First we have to remove the bacterial cells by centrifugation, then add Conc HCl to reduced the pH of supernatant to approximately 2.0 this will precipitate the biosurfactant. Keep the the acidified suspension in the refrigerator overnight. Finally extrate the precipitated biosurfactant by a solvent mixture such as chloroform : methanol (3:1 v:v). evaporated the solvent to obtain the crude biosurfactant.

regarding the activity of biosurfactant in the bacterial suspension you can measure by several assays like drop collapsing, surface tension or drop displacement

1- Samples, 200 ml, of the cell-free broth were each extracted twice

with an equal volume of ethyl acetate. The solvent was removed

by reduced-pressure distillation and the dried product washed

with distilled water.

First we have to remove the bacterial cells by centrifugation, then add Conc HCl to reduced the pH of supernatant to approximately 2.0 this will precipitate the biosurfactant. Keep the the acidified suspension in the refrigerator overnight. Finally extrate the precipitated biosurfactant by a solvent mixture such as chloroform : methanol (3:1 v:v). evaporated the solvent to obtain the crude biosurfactant.

regarding the activity of biosurfactant in the bacterial suspension you can measure by several assays like drop collapsing, surface tension or drop displacement

Broth culture was centrifuged at 6000 rpm and 15°C for 15 min, and the hydrophobic layer located at the surface was extracted using different solvent systems at 27°C on a rotary shaker (200 rpm) for 3 h. Solvent extraction was also carried out on the whole culture broth, and in some cases, the extraction was performed using sonication (Ivshina et al., 1998). The solvent systems used were: MTBE, dichloromethane, chloroform–methanol (1:2 or 2:1) and MTBE–chloroform (1:1). The solvent layer was separated from the aqueous phase, and solvent was removed by rotary evaporation at 50°C under reduced pressure. Resulting crude extracts were freeze-dried and stored under nitrogen.

2.3. Surface and interfacial properties

Surface and interfacial tensions were measured by the ring de Nuoy method using a White digital surface tension balance. As crude biosurfactant was sparingly soluble in water, its surface and interfacial tension measurements were performed immediately after emulsification by ultrasonic treatment in water (23 kHz, 1 min) (Kim et al., 1990). Interfacial tension was determined against n-hexadecane. Critical micelle concentration (CMC) was calculated as the lowest concentration of biosurfactant at which the surface tension value was minimal.

2.4. Determination of surfactant composition

Protein was determined by the Coomassie blue method, with bovine serum albumin (BSA) as a standard (Bradford, 1976). Extractable hexoses were estimated by the phenol–sulphuric acid method, using trehalose as a standard (Dubois et al., 1956).

Separation of lipids was performed by chromatography on a silica gel (60–140 mesh, Merck) column using solvent systems with increasing polarity (Kretschmer et al., 1982). The eluates from the column were collected and dried under nitrogen. Different column fractions were analysed by TLC after dissolving in chloroform. Lipids were subject to chromatography with the following solvent systems: saturated hydrocarbons with n-hexane; unsaturated hydrocarbons with n-hexane–dichloromethane (9:1); acylglycerols and fatty acids with n-hexane–chloroform–acetic acid (20:80:0.5); fatty alcohols with chloroform–methanol (95:5); glycolipids with chloroform–methanol–water (85:15:2); phospholipids with chloroform–methanol–water (65:25:4). Detection of lipids was performed using the following spray reagents: 50% sulphuric acid for n-alkanes; 4-methoxy benzaldehyde (p-anisaldehyde) for unsaturated hydrocarbons and fatty alcohols; hydroxylamine–ferric chloride for acylglycerols; 2,7-dichlorofluorescein–aluminium chloride–ferric chloride for fatty acids; anthrone and phenol–sulphuric acid for glycolipids; ammonium molybdate–perchloric acid and ninhydrin for phospholipids. Corresponding lipids, purchased from Sigma, St. Louis, MO, USA, were used as reference standards.

The fatty acids composition of surfactant extracts was analysed by GC–FID (Chrom-5, Laboratorni Pristroje, Prague, Czech Republic) of fatty acids methyl ethers. Methylation of organic extracts was carried out after alkaline hydrolysis (70°C for 1 h) at 80°C for 4 h, under nitrogen, with methanol–benzene (1:5). Concentrated sulphuric acid was used as a catalyst. The GC capillary column was 3.7 m×4.0 mm containing PEGA (10%) in Intertone N-AWDMCS. The carrier gas was helium at 24 ml/min; evaporator temperature was 310°C; column temperature was 180°C; detector temperature was 210°C. The sample volume was 10 μl. The chromatographic peaks were identified by comparing with the chromatogram of the standard fatty acid methyl ester (FAME) mixture (Sigma). The presence of 10-methyl branched fatty acids was indirectly indicated by bromination of FAME in diethyl ether (Kates, 1988) and by comparison with the retention time data published for Rhodococcus species.

Residual n-hexadecane concentration in crude surfactant was measured by gas chromatography of n-hexane (Sigma GC grade) extracts using capillary GC-FID (Perkin-Elmer 8320). The column was 50 m×0.25 mm containing 0.22 μm CPSIL-8. The carrier gas was helium at 7.5 psi pressure. Oven temperature ramp rate was 40–300°C at 4°C/min. The sample (1 μl) was injected on column at 60°C. The column temperature regime was 60°C/0.1 min at 200–300°C/min.

centrifugation

Hi, I agree with Mr. Haytham M. M. Ibrahim, but i want to add some point. :

First we have to remove the bacterial cells by centrifugation (10,000 rpm 4oC, 20 min ) then add 6N HCl to reduced the pH of supernatant to approximately 2.0 this will precipitate the biosurfactant. Keep the the acidified suspension in the refrigerator  4oC overnight. then centrifuge again and keep the mass ( partially purified biosurfactant) remained at the bottom of tube. now you can dissolve it in ethyl acetate and start thin layer chromatography with different solvent systems including hexane/ ethyl acetate or chloroform/ methanol/distilled water.

thank you everyone

what about cell bound biosurfactant? how can we extract it ?

what is the difference in using chloroform methanol , diethy ether and ethyl acetate.Which of the solvent is better and why is a mixture of methanol used

 Can anyone tell me please why do we Keep the the acidified suspension in the refrigerator 4°C overnight after centrifugation in acide precipitation protocol? another question: if the speed of centrifugation is lower than 10,000 rpm (4800 rpm for exemple) for more than 20min, would i have the separation or not?

separate of biomass from growth medium and further pH of supernatant was adjusted to 2 by 6 M HCl to allow the precipitation of biosurfactant. The precipitates were collected by centrifugation to obtain a crude biosurfactant. For further purification, the crude biosurfactant was dissolved in double-distilled water and was then extracted three times with an equal volume of dichloromethane. The solvent layer was harvested and evaporated. The residue is not still a pure biosurfactant and can be considered as a semi pure biosurfactant.  

does acidification with HCl affect the stability of protein or other compound of the biosurfactant molecules?

I also want to extract bacterial surfactants. After precipitating the surfactants by acidification, I performed the extraction with methanol: chloroform (2: 1). I collect the lower layer, and repeat again. Evaporate the solvent with rotavapor (38 °), however no product is displayed. What do you suggest?

Very easy by C18 syringe cartridge column