Pemencilan dan pencirian bakteria dari telaga minyak tempatan untuk penghasilan asid dan pelarut organik

Abstract

A total of fourty five thermophilic bacteria were successfully isolated from wateroil samples of Malaysia petroleum reservoir using six different types of medium at temperature ranging from 50 to 100oC under strict anaerobic condition. Isolation of potential solvent and acid producer bacteria was performed using spread plate, rolling bottle and shake agar techniques. Sixteen bacteria with high stability were chosen and screened for their capability in producing organic acid, solvent, gas and biosurfactant. Bacteria B160 which isolated from Baram G85L reservoir was the best producer for acid and solvent. Characterization of bacteria B160 using 16S rDNA sequencing showed this strain has 99% similarity with Bacillus licheniformis. Production of organic acid and solvent production by Bacillus licheniformis B160 was conducted in static batch culture using modified Schott bottle. Medium supplemented with 30 g/L of sucrose produced total concentration of acids (acetic, butyric) and solvents (acetone, butanol, and ethanol) at 1.030 g/L and 2.290 g/L respectively. However, medium supplemented with similar concentration of glucose produced 1.3 fold higher acid and 1.2 times lower solvent without butanol. Starchy based materials can also be used as a substrate for acid and solvent production but it does not give a great impact for the enhancement of both products. Approximately only soluble starch produced acetic acid and acetone at concentration of 0.004 g/L and 2.140 g/L, respectively. Starchy materials such as potato, sago and tapioca did give significant effect towards solvent and acid productions. The effect of sucrose on acid and solvent was studies at the range of 10–100 g/L. Sucrose concentration more than 30 g/L favors for cells yield (Yx/s), rather than solvent formation (Yp/s). Yield of cells (Yx/s) at sucrose concentration higher than 30 g/L was comparatively 1-2 times higher than solvent formation per gram of substrate (Yp/s). At low concentration of sucrose (10-30g/L), Yp/s was 1.6 times higher than Yx/s. Sucrose concentration below than 50 g/L was more suitable for enhancement of solvent production. Acid and solvent production preferred organic nitrogen sources rather than inorganic and mixture of both nitrogen sources. Solvent production in medium supplemented with 0.3% (w/v) peptone was comparatively 2 times higher than meat extract and inorganic nitrogen. Solvent production in medium using mixture of nitrogen sources was 4-19 times lower than peptone (5.017 g/L). High C:N ratio was needed for enhancement of biomass and solvent production. The highest solvent production was observed at C:N ratio 44.55. When the C:N ratio increased beyond 44.55, total biomass increased but not for solvent production. Medium with initial pH 7 produced the highest concentration of total acid and solvent (7.504 g/L). At pH lower than 5.5 biomass productions were inhibited, thus decreased total acid and solvent production. Similar phenomena occurred when initial pH 8.0 was used.