Production, purification and characterization of biosurfactant from Geobacillus thermoleovorans and studying its antimicrobial and antitumor activity

    The ten bacterial isolates used in this study have the ability to utilizecrude oil and aromatic compounds. They were isolated in the previousstudy and considered as a novel group of aromatic hydrocarbon degradingextreme thermophillic bacteria. These isolates were screened for theirability to produce biosurfactant depending on the emulsification index(E24%), surface tension measurement (mN/m) and emulsification activity(E.A) using crude oil as a carbon source. The results showed that allisolates were able to produce biosurfactant and Geobacillusthermoleovorans Ir1 (JQ912239) was the most efficient one.The optimum conditions for biosurfactant production by G.thermoleovorans Ir1 (JQ912239) were determined. The results indicatedthat these  conditions are growing this bacterium in mineral salt medium(pH7) containing 1% crude oil as a sole carbon source and 0.3%ammonium chloride as a nitrogen source, incubated in shaker incubatorat 60 ºC, with 200 rpm for 10 days. Biosurfactant was extracted using three methods. The results showed that the extraction with acetone gave maximum biosurfactant activity. The biosurfactant was purified using silica gel column chromatography,
three peaks were obtained and gave emulsification activity. The chemical composition of biosurfactant revealed that it consists of 37.7% lipids, 26.2% carbohydrate and 10.7% protein. The partial and /or purified biosurfactant of G. thermoleovorans Ir1 (JQ912239) was subjected to fourier transform infrared spectrophotometer (FTIR), High Performance Liquid Chromatography (HPLC), gas chromatography (GC)and Nuclear magnetic resonance (NMR) to complete the chemical characterization. The result of FTIR revealed that the biosurfactant contains lipid, carbohydrate and protein.While the HPLC results indicated that the fatty acid components of biosurfactant were palmitic acid, stearic acid and oleic acid, the carbohydrates were xylose, mannose and maltose. While the amino acids were aspartic acid, glutamic acid and glutamine.Gas chromatography analysis of the main part (lipid) of biosurfactant showed that it consists the high percentage of palmitic acid methyl ester (C16:0), Stearic acid (C18:0) and Oleic acid (C18:1n9C), and less percentage of other fatty acids.The 1H Nuclear magnetic resonance  spectrum showed that the partial purified biosurfactant consists of two compounds: the main was
triglycerides and the second may be attributed to fatty acid. The results of purified biosurfactant (the three peaks which obtained with silica gel column chromatography) revealed that all contain only triglycerides and this study may be the first one which is carried out in Iraq, which elucidates the ability of thermophilic bacteria to produce biosurfactant.In order to determine the antitumor activity of the purified biosurfactant, three cell lines (MCF7, Hut78 and Jurkat) were exposed to different concentrations of the biosurfactant. The results showed that the effect was dependent on the type of tumor cell line, biosurfactant concentration, and exposure time. The highest effect (most sensitive) was MCF7 cell line and 100 µg/ml caused 66.55 % inhibition of cell line growth after 72hrs. (incubation period).    MCF7 cell line was subjected to cytotoxicity study. The result showed that 50 and 100µg/ml of purified biosurfactant caused a significant decrease of cell count, with significant increase in cell permeability,releasing cytochrome C from mitochondria, nucleus intensity and a significant reduction in mitochondrial membrane potential. The antimicrobial activity of purified biosurfactant of G.thermoleovorans Ir1 (JQ912239) was applied against some microorganisms, the biosurfactant inhibited the growth of bacteria (Staphylococcus aureus, Streptococcus sp., Pseudomonas aeruginosa) and fungi (Candida albicans).Some physical and chemical properties of biosurfactant were studied. It was found that the biosurfactant was active in a wide range of pH values,thermostable at a high temperatures and stable in a wide range of salt concentrations of NaCl, CaCl  and KCl. In an attempt to detect the gene(s) responsible for biosurfactant produced by G. thermoleovorans, genomic DNA of this bacterium and B.subtilis was extracted and amplified using specific primer for sfp gene (coded for biosurfactant of Bacillus spp.). The results showed that the amplification product (675bp) was obtained with B. subtilis but not with G. thermoleovorans Ir1, that means this gene is not responsible for biosurfactant production in G. thermoleovorans Ir1.