Journal of Colloid and Interface Science

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1 Journal of Colloid and Interface Science 367 (212) Contents lists available at SciVerse ScienceDirect Journal of Colloid and Interface Science Production and characterization of biosurfactant from marine Streptomyces species B3 Abhijit Khopade a, Biao Ren b, Xiang-Yang Liu b, Kakasaheb Mahadik a, Lixin Zhang b, Chandrakant Kokare a,c, a Department of Pharmaceutical Biotechnology, Poona College of Pharmacy, Bharati Vidyapeeth Deemed University, Pune , India b Chinese Academy of Science, Key Laboratory of Pathogenic Microbiology and Immunology, Institute of Microbiology, Chinese Academy of Sciences, Beijing 111, PR China c Department of Pharmaceutics, STES, Sinhgad Institute of Pharmacy, Narhe, Pune , India article info abstract Article history: Received 12 July 211 Accepted 3 November 211 Available online 15 November 211 Keywords: Biosurfactant Streptomyces Surface tension Stability Critical micelle concentration Antimicrobial activity The present study demonstrates the production and properties of a biosurfactant isolated from marine Streptomyces species B3. The production of the biosurfactant was found to be higher in medium containing sucrose and lower in the medium containing glycerol. Yeast extract was the best nitrogen source for the production of the biosurfactant. The isolated biosurfactant reduced the surface tension of water to 29 mn/m. The purified biosurfactant was shown critical micelle concentrations of 11 mg/l. The emulsifying activity and stability of the biosurfactant was investigated at different salinities, ph, and temperature. The biosurfactant was effective at very low concentrations over a wide range of temperature, ph, and salt concentration. The purified biosurfactant was shown strong antimicrobial activity. The biosurfactant was produced from the marine Streptomyces sp. using non-hydrocarbon substrates such as sucrose that was readily available and not required extensive purification procedure. Streptomyces species B3 can be used for microbially enhanced oil recovery process. Ó 211 Elsevier Inc. All rights reserved. 1. Introduction Biosurfactants are a structurally diverse group of surface active molecules synthesized by microorganisms. Virtually, all the surfactants are chemically synthesized [1]. Nevertheless, in recent years, much attention has been directed toward biosurfactants owing to their advantages such as low toxicity, high biodegradability, better environmental compatibility, high foaming capability, higher selectivity, specific activity at extreme temperature, ph, salinity, and the ability to synthesize them from renewable food stocks [2 5]. Surfactants are amphiphilic agents which, by accumulating at interface between immiscible phases, can reduce surface and interfacial tension. The significant reduction of interfacial tension caused by the biosurfactant increases the solubility and emulsification of the immiscible phases and bioavailability of the insoluble substrate for microorganism [3 6]. Biosurfactants with such surface properties make good candidates for enhanced oil recovery (EOR). The most effective biosurfactants reduce the surface tension (ST) of water from 72 dynes/cm to value range of 25 3 dynes/cm [6 9]. Some biosurfactants are known to have therapeutic applications [1,11]. Biosurfactants can also be used in bioremediation of Corresponding author at: Department of Pharmaceutics, STES, Sinhgad Institute of Pharmacy, Narhe, Pune , India. Fax: address: kokare71@rediffmail.com (C. Kokare). soil and sand or in the cleanup of hydrocarbon contamination in groundwater [12 14]. Recently, biosurfactants have been widely used in environmental protection, including EOR, oil spills control, biodegradation, and detoxification of oil contaminated industrial effluents and soil. Another most important applications of a bioemulsifier is the stimulation of oil production in marginal wells that have approached their economic limit of operation [6,12,15]. Biosurfactant production by actinomycetes has been reported in very few cases. Glycolipid from Rhodococcus erythropolis and Rhodococcus aurantiacus and surface active lipid from Nocardia erythropolis were studied in the literature search [16]. Among the many classes of biosurfactants, lipopeptides represent a class of microbial surfactants with remarkable surface properties and biological activities, such as surplus crude oil recovery, food-processing, de-emulsification, antimicrobial, antitumor, antiviral, and antiadhesive activities. Surfactin, produced by various Bacillus subtilis strains, is one of the most powerful and effective lipopeptide-type biosurfactant, which is composed of seven amino acids, linked via a lactone ring to a-hydroxy fatty acid with 13, 14, or 15 carbon atoms. Apart from many characteristic functional activities of biosurfactants, surfactin can also inhibits fibrin clot formation, induces formation of ion channels in lipid bilayer membranes, and inhibits cyclic adenosine monophosphate (camp). In addition, a several authors have documented the antiviral action of surfactin, which is primarily due to a physiochemical interaction between the membrane active /$ - see front matter Ó 211 Elsevier Inc. All rights reserved. doi:1.116/j.jcis

2 312 A. Khopade et al. / Journal of Colloid and Interface Science 367 (212) surfactant and the virus lipid membrane [17,18]. The objective of the present study was production and characterizes the main functional properties of the biosurfactant from marine Streptomyces. Characterization included the determination of minimum surface tension, critical micelle concentration, effect of different hydrocarbon and oil on production, compositional analysis, functional group detection, and stability to different factors such as ph and temperature. Further, the antimicrobial activity of this biosurfactant was assayed against different microorganisms. 2. Materials and methods 2.1. Isolation and identification of marine actinomycetes strain Marine sediment samples were collected from the West coast of India. Different marine actinomycetes species were isolated by using selective media such as glycerol yeast extract agar, starch casein agar, and maltose yeast extract agar [19 22]. The isolated strains were screened for biosurfactant production by using different techniques. Identification of biosurfactant producing strain B3 was done by Scanning Electron Microscopy (SEM), 16S rdna technology, biochemical and cultural characterization. The slide culture preparation for SEM was done as described by Williams and Davies [23,24] Screening methods for potential biosurfactant producers The potential biosurfactant producer was screened by different method such as hemolytic assay, drop collapsing test, oil displacement test, and lipase activity [16,25 27]. Maximum biosurfactant producing marine actinomycetes sp. B3 was maintained on glycerol yeast extract agar medium for further study Inoculum preparation and culture conditions The glycerol yeast extract medium prepared in artificial seawater (ASW) was used for development of inoculum. The seed culture was prepared in 1 ml Erlenmeyer flasks containing 5 ml of medium by inoculating 2. ml of spore suspension containing CFU/ml and cultivated under agitation (15 rpm) at 3 C for 4 days. The seed culture (5 ml) was inoculated in the 1L of fermentation medium containing 2% glycerol,.5% peptone, and 3% yeast extract prepared in artificial sea water, and fermentation was carried out 12 days under agitation at 15 rpm at 28 C [18,19] Medium optimization The medium optimization was conducted in a series of experiments changing one variable at a time, keeping the other factors fixed at a specific set of conditions. Two factors were chosen aiming to obtain higher productivity of the biosurfactant: carbon source (C) and nitrogen source (N). The carbon sources were used as glycerol, xylose, starch, mannitol, hexadecane (2% w/v), sucrose, trehalose, maltose, dextrose, and glucose (2 g/l), with ammonium chloride (NH 4 Cl) as nitrogen source. For evaluation of the most appropriate nitrogen sources for the production of biosurfactants, yeast extract, phenyl alanine, alanine, urea, peptone, tryptone, ammonium sulfate, ammonium chloride, and beef extract were employed at a concentration of 1 g/l with the optimum carbon source. All the media and reagents used for study were procured form Himedia, India [2,28 3] Time course of biosurfactant production The kinetics of biosurfactant production was followed in batch cultures at optimum conditions. The experiment was designed for 12 days starting from the log phase to stationary phase under submerged culture conditions. The resultant cell free supernatant was removed by filtration followed by cold centrifugation at 1, rpm at 4 C for 2 min. The supernatant was analyzed for biosurfactant production [11,28] Effect of ph, temperature, and sodium chloride on biosurfactant production Effect of ph and temperature on production of biosurfactant was studied by adjusting the ph and temperature of the basal medium to different levels. Effect of NaCl on biosurfactant production was studied by varying the concentrations of NaCl% (w/v) added to the basal medium. Biosurfactant activity was expressed as percentage relative activity [3] Effect of oils, surfactants, and hydrocarbon on biosurfactant production The effect of crude oil and surfactant was evaluated for biosurfactant production. The different oils such as castor oil, cod-liver oil, clove oil, coconut oil, eucalyptus oil, senamom oil, (commercial grade), and surfactants such as EDTA, CTAB, SDS, tween 2, 4, 8 were added separately in 1% (v/v) in optimized medium, and emulsification activity of medium was measured. The effect of different hydrocarbons (1% v/v) were observed on production of biosurfactant by using diesel, petrol, toluene, xylene, hexadecane, octadecane, cyclohexane and kerosene [16,3] Production and purification of biosurfactant Study on growth of the organism and biosurfactant production in glycerol yeast extract medium with crude oil as sole carbon source was carried out as described by Ilori et al. [3]. The growth medium contained grams/1 ml: sucrose, 2. g; yeast extract, 1 g; KH 2 PO 4,.53 g; NaCl, 3. g; 7H 2 O and crude oil (2%, v/v) [31]. The ph of the medium was adjusted to 7. before sterilization. Trace elements solution (1 ml) of Bauchop and Elsden [32] was sterilized separately and added aseptically to the medium. The medium (1 ml) contained in an Erlenmeyer flask (25 ml) was inoculated with the organism and incubated at 28 C with shaking at 15 rpm for 12 days. The culture broth was centrifuged (1, rpm, 2 min, 4 C) to remove the cells. The biosurfactant was recovered from the cell free culture supernatant acid precipitation as described by Ilori et al. [3]. To purify the surface active compound, the concentrated extract was subjected to column chromatography on reverse phase silica gel (6 12 mesh) with step wise elution using methanol 1% at a flow rate of 1 ml/min at room temperature. The active fraction was confirmed by the emulsification activity, and the purity was checked by thin layer chromatography. Fractions of 1 ml each were collected and used for reading the optical density at 28 nm using a UV-Spectrophotometer (JASCO, Japan) Determination of the critical micelle concentration (CMC) The surface tension of the biosurfactant was measured by the Du-Nouy ring method [2] at room temperature. The concentration at which micelles began to form was represented as the CMC. At the CMC, a sudden change in surface tension was observed. The CMC was determined by plotting the surface tension as a function of the biosurfactant concentration [33,34].

3 A. Khopade et al. / Journal of Colloid and Interface Science 367 (212) Compositional analysis of purified biosurfactant The total carbohydrate content of purified biosurfactant was assayed by phenol sulfuric acid method using glucose as standard [35]. Protein content was determined by Lowry method [36]. Bovine serum albumin (BSA) was used as calibration standard. The lipid content of biosurfactant was determined by gravimetric estimation [37,38] Determination of the effect of temperature, ph, and sodium chloride on the activity of the biosurfactant The thermal stability of the biosurfactant was determined by maintaining the supernatant at constant different range of temperature from 3 1 C for 15 min and cooled at room temperature. To determine the effect of ph on activity, the ph of the cell free broth was adjusted to different values using 1 N NaOH or 1 N HCl. The effect of addition of different concentration of NaCl on the activity of the biosurfactant was investigated. The biosurfactant was re-dissolved after purification with distilled water containing the specific concentration of NaCl ( 9%, w/v) [18] Antimicrobial activity The crude biosurfactant was tested for antimicrobial activity using well diffusion method [18], and area of the zone was calculated. Extracted active compounds were tested against human pathogens such as Escherichia coli, B. subtilis, Pseudomonas aeruginosa, Staphylococcus aureus, and Candida albicans [29] Fourier transforms infrared spectroscopy Fourier transform infrared (FTIR) spectroscopy of the biosurfactants obtained from Streptomyces sp. B3 was done on a Jasco FT-IR 41 spectrometer by KBr pellet method [34]. The molecular characterization was performed using lyophilized sample of biosurfactant with 4 cm 1 resolution yielding IR traces over the range of 4 4 cm Results and discussions 3.1. Characterization of strain B3 The strain B3 shows good growth in temperature range C in 7 days on glycerol yeast extract agar medium [19,22]. The aerial mycelium at maturity formed chains of three to several spores. Spores were non-motile. Initially, colonies were relatively smooth surfaced but later they developed a weft of aerial mycelium that appears to be granular, powdery, or velvety and produce a wide variety of pigments responsible for the color of the vegetative and aerial mycelia. Spores were oval and warty, seen like hairy (Fig. 1A and B). By morphological, SEM, and 16S rdna sequencing, the isolated strain was found to be a member of Streptomyces genus (Fig. 2) [23,24] Screening of biosurfactant production Hemolytic activity of strain B3 showed zone with diameter 23 mm around the colony. In the present study, a significant correlation was established between the hemolytic activity and biosurfactant production. According to Carrillo et al. [39] and Banat [5], biosurfactant production of the new isolates was preliminary screened by hemolytic activity. Blood agar lysis has been used to quantify surfactant and rhamnolipids [19]. Carrillo et al. [39] found an association between hemolytic activity and surfactant production, and they recommended the use of blood agar lysis as a primary method to screen biosurfactant production. In drop collapsing test a flat drop and in oil displacement method, a clear zone of mm 2 was observed (data not shown). From the above observation, it was confirmed that the Streptomyces sp. B3 was a potent biosurfactant producer. Both the techniques have several advantages such as small volume of samples was required, rapid and easy to carry out and also do not require specialized equipment [29] Cultivation conditions and biosurfactant production The isolated Streptomyces sp. B3 produces biosurfactant when grown in various nutrients. The amount of biosurfactant production was varied with respect to media composition. However, the maximum biosurfactant production was observed in glycerol yeast extract. The production of biosurfactant was not observed in minimal salt medium and potato dextrose medium. Similar result was observed by Desai and Banat [2]. Therefore, glycerol yeast extract was selected as the biosurfactant production medium for the strain at 28 C for 9 days Optimization of cultivation medium Effect of carbon source The production of biosurfactant was found to be dependent on the composition of the medium. In shake-flask experiments, the change of the carbon source in the medium was affected on both, the amount of biomass produced and biosurfactant secretion. The various carbon sources screened for biosurfactant production, out of this sucrose, trehalose, dextrose, and fructose were found favorable. The highest biosurfactant production was achieved using sucrose (2% w/v) being the sole source of the carbon (Fig. 3A) [38]. Screening of nutrient substrates showed that Streptomyces supports growth on all substrates although the yield was limited with glycerol. The Streptomyces sp. B3 showed growth on starch as a Fig. 1. Scanning electron microscopy of strain B3 (A) 4 magnification, (B) 8 magnification.

4 314 A. Khopade et al. / Journal of Colloid and Interface Science 367 (212) Fig. 2. Neighbor-joining phylogenetic tree of strain B3 made by MEGA 4.. Numbers at nodes indicate levels of bootstrap support (%) based on a neighbor-joining analysis of 1 resampled datasets; only values >5% are given. NCBI accession numbers are given in parentheses. Bar,.5 nucleotide substitutions per site. substrate but did not produce the surfactant under similar conditions. The synthesized biosurfactant decreased the surface tension to 29 mn/m and showed 8% emulsifying activity. Similar results were found with P. aeruginosa 44T1 [29] Effect of nitrogen source The choice of nitrogen source affects the biosurfactant production as shown in Fig. 3B. In order to obtain high concentrations of biosurfactant, it is necessary to have restrained conditions of the macro-nutrients. Yeast extract was found to be the best source of nitrogen for growth and biosurfactant synthesis. Ammonium salts in the form of ammonium chloride was used for growth but not for biosurfactant production and caused a significant decrease in ph that results in decrease biosurfactant production. The maximum emulsifying activity (285 EU/ml) and minimal surface tension (3 mn/m) were reached in media with yeast extract [29] Kinetics of biosurfactant production The biosurfactant production and surface tension was dependent on growth of culture in the fermentation medium. The surface tension dropped rapidly after inoculation, reaching its lowest value (29 mn/m) during exponential phase after about 6 day of growth (Fig. 4). On 4th day of growth, the surfactant concentration starts to increase, reaching its maximum after about 6th day. The increase in surface tension and the decrease in E24 after 12th day of incubation showed that biosurfactant biosynthesis has been stopped and is probably due to the production of secondary metabolites that could interfere with emulsion formation and the adsorption of surfactant molecules at the oil water interface. These results indicate that the biosurfactant biosynthesis occurred predominantly during the exponential growth phase, suggesting that the biosurfactant is produced as primary metabolite accompanying cellular biomass formation (growth-associated kinetics) [22] Effect of ph, temperature, and salinity on biosurfactant production The ph of the medium was important characteristic for cell growth of organism and production of secondary metabolites. The biosurfactant production was affected by initial ph of culture medium. At ph 5, the biosurfactant production was severely decreased and the cell growth was significantly retarded. This low ph created unfavorable growth conditions for the bacterial population. When the initial ph was set to 7, the emulsification activity increases (E24 = 85), if ph of medium increase more than 7 the biosurfactant production decreases [11,16]. The optimum ph for growth and biosurfactant production was determined to be 7 (Fig. 5A). Similar result was observed by biosurfactant production from bacteria P. aeruginosa MR1 by Lotfabada et al. [38]. The strain B3 showed good growth between the temperature range of C. A change in temperature caused alterations in the composition of the biosurfactant in the case of Arthrobacter paraffineus [24] and Pseudomonas sp. [25]. Temperature was one of the critical parameters that have been controlled in bioprocess. The results in the present study revealed that the biosurfactant activity reached the highest when the strain was grown at 3 C (E24 = 8%) (Fig. 5B), and this clearly indicates moderately thermostable nature of biosurfactant. The research was focused on the isolation of alkaline biosurfactant from microbes because there is tremendous potentiality of biosurfactant in detergent industry. The strain B3 was found to be moderately halophilic in nature; maximum biosurfactant production (E24 = 78%) was obtained in presence of 4% (w/v) of NaCl and showed emulsification activity (E24 = 6%) in presence of 9% (w/v) of NaCl (Fig. 5C) Effect of oils, surfactants, and hydrocarbons on production of biosurfactant Fermentation was carried out with addition of different concentrations of oils, surfactant, and hydrocarbons in the fermentation medium. It was observed that olive oil, tween 2, and xylene as a substrate showed maximum activity against all test oils, surfac-

5 A. Khopade et al. / Journal of Colloid and Interface Science 367 (212) Dextrose Glucose Amm Sulphate Amm Chloride Glycerol Maltose Xylose Sucrose Trehalose Carbon sources [A] Alanine Peptone Urea Tryptone Nitrogen source [B] Starch Mannitol Fructose Hexadecane Yeast Extract Aspargine Beef Extract Phenyl Alanine Fig. 3. (A) Effect of carbon source on biosurfactant production, (B) Effect of nitrogen source on biosurfactant Production. tants, and hydrocarbons respectively. The olive oil and tween 2 showed emulsification activity 21 EU/ml and 34 EU/ml respectively (Fig. 6A and B). The organism was able to utilize all the hydrocarbons tested as sources of energy; growth was accompanied with biosurfactant production. The results of the hydrocarbon substrate specificity test revealed that the biosurfactant production had very good emulsification activity with hexadecane (Fig. 6C). The liquid aromatic hydrocarbons were particularly not good substrates for emulsification. On other hand, alkenes were found to be good substrates for emulsification by the biosurfactant. Crude oils and diesel oils are mixtures of hydrocarbons and are known to serve as an excellent sources of carbon and energy for most hydrocarbonoclastic microorganisms. Potential toxicity has been cited as possible reasons for the inability of most microorganisms to grow on toluene [9]. Crude oil and hexadecane were also good substrates for emulsification by the biosurfactant. Most microbial surfactants are substrate specific, solubilizing, or emulsifying different hydrocarbons at different rates. An emulsion is formed when one liquid phase is dispersed as microscopic droplets in another liquid continuous phase [2]. Poor emulsification of some of the hydrocarbons might be due to the inability of the biosurfactant to stabilize the microscopic droplets. Similar result was observed in biosurfactant production by oil degrading Aeromonas sp. by Ilori et al. [3] Critical micelle concentration (CMC) of biosurfactant The CMC value of the biosurfactant was determined by separately measuring the surface tension of different concentrations (log of mg/l) of biosurfactant (Fig. 7). The Mili Q distilled water was found to have the surface tension of 72 mn/m and the addition of biosurfactant reduced its surface tension up to 3 mn/m. It is worth mentioning that the CMC develops at the end of the exponential phase. Although the production of the surfactant continues after the exponential phase, its properties, such as the surface tensions, remain constant. This phenomenon is observed because of the conditions in which CMC is approached. The CMC of biosurfactant isolated from the Streptomyces strain was found to be 11 mg/ l. Rashedi et al. [4], was observed close result to that obtained in the present work Preliminary characterization of biosurfactant Compositional analyses revealed that the biosurfactant produced by Streptomyces might be a glycolipid primarily consisting of lipid with relative percent of 58% (w/w) and 33% (w/w) carbohy- Fig. 4. Growth kinetics of Streptomyces cell growth (OD6) and biosurfactant production by Streptomyces sp. B3.

6 316 A. Khopade et al. / Journal of Colloid and Interface Science 367 (212) E 24 (%) ph [A] Caster oil Oliveoil Eucalyptus oil Clove oil Coconut oil Oils [A] Cod liver oil Senamom oil E 24 (%) E 24 (%) drate. A minor fraction of protein (8% w/w) was found in some of extracted samples possibly arising from the existence of the residual cell debris in broth co-precipitated with biosurfactant during the extraction process. Similar result was observed on the isolation and surfactant production of different species of the genus Pseudomonas [35 38] Stability studies Temperature stability The applicability of biosurfactants in several fields depends on their stability at different temperatures and ph values. The stability of biosurfactant was tested over a wide range of temperatures. The biosurfactant produced by Streptomyces sp. was shown to be thermostable (Fig. 8A). Heating of the biosurfactant to 1 C caused no significant effect on the biosurfactant performance Temperature ( C) 4 [B] 5 6 NaCl (% w/v) [C] Fig. 5. (A) Effect of ph on biosurfactant production, (B) Effect of temperature on biosurfactant production, (C) Effect of NaCl on biosurfactant production EDTA Tween X1 Cyclohexane Octane Tween 2 Tween 4 Tween 8 Surfactants [B] Hexadecane Octadecane Diesel Hydrocarbons [C] The surface tension was quite stable at the temperatures 8 C, but the synthetic surfactants such as sodium dodecyl sulfate exhibits a significant loss of emulsification activity beginning at 7 C. Therefore, it can be concluded that this biosurfactant maintains its surface properties unaffected in the range of temperatures between 3 and 1 C. This activity was discovered indicating the usefulness of the biosurfactant in food, pharmaceutical, and cosmetics industries where heating to achieve sterility is of paramount importance [28,41]. SDS CTAB Petrol Toluene Kerosene Xylene Fig. 6. (A) Effect of oils on biosurfactant production, (B) Effect of surfactants on biosurfactant production (C) Effect of hydrocarbons on biosurfactant production.

7 A. Khopade et al. / Journal of Colloid and Interface Science 367 (212) Surface tension (mn/m) Surface tension (mn/m) Conc. of biosurfactant (log of mg/l) Fig. 7. Critical micelle concentration of biosurfactant Temperature ( C) [A] ph stability The surface activity of the biosurfactant remained relatively stable to ph changes between ph 8 12, showing higher stability at alkaline ph 8 than acidic conditions. At ph 12, the surface tension decreased up to 37 mn/m, whereas at ph 8 activities decreased to 29 mn/m. Fig. 8B shows the effect of ph on the biosurfactant properties. These results indicate that increase ph has a positive effect on surface tension and emulsion stability. This could be caused by a better stability of fatty acids surfactant micelles in the presence of sodium hydroxide and the precipitation of secondary metabolites at higher ph values. The effect of ph on surface activity has been reported for biosurfactants for different microorganisms [41] Effect of salinity The effect of sodium chloride addition on biosurfactant produced from Streptomyces was studied. Maximum stability of biosurfactant observes at 6% NaCl concentration. Little changes were observed in increase concentration of NaCl up to 9% (w/v) (Fig. 8C). At higher concentration of NaCl, the biosurfactant retains 8% activity. The biosurfactant has stability at alkaline ph and high salinity; such a biosurfactant may be useful for bioremediation of spills in marine environment because of its stability in alkaline condition and in the presence of salt [41] Antimicrobial activity of biosurfactant Surface tension (mn/m) Surface tension (mn/m) ph [B] Biosurfactant isolated from Streptomyces species showed a wide activity against the pathogenic strains. The partial purified biosurfactant showed activity against B. subtilis, P. aeruginosa, S. aureus, and E. coli and strong activity toward the yeast C. albicans (Fig. 9). According to Tsuge et al. [42], lipopeptide surfactants are potent antibiotics mainly the surfactin, streptofactin, and gramicidin produced by the microorganism and had the wide antimicrobial activity [43,44] compared to the glycolipid producing strain. A glycolipid surfactant from the C. antartica has demonstrated antimicrobial activity against Gram-positive bacteria [29] Fourier transforms infrared spectroscopy The presence of hydroxyl and amine groups of protein was confirmed by Infrared spectrum (data not showed) of the purified biosurfactant, which showed broad stretching peaks from 3458 to 32 cm 1. Absorption around 3159 cm 1 was assigned to the symmetric stretch ACH of CH 2 and CH 3 groups of aromatic chains. Absorption around 2924 cm 1 was assigned to the symmetric stretch ACH of CH 2 and CH 3 groups of aliphatic chains. Also, an intense absorption band at 1738 cm 1 and a weak symmetric stretching peak around 1645 cm 1 indicate the presence of ester carbonyl groups (C@O in COOH) in the biosurfactant. The ester carbonyl group was also proved from the band at 125 cm 1 that corresponds to C@O deformation vibrations, although other groups also absorb in this region. It might be possible that the additional bands at 1645 cm 1 and 1618 resulted from polypeptides originated from cell debris co-precipitated with the biosurfactant during extraction process. Similarly, another strong IR absorption found at 817 cm 1 was due to out of plane CAH bending, characteristic of aromatic compounds [18,45]. The FTIR spectra of the biosurfactant obtained indicated that the isolated biosurfactant was glycolipid in nature. 4. Conclusions The biosurfactant produced by a marine Streptomyces B3 was complex structure of proteins, carbohydrates, and lipids. The bio- 5 6 NaCl (% w/v) [C] Fig. 8. Effect of (A) Temperature, (B) ph and (C) salinity on biosurfactant stability

8 318 A. Khopade et al. / Journal of Colloid and Interface Science 367 (212) Area of the zone (mm 2 ) B. subtilis surfactant obtained from Streptomyces B3 employing olive oil as substrate and having high lipid content may provide a promising focus for further investigations on its application as a compound with efficient biological activity for enhanced oil recovery [33]. The results highlight the usefulness of regulation of physiological parameters and their effects on the biosurfactant production and further emphasize on the unique biochemical properties of these kinds of microbial natural products [27]. The importance of this biosurfactant for industrial uses was shown by studying different physical properties, such as critical micelle concentration, surface and interfacial tension, emulsification activity, and it s showed high stability to environmental factors such as temperature, ph, and salinity [33]. The isolated biosurfactant reduced the surface tension of water from 72 mn/ m to 29 mn/m. The purified biosurfactant showed critical micelle concentrations of 11 mg/l. The functional characterization of isolated biosurfactant indicated that the biosurfactant produced was glycolipid in nature [27,29]. The production of the biologically active fraction may be preferred for the economical production of these valuable molecules for therapeutic purposes such as biosurfactant as an alternative antimicrobial or anticancer agent in the medical field for applications against microorganisms responsible for diseases [18]. From this study, it can be concluded that the biosurfactant obtained from marine Streptomyces sp. B3 can be used as an alternative to chemical surfactants for bioremediation of spills in marine environments [29]. Acknowledgments E. coli S. aureus The authors would like to acknowledge All India Council for Technical Education (AICTE), HRD Ministry, New Delhi, Govt. of India for financial support to this research project under National Doctoral Fellowship (NDF), Appendix A. Supplementary material P. aeruginosa Pathogenic strains Fig. 9. Antimicrobial activity of biosurfactant. C. albicans References [1] I.M. Banat, Acta Biotechnol. 15 (1995) 251. [2] J.D. Desai, I.M. Banat, Microbiol. Mol. Biol. Rev. 61 (1997) 47. [3] A.A. Bodour, R.M. Miller-Maier, Encyclopedia Environ. Microbiol. 57 (22) 75. [4] S.S. Cameotra, R.S. Makkar, Appl. Microbiol. Biotechnol. 5 (1998) 52. [5] I. Banat, Biotechnol. Lett. 15 (1993) 59. [6] I.M. Banat, R.S. Makkar, S.S. Cameotra, Appl. Microbiol. Biotechnol. 53 (2) 495. [7] G. Georgiou, S.C. Lin, M.M. Sharma, Biotechnology 1 (1992) 6. [8] E. Rosenberg, E.Z. Ron, Appl. Microbiol. Biotechnol. 52 (1999) 154. [9] E.Z. Ron, E. Rosenberg, J. Environ. Microbiol. 3 (21) 229. 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