70
C. Elizabeth Rani et al.
2.5 Separation of Biosurfactant by Solvent Extraction
Method
The entire broth was centrifuged at 10,000 rpm for 10 min to remove the cells. The
supernatant was transferred into a sterile screw cap bottle to which acid precipitation
method was employed by reducing the pH to 2.0 by slowly adding the conc. Hcl.
After the acidification of the cell free extract, it was allowed to incubate overnight in
fridge. After the incubation, the content was centrifuged at 10,000 rpm for 10 min.
The surfactant in the broth was extracted in chloroform: methanol (2:1) solvent
system. The cell free broth was transferred into a sterile separating funnel following
that equal volume of above mentioned solvent mixture was added. The contents were
vigorously agitated for proper mixing. Then it was kept undisturbed for overnight.
Next day all the different layers were collected in different petriplates and allowed
for complete evaporation.
3 Screening of Biosurfactant
3.1 Haemolysis Test
The first screening method for the biosurfactant synthesizing bacteria was the
haemolysis test (Carrillo et al 1996). The selected strain was streaked on to blood
agar medium and the plates were incubated for 24–48 h at room temperature. Physical examination was done to check the clear zone formation around the streaked
areas.
3.2 Oil Spreading Test
Oil spreading assay was conducted by the method described by (Morikawa et al.
1993). In brief, 20 ml of distilled water was poured onto a clean petri plate followed
by the addition of 20 µl of crude oil to the surface of water. To this, 10 µl of cell
free culture broth was overlayed to the crude oil surface. If biosurfactant is present
in the cell free extract, the oil will be displaced and a clear zone and diameter of this
indicates the surfactant activity. The diameter of the clearing zone of the oil surface
indicating the activity of biosurfactant. A negative control was maintained by adding
a drop of distilled water to the oil surface (Morikawa et al. 1993).
C. Elizabeth Rani et al.
2.5 Separation of Biosurfactant by Solvent Extraction
Method
The entire broth was centrifuged at 10,000 rpm for 10 min to remove the cells. The
supernatant was transferred into a sterile screw cap bottle to which acid precipitation
method was employed by reducing the pH to 2.0 by slowly adding the conc. Hcl.
After the acidification of the cell free extract, it was allowed to incubate overnight in
fridge. After the incubation, the content was centrifuged at 10,000 rpm for 10 min.
The surfactant in the broth was extracted in chloroform: methanol (2:1) solvent
system. The cell free broth was transferred into a sterile separating funnel following
that equal volume of above mentioned solvent mixture was added. The contents were
vigorously agitated for proper mixing. Then it was kept undisturbed for overnight.
Next day all the different layers were collected in different petriplates and allowed
for complete evaporation.
3 Screening of Biosurfactant
3.1 Haemolysis Test
The first screening method for the biosurfactant synthesizing bacteria was the
haemolysis test (Carrillo et al 1996). The selected strain was streaked on to blood
agar medium and the plates were incubated for 24–48 h at room temperature. Physical examination was done to check the clear zone formation around the streaked
areas.
3.2 Oil Spreading Test
Oil spreading assay was conducted by the method described by (Morikawa et al.
1993). In brief, 20 ml of distilled water was poured onto a clean petri plate followed
by the addition of 20 µl of crude oil to the surface of water. To this, 10 µl of cell
free culture broth was overlayed to the crude oil surface. If biosurfactant is present
in the cell free extract, the oil will be displaced and a clear zone and diameter of this
indicates the surfactant activity. The diameter of the clearing zone of the oil surface
indicating the activity of biosurfactant. A negative control was maintained by adding
a drop of distilled water to the oil surface (Morikawa et al. 1993).