Biosurfactant extraction and purification.

The method used for biosurfactant extraction and purification was modified from the work of Kim et al. (23). When the maximum oil displacement diameter was obtained, cells from duplicate or triplicate 1-liter cultures were removed by centrifugation at 14,300 g for 15 min at 4C. The pellet was dried at 110C overnight, and the dry weight was determined. Biosurfactant in the supernatant was precipitated with 40% (wt/vol) ammonium sulfate and incubated overnight at room temperature. The precipitate, containing the biosurfactant along with other compounds, was then collected by centrifugation at 14,300 g for 30 min at 4C. The precipitate was extracted with 250 l of chilled acetone to remove most of the proteins. Instead of the column chromatography steps used by Kim et al. (23), further purification was achieved by preparative thin-layer chromatography (TLC) of the acetone extract. The whole acetone extract (250 l) was spotted onto preparative silica gel TLC plates (Whatman, Clifton, NJ) with a solvent system of isopropanolwater28% (wt/vol) ammonium hydroxide (80:11:9). The TLC plates were developed with iodine vapor. Each fraction was scraped off the plate, dissolved in 250 l water, and tested for surface activity by the oil-spreading technique. Surface-active fractions were lyophilized. The weight of the lyophilized biosurfactant was determined and used to calculate the biosurfactant yield (biosurfactant weight/dry weight of cells). Biosurfactant yields of different strains varied from 0.9 to 3.1 mg g1 dry weight of cells. The coefficients of variation of biosurfactant yields between different batches of the same strain ranged from 4.9 to 27%. To compare surface activities of different biosurfactants, 1-g l1 solutions of purified biosurfactants were prepared and tested by the oil-spreading technique. The specific activity of each purified biosurfactant was expressed as the diameter of the clear zone, in millimeters per microgram of purified biosurfactant. The biosurfactant specific activities of different strains varied from 0.7 to 4.5 mm g1. Triplicate samples were done for each culture. The coefficients of variation of specific activities between different batches of the same strain ranged from 4 to 26%, while those for the same batch of the same strain were 5%. Evaluation of a new protocol for biosurfactant purification. The lipopeptide produced by triplicate cultures of Bacillus mojavensis strain ROB-2 was used to compare the efficiencies of two purification methods. Method 1 involved acid precipitation (using 1 N HCl to adjust the pH of the cell-free culture fluid to 2) (41) followed by TLC. Method 2 used ammonium sulfate precipitation followed by acetone extraction and TLC. Seventy-five percent of the biosurfactant activity remained in the cell-free culture fluid after cell removal. The surfaceactive fraction obtained from the TLC plate by method 1 had 23% 7% of the activity originally present in the culture, while the surface-active fraction obtained from the TLC plate by method 2 had 63% 11% of the activity originally present in the culture. The specific biosurfactant activity of the surface-active fraction from the TLC plate for 12 different strains was 0.65 0.07 mm g1 by method 1 and 1.9 0.7 mm g1 by method 2. Method 2, being more efficient, was used to purify the biosurfactants. Relationship between biosurfactant yield and activity. The biosurfactant yields of seven different Bacillus strains (duplicate cultures of each strain) with activities ranging from 0.5 to 4.25 times that of B. mojavensis strain JF-2 were determined. Biosurfactant activity was poorly correlated with the biosurfactant yield (linear correlation coefficient [r2] 0.09 and Pearson correlation coefficient [15]0.29). The biosurfactant activity did not always increase with an increase in biosurfactant yield, i.e., some biosurfactants were produced with high yields but had relatively low activities, while others were produced with low yields but had high activities. Isolation and extraction of crude biosurfactant: The biosurfactant product was isolated by acid precipitation method [13, 16]. Briefly, the cells were removed from the fermentation media by centrifugation (10,000g, 20 min) and the cell free supernatant was acidified to pH 2.0 by adding 6 N HCl. The acidified cell free broth was kept overnight at 4C for the precipitation of biosurfactant. The precipitated biosurfactant was collected by centrifugation and the pH was adjusted to 8.0 and the solution was lyophilized. The methanol extract of lyophilized biosurfactant was used for further analysis and purification [7]. High-performance liquid chromatography A HPLC instrument equipped with a diode array detector system (DAD; Agilent 1100 series, Agilent Technologies, CA, USA) and C-18 reversed phase Zorbax (250 4.6 mm, 5 m particle size) columnwas used for the purification process. The methanol extract was filtered through a 0.2-m membrane filter (Millipore, Billerica, USA) before injection. The filtrate (50l per run) was injected into the column by using a glass microsyringe (Agilent Technologies, CA, USA). The elution system consisted of solvent A [sterilized membrane-filtered Milli-Q water] and solvent B [HPLCgrade acetonitrile with 0.1% TFA (Merck, Germany)]. The solvents were degassed by purging with dry nitrogen before use. The elution of various biosurfactant fractions was monitored at 210 nm [14, 15]. The flow rate 0.4 ml min1 for 60 min was used for the initial method by linearly increasing the percentage of solvent A (1090%) [4]. In stage I, the flow rate was increased to 0.7 ml min1 to reduce the contaminant level in the methanol extract and simultaneously the flow of the solvent B was gradually increased from 0 to 100 % for 50 min [15]. In the intermediate methods like stage II, III, and IV, the flow rate and time was adjusted according to biosurfactant peak profiles obtained, to achieve the final optimized method. In the final method, from 0 to 4 min the flow rate was maintained at 2 ml min1 and at this rate the concentration of the solvent A was 60% with solvent B constituting the remaining 40%. From 4 to 12 min the flow rate was maintained at 0.7 ml min1, with a gradient system in which the concentration of solvent A was decreased from 4010%, whereas the solvent B concentration was increased from 6090%. After 12 min, the flow rate was changed to 1 ml min1 with 100% solvent B and was maintained for 20 min. The compounds were collected manually through the waste line of the instrument. Method validation The validity of the final optimized method was checked by injecting the individual biosurfactant fractions as well as the crude mixture. For this purpose, fractions were collected in stage I and the other intermediate methods (stages IIIV). These fractions were then injected individually in the final optimized HPLC method. The identity of the compound was confirmed by comparing the retention time of the individual fractions with the chromatogram using a crude mixture in the same method. Surface tension measurement The surface-active isoforms were identified by measuring surface tension of the eluted peaks. Approximately 20 ml of the eluent were collected in 50-ml glass tubes and were kept at 60C in hot air oven until the solvent got evaporated (~36 h). The concentrated solution was taken for measurement of the surface tension using a mechanical surface tensiometer (Jencon

Instruments, Kolkata, India). The measured surface tension values were confirmed with the help of a DCAT digital surface tensiometer [Dataphysics, Germany]. All the surface tension readings were obtained in triplicate and the results represent the mean values. Critical micelle concentration The critical micelle concentrations (CMC) of the crude and purified isoforms were determined. The stock solutions (1 mg ml1) of purified isoform were prepared and the CMC of the samples were determined from the stock solutions by serial dilution method followed by surface tension measurement [13, 17]. CMC values were used for the estimation of the percentage purity of the biosurfactant isoforms [13]. Fourier transform-infrared spectrophotometry Fourier transform-infrared spectrophotometry (FTIR) analysis of the HPLC purified isoforms was performed using a Nexus-890 Spectrophotometer (Thermo Electron Co., Yokohama, Japan) in order to reveal the chemical nature of the biosurfactant. The purified samples were dispersed in spectral-grade KBr (Merck, Darmstadt, Germany) and made into pellets by applying 56 tons cm2 of pressure for 10 min using the hydraulic press (Specac, UK) instrument. The spectrum was generated in the range of 400 to 4,000 cm1 with a resolution of 4 cm1. The spectral measurement of the compound was carried out in transmittance mode with average of 32 data scans over the entire range of wave numbers [17, 18]. MALDI-ToF HPLC purified isoforms were analyzed by matrix-assisted laser desorption/ionization time-of-flight analysis (MALDIToF) for molecular mass determination. The matrix used for cocrystallization was 2, 5-dihydroxybenzoic acid (Sigma, USA). A 10-mg ml1 matrix stock solution was prepared in acetonitrile, methanol, TFA (5:4:1). Equal volume (2l) of the purified sample and the matrix were mixed vigorously in the vortex for 5 min. After proper mixing, the sample was spotted on the target plate, dried, and was placed inside the sample cabinet of Voyager DE-Pro MALDI-ToF spectrometer (Applied Biosystems Inc, CA, USA). The nitrogen UV laser (337 nm) was used for desorption and ionization and a voltage of 20 kV was maintained to accelerate the molecules. The molecules were separated according to their mass and were detected by the ion detector set in reflector mode [7, 19]. Results Method development: HPLC separation of lipopeptide isoforms: The biosurfactant was isolated from the clarified fermentation broth by acidification [13]. The crude biosurfactant obtained by this method was soluble in water at neutral and alkaline pH values. The methanol extract of the crude biosurfactant was used for further analysis in HPLC. The individual biosurfactant isoforms present in the crude mixtures was resolved using reverse phase HPLC (RP-HPLC). Stepwise method optimization was carried out by adjusting the solvent gradient program and solvent flow rates. The steps of this method development process were as follows: Initial method In this method, four major fractions (AD; Fig. 1) were observed at the retention time 23.7, 25.6, 27.2 and 32.7 min respectively. These collected fractions showed low values of surface tension (Table 1). As in this method, separation and elution of the surface-active fractions took significantly longer time; we proceeded systematically towards the development of an optimal method that could produce significant reduction in retention time for chromatographic separations. Stages IIV methods In stage I, the run time was reduced by 10 min. Though there was no significant change in the retention time of the four major fractions (AD), a good resolution of the peaks were obtained in this method (Fig. 2). These four surfaceactive fractions (Table 1) had retention times of 27, 28, 29 and 30 min, respectively. However, the antimicrobial action was observed only in the fraction A. The other three surface active fractions did not show any antimicrobial activity. Similarly in stage II, with a reduced run time of 30 min, the HPLC purification of biosurfactant isoforms 847 four surface-active fractions were eluted at retention time of 17.9, 18.5, 19.42 and 20.64 respectively. In stage III, the run time was further reduced to 25 min with the four surface active fractions being eluted earlier at 13, 15, 16, and 16.8 min, respectively. In stage IV, again with a run time of 25 min using a modified gradient program, the elution time of the active fractions were further reduced to 10.24, 11.58, 12.8, and 13.78 min respectively. In all the stages of method development, the antimicrobial action was exclusively displayed by the fraction A while the other fractions did not show any antimicrobial property. Optimized method The final optimized method was developed having a run time of 20 min. In this method, the time of elution was significantly reduced to 8.2, 9.5, 10.4, and 11.2 min, respectively, for the four active fractions. Further reduction in elution time could not be achieved without compromising the peak resolutions. This method showed the best resolution of the peaks (Fig. 3) without any change in their surface or biological activities. The surface tension values for the four fractions were presented in Table 1. As seen in the earlier methods, antimicrobial activity was again retained solely in the fraction A. The biosurfactant isoforms obtained by this method was highly purified as compared to other methods in the previous stages, which was confirmed by the lowering of the CMC values and also enhancement in antimicrobial activity. Validity of the optimized method The surface-active isoforms (AD) collected from the different stages (stage IIV) showed the same retention time as that of the corresponding fractions when using a crude lipopeptide mixture. Thus, the four fractions separated in the final optimized method were same as the corresponding fractions of the other stages. Hence, the identities of the peaks were retained in the final optimizedmethod albeitwith a lesser retention time. Determination of CMC for assessment of purity The surface activity of the surfactant was indicated by CMC. An assessment of the CMC showed a lower value and hence, higher purity level (>85%) for the lipopeptides obtained in the final stage in comparison with stage I and crude biosurfactants (Table 2). CMC results showed that the final optimized method was more suitable for the analysis and the purification purpose. Chemical nature of the biosurfactant isoforms : FTIR analysis The chemical nature of HPLC purified biosurfactant isoforms was confirmed by FTIR analysis. The IR absorption pattern (Fig. 4) for fractions A, B, C, and D revealed the presence of peptide and carboxyl groups that indicated their lipopeptide

nature [4, 15, 17, 18]. The antimicrobial isoform (fraction A) showed a transmittance valley at 3,274 cm1 as a result of N H stretching indicating the peptide groups. Aliphatic chain was indicated by CH weak stretching vibration observed in the range 2,930 cm1. The transmittance occurred in 1,658 cm1 due to the amide I band frequency (CO stretching in the peptide bond) and transmittance at 1,531 cm1 range showed CO bonds. The transmittance at 1,390 cm1 range may be due to the aliphatic chain of CH group. Similarly in fraction B, the NH stretching was observed at 3,477 cm1 with a broad valley of transmittance. The transmittance at 1,670 cm1 range resulted in the stretching mode of CO bond and at 1,143 cm1 resulted in CN stretch. The transmittance at 1,203 cm1 corresponding to CN stretch and at 1,440 cm1 and 2,364 cm1 due to the presence of aliphatic chain (CH stretching mode) was observed. The absorption at 1,024 cm1 indicated a CO stretch. In fraction C, IR spectrum showed absorption at 3,109 cm1, 1,623 cm1, and 1,132 cm1 corresponding to strong absorption band of peptides, resulted from the stretching mode of NH and CO bonds, respectively. The presence of CH stretching at 1,398 cm1 indicated the presence of aliphatic chain. The absorption at 1,198 cm1 indicated a CN stretching. The IR spectrum for fraction D revealed absorption corresponding to the presence of peptides at 3,087 cm1, 1,667 cm1, and 1,198 cm1 respectively. The CH stretching at 1,439 cm1 and 1,398 cm1 indicated the aliphatic chain presence. In all the fractions (AD), the peaks observed at the 800600 cm1 range revealed the presence of CH bend aliphatic chain. MALDI-ToF analysis MALDI-ToF analysis of the RP-HPLC purified isoforms were performed for mass determination. In this analysis, the ionized molecules were separated according to their respective masses and the charge of the molecules. The mass spectral analysis showed that all purified isoforms (fractions AD) were lipopeptides and belonged to fengycin family [8]. The mass spectra of fraction A (Fig. 5a) revealed the presence of a major peak of C16 lipopeptides (1,482 Da and 1,484 Da) in their Na+ adduct form. The other molecules present in this fraction were the C17 lipopeptides (1,496 Da and 1,498 Da) and C17 lipopeptides as their H+ adduct (1,503 Da) and Na+ adduct (1,526 Da), respectively. Similarly, in fraction B (Fig. 5b) showed the presence of a major peak corresponding to protonated C16 lipopeptides (1,464 Da and 1,466 Da). This C16 isoforms was also revealed as its Na+ adduct (1,480 Da and 1,486 Da) and also its K+ adduct (1,502 Da and 1,510 Da), respectively. Other isoforms present in this fraction was C17 lipopeptides as their Na+ adduct (1,496 Da) and K+ adduct (1,524 Da) respectively. In the surface-active fraction C (Fig.5c), C16 lipopeptides were revealed as a major peak in their H+ (1,492 Da) and Na+ (1,514 Da) adduct form, respectively. C17 isoforms were also revealed in their H+ (1,478 Da), Na+ adduct (1,506 Da) and K+ adduct (1,516 Da) forms respectively. In the similar manner mass spectra of fraction D (Fig. 5d), C15 isoform was detected in its protonated form (1,448 Da) and K+ adduct (1,480 Da). C16 fengycins were also found to be present in their protonated form (1,462 Da) and as their K+ adduct (1,493 Da). C17 group was also present in protonated form (1,506 Da and 1,508 Da) and as the Na+ adduct (1,528 Da and 1,530 Da) forms, respectively.

Experimental Approach Extraction: The Aim is to Obtain a Crude Extract Free from Aqueous Culture Medium Bacillus Lipopeptides This extraction technique is a combination of acid precipitation and solvent extraction (Vater et al., 2002) 1. To remove cells centrifuge at 13,000 _ g for 15 min at 4 C 2. Acidify by addition of concentrated HCl to pH 2.0 and allow precipitate to form at 4C Overnight 3. Centrifuge at 13,000 _ g for 15 min at 4 C to obtain pellet 4. Remove supernatant and extract the pellet with methanol for 2 h while stirring continuously 5. Filter methanol to remove remaining material and evaporate to dryness using rotary evaporation Pseudomonas Lipopeptides method for this group of lipopeptides is solvent extraction with ethyl acetate (Kuiper et al., 2004). 1. Extract three times with an equal volume of ethyl acetate1 shaking vigorously each time and allow the two layers to separate in a separating funnel 2. Transfer bottom aqueous layer and the top ethyl acetate layer to separate flasks. Re-extract the aqueous portion twice more with ethyl acetate layer 3. Add a small amount of magnesium sulphate to the ethyl acetate portion, to remove the traces of water present, filter and rotary evaporate to dry extract Lipoproteins and Polymerics Extraction Extraction of the majority of high molecular weight biosurfactants is carried out using ammonium sulphate precipitation, followed by dialysis to remove any small molecules that may be present (Rosenberg et al., 1979). The methods reported for isolation of the high

molecular biosurfactants are quite varied and generally specific to the actual biosurfactant present. Ammonium sulphate precipitation can be used for the majority of extractions, however, for more specific details refer to related literature of the microorganism under investigation. Other techniques for high molecular weight biosurfactant isolation include TCA/acetone precipitation, acid ethanol and chloroform/methanol. The ammonium sulphate precipitation method below is based on using 100 ml of culture broth and should be adjusted according to the starting volume. 1. Take 100 ml of culture broth and remove cells by centrifuging at 10,000 _ g for 15 min 2. Cool the supernatant at 4C and add slowly while stirring 23.34 g of ammonium sulphate to obtain a 40% saturation solution.2 Leave at 4 C overnight 3. Centrifuge at 10,000 _ g for 15 min to obtain pellet 4. Re-suspend in a 40% solution of ammonium sulphate and centrifuge again at 10,000 _ g for 15 min to obtain pellet 5. Dissolve pellet in 20 ml water and extract with equal volume of hexane, three times in a separating funnel to remove residual non-polar lipids 6. Using dialysis tubing with a molecular weight cut off point of 5 kDa clamp one end and rinse the tubing with distilled water to check for leaks 7. Fill tubing with product from step 5 and clamp top. Place in beaker with dialysis buffer of distilled water, place on a stirring plate in a cold room overnight 8. Change dialysis buffer once after 6 h 9. Remove sample after completion of dialysis and lyophilize to obtain biosurfactant product Detection/Purification
A number of impurities are often co-extracted together with the target biosurfactant and may need to be separated. TLC of Lipopeptides Thin layer chromatography (TLC) is a simple method, which can be used to detect the presence of lipopeptides while preparative TLC can be used to purify small quantities (Symmank et al., 2002). 1. Dissolve a small quantity of crude extract in chloroform and apply 10 ml onto a TLC plate (silica gel 60) and apply at point of origin near the bottom of the plate 2. Once dried, develop plate in solvent system of chloroform:methanol:water (65:25:4) 3. When developed remove plate and allow to air-dry in a fume cupboard 4. Spray the plate evenly with water or a solution of 5% sulphuric acid and place in an oven at 110_C for 20 min to visualize spots 5. At preparative scale remove lipopeptide spots by scraping the silica from plate into a flask and extract with chloroform:methanol (2:1) overnight 6. Filter and rotary evaporate to dryness HPLC-UV/HPLC-MS for Purification of Different Types of Peptides/Lipopeptides HPLC is an excellent method for the separation of individual peptide/lipopeptide biosurfactants (Aguilar, 2004). The most commonly employed technique is reversed phase chromatography, which results in the separation of each peptide/lipopeptide structure based on polarity. The separated products are detected by UVabsorbance detection and each individual peak can be collected using a fraction collector for further analysis of their structure. Coupling of HPLC with a mass spectrometer provides preliminary information on the molecular mass of each component. Purification with either HPLC-UV or HPLC-MS using different types of column chemistry is also possible. For the purpose of this report the basic steps in HPLC-MS will be discussed below. 1. Interface HPLC to electrospray ionisation mass spectrometry (ESI-MS) system 2. Use semi-preparative HPLC column such as Luna C18 column (250 mm _ 10 mm id) (Phenomenex, Cheshire, UK) connected 3. Prepare the sample by dissolving in water/TFA (99.95:0.05, v/v). Centrifuge at 13,000 rpm for 5 min to remove particulate matter 4. Prepare mobile phase A (water/TFA (99.95:0.05, v/v)) and B (acetonitrile/water/TFA (80:19.95:0.05)) and perform gradient elution starting with 100% A, 0% B changing to 0% A, 100% B over 80 min5 5. Set flow rate at 1.0 ml min _1 and injection volume of 500 ml min _1 6. Connect fraction collector and set up the effluent flow from the column so that 90% is fraction collected and the remaining 10% sent to the MS detector 7. Collect fractions at a rate of 1 ml per minute. Fraction collection may be automated or peak collection may be used 8. MS scan range should be set at m/z 1502,000 9. MS/MS fragmentation can also be set up on the eluent entering the mass spectrometry. The information obtained can then be used to search a number of databases 10. The individual fraction can then be subjected to Q-TOF MS/MS de novo sequencing or Edman degradation to obtain sequence information on each individual peptide/ lipopeptide. One Dimensional Sodium Dodecyl Sulphate Polyacrylamine Gel Electrophoresis(1D SDS-PAGE) SDS-PAGE is commonly used for proteomic experiments and is extremely useful for separation of protein mixtures6 and for estimating the molecular mass of high molecular weight biosurfactants/proteins. The proteins are applied to one end of a gel in a loading buffer that contains a reducing buffer and SDS. The reducing buffer is used to cleave the disulphide bonds rendering the proteins in a linear form. SDS attaches to the protein relative to its molecular mass, resulting in a net negative charge on the protein. When a voltage is applied to the gel, proteins separate according to molecular mass. The individual bands can then be removed and the proteins extracted (Toren et al., 2001). 1. Prepare 10% running gel7 using the following; 12.3 ml of double distilled water; 7.5 ml of gel buffer which is 1.5 M Tris-HCl at pH 6.6 (6 g of Tris in 100 ml of water adjusted with HCl); 9.9 ml of acrylamide/Bis-acrylamide (30%/0.8% w/v); 0.3 ml of 10% SDS (w/v); 0.15 ml of 10% ammonium persulfate (APS w/v) and finally 0.02 ml of TEMED (N,N,N, N-tetramethylethylethylenediamine). For the last two parts (APS and TEMED) of the running gel add just before pouring the gel 2. For preparation of stacking gel use the following; 3.05 ml of double distilled water; 1.25 ml of stacking buffer (0.5 M Tris-HCl at pH 6.8); 0.665 ml of acrylamide/Bis-acrylamide (30%/0.8% w/v); 50 ml of 10% SDS; 50 ml of 10% APS and 5 ml of TEMED.8 For the last two parts (ABS and TEMED) of the stacking gel add just before pouring gel 3. Running buffer should be prepared as follows; 200 mM glycine, 25 mM Tris-HCl at pH 6.8 and 0.1% (w/v) SDS 4. Prepare Laemmli sample buffer (Laemmli, 1970) by using 0.02 g (w/v) of bromophenol blue; 0.313 ml of stacking buffer (0.5 M Tris-HCl at pH 6.8); 0.5 ml 10% (w/v) SDS; 0.25 ml (v/v) glycerol and make up entire solution to 2.5 ml with double distilled water 5. Set up gel electrophoresis as per standard procedure and pour the running gel prepared in step 1 with APS and TEMED being added just before pouring. Place 1 ml of water saturated with butanol carefully on top of monomer solution and leave for 45 min to allow the gel to polymerize 6. Pour the overlying water off and drain the excess with strips of filter paper 7. Add the stacking gel buffer with APS and TEMED being added just before pouring. Insert sample comb at an angle to allow air bubble to escape and leave to set for 15 min 8. Remove the comb and place gel in the buffer chamber and add running buffer to cover the gel 9. Prepare the protein sample to be investigated by adding 20 ml of proteins to 20 ml of sample buffer (step 4) and 1 ml of beta-mercapto-ethanol9 (used to reduce the disulphide bonds). Incubate at 65 _C for 10 min 10. Load 5 ml molecular weight marker and 10 ml of each sample into separate wells 11. Put on lid and connect power supply with 200 volts and run the gel. Stop the electrophoresis when the marker dye is less than 1 cm from bottom of plate (around 1 h) 12. Remove gel and immerse in Coomassie brilliant blue stain10 (made by adding 1.25 g ofCoomassie R-250, 250 ml methanol, 50 ml of acetic acid and 200 ml of water) and leave overnight 13. Pour off the Coomassie stain and destain using 50% methanol in water containing 10% acetic acid. Cover gel with destain solution and leave for 10 min then remove 14. Record image of gel and determine approximate molecular mass of each protein or high molecular weight biosurfactant present by comparison with the molecular weight ladder