Cellulose hydrolyzing bacteria were isolated from rhinoceros dung and tested for clear zone formation around the colonies on the agar plates containing the medium amended with carboxymethylcellulose as a sole carbon source. Isolates were further screened on the basis of carboxymethylcellulase production in liquid medium. Out of 36 isolates, isolate no. 35 exhibited maximum enzyme activity of 0.079?U/mL and was selected for further identification by using conventional biochemical tests and phylogenetic analyses. This was a Gram-positive, spore forming bacterium with rod-shaped cells. The isolate was identified as Bacillus amyloliquefaciens SS35 based on nucleotide homology and phylogenetic analysis using 16S rDNA and gyrase A gene sequences. 1. Introduction Cellulose, a structural carbohydrate of the plant cell wall, is an abundant and ubiquitous polymer. The use of cellulose for the second generation biofuel production involves the hydrolysis of cellulosic biomass, that is, saccharification, to form simple sugar monomers for the fermentation into bioethanol [1–3]. Cellulases are the group of enzymes involved in the conversion of cellulosic substrates to fermentable sugars. Main members of this group include endoglucanase (EC 3.2.1.4), exoglucanase or cellobiohydrolase (EC 3.2.1.91), and -glucosidase (EC 3.2.1.21) [4]. The endoglucanase hydrolyzes -1,4 bonds in cellulose molecule, whereas exoglucanase cleaves the ends to release cellobiose, and -glucosidase converts cellobiose to glucose [5]. Several cellulase producing fungi such as Aspergillus, Rhizopus, and Trichoderma species [6, 7] and bacteria such as Bacillus, Clostridium, Cellulomonas, Thermomonospora, Ruminococcus, Bacteroides, Erwinia, and Acetivibrio species [8–10] have been identified. However, the isolation and characterization of novel cellulose hydrolyzing enzymes from bacteria are still a highly active research area, because bacteria have a higher growth rate than fungi, leading to greater production of enzymes [11]. Also, the habitat of bacteria covers different environmental niches, which favors the existence of versatile strains such as thermophiles [12], psychrophiles, alkaliphiles, and acidophiles. The culturable cellulase producing bacteria have been isolated from the variety of sources such as composting heaps, decaying agricultural wastes, the feces of cow [13] and elephant, gastrointestinal tract of buffalo and horse [14], soil, and extreme environments like hot-springs [15]. Cellulose degrading bacteria play an important role in energy supply for forage animals. Wahyudi et al.
References
[1]
Y. Sun and J. Cheng, “Hydrolysis of lignocellulosic materials for ethanol production: a review,” Bioresource Technology, vol. 83, no. 1, pp. 1–11, 2002.
[2]
C. Martín, Y. López, Y. Plasencia, and E. Hernández, “Characterisation of agricultural and agro-industrial residues as raw materials for ethanol production,” Chemical and Biochemical Engineering Quarterly, vol. 20, no. 4, pp. 443–447, 2006.
[3]
O. J. Sanchez and C. A. Cardona, “Trends in biotechnological production of fuel ethanol from different feedstocks,” Bioresource Technology, vol. 99, no. 13, pp. 5270–5295, 2008.
[4]
E. A. Bayer, R. Lamed, and M. E. Himmel, “The potential of cellulases and cellulosomes for cellulosic waste management,” Current Opinion in Biotechnology, vol. 18, no. 3, pp. 237–245, 2007.
[5]
M. K. Bhat and S. Bhat, “Cellulose degrading enzymes and their potential industrial application,” Biotechnology Advances, vol. 15, no. 3-4, pp. 583–620, 1997.
[6]
K. Murashima, T. Nishimura, Y. Nakamura et al., “Purification and characterization of new endo-1,4-β-D-glucanases from Rhizopus oryzae,” Enzyme and Microbial Technology, vol. 30, no. 3, pp. 319–326, 2002.
[7]
K. Saito, Y. Kawamura, and Y. Oda, “Role of the pectinolytic enzyme in the lactic acid fermentation of potato pulp by Rhizopus oryzae,” Journal of Industrial Microbiology and Biotechnology, vol. 30, no. 7, pp. 440–444, 2003.
[8]
L. M. Roboson and G. H. Chambliss, “Celluases of bacterial origin,” Enzyme and Microbial Technology, vol. 11, no. 10, pp. 626–644, 1989.
[9]
Y. J. Lee, B. K. Kim, B. H. Lee et al., “Purification and characterization of cellulase produced by Bacillus amyoliquefaciens DL-3 utilizing rice hull,” Bioresource Technology, vol. 99, no. 2, pp. 378–386, 2008.
[10]
B. K. Kim, B. H. Lee, Y. J. Lee, I. H. Jin, C. H. Chung, and J. W. Lee, “Purification and characterization of carboxymethylcellulase isolated from a marine bacterium, Bacillus subtilis subsp. subtilis A-53,” Enzyme and Microbial Technology, vol. 44, no. 6-7, pp. 411–416, 2009.
[11]
L. R. Lynd, P. J. Weimer, W. H. Van Zyl, and I. S. Pretorius, “Microbial cellulose utilization: Fundamentals and biotechnology,” Microbiology and Molecular Biology Reviews, vol. 66, no. 3, pp. 506–577, 2002.
[12]
M. Maki, K. T. Leung, and W. Qin, “The prospects of cellulase-producing bacteria for the bioconversion of lignocellulosic biomass,” International Journal of Biological Sciences, vol. 5, no. 5, pp. 500–516, 2009.
[13]
N. Akhtar, A. Sharma, D. Deka, M. Jawed, D. Goyal, and A. Goyal, “Characterization of cellulase producing Bacillus sp. for effective degradation of leaf litter biomass,” Environmental Progress and Sustainable Energy, 2012.
[14]
A. Wahyudi, M. N. Cahyanto, M. Soejono, and Z. Bachruddin, “Potency of lignocellulose degrading bacteria isolated from Buffalo and horse gastrointestinal tract and elephant dung for feed fiber degradation,” Journal of the Indonesian Tropical Animal and Agriculture, vol. 35, no. 1, pp. 34–41, 2010.
[15]
R. H. Doi, “Cellulases of mesophilic microorganisms: cellulosome and noncellulosome producers,” Annals of the New York Academy of Sciences, vol. 1125, pp. 267–279, 2008.
[16]
G. A. Varga and E. S. Kovler, “Microbial and animal limitation to fiber digestion and utilization,” Journal of Nutrition, vol. 127, no. 5, pp. 819–823, 1997.
[17]
D. O. Krause, S. E. Denman, R. I. Mackie et al., “Opportunities to improve fiber degradation in the rumen: microbiology, ecology, and genomics,” FEMS Microbiology Reviews, vol. 27, no. 5, pp. 663–693, 2003.
[18]
D. L. Bushnell and H. F. Haas, “The utilization of certain hydrocarbons by microorganisms,” Kansas Agricultural Experiment Station, vol. 199, pp. 653–673, 1941.
[19]
Y. C. Lo, G. D. Saratale, W. M. Chen, M. D. Bai, and J. S. Chang, “Isolation of cellulose-hydrolytic bacteria and applications of the cellulolytic enzymes for cellulosic biohydrogen production,” Enzyme and Microbial Technology, vol. 44, no. 6-7, pp. 417–425, 2009.
[20]
R. M. Atlas, Handbook of Microbiological Media, CRC Press, Boca Raton, Fla, USA, 3rd edition, 2004.
[21]
H. J. Ruijssenaars and S. Hartmans, “Plate screening methods for the detection of polysaccharase-producing microorganisms,” Applied Microbiology and Biotechnology, vol. 55, no. 2, pp. 143–149, 2001.
[22]
R. M. Teather and P. J. Wood, “Use of Congo red-polysaccharide interactions in enumeration and characterization of cellulolytic bacteria from the bovine rumen,” Applied and Environmental Microbiology, vol. 43, no. 4, pp. 777–780, 1982.
[23]
N. Nelson, “A photometric adaptation of the Somogyi method for the determination of glucose,” Journal of Biological Chemistry, vol. 153, pp. 375–380, 1944.
[24]
M. Somogyi, “A new reagent for the determination of sugars,” Journal of Biological Chemistry, vol. 160, pp. 61–68, 1945.
[25]
D. R. Boone, G. M. Garrity, R. W. Castenholz, D. J. Brenner, N. R. Krieg, and J. T. Staley, “Genus Bacillus,” in Bergey’s Manual of Systematic Bacteriology: The Firmicutes, vol. 3, pp. 21–128, Springer, New York, NY, USA, 2nd edition, 2001.
[26]
J. C. Cappuccino and N. Sherman, Microbiology—A Laboratory Manual, Pearson Education Publication, New Delhi, India, 7th edition, 2004.
[27]
N. Kannan, Laboratory Manual in General Microbiology, Panima Publishing Incarporation, New Delhi, India, 2002.
[28]
J. Chun and K. S. Bae, “Phylogenetic analysis of Bacillus subtilis and related taxa based on partial gyrA gene sequences,” Antonie van Leeuwenhoek, vol. 78, no. 2, pp. 123–127, 2000.
[29]
J. D. Thompson, D. G. Higgins, and T. J. Gibson, “CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice,” Nucleic Acids Research, vol. 22, no. 22, pp. 4673–4680, 1994.
[30]
N. Saitou and M. Nei, “The neighbor-joining method: a new method for reconstructing phylogenetic trees,” Molecular biology and evolution, vol. 4, no. 4, pp. 406–425, 1987.
[31]
M. Kimura, “A simple method for estimating evolutionary rates of base substitutions through comparative studies of nucleotide sequences,” Journal of Molecular Evolution, vol. 16, no. 2, pp. 111–120, 1980.
[32]
K. Tamura, J. Dudley, M. Nei, and S. Kumar, “MEGA4: Molecular Evolutionary Genetics Analysis (MEGA) software version 4.0,” Molecular Biology and Evolution, vol. 24, no. 8, pp. 1596–1599, 2007.
[33]
G. Immanuel, R. Dhanusha, P. Prema, and A. Palavesam, “Effect of different growth parameters on endoglucanase enzyme activity by bacteria isolated from coir retting effluents of estuarine environment,” International Journal of Environmental Science and Technology, vol. 3, no. 1, pp. 25–34, 2006.
[34]
Y. Liang, J. Yesuf, S. Schmitt, K. Bender, and J. Bozzola, “Study of cellulases from a newly isolated thermophilic and cellulolytic Brevibacillus sp. strain JXL,” Journal of Industrial Microbiology and Biotechnology, vol. 36, no. 7, pp. 961–970, 2009.
[35]
G. Rastogi, G. L. Muppidi, R. N. Gurram et al., “Isolation and characterization of cellulose-degrading bacteria from the deep subsurface of the Homestake gold mine, Lead, South Dakota, USA,” Journal of Industrial Microbiology and Biotechnology, vol. 36, no. 4, pp. 585–598, 2009.
[36]
S. K. Tai, H. P. P. Lin, J. Kuo, and J. K. Liu, “Isolation and characterization of a cellulolytic Geobacillus thermoleovorans T4 strain from sugar refinery wastewater,” Extremophiles, vol. 8, no. 5, pp. 345–349, 2004.
[37]
D. Deka, P. Bhargavi, A. Sharma, D. Goyal, M. Jawed, and A. Goyal, “Enhancement of cellulase activity from a new strain of Bacillus subtilis by medium optimisation and analysis with various cellulosic substrates,” Enzyme Research, vol. 2011, Article ID 151656, 8 pages, 2011.
[38]
H. Ariffin, N. Abdullah, M. S. U. Kalsom, Y. Shirai, and M. A. Hassan, “Production and characterization of cellulase by Bacillus pumilus EB3,” International Journal of Engineering and Technology, vol. 3, pp. 47–53, 2006.
[39]
G. E. Fox, J. D. Wisotzkey, and P. Jurtshuk, “How close is close: 16S rRNA sequence identity may not be sufficient to guarantee species identity,” International Journal of Systematic Bacteriology, vol. 42, no. 1, pp. 166–170, 1992.
[40]
B. J. Kim, S. H. Lee, M. A. Lyu et al., “Identification of mycobacterial species by comparative sequence analysis of the RNA polymerase gene (rpoB),” Journal of Clinical Microbiology, vol. 37, no. 6, pp. 1714–1720, 1999.
[41]
S. Yamamoto and S. Harayama, “Phylogenetic relationships of Pseudomonas putida strains deduced from the nucleotide sequences of gyrB, rpoD and 16S rRNA genes,” International Journal of Systematic Bacteriology, vol. 48, no. 3, pp. 813–819, 1998.
[42]
E. E. Idriss, O. Makarewicz, A. Farouk et al., “Extracellular phytase activity of Bacillus amyloliquefaciens FZB45 contributes to its plant-growth-promoting effect,” Microbiology, vol. 148, no. 7, pp. 2097–2109, 2002.