Production and Characterization of Surfactin-Type

Production and Characterization of Surfactin-Type Lipopeptides as Bioemulsifiers Produced by a Pinctada martensii-Derived Bacillus mojavensis B0621A Zongwang Ma & Jiangchun Hu

Applied Biochemistry and Biotechnology Part A: Enzyme Engineering and Biotechnology ISSN 0273-2289 Appl Biochem Biotechnol DOI 10.1007/s12010-015-1832-7

1 23

Your article is protected by copyright and all rights are held exclusively by Springer Science +Business Media New York. This e-offprint is for personal use only and shall not be selfarchived in electronic repositories. If you wish to self-archive your article, please use the accepted manuscript version for posting on your own website. You may further deposit the accepted manuscript version in any repository, provided it is only made publicly available 12 months after official publication or later and provided acknowledgement is given to the original source of publication and a link is inserted to the published article on Springer's website. The link must be accompanied by the following text: "The final publication is available at link.springer.com”.

1 23

Author's personal copy Appl Biochem Biotechnol DOI 10.1007/s12010-015-1832-7

Production and Characterization of Surfactin-Type Lipopeptides as Bioemulsifiers Produced by a Pinctada martensii-Derived Bacillus mojavensis B0621A Zongwang Ma 1,2 & Jiangchun Hu 1

Received: 25 January 2015 / Accepted: 3 September 2015 # Springer Science+Business Media New York 2015

Abstract Bacillus mojavensis B0621A was isolated from the mantle of a pearl oyster Pinctada martensii collected from South China Sea. Semi-purified surfactins (225 mg L−1) were obtained by acid precipitation and vacuum flash chromatography. The component of the semi-purified surfactins was preliminarily analyzed by liquid chromatography mass spectrometer system, and the results showed that all these surfactins could be a group of homologues. Eight surfactin homologues were isolated and afforded by reversed phase high-performance liquid chromatography. Furthermore, their structure was characterized by mass spectrometry analysis combined with nuclear magnetic resonance spectroscopy techniques. These surfactins shared seven amino acids as peptide backbone and a saturated β-hydroxy fatty acid chain residue (from C13 to C15), differed each other from peptide sequence in the position of Leu7 or Val7. All these surfactins had significant activity and stability of emulsification under various pH (from 7.0 to 12.0), temperature range (from 20 to 115 °C) and sodium chloride concentration (from 2.5 to 20.0 %, w/v). Taken all together, these results indicated that B. mojavensis B0621A have potential to be an alternative source as a biological-derived emulsifying agent. Keywords Bacillus mojavensis . Bacillus surfactins . Bioemulsifiers . Chemical identification . Emulsification activity

Electronic supplementary material The online version of this article (doi:10.1007/s12010-015-1832-7) contains supplementary material, which is available to authorized users.

* Jiangchun Hu [email protected] 1

Institute of Applied Ecology, Chinese Academy of Sciences, Shenyang 110016, China

2

Graduate University of Chinese Academy of Sciences, Beijing 100049, China

Author's personal copy Appl Biochem Biotechnol

Introduction Bacillus species are free living bacteria and have the ability adapting the environment swiftly due to spore-forming specificity. Bacillus lipopeptides are synthesized by nonribosomal peptide synthase and mainly including iturins, fengycins (or plipastatins), and surfactins, all three kinds of lipopeptides showing variant of structure differences within each family, namely, on peptide sequences or on long fatty acid residues [1–3]. Surfactins play important roles on interspecies competition, biofilm formation, cell motility, root colonization, and induced systemic protection of plants against pathogens [4]. Some of these features, more or less, directly regard the production of lipopeptides, such as surfactin produced by B. subtilis that could protect bacterial lawns from feeding nematode Caenorhabditis elegans [5]. Priming technique of imaging mass spectrometry characterized that surfactin produced by B. subtilis play the negative role on the formation of aerial hyphae of Streptomyces sp. Mg1 [6]. Surfactin produced by B. subtilis as one of the important factors relating to bacterial motility on the surface of soft agar plates, biofilm formation in artificial surfaces, root colonization and thus play a vital role in the interaction of Bacillus species, plants and other species, such as, bacterial predators and plant pathogens [7–9]. Moreover, surfactins could trigger plant systemic immunity response in a dosedependent manner and mediate the protection of whole plants from plant pathogens [10, 11]. In our previous work, a new family of iturin-type lipopeptides named mojavensins was isolated and characterized from the fermentation broth of Bacillus mojavensis B0621A, mojavensin variants shared same peptide back bone of L-Asn1, D-Tyr2, D-Asn3, L-Gln4, LPro5, D-Asn6, L-Asn7 [12, 13]. Mojavensin A had mediate and dose-dependent antagonistic activities against plant fungal pathogens, Gram-positive bacterium Staphylococcus aureus, and human leukemia cell line. Interestingly, those mojavensins with longer fatty acid chains, such as C16 and C17 mojavensin, had the ability to reduce surface tension to 50 mN m−1 at concentration of 10–15 mg L−1, compared with the control of 70 mN m−1. The antifungalguided approach demonstrated the main antifungal metabolites characterized from B. mojavensis B0621A were iso-C16 fengycin B and anteiso-C17 fengycin B; these two lipopeptides had strong inhibition activities to tested plant pathogens, the minimum of inhibition concentration to most of tested pathogens even lower than 125 μg mL−1 by discagar diffusion assay. However, excepting iturins and fengycins, B. mojavensis B0621A could secrete the third type of lipopeptides yet not isolated and characterized. In our current report, lipopeptides produced by B. mojavensis B0621A were screened for potential bioemulsifiers by an emulsification-activity-guided approach. The fraction with significant emulsification activity was selected, and each individual component was isolated and purified. Moreover, their structure was determined by multiple spectrometry and spectroscopy analysis. Finally, the emulsification activity of the selected surfactants was evaluated under different conditions (such as pH, temperature, and sodium chloride conditions); nHexadecane was used as oil substrate of emulsification activity evaluation.

Materials and Methods Bacterial Isolate and Growth Medium The strain was isolated from the mantle of a pearl oyster Pinctada martensii collected from Weizhou Island, South China Sea, and was characterized as B. mojavensis


B0621A (GenBank accession number: JN585825). The isolate was collected in 20 % glycerol solution at minus 80 °C and routinely maintained on Luria-Bertani medium at 28 °C. The mineral salt medium (MSM) was chosen for seed culture and further large-scale fermentation, which contained, per liter, 20 g sucrose, 2 g NH4NO3, 3 g KH2PO4, 10 g Na2HPO4, 0.2 g MgSO4·7H2O, 0.2 g yeast extracts, 50 μg CaCl2, 50 μg MnSO4·4H2O, and pH 7.0–7.2 [12].

Production and Purification Procedure Seed culture was prepared in 250-mL flasks, each contained 50 mL MSM medium, kept on a shaker at 180 rpm for 20 h, and then inoculated into 3-L flasks, each contained 1.5 L of MSM medium and kept 180 rpm of stirring rate at 28 °C. The broth was yielded after 48 h incubation and subsequently centrifuged at 5000 g for 30 min. The supernatant was adjust to pH 2.0 by 6 N hydrochloric acid and kept at 4 °C overnight. Then, the precipitate was collected by centrifugation and extracted with methanol (300 mL) for three times. The solution was evaporated to dryness and afforded crude extracts. The crude extracts were preliminarily purified with a vacuum flash chromatography over silica gel (600∼800 mesh) and eluted with a gradient of dichloromethane/methanol system (eight fractions from 100/0 (v/v) to 0/100 (v/v)). The fraction with emulsification activity was further purified by a semi-preparative reversed phase high-performance liquid chromatography (RP-HPLC) system (Dionex U3000, Sunnyvale, CA, USA) using C18 YMC-Pack ODS-A column (5 μm, φ 10 × 250 mm), with a flow rate of 2.5 mL min−1 and ultraviolet detection at the wavelength of 220 nm.

Liquid Chromatography Mass Spectrometry Analysis The fraction with emulsification activity was analyzed by a Waters Alliance 2695/ZQ 4000 liquid chromatography mass spectrometer (LC-MS) (Waters Corporation, MA, USA) equipped with a reversed phase HPLC column (Luna 5 U C18 Column, Phenomenex Inc., Torrance, CA, USA, φ 4.6 × 250 mm, UV detection at 220 nm, flow rate of 0.2 mL min−1), using isocratic elution of 93 % acetonitrile containing 0.05 % trifluoroacetic acid (TFA, v/v). Mass parameters were shown as follows: capillary voltage was 5.00 kV, cone voltage was 32.00 V, source temperature was 120 °C, and flow rate of cone gas was 150 L h−1. All detection was in positive mode.

Tandem Mass Spectrometry Analysis A LTQ XL™ linear ion trap mass spectrometer (Thermo Fisher Scientific Inc., USA) was used for tandem mass spectrometry (MS2 and MS3) analysis of peptide sequence of purified compounds. The ions of mass-to-charge ratio (m/z) of [M + H]+ of the purified compound were chosen as mother ions for further MS2 measurements, and those specific ions were chosen for further MS3 analysis. Mass parameters for MS2 and MS3 were shown as follows: source heater temperature was 220 °C, capillary temperature was 300 °C, source type was heated-electrospray ionization, source voltage was 2.50 kV, and capillary voltage was 23.00 V. Activation type was collision-induced dissociation. All detection was in positive mode.


NMR and High-Resolution Electrospray Ionization Mass Spectrometry Analyses One-dimensional (1H-NMR, 13C-NMR) and two-dimensional NMR spectroscopies of purified compounds were recorded on a Bruker AV600 spectrometer (Germany), 600 and 150 MHz for 1 H and 13C NMR, respectively, using DMSO-d6 (δH 2.49 and δC 39.6 ppm) as solvent. Twodimensional NMR spectroscopy, such as, correlation spectroscopy (COSY), heteronuclear single-quantum correlation spectroscopy (HSQC), heteronuclear multiple-bond correlation spectroscopy (HMBC), and rotating frame nuclear Overhauser effect spectroscopy (ROESY), was recorded when necessary in this study. The high-resolution electrospray ionization mass spectrometry (HRESIMS) experiments were performed on a Bruker micro Q-TOF mass spectrometer (Germany) to determine the element composition of the purified compounds.

Emulsification Assay Purified surfactins and selected artificial surfactants were dissolved in phosphate-buffered saline (PBS, pH = 8.0) solution to the same concentration as semi-purified surfactins in fermentation broth. n-Hexadecane was used as oil substrate to evaluate the emulsification activity of bioemulsifiers; 2 mL volume of n-Hexadecane and 2 mL volume of surfactants solution were added to a vial and vigorously mixed with a vortex [3200 rpm, Vortex-Genie 2, 230 V—No Plug (Model G560E), Scientific Industries, Inc.] for 2 min and subsequently kept for 24 h at room temperature. Afterwards, the emulsification index (EI24) was calculated with the following equation: EI24 = (HEL/HT) × 100, where HEL is the height of the emulsion layer, while HT is the height of the total liquid solution. Furthermore, the emulsification activity of surfactants was tested under various conditions, such as temperature (from 20 to 121 °C), pH (from 5.0 to 13.0), and sodium chloride solution (from 2.5 to 20 %, w/v). The EI24 of purified surfactins or semi-purified surfactins, sodium dodecyl sulfate (SDS), tween-20, and tween-80 was also compared under the same concentrations. Descriptive statistics were computed, and statistical analysis was conducted with SPSS versions 15.0 (SPSS Inc., Chicago IL, 2006). Each measurement was repeated independently for three times.

Results Emulsification-Activity-Guided Screening The emulsification-activity-guided approach showed that the fraction eluted via dichloromethane/methanol (95/5, v/v) had significant emulsification activity among all the fractions. Finally, 5.406 g of semi-purified crude extracts was afforded from the total fermentation broth of 24 L; the final yield was 225 mg L−1. Interestingly, the fraction containing semipurified iturins was not active during this kind of emulsification assay (data not shown).

LC-MS Analysis and RP-HPLC Purification The crude extracts with significant emulsification activity were detected by RP-HPLC purification (Fig. 1a) and LC-MS analysis (Figure S1). The results showed that crude extracts


contained at least 16 components. A series of ion peaks was found at m/z of [M + H]+, [M + Na]+, and [M + K]+, respectively. The analysis showed that molecular weight of the components distributed from m/z 994, 1008, 1022, 1036, and 1050, respectively (Table S1). These ions could be deduced as homologues with a molecule difference of –CH2 (molecular weight is 14) group. And most importantly, these ion data showed very similar results with those reported Bacillus surfactin-type lipopeptides [14]. The major peaks of the fraction were further purified with a semi-preparative reversed phase HPLC system, with isocratic elution of 88 % (v/v) acetonitrile/H2O solvent system. Each peaks were collected and dried under vacuum, finally afforded eight purified compounds, compound 1 (25.0 mg), 2 (15.3 mg), 3 (31.5 mg), 4 (15.6 mg), 5 (10.4 mg), 6 (45.3 mg), 7 (9.8 mg), and 8 (12.6 mg) (Fig. 1a). The structure of the purified compounds will be determined by means of mass spectrometry and nuclear magnetic resonance analysis.

Tandem Mass Spectrometry Analysis The full scan mass spectrometry (MS1) of the eight compounds showed a series of ion peaks of m/z [M + H]+ and [M + Na]+ (Figures S2 and S3). All spectra showed the base peak ion of m/z [M + H]+ and followed with the ion peak of [M + Na]+, which had a molecular mass unit of 22 larger than m/z [M + H]+. The mother ion of m/z [M + H]+ was chosen as ions for further tandem mass spectrometry analysis.

a 200

3

Absorbance [mAU]

UV - wave length: 220 nm 150

6

1 100 5 50

2

4

Peaks 1 2 3 4 5 6 7 8

Compound anteiso-C13 Leu7 surfactin anteiso-C13 Val7 surfactin iso-C14 Leu7 surfactin normal-C14 Leu7 surfactin iso-C14 Val7 surfactin anteiso-C15 Leu7 surfactin anteiso-C15 Val7 surfactin anteiso-C15 Ile7 surfactin

7 8

0 0.0

5.0

10.0 15.0 Retention Time [min]

20.0

25.0

b

Fig. 1 Reversed phase high-performance liquid chromatography (a) of semi-purified surfactins and chemical structure (b) of surfactins homologues isolated and purified from the fermentation broth of B. mojavensis B0621A

Author's personal copy Appl Biochem Biotechnol Table 1 MS/MS ion assignments of surfactins generated from the mother ion peaks at m/z of [M + H]+ (y7) Compound

Ion type y7

y6

y5

y4

y3

y2

y1

y0

1

1008.67

895.50

782.00

667.25

568.25

455.17

341.83

/

3

1022.75

909.50

796.17

681.50

582.25

469.17

355.83

227.18

4

1022.92

909.50

796.08

681.42

582.17

/

355.80

226.92

6

1036.75

923.67

810.75

695.42

596.42

483.42

/

/

2

994.50

895.50

782.50

667.25

568.17

/

342.17

213.08

5

1008.67

909.50

796.58

681.17

582.17

469.20

356.08

227.17

7 8

1022.58 1036.58

923.33 923.42

810.50 810.75

695.33 695.58

596.42 596.42

483.42 483.42

370.00 /

241.33 /

All the spectra showed a series of y-type ion fragments which provided the information for the peptidic sequence deduction, always accompany with ion peaks with a molecular weight loss of 18 (–H2O) on these y-type ion fragments. Some ions of y-type were missing in the MS2 spectra, while the neutral loss of the molecule H2O or CO was found in the mass spectra, such as m/z 352.17 (y1–H2O) of 6, m/z 352.08 (y1–H2O) of 8, m/z 437.17 (y2– H2O) of 2, m/z 441.00 (y2–CO) of 4 (Figures S3 and S4). Until now, all y-type ions were assigned except y0 ions of 1, 6, and 8, which were not observed in tandem mass spectra. The slash (/) represented no ion peak observed in the MS/MS spectra

The spectra of MS2 of eight compounds showed a series of ion peaks which were the very characteristics of surfactin-type peptide fragmentations (Figure S3 and S4). MS2 data of these compounds generated a series of y-type ions, leading to the deduction of amino acid sequences. The base peak ion of the MS2 spectra showed that the structural similarities of these compounds, more specifically five (compound 1, 3, 4, 6 and 8) of which, showed the base peak ion of m/z 685 and their peptide sequence could be an assignment to the sequence of E1-L/I2-L/I3-V4-D5-L/I6-L/I7 via the analysis of the different y-type ions fragmented from m/z [M + H]+, where leucine could be replaced with isoleucine (Figure S4), while the remained three compounds (2, 5 and 7) showed the same base peak ion of m/z 671 and their amino acid sequence could be deduced as E1-L/I2-L/I3-V4-D5-L/I6-V7 by y-type ions resulting from ion of m/z [M + H]+, whereas leucine could be replaced with isoleucine attributing to the same molecular weight found in the MS2 spectra. Moreover, some ion peaks of MS/MS spectra of the compounds showed neutral loss of the molecule –H2O (with a molecular unit loss of 18) or –CO (with a molecular unit loss of 28), or both of them (Figure S3 and S4). Table 1 shows the full assignments of y-type ions of these surfactins. Since the base peak ions of 685 and 671 were very specific in the MS2 spectra of these surfactins, these two base peak ions were further chosen as mother ions and fragmented by MS3. The main product ions of m/z 685 showed sequence of [H-L/I2-L/I3-V4-D5-L/I6-L/I7OH]+; the sequence was deducted from the y-type ions from the mass data, such as ions of m/z 667.42, 554.25, 441.25, and 228.75, and b-type ions, such as m/z 245.17 and 360.25 (Figure S4e). While tandem mass data of m/z 671 showed sequence of [H-L/I2-L/I3-V4-D5L/I6-V7-OH]+, the amino sequence was supported by y-type ions, such as m/z 653.42, 554.17, 441.25, 326.00, and 227.17, and b-type ion of m/z 345.83 (Figure S5d); whereas, leucine could be replaced with isoleucine attributing to the same molecular weight of these two amino acids in MS3 data. Therefore, to assign the leucine and isoleucine properly, other analytical techniques, for instance, NMR spectroscopy, still needed in further experiments.


NMR and HRESIMS Analyses One-dimensional 13C NMR data (150 MHz, DMSO-d6) of these surfactins further demonstrated the presence of amino acids matched with those tandem mass data (Table S2, S3, and S4). Those valine-contained surfactins (compounds 2, 5, and 7) showed very high similarity of one-dimensional 1H NMR data (600 MHz, DMSO-d6) (Figures S8, S14, and S18), similar results could also be found in the leucine- (4×) contained surfactins (compound 1, 3, 4 and 6) (Figures S6, S10, S12, and S16). Since isoleucine and leucine had the same molecular weight and composition of chemical elements, the differences of the two amino acids could not be differentiated from the mass to charge ratio (m/z) of fragments from tandem mass spectrometry data. However, the chemical shift (δC, ppm) of the two terminal methyl carbons of leucine and isoleucine was quite distinguished in the high field range from one-dimensional 13 C NMR spectrum (150 MHz) of the purified compounds. These traits from NMR spectra led the absolute identification of leucine and isoleucine. More specifically, the chemical shift (δC, ppm) of two terminal groups of methyl carbons of leucine were almost identical, about 22 to 23 ppm in DMSO-d6 (Table S2 and S3). In contrast, the two terminal methyl carbons of isoleucine were far from identical; the chemical shift of one methyl carbon was 11.2 ppm, and the other was 15.6 ppm in DMSO-d6 (Table S4) [15]. The result showed that the composition of compound 8 contained leucine (3×) and isoleucine (1×); the other compounds (1, 3, 4 and 6) contained four leucines, at the position of Leu2, Leu3, Leu6, and Leu7, respectively (Figures S7, S11, S13, S17, and S21). It was hard to place the position of leucine and isoleucine in compound 8. To make the structure elucidated clearly, compound 8 was conducted on further two-dimensional NMR analysis (Figures S22, S23, S24, and S25). Finally, the structure of compound 8 was completely elucidated and NMR data was assigned based on 1H-1H COSY, HSQC, HMBC, and ROESY experiments (Table S4). The 13 C NMR data of other seven compounds was assigned based on comparison of those reported data [15]. The saturated β-hydroxy fatty acid chain residues of these surfactins had three kinds of terminal methyl branches, namely normal-, iso-, anteiso-types [1]. These three types of saturated branches could not be determined by tandem mass data from this experiment. However, these terminal branches of fatty acid part could be differentiated from chemical shift (δC, ppm) in the high field range of 13C NMR (150 MHz, DMSO-d6) spectra. The chemical shift of δC 11 ppm (compounds 1, 2, 6, 7 and 8) (Figures S7, S9, S17, S19, and S21), δC 14 ppm (compound 4) (Figure S13), and 22 ppm (compounds 3 and 5) (Figures S11 and S15) indicated the terminal methyl carbon of anteiso-, normal-, and iso-types within the fatty acid chains, respectively. In terms of these special traits, the structure of fatty acid residue was determined by analysis of 13C NMR data (Tables S2, S3, and S4). The results of HRESIMS of these compounds (Table S5) showed exact mass information which supported previous LC-MS, tandem mass spectrometry, and NMR spectroscopy analysis. To sum up, those spectroscopic and spectrometric data led to elucidate the structure of these surfactins, namely, anteiso-C13 Leu7 surfactin (1), isoC14 Leu7 surfactin (3), normal-C14 Leu7 surfactin (4), anteiso-C15 Leu7 surfactin (6), anteiso-C13 Val7 surfactin (2), iso-C14 Val7 surfactin (5), anteiso-C15 Val7 surfactin (7), and anteiso-C15 Ile7 surfactin (8).


Emulsification Activity Except natural-derived surfactins purified from B. mojavensis B0621A, including eight purified surfactins and semi-purified surfactins, selected artificial surfactants, such as tween-20, tween-80, and SDS, were selected and evaluated together with these biosurfactants. All these surfactants, excepting SDS, showed very good emulsification activities after mixing with nHexadecane after 24 h incubation at room temperature. Statistical analysis results showed that SDS has significant difference comparing with other surfactants, while other tested surfactant did not show significant difference among each other (p < 0.05). Since there were no differences of EI24 value between purified surfactins and semi-purified surfactins (p < 0.05), the latter was chosen for further stability of EI24 value under different conditions. The results showed that semi-purified surfactins showed stability of EI24 value between pH 7 to 12, when pH out of this range could lead the significant differences on emulsification activities (p < 0.05). To test temperature stability, semi-purified surfactins were kept in water bath at various temperatures for 30 min or kept in autoclave for 20 min at 121 °C. Surprisingly, semi-purified surfactins showed stable emulsification activities from 20 to 121 °C, even kept in autoclave for 20 min. Furthermore, emulsification activities of semipurified surfactins were conducted in 2.5 to 20 % (w/v) of sodium chloride solutions and semipurified surfactins showed very stable value of EI24.

Discussion Bacillus surfactins are a group of biosurfactants that shared various structures variants, and their structure could be elucidated by different spectrometric and spectroscopic methods. In this study, the structure of surfactins produced by B. mojavensis B0621A was completely elucidated by multiple mass spectrometry and NMR spectroscopy. Surfactins characterized by this study showed two main groups by comparing tandem mass data; one group showed base peak ion of m/z 671, which means the seventh position of peptide sequence is valine, while the other group of surfactin showed base peak ion of m/z 685, which means the seventh position of peptide sequence is leucine or isoleucine. Furthermore, the tandem mass data of these surfactins showed very special characteristics from other two groups of Bacillus lipopeptides, iturins, and fengycins. More specifically, tandem mass spectrometry of fengycins showed base peaks ion of m/z 1080, which belongs to fengycin A family [16], while those tandem mass data show m/z 1108 as the main peak belonged to fengycin B family [12]. Different from surfactins and fengycins, iturin family lipopeptides have more diversity and complexity of amino acid sequences and could most likely show more ion fragments of the tandem mass data, which lead to much difficulty on the elucidation of amino acid sequence. Surfactin defined as one of the important microbial derived surface active compounds because of the ability of greatly decreasing surface tension has potential to be derived as industrial raw material, such as in environmental and medical processes [17, 18]. Most importantly, surfactin has some advantages to be developed as commercial surfactants. These traits include environmental friendly characteristic, relative stability under various conditions, and can be easily recovered. Fortunately, a recent study on the surfactin production strain Bacillus subtilis HSO121 showed the yield of surfactins even reached 48.75 g L−1 in optimized medium [19]. This report shed new lights on the way of applying purified (semipurified) surfactins to multiple purposes, instead of Bacillus itself. Intriguingly, it seems that


some natural biosurfactants are not bioemulsifiers, for instance, purified iturins produced by B. mojavensis B0621A did not show any emulsification activities in this study. However, iturin family produced by B. mojavensis B0621A did show surface tension activities in the previous study [17]. Taking consideration of many results have been reported on emulsification activities and stability evaluation of some artificial surfactants. Therefore, instead of studying emulsification activities and stability of artificial surfactants, such as tween-20, SDS, and tween-80, only natural-derived surfactants (purified or semi-purified surfactins) were studied mainly in this report. From a stability evaluation, SDS possessed same emulsification activities at pH that varied from 2 to 13 and temperature from 0 to 100 °C; however, the emulsification activities of SDS was significantly decreased when concentration of sodium chloride is over than 10 % (w/ v), or even completely lost emulsification activities when concentration of sodium chloride is over than 15 % (w/v) [20]. From this study, biological-derived surfactants, such as surfactins, at least showed more tolerance to the emulsification activities in sodium chloride solution, compared with the artificial surfactant SDS. On the other hand, artificial surfactant SDS showed more resistant to pH changes than biosurfactants. These differences showed both biosurfactants and artificial surfactants have their own advantages comparing with each other, which means both of them have potential utilization on specific conditions, in single or combination ways. This study focused on chemical and biological characterization of surfactins from B. mojavensis B0621A, which was isolated from the mantle of a pearl oyster Pinctada martensii collected from South China Sea. These surfactins, to some extent, showed significant and stable emulsification activities against n-Hexadecane. The recovery of surfactins from cultural broth of B. mojavensis B0621A was relatively high (225 mg L−1) in this study; however, future study should conduct on the optimization of the cultural medium of the bacterium, to get a high yield of surfactins production. This study, at least, provided surfactins could be alternative biological emulsifiers; hence, it could provide some new perspectives of applying either purified (semi-purified) surfactins or bacterial supernatant to industrial processes.

Acknowledgments This work was supported by grants from the National Science & Technology Pillar Program (No. 2011BAE06B04), Chinese Academy of Sciences Innovation Project (No. KSCX2-EW-G-16), the National High Technology Research and Development Program of China (863 Program) (No. 2011AA09070404), and Liaoning Province Scientific and Technological Project (No. 2014214004).

References 1. Ongena, M., & Jacques, P. (2008). Bacillus lipopeptides: versatile weapons for plant disease biocontrol. Trends in Microbiology, 16, 115–125. 2. Cochrane, S.A. and Vederas, J. C. (2014) Lipopeptides from Bacillus and Paenibacillus spp.: a gold mine of antibiotic candidates. Medicinal Research Reviews. DOI: 10.1002/med.21321 3. Villegas-Escobar, V., Ceballos, I., Mira, J. J., Argel, L. E., Orduz Peralta, S., & Romero-Tabarez, M. (2013). Fengycin C produced by Bacillus subtilis EA-CB0015. Journal of Natural Products, 76, 503–509. 4. Raaijmakers, J. M., De Bruijn, I., Nybroe, O., & Ongena, M. (2010). Natural functions of lipopeptides from Bacillus and Pseudomonas: more than surfactants and antibiotics. FEMS Microbiology Reviews, 34, 1037–1062. 5. Pradel, E., Zhang, Y., Pujol, N., Matsuyama, T., Bargmann, C. I., & Ewbank, J. J. (2007). Detection and avoidance of a natural product from the pathogenic bacterium Serratia marcescens by Caenorhabditis elegans. Proceedings of the National Academy of Sciences of the United States of America, 104, 2295–2300.

Author's personal copy Appl Biochem Biotechnol 6. Hoefler, B. C., Gorzelnik, K. V., Yang, J. Y., Hendricks, N., Dorrestein, P. C., & Straight, P. D. (2012). Enzymatic resistance to the lipopeptide surfactin as identified through imaging mass spectrometry of bacterial competition. Proceedings of the National Academy of Sciences of the United States of America, 109, 13082–13087. 7. Zeriouh, H., Vicente, A., Pérez-García, A., & Romero, D. (2013). Surfactin triggers biofilm formation of Bacillus subtilis in melon phylloplane and contributes to the biocontrol activity. Environmental Microbiology, 16, 2196–2211. 8. Debois, D., Hamze, K., Guérineau, V., Le Caër, J. P., Holland, I. B., Lopes, P., Ouazzani, J., Séror, S. J., Brunelle, A., & Laprévote, O. (2008). In situ localisation and quantification of surfactins in a Bacillus subtilis swarming community by imaging mass spectrometry. Proteomics, 8, 3682–3691. 9. Bais, H. P., Fall, R., & Vivanco, J. M. (2004). Biocontrol of Bacillus subtilis against infection of Arabidopsis roots by Pseudomonas syringae is facilitated by biofilm formation and surfactin production. Plant Physiology, 134, 307–319. 10. Ongena, M., Jourdan, E., Adam, A., Paquot, M., Brans, A., Joris, B., Arpigny, J. L., & Thonart, P. (2007). Surfactin and fengycin lipopeptides of Bacillus subtilis as elicitors of induced systemic resistance in plants. Environmental Microbiology, 9, 1084–1090. 11. Cawoy, H., Mariutto, M., Henry, G., Fisher, C., Vasilyeva, N., Thonart, P., Dommes, J., & Ongena, M. (2014). Plant defense stimulation by natural isolates of Bacillus depends on efficient surfactin production. Molecular Plant-Microbe Interactions, 27, 87–100. 12. Ma, Z., Wang, N., Hu, J., & Wang, S. (2012). Isolation and characterization of a new iturinic lipopeptide, mojavensin A produced by a marine-derived bacterium Bacillus mojavensis B0621A. The Journal of Antibiotics, 65, 317–322. 13. Ma, Z., & Hu, J. (2014). Production and characterization of iturinic lipopeptides as antifungal agents and biosurfactants produced by a marine Pinctada martensii-derived Bacillus mojavensis B0621A. Applied Biochemistry and Biotechnology, 173, 704–715. 14. Tang, J. S., Zhao, F., Gao, H., Dai, Y., Yao, Z. H., Hong, K., Li, J., Ye, W. C., & Yao, X. S. (2010). Characterization and online detection of surfactin isomers based on HPLC-MS(n) analyses and their inhibitory effects on the overproduction of nitric oxide and the release of TNF-alpha and IL-6 in LPSinduced macrophages. Marine Drugs, 8, 2605–2618. 15. Tang, J. S., Gao, H., Hong, K., Yu, Y., Jiang, M. M., Lin, H. P., Ye, W. C., & Yao, X. S. (2007). Complete assignments of H-1 and C-13 NMR spectral data of nine surfactin isomers. Magnetic Resonance in Chemistry, 45, 792–796. 16. Chen, L. L., Wang, N., Wang, X. M., Hu, J. C., & Wang, S. J. (2010). Characterization of two anti-fungal lipopeptides produced by Bacillus amyloliquefaciens SH-B10. Bioresource Technology, 101, 8822–8827. 17. Liu, X., Ren, B., Chen, M., Wang, H., Kokare, C. R., Zhou, X., Wang, J., Dai, H., Song, F., & Liu, M. (2010). Production and characterization of a group of bioemulsifiers from the marine Bacillus velezensis strain H3. Applied Microbiology and Biotechnology, 87, 1881–1893. 18. Banat, I. M., Franzetti, A., Gandolfi, I., Bestetti, G., Martinotti, M. G., Fracchia, L., Smyth, T. J., & Marchant, R. (2010). Microbial biosurfactants production, applications and future potential. Applied Microbiology and Biotechnology, 87, 427–444. 19. Haddad, N., Gang, H., Liu, J., Mbadinga, S., & Mu, B. (2014). Optimization of surfactin production by Bacillus subtilis HSO121 through Plackett-Burman and response surface method. Protein and Peptide Letters, 21, 885–893. 20. de Sousa, T., & Bhosle, S. (2012). Isolation and characterization of a lipopeptide bioemulsifier produced by Pseudomonas nitroreducens TSB. MJ10 isolated from a mangrove ecosystem. Bioresource Technology, 123, 256–262.