Biological Activity of BiosurfactantsProduced by Locally Isolated Bacillus spp.

Eighty eight local bacterial isolates of Bacillus spp. were obtained from 45 oil contaminated soil samples from different fuel stations in addition to Al- Dorah oil refinery in Baghdad Province /Iraq. All these isolates were screened for their ability for biosurfactant production by surface tension measurement of cell free supernatant after cultivation in E medium , which referred that 81 of the 88 isolates were biosurfactant producer , while screening depended on the blood haemolysis activity on sheep blood agar indicated that only 49 isolates were biosurfactant producers. Molecular identification based on the 16S rDNA by two sets of primers (27F and 1492R) , (B16SF and B16SR) of the best ten biosurfactant producer isolates (B1,B2, B3, B4, B5, B6 , B7, B8, B9, B10) revealed that all isolates were belonged to Bacillus spp. with 100% similarity, and to Bacillus subtilis group, with similarity ranged between (97-100)% to Bacillus subtilis
All the selected ten isolates were capable of producing biosurfactant with highest efficiency (40 -43.1)% in Jacques medium than in E medium (28-30)%. Biosurfactants produced by the selected isolates were purified by acid precipitation followed by ultrafiltration with centricons. Purified biosurfactants were found to be member of the lipopeptide family when characterized by the TLC that sprayed with water or with Rhodamine B, and all isolates were capable of producing surfactin with or without other types of biosurfactants depending on the isolate as showed by comparison with standard surfactin. HPLC/MS analysis of the purified biosurfactant from B. subtilus B6 revealed that it is a surfactin with partial sequence determination as follows: Val-Asp -Leu-Leu-OH2. Cytotoxicity assay of the purified biosurfactant from the selected isolates on ( K562, Hep-2, L1210 ) were tested , results showed that lipopeptides cytotoxicity were dependent on the type of tumor cell line, type of isolate, biosurfactant concentration and exposure time Purified biosurfactant produced by Bacillus B7 was found to be the most cytotoxic one to K562 cell line that caused 57.17% inhibition of the cell line growth at concentration (80) mg/l, while purified biosurfactant produced by isolate Bacillus B9 was the most cytotoxic one to L1210 cell line that caused 58.62% inhibition of the cell line growth at the same concentration , however, purified biosurfactans produced by ( B. subtilis isolates B6, B8, B9) were the most cytotoxic on Hep 2 proliferation with inhibition rate (69.219,86.043,64.6)% at concentration 80mg/l , while purified biosurfactat produced by B.B6 had low effect on the proliferation of normal cell line L02. It was found that it caused depolarization of mitochondria membrane which finally led to cell death. In addition, the caspase activity assay revealed that surfactin induced apoptosis in Hep-2 cells that associated with caspase 3. Optimum conditions for biosurfactants production from B. subtilis B6 were determined . Results indicated that maximum biosurfactant production from this isolate was achieved by using Sucrose (1%) v/v , Potassium nitrate (0.3%) and Potassium dihydrogen phosphate (1 g/l), at pH 8, 30 °C, 180 rpm of shaking for 72 hrs. The biosurfactant yield was 1.4 g/l of culture medium under the optimum conditions.