Isolation, Identification and Characterization of Cellulolytic Bacteria from Agricultural Waste Residues: Unlocking Microbial Solutions for Organic Waste Utilization

H. M. Rakib Hossain; Anti Islam; Tauhidur Rahman Nurunnabi; S. M. Mahbubur Rahman; Md Emdadul Islam; Kazi Mohammed Didarul Islam; Md. Morsaline Billah

doi:10.53808/KUS.2026.23.01.1441-ls

Authors

H. M. Rakib Hossain Biotechnology and Genetic Engineering Discipline, Khulna University, Khulna-9208, Bangladesh
Anti Islam Institute for Integrated Studies on the Sundarbans and Coastal Ecosystems (IISSCE), Khulna University, Khulna-9208, Bangladesh
Tauhidur Rahman Nurunnabi Biotechnology and Genetic Engineering Discipline, Khulna University, Khulna-9208, Bangladesh
S. M. Mahbubur Rahman Biotechnology and Genetic Engineering Discipline, Khulna University, Khulna-9208, Bangladesh
Md Emdadul Islam Biotechnology and Genetic Engineering Discipline, Khulna University, Khulna-9208, Bangladesh
Kazi Mohammed Didarul Islam Biotechnology and Genetic Engineering Discipline, Khulna University, Khulna-9208, Bangladesh
Md. Morsaline Billah Biotechnology and Genetic Engineering Discipline, Khulna University, Khulna-9208, Bangladesh

DOI:

https://doi.org/10.53808/KUS.2026.23.01.1441-ls

Keywords:

Cellulase, Agricultural Residues, Cellulose-Degrading Bacteria, Molecular Analysis, 16S rRNA, Phylogenetic Tree

Abstract

Cellulases, the enzymes that catalyze the breakdown of cellulose, have emerged as critical players in the quest for sustainable energy solutions and the bioconversion of lignocellulosic biomass into valuable products. This study investigates the potential of novel, cellulase-producing bacteria isolated from soils amended with agricultural residues with the goal of improving biorefinery efficiency and reducing biofuel production costs. Through the selective screening of cellulose-degrading bacteria on carboxymethyl cellulose (CMC) agar, we identified a range of facultative anaerobic, Gram-negative, motile rod-shaped isolates. Morphological, cultural, and biochemical analyses suggested affiliations with the genera Enterobacter and Pseudomonas. We further elucidated the taxonomic identities of these isolates by amplifying and sequencing the 16S ribosomal ribonucleic acid (16S rRNA gene) using universal primers (27F and 1492R). The resulting 1465 bp nucleotide sequences were subjected to a comprehensive comparison with the NCBI nucleotide database, leading to the identification of Enterobacter cancerogenus, Pseudomonas aeruginosa, and Enterobacter cloacae. Phylogenetic analysis via the maximum likelihood method Phylogenetic analysis was conducted using the Maximum Likelihood method based on the Tamura-Nei model in MEGA 7.0 confirmed the evolutionary relationships among the isolates. Our findings demonstrate the cellulose-degrading potential of these strains based on qualitative screening assays. However, quantitative cellulase activity assays are required to fully evaluate their industrial applicability and enzyme production capacity.

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References

Ahmad, B., Nigar, S., Shah, S. S., Bashir, S., Ali, J., Yousaf, S., et al. (2013). Isolation and Identification of Cellulose Degrading Bacteria from Municipal Waste and Their Screening for Potential Antimicrobial Activity. World Applied Sciences Journal, 27 (11), 1420-1426. https://doi.org/10.5829/idosi.wasj.2013.27.11.81162

Bai, Y., D’Aoust, F., Smith, D. L., & Driscoll, B. T. (2012). Isolation of plant-growth-promoting Bacillus strains from soybean root nodules. Canadian Journal of Microbiology, 58(8), 933–941. https://doi.org/10.1139/w2012-065

Bautista-Cruz, A., Aquino-Bolaños, T., Hernández-Canseco, J., & Quiñones-Aguilar, E. E. (2024). Cellulolytic Aerobic Bacteria Isolated from Agricultural and Forest Soils: An Overview. Biology, 13(2), 102. https://doi.org/10.339 0/biology13020102

Bahatkar, B. P., Gahukar, S. J., Akhare, A. A., Ingle, Y. V., Rathod, D. R., & Charpe, A. M. (2023). Decomposition of Agriculture Farm Wastes by Cellulolytic Bacteria. International Journal of Environment and Climate Change, 13(10), 411–421. https://doi.org/10.9734/IJECC/2023/v13i102658

Basera, P., Chakraborty, S., & Sharma, N. (2024). Lignocellulosic biomass: insights into enzymatic hydrolysis, influential factors, and economic viability. Discover Sustainability, 5, 311. https://doi.org/10.1007/s43621-024-00543-5

Benatti, A. L. T., & Polizeli, M. L. T. M. (2023). Lignocellulolytic Biocatalysts: The Main Players Involved in Multiple Biotechnological Processes for Biomass Valorization. Microorganisms, 11(1), 162. https://doi.org/10.33 90/microorganisms11010162

Bhatia, T., Bose, D., Sharma, D., & Patel, D. (2024). A Review on Cellulose Degrading Microbes and Its Applications. Industrial Biotechnology, 20(1). https://doi.org/10.1089/ind.2023.0025

Dobrzyński, J., Wróbel, B., & Górska, E. B. (2023). Taxonomy, Ecology, and Cellulolytic Properties of the Genus Bacillus and Related Genera. Agriculture, 13(10), 1979. https://doi.org/10.3390/agriculture13101979

Gaur, R., & Tiwari, S. (2015). Isolation, production, purification and characterization of an organic-solvent-thermostable alkalophilic cellulase from Bacillus vallismortis RG-07. Gaur and Tiwari BMC Biotechnology, 15 (19). https://doi.org/10.1186/s12896-015-0129-9

Gocheva, Y., Dimitrova, L., Hubenov, V., Kabaivanova, L., et al. (2023). Cellulolytic Microorganisms: Aerobic, Microaerophilic, Anaerobic Bacteria and Microbial Consortia (Part II). Ecological Engineering and Environment Protection, 1, 36–53. https://doi.org/10.32006/eeep.2023.1.3653

Gupta, P., Samant, K., & Sahu, A. (2012). Isolation of Cellulose-Degrading Bacteria and Determination of Their Cellulolytic Potential. International Journal of Microbiology, 1-5. https://doi.org/10.1155/2012/578925

Hatami, Siavash & Alikhani, Hossein & Besharati, Hosin & Salehrastin, N. & Afrousheh, Maryam & Jahromi, Z. (2008). Investigation on Aerobic Cellulolytic Bacteria in Some of North Forest and Farming Soils. American-Eurasian J. Agric. & Environ. Sci., 3 (5), 713-716. https://www.researchgate.net/publication/242146405

http://www.ncbi.nlm.nih.gov/blast/

Huang, J., Wang, J., & Liu, S. (2023). Advances in the Production of Fungi-Derived Lignocellulolytic Enzymes Using Agricultural Wastes. Mycology, 15, 1–15. https://doi.org/10.1080/21501203.2023.2253827

Ilić, N., Milić, M., Beluhan, S., & Dimitrijević-Branković, S. (2023). Cellulases: From Lignocellulosic Biomass to Improved Production. Energies, 16(8), 3598. https://doi.org/10.3390/en16083598

Immanuel, G., Dhanusha, R., Prema, P., & Palavesam, A. (2006). 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, 3(1), 25–34. https://doi.org/10.1007/BF03325904

Irfan, M., Safdar, A., Syed, Q., & Nadeem, M. (2012). Isolation and screening of cellulolytic bacteria from soil and optimization of cellulase production and activity. Turkish Journal of Biochemistry–Turk J Biochem, 37 (3), 287–293. http://doi.org/10.5505/tjb.2012.09709

James G. Cappuccino and Natalie Sherman. (2005). Microbiology Laboratory Manual seventh edition. https://faculty.washington.edu/korshin/Class-486/MicrobiolTechniques.pdf

Jasdeep Singh, Chandra Sekhar Mukhopadhyay, Jaspreet Singh Arora and Simarjeet Kaur, (2015). Biocomputational Characterization and Evolutionary analysis of Bubaline Dicer1 enzyme. Asian Australas. J. Anim. Sci., vol 28, 876-887. https://doi.org/10.5713/ajas.14.0767

Jayakumar, M., Gindaba, G. T., Gebeyehu, K. B., Periyasamy, S., Jabesa, A., Baskar, G., John, B. I., & Pugazhendhi, A. (2023). Bioethanol Production from Agricultural Residues as Lignocellulosic Biomass Feedstock's Waste Valorization Approach: A Comprehensive Review. Science of the Total Environment, 879, 163158. https://doi.org/10.1016/j.scitotenv.2023.163158

Lu, W.J., Wang, H.T., Yang, S.J., Wang, Z.C. and Nie, Y.F, “Isolation and characterisation of mesophilic cellulose degrading bacteria from flower stalks-vegetable waste co-composting system,” Journal of General Applied Microbiology, 51. 353-360. Dec. 2005. https://www.sciepub.com/reference/37498

MacWilliams, M. P. (2009). Indole Test Protocol. Retrieved from Microbe Library: 10. http://www.microbelibrary.org/component/resource/laboratory-test/3202-indole-testprotocol

McDevitt, S. (2009). Methyl red and Voges Proskauer test Protocol. Retrieved from Microbe Library: https://asm.org/protocols/methyl-red-and-voges-proskauer-test-protocols

Nargotra, P., Sharma, V., Lee, Y.-C., Tsai, Y.-H., Liu, Y.-C., Shieh, C.-J., Tsai, M.-L., Dong, C.-D., & Kuo, C.-H. (2023). Microbial Lignocellulolytic Enzymes for the Effective Valorization of Lignocellulosic Biomass: A Review. Catalysts, 13(1), 83. https://doi.org/10.3390/catal13010083

R. M. Teather and P. J.Wood. (1982). 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. https://doi.org/10.1128/aem.43.4.777-780.1982

Reiner, K. (2010). Catalase test protocol. Retrieved from Microbe library: http://www.microbelibrary.org/library/ laboratory+test/3226-catalase-test-protocol

Research advances on the consolidated bioprocessing of lignocellulosic biomass. (2024). Engineering Microbiology, 4(2), 100139. https://doi.org/10.1016/j.engmic.2024.100139

Rimi, A. A., Islam, A., Hossain, N., Mostofa, M. A., Mia, M., Anim, S. M. R., ... & Rahman, S. M. (2026). Isolation, Identification, and Application of Rhizobial Inoculants to Enhance Mung Bean (Vigna radiata L.) Yield in Bangladesh. http://dx.doi.org/10.48022/mbl.2510.10013

Rowińska, P., Gutarowska, B., Janas, R., & Szulc, J. (2024). Biopreparations for the decomposition of crop residues. Microbial Biotechnology, 17(8), e14534. https://doi.org/10.1111/1751-7915.14534

Sadhu, S., Saha, P., K Sen, S., Mayilraj, S., & Maiti, T. K. (2013). Production, purification and characterization of a novel thermotolerant endoglucanase (CMCase) from Bacillus strain isolated from cow dung. SpringerPlus, 2 (10). https://doi.org/10.1186/2193-1801-2-10

Sauera, P., Gallob, J., Kesselováa, M., Kolářa, M., & Koukalováa, D. (2005). Universal primers for detection of common bacterial Pathogens causing prosthetic joint infection. Biomed Pap Med Fac Univ Palacky Olomouc Czech Repub., 149 (2), 285–8. https://doi.org/10.5507/bp.2005.043

Sohini R. C., Averi D., Rajat C. (2024). Review on progress in cellulase catalyzed saccharification of agricultural lignocellulosic biomass towards fermentable sugar and bioethanol: Kinetics & reactor configurations. Biocatalysis and Agricultural Biotechnology, vol 58, 103142, ISSN 1878-8181. https://doi.org/10.1016/j.bcab.2024.103142

Tabao, Nik Shawn & Monsalud, Rosario. (2010). Characterization and Identification of High Cellulase-Producing Bacterial Strains from Philippine Mangroves. Philippine Journal of Systematic Biology. IV. 13-20. doi: 10.3860/pjsb.v4i0.1562

Tamura K. and Nei M. (1993). Estimation of the number of nucleotide substitutions in the control region of mitochondrial DNA in humans and chimpanzees. Molecular Biology and Evolution, 10: 512-526. https://doi.org/10.1093/oxfordjournals.molbev.a040023

Vijayaraghavan, P., & Vincent, S. G. (2012). Purification and characterization of carboxymethyl cellulase from Bacillus sp. isolated from a paddy field. Polish journal of microbiology, 61(1), 51–55. https://pubmed.ncbi.nlm.nih. gov/22708346/

Zhang, Z., Xing, J., Li, X., Lu, X., Liu, G., Qu, Y., & Zhao, J. (2024). Review of research progress on the production of cellulase from filamentous fungi. International Journal of Biological Macromolecules, 277(Pt 4), 134539. https://doi.org/10.1016/j.ijbiomac.2024.134539