly isolated and characterized biosurfactants. They have received great attention due
to their medical, food and biotechnological applications. Further,
they were found to remove efciently petroleum hydrocarbons and

Corresponding author. Tel.: +216 22 76 31 14; fax: +216 74 275 595.

E-mail address: hmidet noomen@yahoo.fr (N. Hmidet).
http://dx.doi.org/10.1016/j.procbio.2014.07.001
1359-5113/ 2014 Elsevier Ltd. All rights reserved.

heavy metals from contaminated soils [4]. The lipopeptides produced by numerous Bacillus spp. are classied into three families
depending on their amino acids sequence: surfactins, iturins and
fengycins [5] and are considered as safe. These advantages make
lipopeptides potential alternatives to chemically synthesized surfactants. From another side, the fast progress of biotechnology has
accelerated the research and development of new and more effective lipopeptides.
These molecules are synthesized by multimodular enzymes
complexes known as nonribosomal peptides synthetases (NRPSs)
[5,6]. Lipopeptides contain hydrophilic peptides, which differ in
amino acid composition and sequence (seven to ten amino acids)
linked to a hydrophobic fatty acid with different chain lengths and
isomeries [5].
Among the Bacillus species, Bacillus subtilis is best known for the
production of lipopeptides, mainly surfactin and fengycin [7]. Surfactin is one of the most effective biosurfactants and shows several
pharmacological activities including the antimicrobial, antiviral,
antitumoral and antibrinolytic ones. It is a cyclic lipoheptapeptide which contains a -hydroxy-fatty acid with a chain length of
1315 carbon atoms [8]. Several variants of surfactin have been
described such as lichenysin from Bacillus licheniformis or pumilacidin from Bacillus pumilus. In addition to surfactin, fengycin is

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H. Ben Ayed et al. / Process Biochemistry 49 (2014) 16991707

another best-known lipopeptide, which exhibits several biological

activities, including activity against lamentous fungi [9]. Fengycin
is a cyclic lipodecapeptide containing a -hydroxy-fatty acid with
a length of 1518 carbon atoms [9]. Two variants of fengycins (A
and B) have been already described in the literature. They differ
by the amino acid residue in position 6. Few studies reported the
production of lipopeptides by Bacillus mojavensis strains [10,11].
In this study, B. mojavensis A21 isolated on the basis of its proteolytic activity [12] was also found to produce high amounts of
biosurfactants (based on their ability to reduce surface tension from
71 to 31 mN m1 ) [13]. The biosurfactant mixture showed high
physico-chemical properties in terms of the surface activities and
emulsication index. Further, the mixture was found to remove
diesel more effectively than synthetic surfactants. Therefore, this
study reports on (i) the detection of NRPS genes by PCR and (ii)
structural characterization of lipopeptides by MALDI-TOF-MS. In
addition, the stability of the produced lipopeptides under extreme
conditions was investigated in order to estimate their potential
applications.

Glucose concentration was analyzed in the ltered samples by

HPLC Spectra SYSTEM P1000 XR supplied by Thermoelectron Corporation (Thermo Fisher Scientic Inc., Waltham, MA, USA) using a
Fast Fruit Juice column (150 7.8 mm, Waters Corp., Milford, MA,
USA). The ow rate is set at 0.8 ml/min and the column was maintained at 55 C. Elution was achieved under 0.05% H3 PO4 . Detection
was carried out by using a refractometer Spectra System RI-150.
2.2. Extraction and quantication of lipopeptides

2. Materials and methods

Lipopeptides were extracted from culture supernatant by solid

phase extraction using C18 Maxi clean cartridges (Extract Clean
SPE 500 mg, Grace Davison-Alltech, Deereld, IL, USA) according to
Guez et al. [16]. The supernatant was applied to a C18 cartridge,
which retained lipopeptides. The cartridge was rinsed with 8 mL of
bi-distilled water and lipopeptides were then eluted with 8 mL of
100% methanol. After evaporation of the solvent, the crude lipopeptide mixture was dissolved in 200 L methanol.
Lipopeptides were analyzed and quantied by reversed-phase
high-performance liquid chromatography (600 s, Waters Corp.)
using a C18 column (5 m, 250 mm 4.6 mm, 218 TP, VYDAC, Hesperia, CA, USA).

2.1. Bacterial strain and lipopeptides production

2.3. Detection of NRPS genes by PCR and DNA sequencing

The microorganism used in this study was isolated in our laboratory from marine water in Sfax, Tunisia. It was identied as B.
mojavensis A21 based on its biochemical and physiological characteristics, and on the 16S rRNA gene sequence analysis. It was
assigned the accession number EU366229 [12].
The strain B. mojavensis A21 was inoculated in 250 mL Erlenmeyer ask containing 25 mL LuriaBertani (LB) broth medium
(10 g/L tryptone, 5 g/L yeast extract, 5 g/L NaCl) and cultivated at
37 C for 24 h under agitation at 200 rpm. For lipopeptides production, culture was conducted in 1 L Erlenmeyer ask containing
100 mL of Landy medium [14] consisting of: glucose, 20 g/L; l-g
lutamic acid, 5 g/L; yeast extract, 1 g/L; K2 HPO4 , 1 g/L; MgSO4 7H2 O,
0.5 g/L; KCl, 0.5 g/L; CuSO4 , 1.6 mg/L; Fe2 (SO4 )3 , 1.2 mg/L and
MnSO4 , 0.4 mg/L. The medium was complemented by 100 mM
MOPS and the initial pH was adjusted to 7.0 with 3 M KOH. Culture
was carried out for 72 h at 30 C under shaking at 160 rpm. After
fermentation, the culture broth was centrifuged at 13,000 g for
30 min at 4 C, and the supernatant containing the crude lipopeptide was collected. Lipopeptide molecules were partially puried
from the cell-free supernatant by different steps of ultraltration/dialtration. The crude lipopeptide mixture obtained was
evaluated for its antimicrobial activity and its stability against
extreme conditions. All experiments were carried out in triplicates.
Lipopeptide production was also analyzed by measurement of
surface tension during the growth of the strain. The surface tension of the cell-free supernatant was determined according to the
Du Noy ring method in a TDI tensiometer (Lauda, Knigshofen,
Germany) as described by Leclre et al. [15]. The values obtained
are the mean of three measurements.

Two pairs of degenerate primers (Af2-F/Tf1-R and As1-F/Ts2-R),

designed using the alignment of adenylation or thiolation domains
that compose lipopeptide synthetases, were used for the detection
by PCR of the NRPS genes (Table 1) [17]. DNA was extracted from
overnight culture of A21 strain using the Wizard Genomic DNA
Purication Kit and protocol (Promega Corp., Madison, USA).
The PCR thermal cycle program included an initial denaturation
step at 94 C for 3 min, followed by 30 cycles, with denaturation
at 94 C for 1 min, annealing at 43 C for 30 s, with As1-F/Ts2-R,
and at 45 C with Af2-F/Tf1-R primers, and extension at 72 C for
45 s. Final extension was performed at 72 C for 10 min. The Taq
polymerase used was Master Mix (Thermo Scientic Fermentas,
Illkirch, France) with a nal primer concentration of 1.2 M. PCR
products were analyzed by 0.7% (w/v) agarose gel electrophoresis.
PCR products were puried with QIAquick Gel Extraction Kit
(Qiagen, Hilden, Germany) and then cloned into pGEM-T Easy Vector (Promega Corp.). Recombinant plasmids were introduced into
Escherichia coli JM109 cells by heat shock, according to the manufacturers protocol (Promega). Transformants were selected on
LuriaBertani (LB) solid medium supplemented with ampicillin,
IPTG (isopropyl--d-thiogalactopyranoside) and X-Gal (5-bromo4-chloro-3-indolyl--D-galactopyranoside) at nal concentrations
of 100 g/mL, 200 g/mL and 20 g/mL, respectively. White
colonies were picked and then cultivated in LB medium supplemented with 100 g/mL ampicillin for 24 h at 37 C. Plasmids
were isolated from the transformed cells using the QIAprep
Spin Miniprep kit (Qiagen, Germany). Cloned PCR products were
sequenced using the universal primers pUC-M13-R/F (Eurons
MWG Operon, Ebersberg, Germany). DNA sequences were analyzed

Table 1
List and characteristics of primers used in this study.
Primer names

Sequence of primersa

HyCb

Expected fragment size (bp)

Identied nonribosomal lipopeptides

References

Af2-F
Tf1-R

GAATAYMTCGGMCGTMTKGA
GCTTTWADKGAATSBCCGCC

34
72

443, 452,455

Fengycins

[17]

As1-F
Ts2-R

CGCGGMTACCGVATYGAGC
ATBCCTTTBTWDGAATGTCCGCC

12
36

419, 422, 425, 431

Surfactins

[17]

bp, base pair.

a
Using IUPAC DNA code: Y = C or T, M = A or C, K = G or T, W = A or T, D = G, A or T, S = G or C, B = G, T or C, R = A or G.
b
Coefcient of hybridization (HyC) calculated as described by Tapi et al. [17].

H. Ben Ayed et al. / Process Biochemistry 49 (2014) 16991707

with the GenBank databases using the BLAST (Basic Local Alignment
Search Tools) software provided online by the National Center for
Biotechnology Information (Bethesda, MD, USA).
2.4. Lipopeptides detection by MALDI-TOF-MS analysis
MALDI-TOF-MS was used to detect and characterize lipopeptides from whole cells of strain A21. Individual colonies, grown
on LB agar plate at 30 C for 72 h, were carefully suspended in
Eppendorf tube containing a matrix solution (10 mg/mL cyano-4hydroxycinnamic acid in 70% water, 30% acetonitrile, and 0.1% TFA).
The sample was homogenized and then centrifuged at 5000 rpm.
For classical analysis, 1 L of the sample solution was spotted onto
a MALDI-TOF MTP 384 target plate (Bruker Daltonik GmbH, Leipzig,
Germany) and let dry before analysis.
Mass proles experiments were also analyzed with an Ultraex
MALDI-TOF/TOF mass spectrometer (Bruker, Bremen, Germany)
equipped with a smartbeam laser. Samples were analyzed using
an accelerating voltage of 25 kV and matrix suppression in deexion mode at m/z 750. The laser power was set to just above the
threshold of ionization (around 35%). Spectra were acquired in
reector positive mode in the range from 800 to 3000 Da. Each
spectrum was the result of 1000 laser shots per m/z segment per
sample delivered in 10 sets of 50 shots distributed in three different locations on the surface of the matrix spot. The instrument was
externally calibrated in positive reector mode using Bradykinin
[M+H]+ 757.3991, Angiotensin II [M+H]+ 1046.5418, Angiotensin
I [M+H]+ 1296.6848, Substance P [M+H]+ 1347.7354, Bombesin
[M+H]+ 1619.8223, and ACTH [M+H]+ 2093.0862.
MS/MS spectra were obtained using the LIFT technique
described elsewhere (LIFT-TOF/TOF). In brief, fragment ions are
generated by a selection of the precursor ions produced during the
MALDI process, similar to MS analysis; the isolation mass window
was set to 1% of parent mass and the laser power boost to induce
fragmentation was 80%.
2.5. Antimicrobial activity
Antibacterial activities were tested against Gram-positive and
Gram-negative bacterial strains. The bacteria used were Staphylococcus aureus (ATCC 25923), Bacillus cereus (ATCC 11778),
Enterococcus faecalis (ATCC 29212), Micrococcus luteus (ATCC 4698),
Pseudomonas aeruginosa (ATCC 27853), Salmonella typhimurium
(ATCC 19430), Klebsiella pneumoniae (ATCC 13883) and E. coli (ATCC
25922). Antifungal activities were tested against Aspergillus niger I1,
Mucor rouxii DSM 1191 and Botrytis cinerea.
Antimicrobial activity of B. mojavensis A21 lipopeptides was
assessed by the agar well diffusion method [18]. Culture suspension (100 L) of the indicated strains (about 106 colony forming
units (Cfu)/mL for bacterial cells and 5 104 spores/mL for fungal
strains) were spread over Luria-Bertani (LB) agar or Sabouraud dextrose agar, respectively. Then, wells (3 mm depth, 6 mm diameter)
were made in the agar plates using a sterile borer. A 60 L sample of the crude lipopeptides mixture was loaded into the wells.
The plates were then incubated for 24 h at 37 C for bacteria and
72 h at 30 C for fungal strains. The diameter of the inhibition zone
was measured and the results reported in mm. The experiment was
conducted in triplicate.

1701

residual antibacterial activity using S. aureus as indicator strain was

measured by the agar well diffusion method.
The effect of pH on lipopeptide activity was examined by
assaying antimicrobial activity using S. aureus after incubation for
2 h at 4 C in the pH range of 3.011.0. Samples were neutralized
to pH 7.0 before measurement of the antimicrobial activity. The
following buffer systems were used: 100 mM glycineHCl buffer,
pH 2.04.0; 100 mM acetate buffer, pH 4.06.0; 100 mM TrisHCl
buffer, pH 7.08.0 and 100 mM glycineNaOH buffer, pH 9.0 11.0.
In order to evaluate stability to proteolytic enzymes, the crude
lipopeptide was incubated with each enzyme for 2 h at 37 C with
1 mg/mL (nal concentration) of the following enzymes: trypsin,
chymotrypsin, bromelain and alcalase in 0.05 M TrisHCl buffer (pH
8.0) and pepsin in 0.05 M glycineHCl buffer (pH 2.0). The residual
antibacterial activity was then evaluated by the agar-well diffusion
method against S. aureus.
Organic solvent stability of the crude lipopeptide A21 was carried out by incubating the lipopeptide solution for 1 h at 37 C in
the presence of various organic solvents (50%, v/v). Residual activity
was then determined against S. aureus as indicator strain.

3. Results and discussion

3.1. Detection by PCR of NRPS lipopeptides genes in B. mojavensis
A21
Lipopeptides are synthesized by NRPSs encoded by operons with
different open reading frames. For example, the operon of surfactin
family contains four open reading frames (ORFs) coding for surfactin synthetase, which are designated srfA-A, srfA-B, srfA-C and
srfA-D. The comparative bioinformatics analyses of each operon led
to the design of different primer pairs for the three families taking
into account the differences between open reading frames of each
synthetase gene [17].
In this study, PCR, with two primers pairs (Af2-F/Tf1-R and
As1-F/Ts2-R), was used initially for the detection of NRPS genes,
involved in the biosynthesis of fengycin and surfactin, respectively.
Both primers pairs gave amplicons with the expected sizes (Fig. 1).
The PCR products were cloned into pGEM-T Easy vector and then
sequenced as described in Section 2. The Blast n results obtained
with the two sequenced fragments are presented in Table 2.

2.6. Effects of heat, pH, proteolytic enzymes and organic solvents

on crude lipopeptide antibacterial activity
To investigate thermal stability, the crude lipopeptide mixture
was incubated in water bath at different temperatures (60100 C)
for 15 min. After cooling the treated samples to room temperature,

Fig. 1. PCR amplication by degenerate primers pairs with B. mojavensis A21. M:

molecular weight marker, Af2-F/Tf1-R (lane 1), As1-F/Ts2-R (lane 2).

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H. Ben Ayed et al. / Process Biochemistry 49 (2014) 16991707

Table 2
Blast results of the sequenced products obtained from PCR amplication with degenerate primers.
Primer names

Product size (bp)

GenBank accession number

Probable proteins

Identity (%)

Af2-F
Tf1-R
As1-F
Ts2-R

443

AAB80955.2

Fengycin synthetase of B. subtilis. subsp. str 168

97

421

ZP03590011.1

Surfactin synthetase of B. subtilis.

89

The sequence of the fragment amplied by Af2-F/Tf1-R primers

showed high homology (97%) with the plipastatin synthetase
operon of B. subtilis subsp. subtilis strain 168. Further, As1-F/Ts2-R
primers amplify fragment which showed a high similarity with the
surfactin synthetase operon of B. subtilis 168 (89%). The high homology indicated that surfactin and fengycin synthetases are nearly
identical between Bacillus strains.
Few studies reported the production of lipopeptides by B.
mojavensis strains. Snook et al. [11] reported the production, from
the endophytic bacterium B. mojavensis RRC1001, of Leu7-surfactin
which was found to suppress the growth of Fusarium verticillioides.
More recently, a marine-derived bacterium B. mojavensis B0621A
was found to produce a new iturinic lipopeptide called mojavensin
A [12]. The presence of surfactin and fengycin synthetase genes
was also detected in several Bacillus strains [7]. The production of
this lipopeptide mixture by the same strain could be advantageous,
since a synergistic effect may occur and improve the biological
properties.
3.2. Detection of lipopeptides production by MALDI-TOF analyses
MALDI-TOF mass spectrometry has been largely used as an efcient tool for the detection and identication of lipid molecules
from whole microbial cells cultivated on a solid medium [19,20].
Therefore, in order to check that the presence of both surfactin
and fengycin operons detected by PCR was related to an effective
expression, MALDI-TOF-MS analysis was performed on whole cells
of B. mojavensis A21 cultivated on LB solid medium. Fig. 2 shows the
MALDI-TOF mass spectra of intact cells of this strain. Mass spectra analysis showed two well resolved clusters of peaks, one at
m/z values between 1045 and 1080 (Fig. 2A) and the other one in
the range of 14801550 (Fig. 2B). By comparing the mass with the
mass numbers reported for the lipopeptide complexes from other
Bacillus strains [21], the rst group of peaks could be attributed
to surfactin isomers, while the second cluster of peaks could be
assigned to fengycin isomers. Our results are in line with previous
works reported by Coutte et al. [22] and Kim et al. [23] that showed
the co-production of different homologous compounds for each
lipopeptide family. These isoforms vary according to the length
of their fatty acid side chains as well as the peptide amino acid
composition.
The mass spectra reported in Fig. 2A revealed the presence of
three major [M+K]+ peaks at m/z 1046.66, 1060.69 and 1074.73.
The peak with a m/z 1046.66 corresponded to the mass of [M+K]+
ion of surfactin with a fatty acid chain length of 13 carbon atoms,
or pumilacidin C12. The peaks at m/z 1060.69 and 1074.73 could
be assigned as potassium adduct of surfactin C14 and C15, respectively, or pumilacidin C13 and C14, respectively (Fig. 2A).
On the other hand, the mass spectra reported in Fig. 2B show
the presence of 4 major peaks at m/z 1487.14, 1501.83, 1515.88
and 1529.88. These peaks could be attributed to [M+K]+ forms
of fengycin AC15, AC16/BC14, AC17/BC15 and AC18/BC16, respectively (Fig. 2B). For each group, the different peaks differ by 14 Da,
which corresponds to the molecular weight of one CH2 group.
The results obtained with the PCR and MALDI-TOF-MS techniques conrmed the complementarities of these approaches.
This is in contrast with results obtained by Leenders et al. [24]
who detected by PCR conserved genes encoding surfactin and

fengycin synthetase in B. subtilis 168; however, no lipopeptides

were detected using MALDI-TOF-MS, owing to the presence of a
mutated sfp- gene.
3.3. Characterization of surfactin/pumilacidin lipopeptides by
MS-MS analysis
MALDI-TOF MS2 analysis was also used in order to obtain more
precise information on the chemical structure of some lipopeptides. The fragment ion patterns of the parent ions at m/z 1060.77
and 1074.68, reported in Fig. 3, shows fragments that can correspond to differences among some amino acids in the peptide
moiety. A set of daughter ions was observed in the MS/MS spectrum of the parent ion at m/z 1060.77. The fragmentation pattern
of the peak 1060.77, illustrated in Fig. 3A, resulted in the appearance of product ions at m/z 947.68 [M(A7 = 113)] and 834.68
[M(A7 + A6)]. These two peaks are the results of the consecutive losses of two Leu (or Ile) residues. Therefore, the amino acid
residue at position 7 is a Leu or Ile and not a Val residue. Other
peaks were observed at m/z 737.64 [M(AA1 + FA)] and 624.73
[M(AA1 + AA2 + FA)]. The obtained results indicated that the peak
at m/z 1060.77 is unambiguously a pumilacidin, a cyclic lipopeptide with a fatty acid chain of 13 carbons, and Leu or Ile residue at
position 7.
However, there is more ambiguity with the parent ion at m/z
1074.68. Indeed, on the basis of the obtained fragmentation, displayed in Fig. 3B, this peak could be made up of three different
molecules: surfactin Ile7/Leu7 C15, pumilacidin Ile7/Leu7 C14 and
pumilacidin Val7 C15 (Fig. 3B).
The results obtained with the PCR and MALDI-TOF-MS techniques demonstrated that the strain B. mojavensis A21 produces
two families of lipopeptides with different homologous compounds. Lipopeptides belonging to surfactin and fengycin families
were reported both in B. subtilis and Bacillus amyloliquefaciens
strains, together often with a third lipopeptide of the iturins
group. The co-production of surfactins and fengycins families by
B. mojavensis A21 is an interesting characteristic which could support their potential applications in many biotechnological elds
[25]. Further, the structural diversity of the produced lipopeptides may offer several potential applications. Indeed, it is well
known that different isoforms and homologues exhibit different
properties and activities, which depend in particular on the chain
length. Surfactins are known for their ability to act in a synergistic manner with fengycin which may improve their activities
[25]. The increasing interest in surfactin and fengycin is because of
their amphiphilic character, which is responsible for their excellent surface-active properties. Surfactin, one of the most effective
biosurfactants, showed several pharmacological activities including, antimicrobial, antiviral, antitumoral and antibrinolytic [26].
Fengycin shows specic antifungal activity against lamentous
fungi and inhibits phospholipase A2 [27]. In another study reported
by Ongena et al. [28] the lipopeptide mixture contained surfactin
and fengycin was found to be effective in the induction of systemic resistance in plants which makes them interesting for plant
protection.
Several works reported also the co-production of different
lipopeptides families. In this context, B. subtilis strains are known
to produce up to three families of lipopeptides [5]. For example,

H. Ben Ayed et al. / Process Biochemistry 49 (2014) 16991707

1703

Fig. 2. Mass spectroscopy (MALDI-TOF-MS) spectra of molecular mass of strain A21 lipopeptides. Spectra of surfactin (A) and fengycin (B) produced by B. mojavensis A21.

B. subtilis ATCC 6633 and its derivative BBG100 were found to

produce surfactins and mycosubtilin, while ATCC 9943 secretes
three lipopeptides families: surfactins, iturin A and fengycins [15].
The simultaneous production of two different lipopeptides has
also been reported by Ahimou et al. [29] for B. subtilis. Another
B. amyloliquefaciens strain was also found to produce surfactin and
iturin-like compounds [30].
Although the surfactin production by B. mojavensis strain has
been reported [10], the coproduction of surfactin and fengycin has
been reported only by few species, in particular by B. subtilis strains
[31]. Both of these lipopeptides are of great pharmaceutical and
biotechnological interest [26,32].

3.4. Lipopeptide production, antimicrobial activity and stability

studies of the crude lipopeptide mixture
3.4.1. Lipopeptide production
B. mojavensis A21 was cultivated in Landy medium for 72 h at
30 C. In this medium, glucose is used mainly as carbon source
for bacteria growth and lipopeptides production. The fermentation
data with respect to time indicating the changes in glucose concentration, biomass, and products, as well as surface tension was
shown in Fig. 4. A direct relationship between microbial growth,
surface tension reduction, glucose consumption and lipopeptide
production was observed. In fact, the surface tension of the culture

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H. Ben Ayed et al. / Process Biochemistry 49 (2014) 16991707

Fig. 3. MALDI-TOF MS/MS spectrum of lipopeptide produced by B. mojavensis A21. (A) MALDI-TOF MS/MS at m/z = 1074.9; and (B) MALDI-TOF MS/MS at m/z = 1060.88.

H. Ben Ayed et al. / Process Biochemistry 49 (2014) 16991707

1705

Fig. 4. Kinetics of growth, surface tension reduction, lipopeptide production and glucose consumption by B. mojavensis A21 strain. Culture was performed in Landy medium
at 30 C for 72 h.

was reduced from 71 at the beginning of growth to 31 mN m1

after 48 h of cultivation and then remained nearly constant until
the end of fermentation (72 h). Based on HPLC analysis the concentration of fengycin produced was clearly higher than the surfactin
one. Indeed, fengycin concentration was about 2.5 fold higher than
surfactin after 72 h of cultivation. The surfactin production was
growth-associated and began early in the exponential phase up to
the stationary phase. However, the fengycin production began after
12 h till the end of the culture.
The growth-associated relationship in surfactin production was
reported in some other B. subtilis strains [33]. Deleu et al. [34]
showed that surfactin is usually produced earlier than fengycin.
A direct relationship between glucose consumption and
lipopeptide production was observed and at the end of fermentation (72 h), the total amounts of glucose was consumed. Indeed,
the complete consumption of glucose occurred coinciding with the
maximum production of lipopeptide.
In this study, lipopeptides produced by B. mojavensis A21 strain
were isolated from the cell-free supernatant by different steps
of ultraltration/dialtration based on their aggregation behavior.
Ultraltration (UF) was carried out with 10 kDa cut-off membrane.
The partially puried lipopeptide was tested for its antimicrobial
activity and its stability against extreme conditions was investigated.
3.4.2. Antimicrobial activity
The antibacterial activity of the crude lipopeptide produced
by B. mojavensis A21 was tested against a variety of microorganisms. Inhibition zones diameters were measured using the
agar well diffusion method. Table 3 summarizes the antimicrobial
activity spectrum of the crude partially puried lipopeptide. The
crude lipopeptide exhibited varying degrees of antibacterial activity against all strains tested. Further, this inhibitory activity was
more effective against Gram-positive bacteria compared to Gramnegative bacteria. Indeed, the diameters of the inhibition zones
were in the range of 711 mm and 1322 mm with Gram-negative
and Gram-positive bacteria, respectively. M. luteus and S. aureus
were the most sensitive bacteria. Furthermore, lipopeptide A21 was
found to exhibit inhibitory activity against M. rouxii and A. niger, but
had no effect on B. cinerea.

Table 3
Antimicrobial activity spectrum of B. mojavensis A21 crude lipopeptides.

Gram (+)

Gram ()

Fungi

Indicator organism

Inhibition Zone
diameter (mm)

S. aureus (ATCC 25923)

B. cereus (ATCC 11778)
E. faecalis (ATCC 29212)
M. luteus (ATCC 4698)
P. aeruginosa (ATCC 27853)
K. pneumoniae (ATCC 13883)
E. coli (ATCC 29212)
S. typhimurium (ATCC 19430)
A. niger
B. cinerea
M. rouxii

21 1.0
13 1.0
16 2.0
22 1.0
7 2.0
9 1.0
11 1.0
11 1.1
+

++

Determinations were performed in triplicate and data correspond to mean values standard deviations.

Although antimicrobial activity was detected for most of Bacillus strains, there was considerable difference in their spectra and
degrees of inhibition. The high antibacterial and anti-fungal activities of A21 crude lipopeptide may be related to a synergistic effect
of both surfactins and fengycins.
3.4.3. Effects of proteolytic enzymes, heat, pH and organic
solvents on antibacterial lipopeptide activity
The resistance of lipopeptides against proteases and extreme
conditions, including pH and temperature was a prerequisite
for their potential biomedical and biotechnological applications.
Therefore, to check the stability, the crude lipopeptide mixture
was subjected to different conditions and its residual antimicrobial activity was evaluated using S. aureus as indicator strain. The
obtained results are summarized in Table 4 and Fig. S1. The stability of the crude lipopeptide against several proteolytic enzymes
was evaluated. Interestingly, all proteases tested had no effect on
the antimicrobial activity of the crude lipopeptide after incubation for 2 h at 37 C, since there was no signicant difference in
the inhibition zone diameter compared to control (without enzymatic treatment). This indicates that the antimicrobial compounds
could be cyclic peptides containing unusual amino acids [35]. These
results suggest that A21 lipopeptides possibly can survive at the

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H. Ben Ayed et al. / Process Biochemistry 49 (2014) 16991707

Table 4
Effects of enzymes, heat and organic solvents on antimicrobial activity.
Treatment

Inhibition Zone diameter (mm)

Enzyme (1 mg/ml)
Trypsin
Pepsin
Chymotrypsin
Bromelain
Alcalase

21
19
18
19
17

1.0
1.0
1.0
2.0
1.1

Temperature ( C) a
60
70
80
90
100
Freeze-dried

21
21
19
18
18
21

2.0
1.0
1.0
1.0
2.0
1.0

Organic solvents
Chloroform
Acetonitrile
Methanol

13 2.0
21 1.0
21 1.0

Determinations were performed in triplicate and data correspond to mean values standard deviations.
a
Incubations at different temperature were realized during 15 min.

intestinal environment and could be administered with feed. The

obtained results exclude the eventual existence of bacteriocin-like
substances, which may contribute to the inhibitory activities;
since the crude lipopeptide mixture was resistant to proteolytic
enzymes. Further, the stability of lipopeptides against proteolytic
enzymes is also supported by its high stability against proteases (at
least six) produced by A21 strain [12].
Moreover, one of the signicant ndings of this study was the
thermostability of the antimicrobial activity of lipopeptides produced by B. mojavensis A21. Antimicrobial activity of the crude
lipopeptide against S. aureus remained highly stable after 15 min
incubation at temperatures from 60 to 80 C, while a slight decrease
in activity was observed at 90 and 100 C (Table 4).
The activity of lipopeptides A21 at different pH values was also
determined (Fig. S1). The obtained results suggested that pH has no
signicant effect on the antimicrobial activity. The highest activity was observed at pH 7.0 and 8.0. The activity decreased slightly
below pH 6.0 and above pH 9.0. A similar stability prole was
also observed for lipopeptide biosurfactant produced by Serratia
marcescens NSK-1 [36].
In order to provide some information about the organic solvent
which can be used for lipopeptide extraction, the crude lipopeptide produced by B. mojavensis A21 was incubated in the presence
of several organic solvents. It was observed that the addition of
methanol and acetonitrile, during 15 min at room temperature,
showed no appreciable effect on lipopeptide activity. However, the
activity decreased after incubation with chloroform, with an inhibition zone of 16 mm, while that of control was 21 mm (Table 4).
Further, lipopeptides produced by B. mojavensis A21 exhibited high pH and thermal stability. Heat stability is potentially a
useful characteristic during food preservation because many foodprocessing procedures involve a heating step.
4. Conclusion
The presence of NRPS genes was detected by PCR using degenerate primers. In addition, the produced lipopeptides were detected
and their structures were more precisely characterized by MALDITOF-MS on whole cells of A21 strain. The crude lipopeptide mixture
was found to be mainly constituted of surfactins, pumilacidins and
fengycins.
The lipopeptide mixture exhibited a strong antimicrobial activity. Further, lipopeptides showed high stability under various

extreme conditions. They were quite stable over a wide temperature range from 60 to 100 C, and were highly active over a wide
range of pH from 3.0 to 11.0. In addition, the antimicrobial activity
was not affected by exposure to organic solvents for 1 h at 37 C.
These results indicated the robust stability of lipopeptides A21
under extreme conditions.
In the light of all these results, the lipopeptides from strain A21
could be excellent candidates for application in food and pharmaceuticals industries.
Acknowledgement
This work was funded by the Ministry of Higher Education and
Scientic Research-Tunisia.
Appendix A. Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.procbio.2014.07.001.
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