Philippine Science Letters
vol. 5 | no. 2 | 2012
published online December 19, 2012


Isolation of thermophilic bacteria (Bacillus AND Ureibacillus ) and amplification of genes for selected enzymes

by Cynthia T. Hedreyda*, John Jewish A. Dominguez, and Karen Rosal

National Institute of Molecular Biology and Biotechnology, College of Science, University of the Philippines, Diliman, Quezon City, Philippines 1101



Because catalyses often employ or produce high temperature environment, isolation of bacterial thermophiles that produce thermostable enzymes is important in industry. This study was focused on the isolation of bacteria that can grow at 55°C and higher, by using hot spring, mud spring, and solid oil sludge samples as sources of inocula added to Luria Bertini broth that were incubated at 55 to 70ēC with shaking (25 rpm). One isolate from the hot spring sample that grew at 60°C and another isolate from oil sludge sample that grew at 55ēC, were identified to belong to Bacillus licheniformis. An isolate from mud spring that grew at 55°C was identified to belong to Bacillus subtilis. Pure culture of two isolates from the oil sludge sample that could survive up to 60ēC exhibited 99% 16s rRNA gene sequence similarity with two relatively new species, Ureibacillus suwonensis and Ureibacillus thermosphaericus. The apr for alkaline protease was detected from the mud spring B. licheniformis while a fragment of a gene with 98% sequence similarity with the gene for Bacillopetidase F was amplified from the B. licheniformis isolated from oil sludge. PCR using gene-targeted primers and template DNA of the Bacillus subtilis isolate from mudspring, resulted in the amplification of expected size amplicons for a neutral protease, ß-glycosidase, α-amylase and xylanase genes. Amplification of target gene fragments was confirmed by gene sequence analysis. Target amplicons were not generated from the Ureibacillus suwonensis and Ureibacillus thermosphaericus isolates, suggesting the need for further studies using new or degenerate primers to amplify genes for enzymes with industrial applications. These relatively two new bacterial species that are not yet well studied, particularly at the molecular level, could be potential sources of important enzymes that allow the bacteria to survive and grow in extreme environments such as the oil sludge.

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Submitted: August 24, 2012
Revised: November 5, 2012
Accepted: November 5, 2012
Published: December 19, 2012
Editor-in-charge: Eduardo A. Padlan
Reviewer: Esperanza C. Cabrera