Optimization of Cultural Condition and Synergistic Effect of Lactose with Carboxymethyl Cellulose on Cellulase Production by Bacillus sp. Isolated from Fecal Matter of Elephant (Elephas maximus)


A cellulase producing bacterium (E3 strain) was isolated from fecal matter of elephant and identified as Bacillus sp. using 16S rDNA sequenced based molecular phylogenetic approach. While studying the effect of substrates like Carboxymethyl cellulose (CMC), avicel, starch, maltose, sucrose, glucose, fructose, galactose and lactose on cellulase production, it was found that CMC was best carbon source induced cellulase production followed by lactose in this bacterial strain. A positive synergistic effect of lactose with CMC was also observed with enhancement of 5 - 6 times in cellulase production. The optimum cellulase production was recorded with 1% CMC and 1% lactose when added individually in the Omeliansky’s medium. The results showed that addition lactose with CMC greatly enhances the production and activity of various cellulase enzymes. The optimal fermentation conditions for the biosynthesis of cellulase by this strain were found to be temperature: 37, pH 7.0. The nitrogen source NH4Cl at 0.15% was optimum for cellulase production by this bacterium.

Share and Cite:

S. Sadhu, P. Ghosh, T. De and T. Maiti, "Optimization of Cultural Condition and Synergistic Effect of Lactose with Carboxymethyl Cellulose on Cellulase Production by Bacillus sp. Isolated from Fecal Matter of Elephant (Elephas maximus)," Advances in Microbiology, Vol. 3 No. 3, 2013, pp. 280-288. doi: 10.4236/aim.2013.33040.

Conflicts of Interest

The authors declare no conflicts of interest.


[1] P. Beguin and J. P. Aubert, “The Biological Degradation of Cellulose,” FEMS Microbiology Reviews, Vol. 13, No. 1, 1994, pp. 25-58. doi:10.1111/j.1574-6976.1994.tb00033.x
[2] M. P. Coughlan, “Cellulose Degradation by Fungi, in Microbial Enzymes and Biotechnology,” In: W. Fogarty and C. Kely, Eds., Microbial Enzymes and Biotechnology, Elsevier, London, 1990, pp. 1-36. doi:10.1007/978-94-009-0765-2_1
[3] A. Cavaco-Paulo, “Mechanism of Cellulose Action in Textile Processes,” Carbohydrate Polymers, Vol. 37, No. 3, 1998, pp. 273-277. doi:10.1016/S0144-8617(98)00070-8
[4] E. P. S. Bon and A. F. Maria, “Bioethanol Production via Enzymatic Hydrolysis of Cellulosic Biomass,” FAO Seminar on the Role of Agricultural Biotechnologies for Production of Bioenergy in Developing Countries, Rome, 12 October 2007, pp. 1-11. http://www.fao.org/biotech/seminaroct 2007.htm
[5] Y. H. P. Zhang, M. E. Himmel and J. R. Mielenz, “Outlook of cellulase Improvement: Screening and Selection Strategies,” Biotechnology Advances, Vol. 24, No. 5, 2006, pp. 452-481. doi:10.1016/j.biotechadv.2006.03.003
[6] R. B. Costa, M. V. A. Silva, F. C. Freitas, V. S. F. Leitao, P. S. B. Lacerda, M. A. Ferrara and E. P. S. Bon, “Mercado e Perspectivas de Uso de Enzimas Industriais e Especiais no Brasil,” In: E. P. S. Bon, M. A. Ferrara, M. L. Corvo, A. B. Vermelho, C. L. A. Paiva, R. B. Alencastro and R. R. R. Coelho, Eds., Enzimas em Biotecnologia, Producao, Aplicac oes e Mercados, Interciencia, Rio de Janeiro, 2008, pp. 463-488.
[7] N. Moreira, “Growing Expectations: New Technology Could Turn Fuel into a Bump Crop,” Science News Online, Vol. 168, No.14, 2005, pp. 209-224.
[8] M. Knauf and M. Moniruzzaman, “Lignocellulosic Biomass Processing: A Perspective,” International Sugar Journal, Vol. 106, No. 1263, 2004, pp. 147-150.
[9] K. E. Erikson and B. Patterson, “Extra Cellular Enzyme System Produced by the Fungus Sporotrichum pulverulentum,” Biotechnology and Bioengineering, Vol. 20, No. 3, 1975, pp. 317-332.
[10] R. Mullings, “Measurement of Sacharification by Cellulases,” Enzyme and Microbial Technology, Vol. 7, No. 12, 1985, pp. 586-591. doi:10.1016/0141-0229(85)90025-0
[11] Y. H. P. Zhang and L. R. Lynd, “Toward an Aggregated Understanding of Enzymatic Hydrolysis of Cellulose Systems,” Biotechnology and Bioengineering, Vol. 88, No. 7, 2004, pp. 797-824. doi:10.1002/bit.20282
[12] C. S. Stewart, H. J. Flint and M. P. Bryant, “The Rumen Bacteria,” In: P. N. Hob-son and C. S. Stewart, Eds., The Rumen Microbial Ecosystem, Blackie Academic and Professional Publishers, London, 1997, pp. 10-72.
[13] V. Omeliansky, “Ueber die Garung der Cellulose,” Proceedings of Indiana Academy of Science, Vol. 8, 1902, pp. 225-231.
[14] 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, 1982, pp. 777-780.
[15] J. Sambrook and D. W. Russell, “Molecular Cloning: A Laboratory Manual,” 3rd Edition, Cold Spring Harbor Laboratory Press, New York, 2001.
[16] 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, 1980, pp. 111-120. doi:10.1007/BF01731581
[17] J. Felsenstein, “Confidence Limits on Phylogenies: An Approach Using the Bootstrap,” Evolution, Vol. 39, No. 4, 1985, pp. 783-791. doi:10.2307/2408678
[18] T. K. Ghosh, “Measurement of Cellulase Activities,” Pure and Applied Chemistry, Vol. 59, No. 2, 1987, pp. 257-268. doi:10.1351/pac198759020257
[19] G. L. Miller, “Use of Dinitrosalicylic Acid Reagent for Determination of Reducing Sugars,” Journal of Analytical Chemistry, Vol. 31, No. 3, 1959, pp. 426-428. doi:10.1021/ac60147a030
[20] O. H. Lowry, N. J. cRosebrough, A. L. Farr and R. J. Randall, “Protein Measurement with the Folin-Phenol Reagent,” The Journal of Biological Chemistry, Vol. 193, No. 1, 1951, pp. 265-275.
[21] S. Sadhu, P. Saha, S. Mayilraj and T. K. Maiti, “Lactose-Enhanced Cellulase Production by Microbacterium sp. Isolated from Fecal Matter of Zebra (Equus zebra),” Current Microbiology, Vol. 62, No. 3, 2011, pp. 1050-1055. doi:10.1007/s00284-010-9816-x
[22] S. Sadhu, P. Saha, S. Mayilraj and T. K. Maiti, “Characterization of a Bosea sp. Strain SF5 (MTCC 10045) Isolated from Compost Soil Capable of Producing Cellulase,” The Journal of Microbiology, Biotechnology and Food Sciences, Vol. 2, No. 2, 2012, pp. 576-591.
[23] P. Chellapandi and M. J. Himanshu, “Production of endoglucanase by the native strains of Streptomyces isolates in submerged fermentation”, Brazilian Journal of Microbiology, Vol. 39, 2008, pp. 122-127. doi:10.1590/S1517-83822008000100026
[24] E. Stackebrandt and B Goebel, “Taxonomic Note: A Place for DNA-DNA Reassociation and 16S rRNA Sequence Analysis in the Present Species Definition in Bacteriology”, International Journal of Systematic Bacteriology, Vol. 44, No. 4, 1994, pp. 846-849. doi:10.1099/00207713-44-4-846
[25] J. Gallagher, A. Winters, N. Barron, L. McHale and A.P McHale, “Production of Cellulase and β-Glucosidase Activity during Growth of the Actinomycete Micromonospora chalcae on Cellulose-Containing Media,” Biotechnology Letters, Vol. 18, No. 5, 1996, pp. 537-540. doi:10.1007/BF00140199
[26] M. Tuncer, A. Kuru, M. Isikli, N. Sahin and F. G. Celenk, “Optimization of Extra-Cellular Endoxylanase, Endoglucanase and Peroxidase Production by Streptomyces sp. F2621 Isolated in 17 Turkey,” Journal of Applied Microbiology, Vol. 97, No. 4, 2004, pp. 783-791. doi:10.1111/j.1365-2672.2004.02361.x
[27] B. H. Lee and T. H. Blackburn, “Cellulase Production by a Themophilic Clostridium sp.,” Applied Microbiology, Vol. 30, No. 3, 1975, pp. 346-353.
[28] M. A. T. Amira, A. R. Shatha, S. A. A. Siham and A. N. Basima, “Cellulase Production from Actinomycetes Isolated from Iraqi Soils: II Cell Growth and Cellulase Activity of Streptomyces sp. Strain AT7 at Different Temperatures,” Journal of Islamic Academy of Sciences, Vol. 2, No. 3, 1989, pp. 185-188.
[29] A. S. S. Ibrahim and I. Ahmed, “Isolation and Identification of New Cellulases Producing Thermophilic Bacteria from an Egyptian Hot Spring and Some Properties of the Crude Enzyme,” Australian Journal of Basic and Applied Science, Vol. 1, No. 4, 2007, pp. 473-478.
[30] M. I. Rajoka, “Influence of Various Fermentation Variable on Exoglucanase Production in Cellulomonas flavigena,” Electronic Journal of Biotechnology, Vol. 7, No. 3, 2004, pp. 259-266. doi:10.2225/vol7-issue3-fulltext-2
[31] S. Acharya and A. Choudhury, “Effect of Nutritional and Environmental Factors on Cellulose Activity by Thermophillic Bacteria Isolated from Hot Spring,” Journal of Scientific and Industrial Research, Vol. 70, No. 2, 2011, pp. 142-148.
[32] N. A. Spiridonov and D. B. Wilson, “Regulation of Biosynthesis of Individual Cellulases in Thermomonospora fusca,” Journal of Bacteriology, Vol. 180, No. 4, 1998, pp. 3529-3532.
[33] S. Thirumale, R. D. Swaroopa and K. Nand, “Control of Cellulose Formation by Trehalose in Clostridium Papyrosolvens CFR-703,” Process Biochemistry, Vol. 37, No. 3, 2001, pp. 241-245. doi:10.1016/S0032-9592(01)00212-6
[34] M. Shiang, J. C. Liden, A. Mohagheghi, K. Grohmann and M. E. Himmel, “Regulation of Cellulose Synthesis in Acidothermus cellulolyticus,” Biotechnology Progress, Vol. 7, No. 4, 1991, pp. 315-322. doi:10.1021/bp00010a005
[35] L. M. Robson and G. H. Chambliss, “Characterization of the Cellulolytic Activity of a Bacillus Isolate,” Applied and Environmental Microbiology, Vol. 47, No. 5, 1984, pp. 1039-1046.
[36] L. Y. Liang, Z. Feng, J. Yesuf and J. W. Blackburn, “Optimization of Growth Medium and Enzyme Assay Conditions for Crude Cellulases Produced by a Novel Thermophilic and Cellulolytic Bacterium Anoxybacillus sp. 527.” Applied Biochemistry and Biotechnology, Vol. 160, No. 6, 2010, pp. 1841-1852. doi:10.1007/s12010-009-8677-x
[37] B. Seiboth, G. Hofmann and C. P. Kubicek, “Lactose Metabolism and Cellulase Production in Hypocrea jecorina: The Gal 7 Gene, Encoding Galactose-1-phosphate Uridylyltransferase, Is Essential for Growth on Galactose but Not for Cellulase Induction,” Molecular Genetics and Genomics, Vol. 267, No. 1, 2002, pp. 124-132. doi:10.1007/s00438-002-0654-9
[38] M. Ilman, A. Saloheimo, M. L. Onnela and M. E. Penttila, “Regulation of Cellulase Gene Expression in the Filamentous Fungus Trichoderma reesei,” Applied and Environmental Microbiology, Vol. 63, No. 4, 1997, pp. 1296-1306.
[39] L. Karaffa, E. Fekete, C. Gamauf, A. Szentirmai, C. P. Kubicek and B. Seiboth, “Dgalactose Induces Cellulase Gene Expression in Hypocrea jecorina at Low Growth Rates,” Microbiology, Vol. 152, No. 5, 2006, pp. 1507-1514. doi:10.1099/mic.0.28719-0
[40] M. Schmoll and C.P. Kubicek, “Regulation of Trichoderma Cellulose Formation: Lessons in Molecular Biology from an Industrial Fungus,” Acta Microbiologica et Immunologica Hungarica, Vol. 50, No. 2, 2003, pp. 125-145. doi:10.1556/AMicr.50.2003.2-3.3

Copyright © 2023 by authors and Scientific Research Publishing Inc.

Creative Commons License

This work and the related PDF file are licensed under a Creative Commons Attribution 4.0 International License.