Preparation and characterization of silver-modified La0.8Sr0.2MnO3 cathode powders for solid oxide fuel cells by chemical reduction method

Accepted Manuscript Investigation of the structural, physicochemical properties, and aggregation behavior of lipopeptide biosurfactant produced by Acinetobacter junii B6

Mandana Ohadi, Gholamreza Dehghannoudeh, Forootanfar, Mojtaba Shakibaie, Majid Rajaee PII: DOI: Reference:

Hamid

S0141-8130(17)33272-5 https://doi.org/10.1016/j.ijbiomac.2018.01.209 BIOMAC 9041

To appear in: Received date: Revised date: Accepted date:

28 August 2017 23 January 2018 31 January 2018

Please cite this article as: Mandana Ohadi, Gholamreza Dehghannoudeh, Hamid Forootanfar, Mojtaba Shakibaie, Majid Rajaee , Investigation of the structural, physicochemical properties, and aggregation behavior of lipopeptide biosurfactant produced by Acinetobacter junii B6. The address for the corresponding author was captured as affiliation for all authors. Please check if appropriate. Biomac(2018), https://doi.org/10.1016/j.ijbiomac.2018.01.209

This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

ACCEPTED MANUSCRIPT Investigation of the structural, physicochemical properties, and aggregation behavior of lipopeptide biosurfactant produced by Acinetobacter junii B6

T

Mandana Ohadia, Gholamreza Dehghannoudeha,c*, Hamid Forootanfara,b**, Mojtaba

Pharmaceutics Research Center, Institute of Neuropharmacology, Kerman University of

US

a

CR

IP

Shakibaiea,b, Majid Rajaeec

Department of Pharmaceutical Biotechnology, Faculty of Pharmacy, Kerman University of

Medical Sciences, Kerman, Iran

Department of Pharmaceutics, Faculty of Pharmacy, Kerman University of Medical Sciences,

ED

c

M

b

AN

Medical Sciences, Kerman, Iran

CE

PT

Kerman, Iran

AC

-----------------------------------------------*Corresponding author: Tel.: +98-34-31325015; fax: +98-34-31325003. E-mail: addresses: [email protected] (G. Dehghan-Noudeh) [email protected]. **Corresponding author: Tel: +98-34-31325238; Fax: +98-34-31325003; E-mail: [email protected] (H. Forootanfar).

1

ACCEPTED MANUSCRIPT Abstract In the present study the produced biosurfactant of Acinetobacter junii B6 (recently isolated from Iranian oil excavation site) were partially purified and identified by high performance thin layer chromatography (HPTLC), Fourier transform infrared spectroscopy (FTIR), and proton nuclear

IP

T

magnetic resonance (1H NMR). Elemental analysis of the biosurfactant by energy dispersive X-

CR

ray spectroscopy (EDS) revealed that the biosurfactant was anionic in nature. The physiochemical properties of the lipopeptide biosurfactant were evaluated by determination of its

US

critical micelle concentration (CMC) and hydrophile-lipophile balance (HLB). The produced biosurfactant decreased the surface tension of water to 36 mN m−1 with the CMC of

AN

approximately 300 mg/l. Furthermore, the solubility properties of the biosurfactant (dissolved in

M

phosphate-buffer saline solution, pH 7.4) were investigated by turbidity examination, dynamic light scattering (DLS) measurements, and transmission electron microscopy (TEM) inspection. It

ED

could be concluded that the biosurfactant showed the spherical-shaped vesicles at a concentration

PT

higher than its CMC and the circular dichroism (CD) spectra showed that the secondary structure

CE

of the biosurfactant vesicles is dominated by the β sheet.

AC

Keywords: Biosurfactant, Lipopeptide, Acinetobacter junii, Aggregation behaviors, Physicochemical properties

2

ACCEPTED MANUSCRIPT 1. Introduction Biosurfactants as naturally derived surfactants are produced by different microorganisms such as bacteria, fungi, and yeasts [1]. Because of higher biodegradability, more environmental acceptability, lesser toxicity, and lower CMC, biosurfactants have been mentioned as an

IP

T

alternative for chemically derived and conventional surfactants [2]. Biosurfactants reduce the

CR

surface tension and interfacial tension while increase the stability of the emulsions due to their amphiphilic (hydrophobic and hydrophilic moieties) structures [3]. These amphipathic

US

compounds form micelles, vesicles, and cylindrical fibrils because of their self-assembling and aggregation behaviors in water and a few other polar solvents [4]. One of the most important

AN

functions of these molecular aggregates is the enhancement of the solubilization capacity [5]

M

which has been applied in manufacturing of many products such as cleansing products, pharmaceuticals, cosmetics, and foods [6]. Furthermore, other physiochemical properties of

ED

biosurfactants such as HLB and CMC that are defined as the point at which micelles start to form

PT

influences their applications [7]. Currently, the potential industrial applications of two classes of biosurfactants including glycolipids and lipopeptide have been widely considered [8]. The

CE

hydrophobic moieties of these two types of biosurfactant are similar (unsaturated or saturated

AC

hydrocarbon chains or fatty acids) while their hydrophilic moieties are different (sugar ring and peptide chain in the glycolipids and lipopeptide, respectively). Therefore, they represent different physical and biological properties because of their chemical structures [9]. For example, cyclic lipopeptide is a remarkable class of self-assembling molecules which exhibits notable bioremediation [10], antimicrobial [11], antifungal [12], anticancer [13], and wound healing activities [14]. Cyclic lipopeptide biosurfactants are mainly produced by Bacillus spp. that are classified into three families of Surfactin, Iturin, and Fengycin [15]. In a few studies, 3

ACCEPTED MANUSCRIPT Acinetobacter genus has been also reported as lipopeptide biosurfactant producer [16, 17]. The self-assembly characteristic of the lipopeptide biosurfactants was rarely studied [18]. Therefore, the main aim of the present study was to investigate and characterize the chemical structure of the biosurfactant produced by A. junii B6 (recently isolated from Iranian oil excavation site) via

T

analytical methods such as HPTLC, FTIR spectroscopy, scanning electron microscopy (SEM)

IP

equipped by EDS spectroscopy and 1H-NMR. In addition, the secondary structure of the

CR

biosurfactant was estimated by using CD spectroscopy and its thermostability was determined by thermal gravimetric analysis (TGA) and differential thermal analysis (DTA). Then, its

US

aggregation behaviors such as micellization of the biosurfactant in a phosphate-buffer saline

AN

(PBS) solution (pH 7.4) were investigated using DLS technique, turbidity measurement, and

PT

2. Materials and methods

ED

M

TEM inspection.

2.1. Cultivation of the biosurfactant-producing bacterial strain

CE

The strain used in this research was A. junii B6 (GenBank accession number KT946907) which

AC

previously isolated from oil contaminated soils in the southwest part of Iran [19]. Biosurfactant production was carried out by culturing of the strain in an optimized mineral salt medium (MSM) containing (g/L): MgSO4, 0.1; KH2PO4, 0.5; CaCl2.2H2O, 0.01; FeSO4.7H2O, 0.001; NaNO3, 2; and K2HPO4, 0.5 supplemented with Iranian light crude oil (ILCO, 5%, v/v) as the sole carbon source and incubation at 25 °C and 300 rpm for 48 h [19].

4

ACCEPTED MANUSCRIPT 2.2. Extraction and recovery of the biosurfactant The organic solvent extraction method was performed in order to extract the produced biosurfactant as described by Aparna et al. [2]. Briefly, the culture broth (prepared as described in the previous section) was centrifuged (8000 rpm for 20 min) using a 5810 centrifugation

IP

T

apparatus (Eppendorf Inc., Hamburg, Germany) to remove the bacterial biomass. Then, the

CR

supernatant was adjusted to pH 2.0 using HCl (6 N) and maintained at 4 °C overnight. On the next day, the acidified supernatant was treated with the equal volume of ethyl acetate-methanol

US

(3:1 v/v) to separate the biosurfactant. Finally, the organic phase of the obtained extract was

AN

separated by a vacuum rotary evaporator to concentrate the biosurfactant.

M

2.3. Analytical characterization of the biosurfactant

ED

Analytical HPTLC was performed using the HPTLC system (CAMAG, Muttenz, Switzerland)

PT

that was controlled by winCATS Software. An amount of 20 µl sample (the obtained biosurfactant dissolved in methanol as described in the previous section) were automatically

CE

injected to a 20×10 cm silica gel HPTLC plate (Merck, Darmstadt, Germany) containing green

AC

fluorescent F254. The plates were then developed with solvent mixture containing chloroform: methanol: water (65:25:4, v/v/v) up to a migration distance of 60 mm. Thereafter, the quantitative and qualitative characteristics of the HPTLC plate were investigated by TLC scanner and TLC visualizer under direct UV 254 nm and UV 366 nm light. The UV-Visible spectra of the biosurfactant (final concentration of 1 mg/ml) were recorded in the range of 200 to 600 nm using a UV-Visible spectrophotometer (PerkinElmer, Waltham, MA, USA). The FTIR spectra of 5

ACCEPTED MANUSCRIPT the biosurfactant were recorded as KBr discs in the range 4000–400 cm-1 using a Bruker ALPHA FTIR spectrometer (Bruker Inc., Massachusetts, USA). 1H-NMR spectroscopy was performed on the biosurfactant sample dissolved in DMSO using Bruker Avance 300 spectrometer operating at 300.13 MHz. (Bruker Inc., Massachusetts, USA). Quantitative elemental analysis of the

T

biosurfactant was carried out using a MIRA 3 XM SEM-EDX apparatus (Tescan Inc., Brno,

IP

Czech Republic). Samples weighing between 5-15 mg were mounted on an aluminum grid and

CR

examined at an acceleration voltage of 15 keV. Thermal properties of the biosurfactant were demonstrated using a BAHR STA 503 thermo analyzer (Hullhorst, Germany) after loading of

US

about 10 mg of the biosurfactant sample on an aluminum pans and heating from 10 °C to 500 °C

AN

at a heating rate of 10 °C/min in air. The zeta potential (ζ) of the biosurfactant was determined by laser doppler anemometry (LDA) (Zetasizers 3000 HS particle size analyzer, Malvern

M

Instruments, Welwyn Garden City, UK). The CD spectrum of the biosurfactant was measured at

ED

25 °C using an AVIV 215 spectropolarimeter (AVIV Inc., Lakewood, USA) after preparation of

CE

length cuvette [20].

PT

the biosurfactant solution in PBS (pH 7.4, 1 mg/ml) and placing the sample in a 1 mm path-

AC

2.4. Physicochemical properties of the biosurfactant 2.4.1. CMC determination The CMC of the biosurfactant was calculated compared to those of three commercial surfactants including Tween 80 (T80, a nonionic surfactant), benzalkonium chloride (BC, a cationic surfactant) and sodium dodecyl sulfate (SDS, an anionic surfactant). The CMC were determined 6

ACCEPTED MANUSCRIPT by measuring the surface tension of each sample (prepared in deionized water) at different concentrations (0–400 mg/l) using the Du Nouy ring method (Tensiometer K100, KRUSS, Hamburg, Germany) at room temperature (25 °C) [21].

T

2.4.2. Determination of required HLB

IP

HLB value of the biosurfactant was determined as previously described [20]. Firstly, the

CR

emulsifier solutions (0.5% in liquid paraffin) of Span 80 (emulsion A, an oil soluble emulsifier with HLB of 4.3) and Tween 80 (emulsion B, a water soluble emulsifier with HLB of 15) was

US

prepared. Thereafter, a series of emulsions were made by mixing of emulsions A and B in

AN

different ratio to prepare HLB values ranging from 4.3–15. A second set of emulsions were consequently prepared by addition of the biosurfactant (0.5% in liquid paraffin) to the above

M

prepared emulsions (in graduated tubes) followed by centrifugation (1500 rpm, 2 min) and

ED

measuring the amount of separated aqueous phase in milliliters at two minute intervals. The HLB value of the biosurfactant emulsion was equal to the HLB of emulsion with the same value of the

PT

separated phase. The required HLB (RHLB) was then calculated according to the following

CE

equation [22].

% (A) =100 (X -HLB (B)) / HLB (A) – HLB (B)

AC

(1)

% (B) = 100 -% (A)

Where: X is the RHLB; HLB (A) is the HLB of span 80 (4.3); HLB (B) is the HLB of Tween 80 (15). All above mentioned procedures were repeated for four times.

7

ACCEPTED MANUSCRIPT 2.5. Rheological properties Rheological measurements of the biosurfactant sample (0.5%, w/v) were performed using a DVIII Ultra Stoughton Rheometer (Brookfield Engineering Labs, MA, USA). Rheological properties including shear stress, shear rate, and viscosity were obtained directly from the

IP

T

instrument while the SC4-61 spindle was selected for the measurement. Shear stress was

CR

calculated by the following equation (2) [23]: τ = ηδ

(2)

AN

US

Where τ is the shear stress, δ is shear rate and η is apparent viscosity.

M

2.6. Aggregation behavior and vesicle formation

ED

2.6.1. Preparation of biosurfactant solution

PT

The biosurfactant solutions of different concentration were prepared by diluting the required

CE

amount of the stock solution (1 mg/ml) in PBS (pH 7.4) and then samples were homogenized by ultrasonic bath for 15 min at room temperature and then filtered through a 0.45 µm pore size

equilibrate.

AC

nylon filter. The samples were incubated at room temperature one day before usage to

2.6.2. Turbidity measurement Biosurfactant turbidity was determined using a UV/Vis spectrophotometer (PerkinElmer, Waltham, MA, USA) at 237 nm [20].

8

ACCEPTED MANUSCRIPT 2.6.3. Dynamic light scattering measurement Particles size and size distribution pattern were measured by employing dynamic light scattering (DLS) technique (Zetasizers 3000 HS particle size analyzer, Malvern Instruments, Welwyn

T

Garden City, UK) at various conditions.

IP

2.6.4. Transmission electron microscopy (TEM) examination

CR

TEM negative staining technique was applied to investigate the morphology of the biosurfactant

US

vesicles. A drop of the biosurfactant solution was placed on a copper grid and was stained with 1% uranyl acetate aqueous solution. The excess of the biosurfactant solution was removed by

AN

adsorbing the drop with a piece of filter paper. Then, the grid was dried in a vacuum desiccator

ED

(Philips, Eindhowen, Nederland).

M

for at least 6 h. Thereafter, the TEM images were taken assisting by a CM10 TEM apparatus

CE

2.7.1. RBC preparation

PT

2.7. Red blood cell (RBC) hemolytic assay

AC

Ten ml blood from a healthy volunteer was collected in a heparinized tube. RBC was then separated by centrifugation at 4000 rpm for 10 min followed by washing them three times by McIlvaine's buffer (0.2 M Na2HPO4, 0.1 M citric acid adjusted to pH 7.0) and suspended in the same buffer to give 12% hematocrit [20]. 2.7.2. Hemolytic activity of the biosurfactant

9

ACCEPTED MANUSCRIPT In order to determine the percentage of hemolysis, 200 µl of the biosurfactant sample (0–500 mg/L) was added to equal volume of RBC suspension and incubated at 37 °C for 30 min. The mixture was then centrifuged at 300 rpm for 15 Sec and 200 µl of the resulting supernatant was consequently added to 3 ml Drabkin's reagent (120 mM potassium ferricyanide, 150 mM

T

potassium cyanide, and 0.1% non-ionic detergent in 200 mM potassium dihydrogen phosphate)

IP

followed by measuring the absorbance of the test sample at 540 nm using a Synergy 2 microplate

CR

ELISA reader (BioTek Inc., Winooski, USA). Positive control (100% hemolysis) was designed by lysing the RBC suspensions with Drabkin's reagent and negative control consisted of sample

US

taken from centrifuged (300 rpm for 15 Sec) mixtures of RBC suspensions and the McIlvaine's

AN

buffer [20, 24]. Afterwards, the percentage of hemolysis was calculated using the following

M

equation (3) [25]:

(3)

ED

Hemolysis% = [(Abst –Absneg)/ (Abspos – Absneg)] ×100

Where the Abst was absorbance of the test sample, Absneg was the absorbance of negative control

CE

PT

and Abspos is the absorbance of positive control.

AC

3. Results and discussion

3.1. Structural characterization of the biosurfactant 3.1.1. HPTLC of the biosurfactant According to the HPTLC chromatogram (Fig. 1a) the zones were well separated and there are two clear peaks with RF values of 0.07 and 0.4. Our results were in accordance with the results 10

ACCEPTED MANUSCRIPT previously reported by Geissler et al. [26] who displayed similar HPTLC chromatograms using the similar mobile phase of chloroform/methanol/water (65:25:4, v/v/v) for identification and simultaneous quantification of Surfactin, Iturin A, and Fengycin in Bacillus culture broth. They reported that samples of Fengycin and Surfactin exhibited the RF values of 0.07 and 0.4,

T

respectively. In another study, Al-Wahaibi et al. [27] applied two solvent systems for HPTLC

IP

analysis of the biosurfactants produced by B. subtilis B30. They found that the MP1 solvent

CR

system [butanol: acetic acid: water (8:2:2)] was more efficiently separated the fractions of culture

US

broth compared to that of MP2 solvent system [chloroform: methanol: water (65:25:4)] [28].

AN

3.1.2. FTIR and 1H NMR spectra

The FTIR spectra of the biosurfactant are showed in the Fig. 1b from which two strong

M

absorption bands at 1655 cm−1 and one at 1546 cm−1 are characterized as amide I and amide II

ED

vibrations in peptides, respectively. Thus, this finding confirms the presence of the peptide groups in the biosurfactant structure [18, 28]. Also, the band observed at 3284 cm−1 is indicative

PT

of the N-H stretching bond [28, 29]. The presence of aliphatic chains (-CH3, CH2-) in the

CE

biosurfactant structure was confirmed by the bands between 2927–2855 cm−1 [16, 28]. Moreover, the presence of an ester and a carbonyl group were indicated by the absorption regions at 1720–

AC

1665 cm−1. The related stretching band of C=O appeared at 1720 cm−1 [18, 28] as represented in Fig. 1b. The obtained results of 1H NMR analysis are presented in Fig. 1c. The presence of the terminal branching in the fatty acid component (-CH (CH3)2) is confirmed at 0.97–0.81 ppm. The long aliphatic chain (-CH2) is displayed at 1.55–1.24 ppm. The chemical shift at 8.2–7.2 ppm was consistent with a peptide backbone (N-H) (Fig. 1c). Also, the presence of the ester carbonyl group is indicated at 5.4–4.9 ppm, which may be a part of lactone ring [16, 30, 31]. The 11

ACCEPTED MANUSCRIPT intense singlet at 3.47 ppm is similar with that of 1H NMR spectrum of the lipopeptide monoesters previously reported in the literature [30, 32] which suggests the existence of one methoxy group on the aspartic acid and glutamic acid amino residues (Fig. 1c). The singlet at 2.51–2.72 ppm is accordance with the presence of the DMSO as a solvent (Fig. 1c). Therefore,

T

both the FTIR and 1H NMR spectra as well as the related Rf value of the biosurfactant obtained

IP

in the present study suggests the similarity in its structure with those of lipopeptides previously

AC

CE

PT

ED

M

AN

US

CR

reported in the literature. [27, 32, 33].

12

AC

CE

PT

ED

M

AN

US

CR

IP

T

ACCEPTED MANUSCRIPT

Fig.1. a); 3D graph HPTLC chromatograms of the biosurfactant after development with chloroform/methanol/water (65:25:4, v/v/v), b); FT-IR , and c); H NMR spectrums of biosurfactant produced by A. junii B6.

13

ACCEPTED MANUSCRIPT 3.1.3. EDX analysis The qualitative elemental analysis done by SEM-EDX revealed the weight and atomic percentage of six elements (C, N, O, Na, P, and Cl) in the biosurfactant structure (Fig. 2a). The presence of carboxyl functional group in the biosurfactant that was previously confirmed by

IP

T

FTIR and 1H NMR analysis is an indicative of the anionic character of the biosurfactant [34, 35].

CR

Moreover, the presence of cation such as sodium (Na) in the biosurfactant suggested their binding to the negative charges of the carboxyl groups [37]. This phenomenon resulted in the

US

greater availability of these elements to the microbes which is essential for their survival and growth [34, 35]. Zeta potential measurement of the biosurfactant in the present study revealed a

AN

negative charge of -24 mV. In general, zeta potential is obtained from the measurement of the

M

electrophoretic mobility and is related to the electric charge of the particles [36]. The negative sign of the biosurfactant zeta potential might be confirmed the presence of the negatively-

ED

charged carboxyl group in the hydrophilic moiety of the biosurfactant structure [4, 37].

PT

3.1.4. UV-Vis and CD spectroscopy

CE

The UV-Vis spectrum of the biosurfactant is illustrated in Fig. 2b. The maximum wavelength area of the absorption spectra was 200-240 nm which characterizes the presence π–σ* and/or π –

AC

π* transitions of the functional groups such as amine, carboxyl, carbonyl, and ester [35]. The broadness of this peak might be due to an overlap with another peak at 237 nm characteristic of lipopeptide biosurfactants (Fig. 2b) [20]. Moreover, the presence of substituted aromatic rings is indicated around 260-280 nm [28]. Biosurfactant’s CD spectrum was measured by monitoring the changes of the signal from 190 to 260 nm (Fig. 2c). The spectrum shows a typical shape and features of a mainly β-sheet secondary structure, i.e. negative bands at about 220 nm and a 14

ACCEPTED MANUSCRIPT positive band at 195 nm (Fig. 2c). Generally, the peaks at about 216-220 nm correspond to the absorption due to the presence of β-sheet structure [38, 39]. To estimate the structural composition, the obtained spectra were analyzed by the software CDNN (Circular Dichroism Nerve Network). The calculations gave estimates of secondary structural components of 16% α-

AC

CE

PT

ED

M

AN

US

CR

IP

T

helix, 24% β-sheet, 32% antiparallel, 5 % parallel, and 23% random coil structure.

15

AC

CE

PT

ED

M

AN

US

CR

IP

T

ACCEPTED MANUSCRIPT

Fig. 2. a); EDX Elemental analysis, b); Far UV, and c); CD spectrum of biosurfactant.

16

ACCEPTED MANUSCRIPT 3.1.5. Simultaneous thermal analysis TGA and DTA profiles of the biosurfactant are presented in Fig. 3. TGA profile showed that the biosurfactant had an onset temperature (To) of 50–150 °C and a peak temperature (Tp) of 185 °C (Fig. 3a). These were resulted in a weight loss of approximately 90%, possibly attributed to the

IP

T

loss of moisture and pyrolysis of the biosurfactant during heating, which were consistent with

CR

other studies [8, 40]. An endothermic and exothermic events at Tp of 189 and 375 °C were seen in the DTA graph (Fig. 3b), which endothermic event is in good correlation with the TG peak

AC

CE

PT

ED

M

AN

US

temperature (Fig. 3a and Fig. 3b) [41].

Fig. 3. a); TGA and b); DTA thermograms of biosurfactant.

3.2. Physicochemical characterization 17

ACCEPTED MANUSCRIPT The significant reduction in water surface tension was observed with increasing the concentration of the biosurfactant (from 70 ± 0.1 to 36± 0.08 mN m-1) (Fig. 4a). Results confirmed that at concentrations higher than 300 mg/l, water surface tension became stable. Thus, the CMC was found to be approximately 300 mg/l. The spontaneous aggregation and

T

formation of well-known structures (spherical micelles) is the most important properties of a

IP

biosurfactant [42]. The obtained results are consistent with the previously reported values [10,

CR

20, 28, 43]. The nature of the solvent as well as the purity of biosurfactant affect the variation in the CMC value [44]. The results showed that the biosurfactant CMC was near to that of SDS as

US

anionic surfactant while it was too much higher than cationic (BC) and non-ionic surfactant

AN

(Tween 80) [3].

M

According to the Griffin’s HLB scale, the biosurfactant showed an HLB value of 10 that reflects suitable O/W emulsifying property [45]. HLB is known as emulsion stability that represents the

ED

relative contribution of the hydrophilic and lipophilic groups of the surfactant to the emulsion.

PT

Similar results reported by Burch et al. [46] who suggested that Surfactin may also have an HLB

CE

near 10.

AC

3.3. Rheological properties

The pseudo-plastic fluids behavior was exhibited for the biosurfactant according to the flow curve (Fig. 4b). An increase in the shear rate, showed a decreasing rate for viscosity [47]. Similarly, biosurfactants produced by Cronobacter sakazakii and Klebsiella sp. exhibited pseudo-plastic performance and viscosity decreased with increasing shear rate [23, 42]. 18

AC

CE

PT

ED

M

AN

US

CR

IP

T

ACCEPTED MANUSCRIPT

Fig. 4. a); Evaluation of the surface tension changes versus concentration amount of the biosurfactant compared with chemical surfactants, b); Rheological studies of the biosurfactant (0.5%, w/v) with shear rate stress changes from 0-200. 19

ACCEPTED MANUSCRIPT 3.4. Aggregation behavior and vesicle formation Results showed that the aggregation behaviors depended on the concentration changes. Aggregation behaviors were investigated as a function of biosurfactant concentration. In the selfassembly systems, concentration roles as a key parameter [48]. According to the CMC data of

IP

T

the biosurfactant (300 mg/l), the idea was proposed that biosurfactant might exhibit the

CR

aggregation behaviors above the CMC point [49]. Turbidity measurements were carried out to further study the self-association process of the biosurfactant. Fig. 5a showed that increasing the

US

concentration of biosurfactant beyond the CMC (300 mg/l) led to rising of the turbidity and then until leveled off. Normally, this increase in the turbidity is related to the size or amount of the

AN

biosurfactant aggregates in the buffer solution [49, 50]. Variation of the hydrodynamic diameters

M

was evaluated by determination of DLS at various concentrations (Fig. 5b). The attained results showed that in concentrations greater than the CMC point, the aggregate size was more than 280

ED

nm. Thereof, DLS measurements support the trends in the variation of aggregate sizes that

PT

showed in the turbidity measurement results. Based on the work of Champion et al. [51] the aggregates with sizes larger than 250 nm are considered as large vesicular structures. Thereafter,

CE

TEM inspection was used to observe the morphology of the vesicles formed by the biosurfactant

AC

(Fig. 5c). The TEM micrographs confirmed the presence of large spherical vesicles >250 nm above CMC point, that are consistent with the findings of Ishigami et al. [38]. Their results confirmed the large rod-shaped micelles by Surfactin with concomitant β-sheet formation in bicarbonate buffer. Additionally, Hamley et al.[18] showed that both the Surfactin and Plipastatin formed the spherical micelles in aqueous solution. Thus, the presence of the β-sheet conformation in the secondary structure was proved as mentioned above (section 3.1.4). On the other hand, micelle formation easily reflect the piling of the biosurfactant molecules that are 20

ACCEPTED MANUSCRIPT organized by β-sheet formation [38]. Additionally, it has been reported that Surfactin undergoes a conformational change from linear to α-helix (below CMC point) or β-sheet (above CMC

CE

PT

ED

M

AN

US

CR

IP

T

point), depending on its concentration [52].

AC

Fig. 5. Aggregation behavior and vesicle formation of the biosurfactant solution at different concentrations including: a); Variation of turbidity, b); aggregate sizes and c); TEM micrographs were characterized.

3.5. Hemolytic activity measurement 21

ACCEPTED MANUSCRIPT According to the obtained results the hemolytic activity of the biosurfactant was much lower than the chemical surfactants (SDS, BC) while, it was similar to that of the Tween 80 (Fig. 6). The hemolytic activity of SDS and BC rose with concentration increasing and showed the highest hemolytic activity above the CMC point. It was reported that non-ionic surfactants (like

T

Tween 80) with moderate polarity and low toxicity could be widely used in the pharmaceutical

CE

PT

ED

M

AN

US

CR

IP

formulations as solubilizing agent and enhancer [53].

AC

Fig. 6. Hemolysis activities of the biosurfactant exposed to the human erythrocytes compared to the chemical surfactants.

4. Conclusion

22

ACCEPTED MANUSCRIPT To sum up, the biosurfactant produced by A. junii B6 exhibited a lipopeptide structure and anionic nature regarding to HPTLC, FT-IR, 1H-NMR, UV-vis spectrum, and EDS analysis results. The estimated HLB value (10) of the biosurfactant reflects its suitable O/W emulsifying property. In addition, determination of aggregation behavior of the biosurfactant revealed that

T

spontaneous formation of vesicles was observed above the CMC point (300 mg/l). Assessment of

IP

the hemolytic feature of the biosurfactant represented its low hemolytic activity. The secondary

CR

structure of the biosurfactant vesicles was found to be β sheet according to the CD spectra. Behind all mentioned characteristics, the pseudo-plastic rheology behavior of the biosurfactant

US

were also evaluated in the present study. Thermostability, low toxicity (hemolytic activity), and

AN

pseudo-plastic rheology behavior made this biosurfactant as a probable promising candidate for

ED

M

biotechnological and pharmaceutical applications. However, it merits further investigations.

PT

Acknowledgments

The present work was part of a Ph.D. thesis financially (Grant No. 94/417) supported by the

AC

CE

Kerman University of Medical Sciences (Kerman, Iran).

References

[1] I.M. Banat, S.K. Satpute, S.S. Cameotra, R. Patil, N.V. Nyayanit, Cost effective technologies and renewable substrates for biosurfactants’ production, Front Microbiol. 5 (2014) 697.

23

ACCEPTED MANUSCRIPT [2] A. Aparna, G. Srinikethan, H. Smitha, Production and characterization of biosurfactant produced by a novel Pseudomonas sp. 2B, Colloids Surf. B. Biointerfaces 95 (2012) 23-29.

[3] O. Pornsunthorntawee, P. Wongpanit, S. Chavadej, M. Abe, R. Rujiravanit, Structural and

T

physicochemical characterization of crude biosurfactant produced by Pseudomonas aeruginosa

CR

IP

SP4 isolated from petroleum-contaminated soil, Bioresour. Technol. 99(6) (2008) 1589-1595.

[4] H. Zhong, L. Yang, X. Yang, G. Zeng, Z. Liu, Y. Liu, X. Yuan, Aggregation of low-

US

concentration dirhamnolipid biosurfactant in electrolyte solution, RSC Advances 5(108) (2015)

AN

88578-88582.

ED

Pharm. Sci. 8(2) (2005) 147-163.

M

[5] C.O. Rangel-Yagui, A. Pessoa Jr, L.C. Tavares, Micellar solubilization of drugs, J. Pharm.

PT

[6] I.M. Banat, R.S. Makkar, S.S. Cameotra, Potential commercial applications of microbial

CE

surfactants, Appl. Microbiol. Biotechnol. 53(5) (2000) 495-508.

AC

[7] L. Sang-Cheol, S.-H. Kim, I.-H. Park, S.-Y. Chung, M.S. Chandra, C. Yong-Lark, Isolation, purification, and characterization of novel fengycin S from Bacillus amyloliquefaciens LSC04 degrading-crude oil, Biotechnol. Bioprocess Eng. 15(2) (2010) 246-253.

[8] R. Chandankere, J. Yao, M. Cai, K. Masakorala, A.K. Jain, M.M. Choi, Properties and characterization of biosurfactant in crude oil biodegradation by bacterium Bacillus methylotrophicus USTBa, Fuel 122 (2014) 140-148. 24

ACCEPTED MANUSCRIPT [9] I.M. Banat, A. Franzetti, I. Gandolfi, G. Bestetti, M.G. Martinotti, L. Fracchia, T.J. Smyth, R. Marchant, Microbial biosurfactants production, applications and future potential, Appl. Microbiol. Biotechnol. 87(2) (2010) 427-444.

T

[10] F.A. Bezza, M. Beukes, E.M.N. Chirwa, Application of biosurfactant produced by

IP

Ochrobactrum intermedium CN3 for enhancing petroleum sludge bioremediation, Process

CR

Biochem. 50(11) (2015) 1911-1922.

US

[11] Y.-C. Chen, T.-J. Chiang, T.-W. Liang, I.-L. Wang, S.-L. Wang, Reclamation of squid pen

AN

by Bacillus licheniformis TKU004 for the production of thermally stable and antimicrobial

M

biosurfactant, Biocatal Agric Biotechnol. 1(1) (2012) 62-69.

ED

[12] A. Roy, D. Mahata, D. Paul, S. Korpole, O.L. Franco, S.M. Mandal, Purification, biochemical characterization and self-assembled structure of a fengycin-like antifungal peptide

PT

from Bacillus thuringiensis strain SM1, Front Microbiol. 4 (2013) 332.

CE

[13] G. Dey, R. Bharti, R. Sen, M. Mandal, Microbial amphiphiles: a class of promising new-

AC

generation anticancer agents, Drug Discov. Today 20(1) (2015) 136-146.

[14] R. Zouari, D. Moalla-Rekik, Z. Sahnoun, T. Rebai, S. Ellouze-Chaabouni, D. Ghribi-Aydi, Evaluation of dermal wound healing and in vitro antioxidant efficiency of Bacillus subtilis SPB1 biosurfactant, Biomed. Pharmacother. 84 (2016) 878-891.

25

ACCEPTED MANUSCRIPT [15] S.S. Cameotra, R.S. Makkar, Recent applications of biosurfactants as biological and immunological molecules, Curr. Opin. Microbiol. 7(3) (2004) 262-266.

[16] C. Zou, M. Wang, Y. Xing, G. Lan, T. Ge, X. Yan, T. Gu, Characterization and

T

optimization of biosurfactants produced by Acinetobacter baylyi ZJ2 isolated from crude oil-

IP

contaminated soil sample toward microbial enhanced oil recovery applications, Biochem. Eng. J.

CR

90 (2014) 49-58.

US

[17] J. Chen, P. Huang, K. Zhang, F. Ding, Isolation of biosurfactant producers, optimization and

M

Appl. Microbiol. 112(4) (2012) 660-671.

AN

properties of biosurfactant produced by Acinetobacter sp. from petroleum‐contaminated soil, J.

ED

[18] I.W. Hamley, A. Dehsorkhi, P. Jauregi, J. Seitsonen, J. Ruokolainen, F. Coutte, G. Chataigné, P. Jacques, Self-assembly of three bacterially-derived bioactive lipopeptides, Soft

PT

Matter 9(40) (2013) 9572-9578.

CE

[19] M. Ohadi, G. Dehghannoudeh, M. Shakibaie, I.M. Banat, M. Pournamdari, H. Forootanfar,

AC

Isolation, characterization, and optimization of biosurfactant production by an oil-degrading Acinetobacter junii B6 isolated from an Iranian oil excavation site, Biocatalysis and Agricultural Biotechnology 12 (2017) 1-9.

[20] G. Dehghan-Noudeh, M. Housaindokht, B.S.F. Bazzaz, Isolation, characterization, and investigation of surface and hemolytic activities of a lipopeptide biosurfactant produced by Bacillus subtilis ATCC 6633, J Microbiol 43(3) (2005) 272-276. 26

ACCEPTED MANUSCRIPT [21] R.M. Maier, Biosurfactants: evolution and diversity in bacteria, Adv. Appl. Microbiol. 52 (2003) 101-121.

[22] I. Americas, The HLB System: A Time-saving Guide to Emulsifier Selection, ICI Americas,

IP

T

Incorporated1984.

CR

[23] R.M. Jain, K. Mody, A. Mishra, B. Jha, Isolation and structural characterization of biosurfactant produced by an alkaliphilic bacterium Cronobacter sakazakii isolated from oil

AN

US

contaminated wastewater, Carbohydr. Polym. 87(3) (2012) 2320-2326.

[24] N.I. Kalinovskaya, E.P. Ivanova, Y.V. Alexeeva, N.M. Gorshkova, T.A. Kuznetsova, A.S.

M

Dmitrenok, D.V. Nicolau, Low-molecular-weight, biologically active compounds from marine

ED

Pseudoalteromonas species, Curr. Microbiol. 48(6) (2004) 441-446.

PT

[25] B. Balakrishnan, D. Soman, U. Payanam, A. Laurent, D. Labarre, A. Jayakrishnan, A novel

AC

343-354.

CE

injectable tissue adhesive based on oxidized dextran and chitosan, Acta Biomater. 53 (2017)

[26] M. Geissler, C. Oellig, K. Moss, W. Schwack, M. Henkel, R. Hausmann, High-performance thin-layer chromatography (HPTLC) for the simultaneous quantification of the cyclic lipopeptides Surfactin, Iturin A and Fengycin in culture samples of Bacillus species, J. Chromatogr. B 1044 (2017) 214-224.

27

ACCEPTED MANUSCRIPT [27] Y. Al-Wahaibi, S. Joshi, S. Al-Bahry, A. Elshafie, A. Al-Bemani, B. Shibulal, Biosurfactant production by Bacillus subtilis B30 and its application in enhancing oil recovery, Colloids Surf. B. Biointerfaces 114 (2014) 324-333.

T

[28] W. Ismail, I.S. Al-Rowaihi, A.A. Al-Humam, R.Y. Hamza, A.M. El Nayal, M. Bououdina,

IP

Characterization of a lipopeptide biosurfactant produced by a crude-oil-emulsifying Bacillus sp.

CR

I-15, Int. Biodeterior. Biodegrad. 84 (2013) 168-178.

US

[29] C. Sivapathasekaran, S. Mukherjee, R. Samanta, R. Sen, High-performance liquid

AN

chromatography purification of biosurfactant isoforms produced by a marine bacterium, Anal.

M

Bioanal. Chem. 395(3) (2009) 845-854.

ED

[30] T. Fooladi, N. Moazami, P. Abdeshahian, A. Kadier, H. Ghojavand, W.M.W. Yusoff, A.A. Hamid, Characterization, production and optimization of lipopeptide biosurfactant by new strain

PT

Bacillus pumilus 2IR isolated from an Iranian oil field, J Petrol Sci Eng. 145 (2016) 510-519.

CE

[31] H. Sharafi, M. Abdoli, H. Hajfarajollah, N. Samie, L. Alidoust, H. Abbasi, J. Fooladi, H.S.

AC

Zahiri, K.A. Noghabi, First report of a lipopeptide biosurfactant from thermophilic bacterium Aneurinibacillus thermoaerophilus MK01 newly isolated from municipal landfill site, Appl. Biochem. Biotechnol. 173(5) (2014) 1236-1249.

[32] J.F. Pereira, E.J. Gudiña, R. Costa, R. Vitorino, J.A. Teixeira, J.A. Coutinho, L.R. Rodrigues, Optimization and characterization of biosurfactant production by Bacillus subtilis isolates towards microbial enhanced oil recovery applications, Fuel 111 (2013) 259-268. 28

ACCEPTED MANUSCRIPT [33] M. Ibrahim, U. Ijah, S. Manga, L. Bilbis, S. Umar, Production and partial characterization of biosurfactant produced by crude oil degrading bacteria, Int. Biodeterior. Biodegrad. 81 (2013) 28-34.

T

[34] R. Chandankere, J. Yao, M.M. Choi, K. Masakorala, Y. Chan, An efficient biosurfactant-

IP

producing and crude-oil emulsifying bacterium Bacillus methylotrophicus USTBa isolated from

CR

petroleum reservoir, Biochem. Eng. J. 74 (2013) 46-53.

US

[35] C. Hazra, D. Kundu, A. Chaudhari, Lipopeptide biosurfactant from Bacillus clausii BS02

AN

using sunflower oil soapstock: evaluation of high throughput screening methods, production,

M

purification, characterization and its insecticidal activity, RSC Advances 5(4) (2015) 2974-2982.

ED

[36] N.R. Andrade Silva, M.A. Luna, A.L. Santiago, L.O. Franco, G.K. Silva, P.M. de Souza, K. Okada, C.D. Albuquerque, C.A. Silva, G.M. Campos-Takaki, Biosurfactant-and-bioemulsifier

PT

produced by a promising Cunninghamella echinulata isolated from caatinga soil in the northeast

CE

of Brazil, Int. J. Mol. Sci. 15(9) (2014) 15377-15395.

AC

[37] C. Burgos-Díaz, R. Pons, J.A. Teruel, F.J. Aranda, A. Ortiz, A. Manresa, A.M. Marqués, The production and physicochemical properties of a biosurfactant mixture obtained from Sphingobacterium detergens, JCIS 394 (2013) 368-379.

[38] Y. Ishigami, M. Osman, H. Nakahara, Y. Sano, R. Ishiguro, M. Matsumoto, Significance of β-sheet formation for micellization and surface adsorption of surfactin, Colloids Surf. B. Biointerfaces 4(6) (1995) 341-348. 29

ACCEPTED MANUSCRIPT [39] H. Kwaambwa, R. Maikokera, Infrared and circular dichroism spectroscopic characterisation of secondary structure components of a water treatment coagulant protein extracted from Moringa oleifera seeds, Colloids Surf. B. Biointerfaces 64(1) (2008) 118-125.

T

[40] H. Abbasi, M.M. Hamedi, T.B. Lotfabad, H.S. Zahiri, H. Sharafi, F. Masoomi, A.A.

IP

Moosavi-Movahedi, A. Ortiz, M. Amanlou, K.A. Noghabi, Biosurfactant-producing bacterium,

CR

Pseudomonas aeruginosa MA01 isolated from spoiled apples: physicochemical and structural

US

characteristics of isolated biosurfactant, J. Biosci. Bioeng. 113(2) (2012) 211-219.

AN

[41] H.-l. Jian, X.-x. Liao, L.-w. Zhu, W.-m. Zhang, J.-x. Jiang, Synergism and foaming properties in binary mixtures of a biosurfactant derived from Camellia oleifera Abel and

ED

M

synthetic surfactants, JCIS 359(2) (2011) 487-492.

[42] R.M. Jain, K. Mody, A. Mishra, B. Jha, Physicochemical characterization of biosurfactant

PT

and its potential to remove oil from soil and cotton cloth, Carbohydr. Polym. 89(4) (2012) 1110-

CE

1116.

AC

[43] M. Abouseoud, A. Yataghene, A. Amrane, R. Maachi, Biosurfactant production by free and alginate entrapped cells of Pseudomonas fluorescens, J. Ind. Microbiol. Biotechnol. 35(11) (2008) 1303-1308.

[44] D.A. Vaz, E.J. Gudiña, E.J. Alameda, J.A. Teixeira, L.R. Rodrigues, Performance of a biosurfactant produced by a Bacillus subtilis strain isolated from crude oil samples as compared to commercial chemical surfactants, Colloids Surf. B. Biointerfaces 89 (2012) 167-174. 30

ACCEPTED MANUSCRIPT [45] W.C. Griffin, Classification of surface-active agents by" HLB", J Soc Cosmetic Chemists 1 (1946) 311-326.

[46] A.Y. Burch, B.K. Shimada, P.J. Browne, S.E. Lindow, Novel high-throughput detection

T

method to assess bacterial surfactant production, Appl. Environ. Microbiol. 76(16) (2010) 5363-

CR

IP

5372.

[47] T. Eslaminejad, S.N. Nematollahi-Mahani, M. Ansari, Cationic β-Cyclodextrin–Chitosan

US

Conjugates as Potential Carrier for pmCherry-C1 Gene Delivery, Mol. Biotechnol. 58(4) (2016)

AN

287-298.

M

[48] I.W. Hamley, Lipopeptides: from self-assembly to bioactivity, ChCom 51(41) (2015) 8574-

ED

8583.

PT

[49] M. Sánchez, F.J. Aranda, M.J. Espuny, A. Marqués, J.A. Teruel, Á. Manresa, A. Ortiz, Aggregation behaviour of a dirhamnolipid biosurfactant secreted by Pseudomonas aeruginosa in

AC

CE

aqueous media, JCIS 307(1) (2007) 246-253.

[50] O. Pornsunthorntawee, S. Chavadej, R. Rujiravanit, Solution properties and vesicle formation of rhamnolipid biosurfactants produced by Pseudomonas aeruginosa SP4, Colloids Surf. B. Biointerfaces 72(1) (2009) 6-15.

31

ACCEPTED MANUSCRIPT [51] J.T. Champion, J.C. Gilkey, H. Lamparski, J. Retterer, R.M. Miller, Electron microscopy of rhamnolipid (biosurfactant) morphology: effects of pH, cadmium, and octadecane, JCIS 170(2) (1995) 569-574.

T

[52] M. Marti, W. Colonna, P. Patra, H. Zhang, C. Green, G. Reznik, M. Pynn, K. Jarrell, J.

IP

Nyman, P. Somasundaran, Production and characterization of microbial biosurfactants for

CR

potential use in oil-spill remediation, Enzyme Microb. Technol. 55 (2014) 31-39.

US

[53] G.D. Noudeh, P. Khazaeli, P. Rahmani, Study of the effects of polyethylene glycol sorbitan

AC

CE

PT

ED

M

AN

esters surfactants group on biological membranes, Int J Pharmacol 4(1) (2008) 27-33.

32

ACCEPTED MANUSCRIPT Highlights: 

The biosurfactant produced by A. junii B6 exhibited a lipopeptide structure and anionic nature.



The estimated HLB value (10) of the biosurfactant reflects its suitable O/W emulsifying

IP

Aggregation behavior of the biosurfactant revealed that vesicles were formed above the

CR



T

property.

CMC point.

US

Assessment of the hemolytic feature of the biosurfactant represented its low hemolytic

CE

PT

ED

M

AN

activity.

AC



33