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Screening and Molecular Identification of Hypercellulase and Xylanase-Producing Microorganisms for Bioethanol Production


Affiliations
1 Microbiology Laboratory, Department of Basic Sciences, Dr Y. S. Parmar University of Horticulture and Forestry, Nauni–Solan 173 230, India
 

The present study projects the baseline work for bioconversion of pine needles to second generation biofuel, which deals mainly with screening, molecular identification and optimization of process parameters for cellulase and xylanase production. In total, 89 hydrolytic enzymes producing isolates were isolated from the soils and ten potential enzyme producers (seven for cellulase and three for xylanase) were subjected to secondary screening by inducing physical and chemical mutation. The wild and mutant strains of hypercellulase producers N12 and Kd1 were identified as Bacillus stratosphericus N12 and Bacillus altitudinis Kd1 using 16S rRNA technique. The fungal isolates RF1 and F2 were identified on the basis of 5.8 rRNA ITS technique and identified as Rhizopus oryzae, RF1 and Rhizopus delemar, F2 respectively. The mutant strains B. stratosphericus N12 (M) and B. altitudinis Kd1 (M) are highly stable till 10 generations. Cellulase activity increased from 3.230 to 5.983 IU, i.e. 85.23% increase in cellulase activity was achieved. Xylanase production increased from 51.32 to 95.25 IU with 85.60% increase in production. Solid-state fermentation was also performed by potential fungal strains, i.e. R. delemar F2 and R. oryzae RF1 using pine needles as the substrate.

Keywords

Bioethanol, Cellulase, Solid-state Fermentation, Submerged Fermentation, Xylanase.
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  • Keshwani, D. R., Modeling changes in biomass composition during microwave based alkali preteatment of switchgrass. Biotechnol. Bioeng., 2010, 105, 88–97.
  • Lynd, L. R., Weimer, P. J., van Zyl, W. H. and Pretorius, I. S., Microbial cellulose utilization: fundamentals and biotechnology. Microbiol. Mol. Biol. Rev., 2002, 66, 506–577.
  • Alam, M. Z., Manchur, M. A. and Anwar, M. N., Isolation, purification, characterization of cellulolytic enzymes produced by the isolate Streptomyces omiyaensis. Pak. J. Biol. Sci., 2004, 7, 1647– 1653.
  • Sharma, V. and Singh, P. K., Strain improvement of Bacillus coagulans and Geobacillus stearothermophilus for enhanced thermostable cellulase production and the effect of different metal ions on cellulase activity. Int. J. Eng. Sci. Technol., 2012, 4, 4704– 4709.
  • Immanuel, G., Dhanusha, R., Prema, P. and Palavesam, A., Effect of different growth parameters on endoglucanase enzyme activity by bacteria isolated from coir retting effluents of estuarine environment. Int. J. Environ. Sci. Technol., 2006, 3, 25–34.
  • Sharma, N. and Sharma, N., Cellulase and xylanase productions from cellulolytic and xylanolytic microorganisms isolated from soils of Northern Himalayas. World J. Pharm. Res., 2016, 5, 754– 764.
  • Kim, J., Hur, S. and Hong, J., Purification and characterization of alkaline cellulase from a newly isolated alkalophilic Bacillus sp. HSH-810. Biotechnol. Lett., 2005, 27, 313–316.
  • Maniatis, T., Fritsch, E. F. and Sambrock, J., Molecular Cloning: A Laboratory Manual, Cold Spring Harbour Laboratory, New York, USA, 1982.
  • Sharma, N., Bansal, K. L. and Neopaney, B., Comparison of forest waste degradation under solid state fermentation and submerged fermentation by co-culture of Bacillus coagulans and Bacillus licheniformis. Asian J. Microbiol. Biotechnol. Environ. Sci., 2005, 7, 51–54.
  • Okoshi, H., Ozaki, K., Shikate, S., Oshoni Kawai, S. and Ito, S., Purification and characterization of multiple carboxymethyl cellulases from Bacillus sp. KSM-522. Agric. Biol. Chem., 1990, 54, 83–89.
  • Li, X. and Gao, P., CMC-liquefying enzyme, a low molecular mass initial cellulose-decomposing cellulase responsible for fragmentation from Streptomyces sp. LX. Appl. Microbiol., 1997, 83, 59–66.
  • Vyas, A., Vyas, D. and Vyas, K. M., Production and optimization of cellulases on pretreated groundnut shell by Aspergillus terreus AV49. J. Sci. Ind. Res., 2005, 64, 281–286.
  • Annamalai, N., Thavasi, R., Jayalakshmi, S. and Balasubramanium, T., Thermostable and alkaline tolerant xylanase production by Bacillus subtilis isolated from marine environment. Indian J. Biotechnol., 2009, 8, 291–297.
  • Garg, S., Ali, R. and Kumar, A., Production of alkaline xylanase by an alkalo-thermophilic bacteria, Bacillus halodurans, MTCC9512 isolated from dung. Curr. Trends Biotechnol. Pharm., 2009, 3, 90–96.
  • Heck, J. X., Hertz, P. F. and Ayub, M. A. Z., Cellulase and xylanase production by isolated amazon Bacillus strains using soyabean industrial residue based solid state cultivation. Braz. J. Microbiol., 2002, 33, 213–218.
  • Bollag, D. M. and Edelstein, S. J., Protein Methods, Wiley Liss, John Wiley, New York, USA, 1993.
  • Chavan, A., Chougale, D., Lakshmikantha, R. Y. and Satwadi, S. P. R, Mutational study of Bacillus species for production, purification and characterization of lipase. Int. J. Pharm. Chem. Biol. Sci., 2012, 2, 545–551.
  • Ghazi, S., Sepahy, A.A., Azin, M., Khaje, K. and Khavarinejad, R., UV mutagenesis for the overproduction of xylanase from Bacillus mojavensis PTCC1723 and optimization of the production condition. Iran. J. Basic Med. Sci., 2014, 17, 844–853.
  • Kuttanpillai, S. K., Permaul, K. and Singh, S., Inducible character of β -xylanase in a hyperproducing mutant of Thermomyces lanuginosus. Eng. Life Sci., 2009, 9, 298–230.
  • Fontana, C. M., Favaro, M., Pelliccioni, E. S. and Favalli, P. C., Use of the Microseq 16S rRNA gene-based sequencing for identification of bacterial isolates that commercial automated systems failed to identify correctly. J. Clin. Microbiol., 2005, 43, 615–619.
  • Chen, J., Zhang, L., Zhan, P., Wang, Y., Ai, B and Wang, G., Optimization of simultaneous saccharification and co-fermentation process for ethanol production from poplar wood. In International Conference Agricultural and Biosystems Engineering, Hong Kong, 2011, vol. 1, pp. 291–294.
  • Willey, J. M., Sherwood, L. M. and Woolverton, C. J., Prescott, Harley and Kleins’ Microbiology, McGraw Hill, Boston, USA, 2008, 7th edn.
  • Hickey, D. A. and Singer, G. A., Genomic and proteomics adaptation to growth at high temperature. Genome Biol., 2004, 5, 1–7.
  • Kumar, D. J. M., Sudha, M., Devika, S., Balakumaran, M. D., Kumar, M. R. and Kalaichelvan, P. T., Production and optimization of β -glucosidase by Bacillus sp. MPTK 121, isolated from dairy plant soil. Ann. Biol. Res., 2012, 3, 1712–1718.
  • Singh, J. and Kaur, P., Optimization of process parameters for cellulase production from Bacillus sp. JS14 in solid substrate fermentation using response surface methodology. Braz. Arch. Biol. Technol., 2012, 55, 505–512.
  • Shanmugapriya, K., Saravana, P. S., Krishnapriya, Manoharan, M., Mythili, A. and Joseph, S., Isolation, screening and partial purification of cellulase from cellulase producing bacteria. Int. J. Adv. Biotechnol. Res., 2012, 3, 509–514.
  • Nirmla, P. and Sindhu, A., Production of endoglucanase by optimizing the environmental conditions of Bacillus circulans on submerged fermentation. Int. J. Appl. Eng. Res., 2011, 2, 472–481.
  • Shankar, T. and Isaiarasu, L., Cellulase production by Bacillus pumilus EWBCM1 under varying conditions. Middle East J. Sci. Res., 2011, 8, 40–45.
  • Seiboth, B., Gamauf, C., Pail, M., Hartl, L. and Kubicek, C. P., The D-xylose reductase of Hypocrea jecorina is the major aldose reductase in pentose and D-galactose catabolism and necessary for beta-galactosidase and cellulase induction by lactose. Mol. Microbiol., 2007, 66, 890–900.
  • Sadhu, S., Saha, P., Sen, S. K., Mayilraj, S. and Maiti, T. K., Production, purification and characterization of a novel thermotolerant endoglucanase (CMCase) from Bacillus strain isolated from cow dung. Springer Plus, 2013, 2, 1–10.
  • Gupta, U. and Kar, R., Optimization and scale up of cellulase free endo-xylanase production by solid state fermentation on corn cob and by immobilized cells of a thermolerant bacterial isolates. Jordan J. Biol. Sci., 2008, 1, 129–134.
  • Kapoor, M., Lavanya, M. N. and Kuhad, R. C., Cost effective xylanase production from free and immobilized Bacillus pumilus strain MK001 and its application in saccharification of Prosopis juliflora. Biochem. Eng. J., 2008, 38, 88–97.

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  • Screening and Molecular Identification of Hypercellulase and Xylanase-Producing Microorganisms for Bioethanol Production

Abstract Views: 208  |  PDF Views: 83

Authors

Nivedita Sharma
Microbiology Laboratory, Department of Basic Sciences, Dr Y. S. Parmar University of Horticulture and Forestry, Nauni–Solan 173 230, India
Nisha Sharma
Microbiology Laboratory, Department of Basic Sciences, Dr Y. S. Parmar University of Horticulture and Forestry, Nauni–Solan 173 230, India

Abstract


The present study projects the baseline work for bioconversion of pine needles to second generation biofuel, which deals mainly with screening, molecular identification and optimization of process parameters for cellulase and xylanase production. In total, 89 hydrolytic enzymes producing isolates were isolated from the soils and ten potential enzyme producers (seven for cellulase and three for xylanase) were subjected to secondary screening by inducing physical and chemical mutation. The wild and mutant strains of hypercellulase producers N12 and Kd1 were identified as Bacillus stratosphericus N12 and Bacillus altitudinis Kd1 using 16S rRNA technique. The fungal isolates RF1 and F2 were identified on the basis of 5.8 rRNA ITS technique and identified as Rhizopus oryzae, RF1 and Rhizopus delemar, F2 respectively. The mutant strains B. stratosphericus N12 (M) and B. altitudinis Kd1 (M) are highly stable till 10 generations. Cellulase activity increased from 3.230 to 5.983 IU, i.e. 85.23% increase in cellulase activity was achieved. Xylanase production increased from 51.32 to 95.25 IU with 85.60% increase in production. Solid-state fermentation was also performed by potential fungal strains, i.e. R. delemar F2 and R. oryzae RF1 using pine needles as the substrate.

Keywords


Bioethanol, Cellulase, Solid-state Fermentation, Submerged Fermentation, Xylanase.

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DOI: https://doi.org/10.18520/cs%2Fv120%2Fi5%2F841-849