Characterization of a novel biosurfactant produced by Staphylococcus sp. strain 1E with potential application on hydrocarbon bioremediation

408 Journal of Basic Microbiology 2012, 52, 408–418

© 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.jbm-journal.com

Research Paper

Characterization of a novel biosurfactant produced by Staphylococcus sp. strain 1E with potential application on hydrocarbon bioremediation

Kamel Eddouaouda*, 1, 2, Sami Mnif*, 1, Abdelmalek Badis1, 2, Sonia Ben Younes1, Slim Cherif1, Samira Ferhat1, 2, Najla Mhiri1, Mohamed Chamkha1 and Sami Sayadi1

1 Laboratoire des Bioprocédés Environnementaux, Pôle d’Excellence Régional AUF (PER-LBPE), Centre de Biotechnologie de Sfax, Tunisie

2 Chemical Industrial Department, Engineering Faculty, University Of Saad Dahlab At Blida-Algeria

A biosurfactant-producing bacterium (Staphylococcus sp. strain 1E) was isolated from an Algerian crude oil contaminated soil. Biosurfactant production was tested with different carbon sources using the surface tension measurement and the oil displacement test. Olive oil produced the highest reduction in surface tension (25.9 dynes cm–1). Crude oil presented the best substrate for 1E biosurfactant emulsification activity. The biosurfactant produced by strain 1E reduced the growth medium surface tension below 30 dynes cm–1. This reduction was also obtained in cell-free filtrates. Biosurfactant produced by strain 1E showed stability in a wide range of pH (from 2 to 12), temperature (from 4 to 55 °C) and salinity (from 0 to 300 g l–1) variations. The biosurfactant produced by strain 1E belonged to lipopeptide group and also constituted an antibacterial activity againt the pathogenic bacteria such as Staphylococcus aureus, Escherichia coli, Pseudomonas aeruginosa and Bacillus subtilis. Phenanthrene solubility in water was enhanced by biosurfactant addition. Our results suggest that the 1E biosurfactant has interesting properties for its application in bioremediation of hydrocarbons contaminated sites.

Keywords: Biosurfactant / Staphylococcus sp. / Emulsification / Solubilisation / Bioremediation

Received: May 30, 2011; accepted: July 11, 2011

DOI 10.1002/jobm.201100268

Introduction*

The microbial surfactants called as biosurfactants are microbial compounds with a distinct surface activity that exhibit a broad diversity of chemical structures such as glycolipids, lipopeptides, lipoproteins, lipopoly-saccharides, phospholipids, fatty acids and polymeric lipids [1]. Therefore, it is reasonable to expect diverse properties and physiological functions of biosurfactants such as increasing the surface area and bioavailability of hydrophobic water-insoluble substrates, heavy metal binding, bacterial pathogenesis, quorum sensing and biofilm formation [2]. A host of interesting features of

* These authors had contributed equally for the realization of this work. Correspondence: Dr. Abdelmalek Badis, Laboratoire de Biochimie et de Microbiologie Industrielle, Département de Chimie Industrielle, Uni-versité Saad Dahlab de Blida, B.P 270, 09000 Blida, Algeria E-mail: [email protected] Phone: +213 7 72 17 44 32 Fax: +213 25 4336 31

biosurfactants have led to a wide range of potential applications in the medical field. They are used as anti-bacterial, antifungal and antiviral agents, and they also have the potential to be used as major immuno-modu-latory molecules and adhesive agents [3]. Microbial biosurfactants are produced by a wide vari-ety of diverse microorganisms and they have different chemical structures and properties [3]. Potential com-mercial applications of microbial surfactants had been previously reported [4]. Diversity of biosurfactant pro-ducing microorganisms isolated from soils contami-nated with diesel oil had been described [5]. In general, bacteria produce low molecular weight molecules that efficiently reduce surface and interfacial tensions such as glycolipids, lipopeptides [6]. They also produce high molecular weight polymers that are efficient emulsifi-ers such as emulsan, alasan or biodispersan [7]. Rham-nolipid biosurfactant produced by Pseudomonas species was composed of two molecules of rhamnose and two

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molecules of 3 hydroxyacids. Also remarkable are the trehalolipids which were produced by several species of Rhodococcus, Arthrobacter and Mycobacterium composed of trehalose, nonhydroxylated fatty acid and mycolic ac-ids. Sophorolipids were produced by Candida and Toru-lopsis, in which sophorose was combined with long chain hydroxyacid [8]. The criterion used for selecting biosurfactant-prod-ucers is the ability to reduce the surface tension below 40 mN m–1 while a criterion cited for emulsion-stabil-izing capacity is the ability to maintain at least 50% of the original emulsion volume for 24 h after formation [9]. Even Staphylococcus genus is known by its pathogenic effect, Staphylococcus strains isolated from natural envi-ronment could be used for biotechnological studies and produced substances with high added values [10]. Pre-vious studies investigated the possibility of biosurfac-tant production by Staphylococcus hominis, Kocuria palus-tris and Pseudomonas aeruginosa LBI, using weathered diesel oil from a long-standing spillage as raw material [11]. However, to our knowledge, no studies had de-scribed the high production and characterization of biosurfactant from strains belonging to Staphylococcus genus. The objective of this study was to characterize for a first time an efficient biosurfactant produced by a newly isolated Staphylococcus sp. strain 1E.

Materials and methods

Screening of biosurfactant producing bacteria Initial screening of biosurfactant producers from vari-ous biotopes was carried out using the oil displacement test [12]. The liquid medium for biosurfactant produc-tion contained: 10 ml olive oil, 5 g yeast extract, 5 g NaCl, 10 g peptone and distilled water 1 l. After exten-sive screening of surfactant producers using LB me-dium supplemented with 1% olive oil, strain 1E, previ-ously isolated from a hydrocarbon contaminated soil (Hassi Massoud, Algeria) had showed an interesting biosurfactant production potentiality. Strain 1E was previously isolated after enrichment culture in mini-mal medium with crude oil as the only carbon source as described by Mnif et al. [12]. The 16S rRNA gene se-quence determination and phylogenetic analysis were realized as described previously by Mnif et al. [12]. The 16S rRNA sequence was deposited in the Genbank nu-cleotide database under accession number HQ699551.

Media and culture conditions Staphylococcus sp. strain 1E was cultivated in 250 ml conical flasks containing 100 ml of Luria-Bertani broth

medium composed (l–1 distilled water): peptone, 10 g; yeast extract, 5 g; NaCl, 5.0; pH, 7). The conical flask was kept in shaker at 200 rpm with 37 °C as incubation temperature. 14 h 1E cultures used as inocula for biosurfactant produced cultures were incubated in LB medium supplemented with 1% (v/v) olive oil (hydro-phobic substrate inducing biosurfactant production) added after 6 h of incubation. The culture was incu-bated aerobically 48 h (correspond to minimal surface tension and maximum biosurfactant production) on a rotary shaker at 200 rpm with 37 °C as incubation tem-perature. The cell-free supernatants were used for biosurfactant extraction.

Selection of optimal carbon source for biosurfactant production Four carbon sources were used in the present study in order to select the optimum source for maximum biosurfactant production: Vegetable oil (1%, v/v), olive oil (1%, v/v), crude oil (1%, v/v) and hexadecane (1%, v/v) (Merck, Darmstadt). All substrates were sterilized using 0.22 μm filter. The reduction in surface tension was measured with a tensiometer (TSD132389, Gibertini, Italy) and was used as a tool for selecting the carbon source for optimal biosurfactant production.

Recuperation of biosurfactant and purification The culture broth was centrifuged at 7000 rpm during 15 min to remove the cells. The clear sterile super-natant served as the source of the crude biosurfactant. In fact, the biosurfactant was recovered from the cell-free culture supernatant by adjusting to pH 2 with 2 N HCl. The precipitate was separated by centrifugation at 7000 rpm. for 20 min at 4 °C and then was extracted with ethyl acetate before being concentrated in rotary evaporator [13]. For purification, crude biosurfactant was subjected to C18 chromatography column (id: 2.5 cm, length: 30 cm) eluted step by step procedure with a gradient of methanol-water from 65%:35% (me-thanol:water) to 100% methanol. Fractions were sub-jected to oil displacement test analysis and OD280nm measurement.

pH, surface tension and emulsification index (E24) measurements The pH of the supernatant was measured with a digi- tal pH-meter (Istek, NeoMet). A digital tensiometer TSD132389 (Gibertini, Milano, Italy) was used to meas-ure the surface tension of the supernatants, using the Wilhelmy standard method. The values reported are the mean of three measurements. All measurements were made on cell-free broth obtained by centrifuging

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the cultures at 8000 rpm for 20 min. A mixture of 4 ml supernatant and 4 ml of hydrophobic substrate was vertically stirred for 2 min and the height of emul-sion layer was measured after 24 h to determine the emulsification index. The equation used to deter-mine the emulsification index (E24 %) is as follows:

24 100E

EE

= ⋅′

with E: The length of the emulsified layer,

and E’: The total length of the mixture.

Biosurfactant characterization Compositions of crude and purified biosurfactant were established using standard methods: Protein was quan-tified according to Bradford’s method [14], using Bovine Serum Albumin (BSA) as standard protein. Carbohy-drates were quantified as reported by Dubois et al. [15]. Lipids were determined by extracting twice with 0.25 g of emulsifier with 4 ml of hexane, then, the solvent was evaporated and the lipid content was quantified accord-ing to standard methods [16]. The infrared spectrum (IR) was obtained by dissolving a quantity of the pre-cipitate in methanol, and then the cell containing this solution was placed in the light path. After solvent evaporation, residual solid biosurfactant was analyzed with a wavelength between 500 and 4000 cm–1. FTIR analyses were conducted with a thermo scientific Nicolet 380 apparatus. HPLC-ESI combined with mass spectrometer (Agilent Technologies, USA) was used to analyze the chemical components of the biosurfactant. The sample used was prepared as follows: the pure biosurfactant was dissolved in acetonitrile (ACN):water (1 :1, v/v), centrifuged and filtered. Chromatographic separation was achieved with a 150 mm × 2.1 mm × 5 mm Eclipse plus C18 reverse-phase column. HPLC analytical conditions: Five micro-liter samples were injected via an autosampler with split injection at split rate of 1:4. A gradient was applied us-ing distilled water and ACN with a flow rate of 250 ml min–1: initially 25% ACN and 75% water, then a linear gradient over 30 min to 50% ACN and 50% water, fur-ther for 15 min to 90% ACN and 10% water and the column was washed for 0.1 min, and ultimately reequi-librated to 25% ACN and 75% water for 30 min. ESI-MS was performed in positive ion mode. The collision volt-age and ionization voltage were 70 V and 4.5 kV, respec-tively, using nitrogen as atomization and desolvation gas. The desolvation temperature was set at 400 °C. The scan range of mass spectrum was 200–1000 m/z.

Functional characterization To asses foaming activity, a separate quantity of the precipitate was dissolved in distilled water in a test

tube. The mixture was shaken manually for 30 s and left to stand for 5 min. In order to study the emulsify-ing potential of 1E biosurfactant against oils and hy-drocarbons, 4 ml of crude biosurfactant were mixed with equal volumes of: diesel oil, crude oil, motor oil, vegetable oil and olive oil, then agitated for 2 min with a maximum speed and left to rest for 24 h. The E24 cor-responded to each tested oils or hydrocarbons was measured after 24 h. The antimicrobial activity was essayed using the NCCLS method [17]. In fact, the bacteria (Table 1) were cultivated in tryptic soy broth (TSB) or agar (TSA) (Sigma) at the appropriate temperature of the strain (30 or 37 °C). Fungi and yeasts were cultured on malt extract broth (MEB) or agar (MEA) (Fluka, Madrid, Spain) at 28 °C. Inocula were prepared by adjusting the turbidity of each bacterial and yeast cultures to reach an optical comparison to that of a 0.5 McFarland stan-dard, corresponding to a cell count of approximately 1–5 × 108 CFU ml–1. The concentration of spore suspen-sions was determined using a hematocytometer (Thoma cell) and adjusted to 1–5 × 108 spores ml–1. Minimal inhibitory concentrations (MICs) and mini-mal cidal concentrations (MCCs) of 1E biosurfactant were determined according to the National Committee for Clinical Laboratory Standard (NCCLS 2002) [17] against a panel of microorganisms representing differ-ent species of different ecosystems. The test was per-formed in sterile 96-well microplates. The inhibitory activity of the biosurfactant was properly prepared and transferred to each microplate well in order to obtain a twofold serial dilution of the original sample. To obtain stable diffusion, a stock solution of biosurfactant was prepared in 0.1% methanol. The inocula (100 μl) con-taining 108 CFU ml–1 approximately of bacteria or fungi were added to each well. Negative control wells con-tained bacteria or fungi only in their adequate medium. Positive control wells contained 10 μg l–1 of chloram-phenicol or gentamycin antibiotics for bacteria and fungi. Thereafter, 30 μl of 0.02% resazurin and 12.5 μl of 20% Tween 80 were added. Plates were aerobically incubated at 30 °C for 16–20 h. After incubation, the wells were observed for a color change from blue to pink. MIC was defined as the lowest concentration at which no growth was observed (blue colored) after in-cubation. To determine MCC values, 10 μl of each cul-ture medium with no visible growth were recovered from each well and inoculated in TSB or MEB plates. After aerobic incubation at the appropriated tempera-ture during 16–20 h, the CFU number of surviving organisms was determined. For stability assay, the crude biosurfactant was incubated at different tem-

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peratures: 4, 25, 37, 45, 55, 75 and 100 °C for 24 h. The surface tensions were measured with these tempera-tures in order to assess the thermal stability of our product [18]. Sodium chloride (purity > 99.5%, SlimLab, Tunisia) was added to samples of the supernatant at different concentrations: 0, 50, 100, 150, 200, 250 and 300 g l–1. After resting for 24 h at room temperature, their surface tensions were assessed [18]. The pH of the supernatant was varied from 2 to 12, using HCl or NaOH solutions, after a rest period of 24 h, the surface tension and emulsifying index were assessed [18]. CMC (critical micelle concentration) is an important parameter during the evaluation of activity of biosur-factant. The surface tension of surfactant reaches the lowest at its CMC. Above this concentration, no further effect can be observed on the surface activity. Measur-ing surface tension of serially diluted biosurfactant solution, the CMC was determined by plotting the sur-face tension versus concentration of biosurfactant in the solution. These experiments were conducted in three independent replicates and the results presented were the average data [13].

Application: phenanthrene solubilisation by 1E biosurfactant Phenanthrene, a three-ring PAH (Polycyclic Aroma- tic Hydrocarbon) with water solubility as low as 6.6 × 10–6 mol l–1 [19], was selected here as representa-tive of PAHs to determine the solubilization caused by 1E biosurfactant solution. Therefore, 0.006 g of phe-nanthrene (purity 98%, Aldrich-Germany) was added into a series of 10 ml biosurfactant solutions at dif- ferent concentrations. This concentration of phenan-threne is much higher than the solubility of phenan-threne in water. The concentrations of biosurfactant solution were adjusted to 62.5, 125, 250, 500 and 1000 mg l–1 individually. Each concentration was per-formed in triplicate and de-ionized water was served as control. The solubilization experiments were per-formed in 20 ml centrifuge tubes. These tubes, which contained the mixture of phenanthrene and water, were agitated in a vertical position at 30 °C with shak-ing (200 rpm.) in the dark for 24 h, followed by cen-trifugation at 12500 rpm, at 4 °C for 30 min. A 5 ml aliquot of supernatant was collected; the pH was ad-justed to 2.0 using 6 M HCl. Then, an equal volume of dichloromethane was added in a separation funnel to extract phenanthrene dissolved in biosurfactant. This process was repeated twice. After extraction, the or-ganic portions were combined, dried with anhydrous sodium sulphate and concentrated to 1 ml. The concen-trated liquid was filtered through a 0.20 mm micro-

pore filter before being analyzed by UV-Vis spectropho-tometer at 254 nm (Shimadzu-1100, Japan) according to Zhu et al. [19].

Results

Strain 1E identification Strain 1E, a gram positive, coccus and catalase positive bacterium was selected as a powerful biosurfactant producing bacterium. The strain was previously iso-lated from Algerian hydrocarbon contaminated soil (heavy crude oil contamination). In fact, the strain was previously isolated after enrichment culture with crude oil as the only carbon and energy sources. The degrada-tion potential of strain 1E in n-alkane fraction present in crude oil (more than 80% of n-alkane present in 1% v/v crude oil) was previously quantified (data not shown). The phylogenetic identification of this strain was based on the nucleotide sequence obtained by us-ing 16S rRNA gene sequencing analysis. The results showed that it was closely related to Staphylococcus ge-nus with Staphylococcus haemolyticus (D83367) and Staphy-lococcus devriesei (FJ938168) being the most closely re-lated species with a similarity of 99.58% and 99.1%, respectively (Fig. 1). Strain 1E also shared 99.03% and 98.95% identity with Staphylococcus croceolyticus (AY953148) and Staphylococcus hominis (L37601), res- pectively. The 16S rRNA gene sequence of strain 1E was deposited in Genbank under Accession Number HQ699551. Strain 1E was conserved at –80 °C. The working culture was maintained at 4 °C in LB agar plates and sub-cultured every week.

Selection of isolate based on surface tension meas-urement and oil displacement test In order to select the most powerful strain for biosur-factant production, the reduction in surface tension and the diameter of clear zone, produced by the oil displacement test during growth on olive oil, were measured. Results obtained clearly demonstrate that the highest reduction of surface tension was achieved with isolate 1E (26 dynes cm–1) and the highest diam-eter of clear zone according to the oil displacement test was about 8 cm (Fig. 2a, b). Based on these results, we selected strain 1E as the best strain for biosurfactant production and characterization.

Production of biosurfactant The influence of the nature of the carbon source on biosurfactant production using the hexadecane (ali-phatic hydrocarbon), olive oil, vegetable oil, and crude

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Figure 1. Phylogenetic tree based on 16S rRNA sequence, constructed by the neighbor-joining method, showing the position of strain 1E among related members of the genus Staphylococcus. Reference strain organisms are included and sequence accession numbers are given in parentheses. Bootstrap values, expressed as percentage of 100 replications, are shown in branching points. Bar, 2 substitution in 100 nt.

oil (Fig. 2c) was studied. Olive oil presented the most reduction in surface tension. Therefore, it constituted the best carbon source for biosurfactant production (Fig. 2c). Even if strain 1E was able to degrade crude oil, the production of biosurfactant using crude oil as in-duced substrate was low (ST = 30.4 dynes cm–1, Diam-eter of oil displacement test = 2.5 cm), compared to that obtained with olive oil (ST = 25.8 dynes cm–1, Diameter of oil displacement test = 8 cm) (Fig. 2c).

Biosurfactant separation, purification and characterization The separation method gave a yield of 2.1 g l–1 of biosurfactant produced by strain 1E. The precipitate obtained is a transparent crystalline powder soluble in distilled water and/or methanol. The main active frac-tions eluted from C18 column chromatography were marked (fraction 1–3) as shown in Fig. 3. Fraction 3 showed effective oil displacement with a diameter of clear zone of about 5 cm and therefore used for struc-tural analysis.

Physico-chemical characterisation The thermal stability showed that above 55 °C, the surface tension was slightly influenced by temperature for 1E biosurfactant. The product was stable during the increase in temperature. The thermal stability of biosurfactant was demonstrated by retaining the same

Figure 2. (a) Kinetic of growth of strain 1E with olive oil as carbon source: (�) OD at 600 nm, (�) surface tension in dynes cm–1, and diameter of oil displacement test in cm (�); (b) Oil displacement test at different time of culture; (c) Optimization of carbon source for biosurfactant production for the selected strain 1E by the determina-tion of surface tension and oil displacement test measurement. c)

b)

a)

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Figure 3. C18 chromatography separation: (�) OD at 280 nm, (�) methanol/water gradient and (�) diameter of oil displacement test.

surface tension for a range of temperature from 4 to 55 °C (average of 31 dynes cm–1). At 75 and 100 °C, the surface tension was increased (more than 35 dynes cm–1) indicated the instability of the product at high temperature. The study of pH stability showed no significant effect on surface tension for Bio-1E during pH variations. In fact, the surface tension was stable from pH 2 (γ = 31.6 dynes cm–1) to pH 12 (γ = 31.45 dynes cm–1) for 1E biosurfactant. The pH variation had no significant effect on the stability and the activity of 1E biosurfactant. The stability against salinity was also evaluated. As evident from results obtained, it was observed that the surface tension of biosurfactant was not affected up to 300 g l–1 of salt (27.3 dynes cm–1 at 300 g l–1 NaCl). This also indicates that biosurfactant produced by 1E is more effective in presence of monovalent ions like Na+. Critical micelle concentration is defined as the concentration of sur-factants above which micelles form and almost all additional surfactants added to the system go to mi-celles. For critical micelle concentration (CMC) deter-mination, the surface tension of serially diluted bio-surfactant solution was measured. In fact, the 1E biosurfactant concentration beyond which the surface tension does not change is defined as CMC. Results showed that the surface tension depending on the con-centration of biosurfactant. In fact, surface tension decreased, reaching a minimal value near 25–26 dynes cm–1 for a 1E biosurfactant concentration greater than

or equal to 750 mg l–1 for biosurfactant produced by isolate 1E. So that, we concluded that the CMC of 1E biosurfactant was about 750 mg l–1.

Functional characterisation 1E biosurfactant had a good foaming power. The emul-sification index (E24) values of the produced biosur-factant against several hydrocarbons and oils showed that the maximum emulsification was obtained with crude oil (E24 = 98.9%). Moreover, motor oil constituted a good substrate for emulsification activity (E24 = 78.57), whereas, diesel oil, olive oil and vegetable oil presented moderate emulsifying activities of 42.85%, 42.48% and 57.14%, respectively. Antimicrobial activity of 1E biosurfactant was tested on bacterial strains including Gram + and Gram – of rods and cocci, and fungal strains representing differ-ent families. Table 1 showed the MICs and MCCs values of biosurfactant 1.68 to 27 mg ml–1 examined by the broth microdilution susceptibility assay NCCLS on the tested microorganisms. The MICs values ranged from 1.68 to 13.5 mg ml–1 indicated that crude biosurfactant was effective antimicrobial agents and the MIC/MCC ratio was very close to 1 (excepted the case of Candida albicans strain) confirming their bactericidal activity against the tested strains (Staphylococcus aureus, Esche-richia coli, Pseudomonas aeruginosa and Bacillus subtilis) (Table 1). However, 1E Biosurfactant had no fungicidal activity against Aspergilus niger strain (Table 1).

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Table 1. Antimicrobial activity of 1E biosurfactant presented by MIC and MCC concentrations intervals against each of the studied bacteria and fungi strains.

Organisms MIC (mg ml–1) MCC (mg ml–1)

Staphylococcus aureus (ATCC 9144)

3.375–6.75 3.375–6.75

Escherichia coli (ATCC 10536)

6.75–13.5 6.75–13.5

Pseudomonas aeruginosa (ATCC 15442)

6.75–13.5 6.75–13.5

Bacillus subtilis (ATCC 6633)

3.375–6.75 3.375–6.75

Aspergillus niger (ATCC 16404)

– –

Candida albicans (ATCC 10231)

1.6875–3.375 13.5–27

Structural characterisation Crude biosurfactant was found to contain about 5% of carbohydrates, 70.58% of lipids and 7.21% of proteins. However, no carbohydrates had been detected in frac-tion 3, the most active biosurfactant fraction obtained after C18 column purification. The lipopeptide nature of the biosurfactant (frac- tion 3) was further confirmed by the IR spectra of the compound (figure not shown). A small peak was seen at 3300 cm–1, corresponding to the presence of N–H bond, a sharp absorbance peak is seen at 2984 cm–1 signifying the presence of C–CH3 bonding or long alkyl chains.

The peak with high absorbance in the spectrum was observed at 1737 cm–1. Absorbance in this region signi-fies the presence of peptide group in the molecule. Absorbance in these regions are because of the pres-ence of C=O bonds and is caused due to C=O stretching vibrations. Other significant peaks observed at 1446 and 1372 cm–1 corresponded to C–H bending vibrations and is common in compounds with alkyl chains. Peaks at 1232, 1097 and 1043 cm–1 were probably because of C–O–C vibrations in esters or C-N stretching vibration. Fraction 3, the most active fraction was subjected to LC-MS analysis. Results obtained showed the presence of two peaks (peak 1 and peak 2) with mass spectra of M + H at m/z 334.6 and 352.7 respectively. Based on lipids, proteins and carbohydrates contents, and FTIR analysis fraction 3 was a lipopeptide in nature. So that, mass fragmentations and fraction 3 basic structure allowed us to conclude that biosurfactant 1E structure was composed of 3 amino-acids which are probably Proline-Glycine-Glycine according to their molecular mass (mz/226) and a long hydrocarbon chain of 9 car-bon atoms. The MS of the second peak, its m/z frag-mentations and predicted structure of Bio-1E frac- tion 3 are shown in Fig. 4. However, further purifica-tion must be done in future to permit NMR structural analysis and to determine the exact structure of our product.

Figure 4. LC-MS analysis of Bio-1E fraction 3: (a): ESI mass spectrum and chemical structure of peak 2 (tr of 21.9 min); (b): MS2 of peak 2; (c): predicted structure based on mass fragmentation.

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Effect of biosurfactant on phenanthrene solubilization PAHs, one of the persistent organic pollutants found in oil containing wastewater, are also a kind of hydropho-bic organic compounds (HOCs) [20]. As well, the water solubility of PAHs decreases with the increasing num-ber of rings in molecular structure. This property induces the low bioavailability of these organic com-pounds that is an important factor in the biodegra-dation of these compounds. The water solubility of some HOCs can be improved by surfactant or biosurfac-tant addition owing to its amphipathic structure. The degree of solubilization caused by biosurfactant can be expressed through the ratio of Sw* (the apparent solubil-ity of certain solute in surfactant solution) to Sw (the solubility of certain solute in pure water). As shown in Fig. 5, biosurfactant has a highly obvious effect on solubilization of phenanthrene. The solubility of phe-nanthrene in biosurfactant solution was about 20 times higher than the control when the concentration of biosurfactant was at its highest point. Fig. 5 also re-vealed that biosurfactant had certain effect on the solubilization of phenanthrene either below or above its CMC (the CMC of this biosurfactant was 750 mg l–1), nevertheless, the solubilization function was much more obvious when the biosurfactant concentration was above its CMC. This is because surfactant molecules exist as monomers when the biosurfactant concentra-tion is below its CMC. Since this kind of monomer has little partition effect on solute, solubility of phenan-threne is very small or only increases slightly. However, the apparent solubility of phenanthrene increases quickly when biosurfactant concentration is higher than 750 mg l–1 (1000 mg l–1).

Discussion

To carry out the bioremediation of hydrocarbon con-taminated sites, it is very important to isolate and se-lect bacteria possessing high capacities to degrade many components of petroleum products but also to be able to produce biosurfactant which can solubilize hydrophobic compounds and render them more acces-sible for biological break down. The potential of strain 1E for hydrocarbon degrada-tion had been previously demonstrated (not published data). Here, in this work we have studied the potential of strain 1E to produce biosurfactant, characteristics of the biosurfactant and its field of applications. Screening programs used here were based on the concept of ob-taining bacterial isolate from the soils and evaluated

Figure 5. Solubilization of phenanthrene by 1E biosurfactant in pure water.

the ability of the isolate to grow and produce biosurfac-tant under laboratory condition. Olive oil constituted the optimal carbon source among the tested substrates. This was not surprising since the knowledge that strain 1E was isolated from hydrocarbons contaminated soils and thus hydrophobic substrate constituted a natural carbon source for this strain in natural environment [12]. The same result was obtained with Snehal et al. [21] where Rhodococcus spp. strains had the ability to grow on hydrophobic substrate coupled with biosurfactant production [21]. Moreover, strain 1E was also able to produce biosurfactant when crude oil and hexadecane were used as induced substrates. In fact, these findings were of great interest making possible the use of strain 1E in bioremediation or bioaugmentation of hydrocar-bon contaminated sites [22]. Surface and interfacial tension, stabilization of an oil and water emulsion is commonly used as a surface activity indicator [23]. 1E biosurfactant showed interest surface tension reduction (25 dynes cm–1), and impor-tant emulsifying activity comparable to that found elsewhere [24]. 1E biosurfactant was stable even at high NaCl concentrations. In fact, there are reports that the presence of salts results in disruption of emulsions of oil and water, thus affecting the emulsification ability and surface tension of surfactants [25]. Therefore, activ-ity of biosurfactant preparation was evaluated in pres-ence of different concentrations of NaCl to deter- mine its field application. Prieto et al. [26] had reported that the biosurfactant produced by P. aeruginosa strain formed stable emulsions at low NaCl concentrations (below 0.05 mol l–1). However, it was capable of main-taining about 80% of its original activity up to a salinity level of 0.3 mol l–1. Abouseoud et al. [18] had reported that little changes were observed in the surface active properties of P. fluorescens biosurfactant with addition of NaCl up to 2.0 mol l–1. There are few reports in the

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literature indicating that in saline soil degradation of parathion and total crude oil was lowered, as compared to non-saline soils [27]. Thus, there is a need for exten-sive studies in order to evaluate the effectiveness of the bioremediation processes, using biosurfactant for pol-luted sites having different levels of salinity. Moreover, 1E biosurfactant was stable at temperatures up to 55 °C and pH variations had no effect on the stability of this biosurfactant. Characteristics of this biosurfactant and its production using hydrophobic substrates constitute original features for its application in bioremediation of hydrocarbons contaminated sites as well as for micro-bial enhanced oil recovery (MEOR). Recently, biosurfac-tants have gained numerous industrial and environ-mental applications which frequently involve exposure to extreme conditions. As a result, researchers have focused on isolating and screening strains that are able to produce biosurfactant under extreme environments, especially for MEOR and bioremediation purposes [28]. It had been reported that Pseudomonas sp. could be used in microbial plugging, a MEOR technique that had been applied in a candidate oil reservoir of Daqing oilfield (China) [29]. These proprieties of salinity, temperature and pH stabilities are of great interest make it possible the use of the strain 1E in microbial enhanced oil re-covery (MEOR) applied to moderate geothermic or mesophilic petroleum reservoir presented high level of salinity. CMC is an important parameter to evaluate the effectiveness of biosurfactant [30]. In our case, pro-duced biosurfactant had a CMC of 750 mg l–1 higher than CMC of other biosurfactants produced by other Pseudomonas strains [31], but lower than chemical sur-factant such as SDS [30]. Although, the largest application of biosurfactant is the oil industry, for petroleum production and incorpo-ration into oil formulations, oil spill bioremediation, removal of oil sludge from storage tanks and enhanced oil recovery [29, 31], biosurfactants from many micro-organisms have demonstrated antimicrobial properties and are currently being extracted and investigated to stem the incidence of antibiotic resistance plaguing the world today [32]. Antimicrobial activities of the biosur-factant produced in this study showed broad spectrum of action having been able to inhibit the total of tested bacterial strains including gram positive and gram negative types (Table 1). MIC/MCC ratio was very close to 1, especially for bacteria test, confirming their bacte-ricidal activity. The ability to produce biosurfactant with antimicrobial property could be a survival strategy allowing this isolate to flourish ahead of other micro-organisms in the environment. In fact, Biosurfactant could inhibit the other microbial growth by affecting

the release of intracellular materials [33]. This property was with great interest making bioaugmentation ap-proach in hydrocarbon contaminated sites (water or soils) more effective and selective. In this study, strain 1E belonging to Staphylococcus genus, produced a biosurfactant with interest proprie-ties. Even if Staphylococcus genus is known by the patho-genic character of the majority of their representative species, the application of strain 1E or its biosurfactant in hydrocarbon remediation is with great interest. In fact, reports on characterization and application of biosurfactant or biosurfactant-producing bacteria clas-sified as pathogen had been previously reported. For example, the pathogens species Pseudomonas aeruginosa and Serratia marescens had been extensively reported to produce biosurfactants [34, 35]. In addition, the use of such strains in environmental purpose could be justi-fied by the fact that they came from nature environ-ment. So that, their application is possible mainly when controlled by molecular tools or by using recombinant strains [36]. Also, there is a possibility to produce biosurfactant under laboratory condition (controlled conditions) followed by its application without the addition of pathogenic strain in bioremediation [37]. To our knowledge, there is no previous study focused in the literature about isolation and partial structural and functional characterization of biosurfactants from staphylococcus genus. Therefore, this study could be considered as first promising research paper for more future interesting researches in this field of study (biosurfactant from Staphylococcus genus). The use of produced biosurfactant obtained from strain 1E for phenanthrene solubilization showed interested results mainly when biosurfactant was applied at concentra-tions above CMC. This is due to the fact that biosurfac-tant can enhance micelle formation and thus cause undissolved organic components to dissolve in micelles [13]. The apparent solubility of phenanthrene increases quickly when biosurfactant concentration is higher than 750 mg l–1 (1000 mg l–1).

Conclusion Staphylococcus sp. strain 1E was able to excrete biosur-factant with interesting surface-active properties. Puri-fied biosurfactant had a CMC 750 mg l–1, which was much lower than that of chemical surfactants. Accord-ing to FTIR and LC-MS analyses, the predicted probable chemical components of this biosurfactant were lipo-peptides compounds. The study on biosurfactant stabil-ity showed that there was no appreciable change in surface activity with the changing pH and salinity of medium. Moreover, the antibacterial activity of this

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biosurfactant was demonstrated. The findings in solubi-lization effect of biosurfactant demonstrated that this biosurfactant could enhance the water solubility of phenanthrene by about 20 times compared with con-trol. Biosurfactant produced by the strain 1E was a kind of preferable surface-active substance, having potential application in bioremediation of hydrocarbon contami-nation or in MEOR.

Acknowledgements

This study was supported by a grant provided by the Tunisian Ministry of Higher Education, Scientific Re-search. Many thanks to AUF (agence universitaire de la francophonie, project contract n° 6313PS652). Many thanks to CRD (centre de recherche et de développe-ment) from SONATRACH companies in Algeria. Thanks to Mr. A. Gargoubi for his technical assistance in FTIR analysis, Miss L. Jlail for her assisatance in LC-MS, Miss T. Yangui for her help in antimicrobial activity analyses and Mr. G. Rigane for his help in LC-MS spectrum in-terpretation.

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((Funded by • Tunisian Ministry of Higher Education, Scientific Research • AUF (agence universitaire de la francophonie; project contract: n°6313PS652 • CRD (centre de recherche et de développement) from SONATRACH companies in Algeria))