Identification of Cellulose-Hydrolytic Thermophiles Isolated from Sg. Klah Hot Spring Based On 16S rDNA Gene Sequence

Authors: M. J. Norashirene, Y. Zakiah, S. Nurdiana, I. Nur Hilwani, M. H. Siti Khairiyah, M. J. Muhamad Arif

Abstract:

In this study, six bacterial isolates of a slightly thermophilic organism from the Sg. Klah hot spring, Malaysia were successfully isolated and designated as M7T55D1, M7T55D2, M7T55D3, M7T53D1, M7T53D2 and M7T53D3 respectively. The bacterial isolates were screened for their cellulose hydrolytic ability on Carboxymethlycellulose agar medium. The isolated bacterial strains were identified morphologically, biochemically and molecularly with the aid of 16S rDNA sequencing. All of the bacteria showed their optimum growth at a slightly alkaline pH of 7.5 with a temperature of 55°C. All strains were Gram-negative, non-spore forming type, strictly aerobic, catalase-positive and oxidase-positive with the ability to produce thermostable cellulase. Based on BLASTn results, bacterial isolates of M7T55D2 and M7T53D1 gave the highest homology (97%) with similarity to Tepidimonas ignava while isolates M7T55D1, M7T55D3, M7T53D2 and M7T53D3 showed their closest homology (97%-98%) with Tepidimonas thermarum. These cellulolytic thermophiles might have a commercial potential to produce valuable thermostable cellulase.

Keywords: Cellulase, Cellulolytic, Thermophiles, 16S rDNA Gene.

Digital Object Identifier (DOI): doi.org/10.5281/zenodo.1096313

References:

[1] H. Zuridah, N. Norazwin, M. Siti Aisyah, M. N. A. Fakhruzzaman and N. A. Zeenathul, "Identification of lipase producing thermophilic bacteria from Malaysian hot springs,” African Journal of Microbiology Research, vol. 5, pp. 3569-3573, 2011.
[2] Haki, G.D. and Rakshit, S.K. (2003). Development in industrially important thermostable enzyme: A review. Biosource Technology, 89: 17-34.
[3] D. Gaur, K. J. Pankaj, S. S. Yamini and B. Vivek, "Estimation of extracellular lipolytic enzyme activity by thermophillic Bacillus sp. isolated from Arid and semi-Arid region of Rajasthan, India,” Journal of Microbiology Biotechnology and Food Sciences, vol. 2 (2), pp. 619-633, 2012.
[4] A. Somen and C. Anita (2012). "Bioprospecting thermophiles for cellulase production,” Brazilian Journal of Microbiology, vol. 43, pp. 164 – 187, 2002.
[5] V. Liisa, A. Marika, P. Terhi, V. Jari and S. Matti, "Thermostable enzymes in lignocellulose hydrolysis,” Journal of Advance Biochem Engineering/Biotechnology, vol 108, pp. 121–145, 2007.
[6] C. Vielle and G. J. Zeikus, "Hyperthermophilic enzymes: sources, uses and molecular mechanisms for thermostability,” Microbiology and Molecular Biology Reviews, vol. 65, pp. 1-43, 2001.
[7] M. Bhat, "Cellulases and related enzymes in biotechnology,” Biotechnol. Adv., vol 18, pp. 355–383, 2000.
[8] R. D., Firn, "Bioprospecting-Why is it so unrewarding?,” Biodiversity and Conservation, vol. 12, pp. 207-216, 2003.
[9] X. Li, H. Yang, B. Roy, D. Wang, W. Yue, L. Jiang, E. Park and Y. Miao, "The most stirring technology in future: Cellulase enzyme and biomass utilization,” African Journal of Technology, vol. 8 (11), pp. 2418-2422, 2009.
[10] S. S. I., Abdelnasser and I. E., Ahmed, "Isolation and identification of new cellulase producing thermophilic bacteria from an Egyptian hot springs and some properties of the crude enzyme,” Australian Journal of Basic and Applied Sciences, vol. 1(4), pp. 473-478, 2007.
[11] H. Yuichi, Y. Hiroe, O. Moriya and K. Toshiaki, ”Evaluation of primers and PCR condition for the analysis of 16S rDNA genes from a natural environment,” FEMS Microbiology Letters, vol. 221, pp. 299-304, 2003.
[12] L. Albuquerque, I. Tiago, A. Verissimo and M. S. Costa, "Tepidimonas thermarum sp. nov., a new slightly thermophilic betaproteobacterium isolated from the Elisenquelle in Aachen and emended description of the genus Tepidimonas,” Systematic and Applied Microbiology, vol. 29, pp. 450 - 456, 2005.
[13] M. Freitas, F. R. Rainey, M. F. Nobre, A. J. D. Silvestre and M. S. da Costa, "Tepidimonas aquatica sp. nov., a new slightly thermophilic b- Proteobacterium isolated from a hot water tank,” System, Issue Series Title: Appl. Microbiol., vol. 26, pp. 376–381, 2003.
[14] T. L. Chen, Y. J. Chou, W. M. Chen, B. Arun, C. C. Young, "Tepidimonas taiwanensis sp. nov., a novel alkalineprotease- producing bacterium isolated from a hot spring,” Application Environment Microbiology, vol. 10, pp. 35 – 40, 2005.
[15] C. Moreira, F. A. Rainey, M. F. Nobre, M. T. Silva, M. S. da Costa, "Tepidimonas ignava gen. nov., sp. nov., a new chemolithoheterotrophic and slightly thermophilic member of the b-Proteobacteria,” Int. J. Syst. Bacteriol. vol. 50, pp. 735–742, 2000.