Bioprospecting for Cellulase-Producing Bacteria from Victoria Falls Rainforest Decaying Logs

Makhosazana Nyathi^1, , Zephaniah Dhlamini¹ and Thembekile Ncube¹

¹Applied Biology and Biochemistry, National University of Science and Technology, Bulawayo, Zimbabwe

Cite this paper:
Makhosazana Nyathi, Zephaniah Dhlamini and Thembekile Ncube. Bioprospecting for Cellulase-Producing Bacteria from Victoria Falls Rainforest Decaying Logs. American Journal of Microbiological Research. 2023; 11(3):73-78. doi: 10.12691/ajmr-11-3-2

Abstract

Bioprospecting for cellulases seeks to find highly active enzymes that are stable, affordable, and can be readily incorporated into industrial processes. Research focus is shifting from fungal to bacterial cellulases due to the latter having higher growth rates, being easy to handle, and being more adaptable to genetic manipulations. In this research, bioprospecting for cellulolytic bacteria was carried out in decaying logs and soils sampled from the Victoria Falls rainforest. A compound sample of decaying logs and soils was inoculated on Mandel's media to isolate cellulase-producing bacteria. Morphological and biochemical analysis of the bacterial isolates was carried out to identify unique isolates showing cellulase production. Two Gram-negative and two Gram-positive isolates were selected and subsequently identified as Acinetobacter sp 9, Enterobacter sp, Bacillus thuringensis, and Bacillus cereus strain HYM88 by 16S rDNA sequencing. Submerged fermentation of the selected cellulase-producing bacteria was carried out for cellulase production. The cellulases were partially characterized to determine their optimum pH and temperature for activity. The optimum cellulase activity from all the bacterial isolates was at pH 5 and 50 °C. The diversity of the cellulases produced was determined. All the bacterial isolates proved to be true cellulolytic bacteria as they produced both exoglucanases and endoglucanases. All the bacterial isolates produced more exoglucanases than endoglucanases by 80 to 90%. The isolated cellulase-producing bacteria are a reflection of Victoria Falls rain forest's potential as an enzyme bioprospecting site which is yet to be tapped.

Keywords:
Victoria Falls rain forest decaying logs cellulases bioprospecting

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References:

[1]	Acharya, S. and Chaudhary, A. (2012).Bioprospecting thermophiles for cellulase production: a review. Brazilian Journal of Microbiology 3: 844-856.
[2]	Lee, RL., Weimer, P.J., Willem, H.and Pretorius, I.S.(2002). Microbial Cellulose Utilization: Fundamentals and Biotechnology. Microbiology and Molecular Biology Reviews 3:506-577.
[3]	Wyman, C.E., Dale, B.E., Elander, R.T., Hotzapple, M., Ladisch, M.R. and Lee, Y.Y. (2005). Coordinated development of leading biomass pre-treatment technologies. Bioresource Technology 96:1959-1966.
[4]	Li, X., Hua-jun, Y., Bhaskar, R., Dan, W., Wan-fu, Y., Li-jun, J., Enoch, Y. and Yun-gen, M. (2009). The most stirring technology in the future; cellulase enzyme and biomass utilization. African Journal of Biotechnology 8: 2418-2422.
[5]	Sukumaran, K.R., Singhania, R.R. and Pandey, A. (2005). Microbial cellulases- Production, applications, and challenges. Journal of Scientific and Industrial Research 64: 832-844.
[6]	Monserrate, E., Leschine, S.B. and Canale-Parola, E. (2001).Clostridium hungatei a mesophillic, N2-fixing cellulolytic bacterium isolated from soil. Evolution and Microbiology 51: 123-132.
[7]	Mrudula, S. and Murugammal, R. (2011). Production of cellulase by Aspergillus niger under submerged and solid-state fermentation using coir waste as a substrate. Brazilian Journal of Microbiology, 42: 1119-1127.
[8]	Sreedevi, S., Sreedharan, S. and Sailas, B. (2013). Cellulase-producing bacteria from the wood yards on Kallai River Bank. Advances in Microbiology 3: 326-332.
[9]	Maki, M., Kam, T. and Wensheng, Q. (2009). The prospects of cellulase-producing bacteria for the bioconversion of lignocellulosic biomass. International Journal of Biological Sciences 5: 500-510.
[10]	Kellner, H., Vandenbol, M. (2010). Fungi unearthed: transcripts encoding lignocellulolytic and chitinolytic enzymes in forest soil. Public Library of Science 6:109-115.
[11]	https://whc.unesco.org/en/list/509 [Accessed Aug. 20, 2021].
[12]	Acharya, P. B., Acharya, D.K. and Modi, H.A. (2008). Optimization for cellulase production by Aspergillus niger using sawdust as substrate. African Journal of Biotechnology 7: 4147-4152.
[13]	Kasana, R.C., Richa, S., Dhar, H., Dutt, S. and Gulati, A. (2008). A rapid and easy method for the detection of microbial cellulases on agar plates using Gram’s iodine. Current Microbiology 5:503-507.
[14]	Sambrook, J., Fritch, E.F. and Maniatis T. (1989) Molecular Cloning. A Laboratory Manual 2nd Ed. Cold Spring Harbor, New York.
[15]	Weisburg, W.G., Barns, S.M., Pelletier, D.A. and Lane, D.J. (1991) 16S Ribosomal DNA Amplification for Phylogenetic Study. Journal of Bacteriology 173(2): 697-703.
[16]	Liang, Y., Zhang, Z., Wu, M. and Feng, J. (2014). Isolation, Screening, and Identification of Cellulolytic Bacteria from Natural Reserves in the Subtropical Region of China and Optimization of Cellulase Production by Paenibacillus terrae ME27-1. BioMedical Research International. 5(7), 144-149.
[17]	Sethi, S., Aparna, D.B., Gupta, L. and Gupta, S. (2013). Optimization of Cellulase Production from Bacteria Isolated from Soil. International Scholarly Research Notices.
[18]	Miller, G.N. (1959). Use of dinitrosalicylic acid reagent for determination of reducing sugar. Analytical Chemistry 3: 426–428.
[19]	King, B.C., Donnelly, M.K., Bergstrom, G.C., Walker, L.P. and Gibson, D.M. (2009). An optimized micro-plate assay system for quantitative evaluation of plant cell wall degrading enzyme activity of fungal culture extracts. Biotechnology and Bio-engineering 102: 1033-1044.
[20]	Xiao, Z., Storms, R. and Tsang, A. (2004). Microplate-Based Filter Paper Assay to Measure Total Cellulase Activity. Biotechnology and Bioengeneering 7: 832-837.
[21]	Quiroz-Castaneda, R.E., Balcazar-Lopez, E., Dantan-Gonzalez, E., Martinez, A., Folch-Mallol, J. and Martinez Anaya, C. (2009).Characterization of cellulolytic activities of Bjerkandera adusta and Pycnoporus sanguineuson solid wheat straw medium. Electronic Journal of Biotechnology 12 (4): 1-8.
[22]	Goyari, S., Shantibala, S. D., Mohan, C. K. and Talukudar, N. C.. (2014). Population, diversity, and characteristics of cellulolytic microorganisms from the Indo-Burma Biodiversity hotspot.Springer Plus 3 (1): 700.
[23]	Behera, B., Parida, S., Dutta, S. and Thatoi, H. (2014). Isolation and identification of cellulose-degrading bacteria from mangrove soil of Mahanadi River Delta and their cellulase production ability. American Journal of Microbiological Research, 2: 41-46.
[24]	Jansen, P.H. (2006). Identifying the dominant soil bacterial taxa in libraries of 16S rRNA and 16S rRNA genes. Applied and Environmental Microbiology 3: 1719-1728.
[25]	Bai, S., Ravi, M., Mukesh, P., Kumar, D.J., Balashanmugam, P. and Bala, M. D. (2012). Cellulase Production by Bacillus subtilis isolated from Cow Dung. Archives of Applied Science Research 4: 269-279.
[26]	Sadhu, S., and Maiti, T. K. (2013). Cellulase Production by Bacteria: A Review British Microbiology Research Journal. 3:235-258.
[27]	Farinas, C., Loyo, M.M., Baraldo, A. and Couri, S. (2010). Finding stable cellulase and xylanase: Evaluation of the synergistic effect of pH and temperature. New Biotechnology. 27: 810-815.
[28]	Kumar, R., Sing, S., Singh, O.V. (2008). Bioconversion of lignocellulosic biomass: biochemical and molecular perspectives. Journal of Industrial Microbiology and Biotechnology 35: 377-391.