Identification, Growth Kinetics And Cell Disintegration Of Mru5, A Bacterium Isolated From An Oil Well In Sarawak, Malaysia

• Main Author: Lee, Sook Fong
• Format: Thesis
• Language: English; English
• Published: 2007
• Online Access: http://psasir.upm.edu.my/id/eprint/4890/1/FBSB_2007_11.pdf; http://psasir.upm.edu.my/id/eprint/4890/

A hyperthermophilic bacterium, coded MRU5 was isolated from an oil-producing well in Sarawak, Malaysia. This microorganism was found to be a strictly anaerobic sulphate-reducing bacteria. Since this bacteria can survive and carry out its functions at extremely harsh conditions (high pressure, high temperature, high salinity), it is postulated that the enzymes isolated would be highly suitable for use in industrial processes, which need enzymes to be robust and stable. In this study, the cell disruption methods of the bacterium were optimized and the activity of possible industrial thermostable extracellular enzymes such as xylanases, lipases and amylolytic enzymes were determined. The bacterium, has an irregular coccoid shape, is Gram negative, and is able to grow at 90oC, pH 7.0 and high salinity (10% NaCl). It is resistant to antibiotic such as ampicillin, chloramphenicol, kanamycin, rifampin, streptomycin, tetracycline and geneticin. Based on this profile, MRU5 is suggested to be a new species under the halothermophile Archaea family. 16S rRNA sequencing failed to identify the taxanomy because of limitation of universal PCR primers. Amylase, xylanase and lipase had been isolated extracellularly with the activity of 11.8 U/L, 82.1 U/L and 7.61 U/L, respectively. Amylase was chosen for cell breakage studies as an indicator. Different methods of cell breakage were applied to obtain a high intracellular enzyme activity. The methods used were bead miller, lysozyme and combinations of bead mill with ultrasonicator and high pressure homogenizer. The breakages of cells were observed under scanning electron microscope. Amylase activity was detected intracellularly as well as on the membrane. Cell disruption treatment with only bead mill with 6g of glass beads recorded the highest specific enzyme activity (1.81 U/mg) at hour 3 where it is almost double compared to combination of bead mill and ultrasonication (0.983 U/mg) at similar time.