Sustainable bioconversion of poultry feather waste into industrial protease by Bacillus licheniformis through optimised fermentation and functional application
Article Main
Abstract
The accumulation of poultry feather waste poses a significant environmental challenge, while its keratin-rich composition offers an opportunity for sustainable bioprocessing into value-added industrial enzymes. Microbial proteases, widely used in detergents and biocatalysis, are predominantly produced from costly conventional substrates, necessitating economical, eco-friendly alternatives. The present study aimed to optimise extracellular protease production from Bacillus licheniformis (NCBI accession OQ552873) using pre-treated chicken feather biomass as the sole substrate under submerged fermentation conditions. Systematic one-factor-at-a-time optimisation was performed for incubation time (24-120 hours), pH (7-11), carbon source supplementation (glucose, maltose, sucrose at 0.1%), and nitrogen source supplementation (peptone, gelatin, sodium nitrate at 0.1%), followed by quantification of enzyme activity, total protein, and specific activity. Optimal protease production (2.833 ± 0.011 µG/mL) was achieved after 72 hours at pH 9 with 0.1% glucose and 0.1% peptone supplementation, yielding a specific activity of 23.01 µG/mg. Feather degradation efficiency reached 12.01 ± 0.74%, and the crude protease demonstrated effective removal of beetroot, grease, blood, and pickle stains from fabric samples. This study presents a dual-purpose bioprocess that simultaneously valorises poultry feather waste into an industrially relevant protease, offering a sustainable, cost-effective alternative to conventional enzyme production. The optimised parameters provide a scalable blueprint for waste-to-value bioconversion, with direct applications in eco-friendly detergent formulations and circular bioeconomy-driven waste management.
Article Details
Article Details
Keywords
Bioconversion, B. licheniformis, Feather, Optimization, Protease
References
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He H, Zhang Y & Shi G, Li Y. (2023). Recent biotechnological advances and future prospective of Bacillus licheniformis as microbial cell factories. Systems Microbiology and Biomanufacturing, 3, 521-532. https://doi.org/10.1007/s43393-023-00162-7
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Lange L, Huang Y & Busk PK. (2016). Microbial decomposition of keratin in nature—a new hypothesis of industrial relevance. Applied Microbiology and Biotechnology, 100, 2083-2096. https://doi.org/10.1007/s00253-015-7262-1
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Mottet A & Tempio G. (2017). Global poultry production: current state and future outlook and challenges. World's Poultry Science Journal, 73, 245-256. https://doi.org/10.1017/S0043933917000071
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Mushtaq H, Ganai SA, Jehangir A, Ganai BA & Dar R. (2023). Molecular and functional characterization of protease from psychrotrophic Bacillus sp. HM49 in North-western Himalaya. PLoS One, 18, e0283677. https://doi.org/10.1371/journal.pone.0283677
Nguyen V-M-L, Ndao A, Peterson EC, Blais J-F & Adjallé K. (2025). Bacillus species: evolving roles in bio-based detergents. Processes, 13, 1885.
Okoroma EA, Garelick H, Abiola OO & Purchase D. (2012). Identification and characterisation of a Bacillus licheniformis strain with profound keratinase activity for degradation of melanised feather. International Biodeterioration & Biodegradation, 74, 54-60. https://doi.org/10.1016/j.ibiod.2012.07.013
Olajuyigbe FM. (2013). Optimized production and properties of thermostable alkaline protease from Bacillus subtilis SHS-04 grown on groundnut (Arachis hypogaea) meal. Advances in Enzyme Research, 1, 112.
Olajuyigbe FM, Ajele JO. (2005). Production dynamics of extracellular protease from Bacillus species. African Journal of Biotechnology, 4, 776-779.
Park G-T & Son H-J. (2009). Keratinolytic activity of Bacillus megaterium F7-1, a feather-degrading mesophilic bacterium. Microbiological Research, 164, 478-485. https://doi.org/10.1016/j.micres.2007.02.004
Peydayesh M, Bagnani M, Soon WL & Mezzenga R. (2022). Turning food protein waste into sustainable technologies. Chemical Reviews, 123, 2112-2154. https://doi.org/10.1021/acs.chemrev.2c00236
Rao Y, Ishaq AR, Zhang Y, Zhang R, Cai D, He P & Chen S. (2025). Cell membrane engineering of Bacillus licheniformis for the enhancement of heterologous protein production. International Journal of Biological Macromolecules, 305, 141178.
Sellami-Kamoun A, Haddar A, Ali NE-H, Ghorbel-Frikha B, Kanoun S & Nasri M. (2008). Stability of thermostable alkaline protease from Bacillus licheniformis RP1 in commercial solid laundry detergent formulations. Microbiological Research, 163, 299-306. https://doi.org/10.1016/j.micres.2006.06.001
Sharma S & Gupta A. (2016). Sustainable management of keratin waste biomass: applications and future perspectives. Brazilian Archives of Biology and Technology, 59, e16150684. https://doi.org/10.1590/1678-4324-2016150684
Tyndall JD, Nall T & Fairlie DP. (2005). Proteases universally recognize beta strands in their active sites. Chemical Reviews, 105, 973-1000. https://doi.org/10.1021/cr040669e
Waterborg JH. (2009). The lowry method for protein quantitation. The protein protocols handbook. 7-10. https://doi.org/10.1007/978-1-59745-198-7_2
Zhang Y, Liu Z, Hu J, Yao Q, Xu H, Zhang Q, Chen S & Wang Y. (2025). Elucidating metabolic mechanisms underlying the influence of specific growth rate on alkaline protease synthesis in Bacillus licheniformis through combined omics and computational modeling analysis. Bioresource Technology, 132762.
Zilda DS, Harmayani E, Widada J, Asmara W, Irianto HE, Patantis G & Fawzya YN. (2012). Screening of thermostable protease producing microorganisms isolated from Indonesian hotspring. Squalen Bulletin of Marine and Fisheries Postharvest and Biotechnology, 7, 105-114.
Zubair M, Sultan HM, Ghazanfar S, Khalid S, Rehman MZu, Islam A & Ashique S. (2025). Advances in the production of recombinant protease through expression systems: an updated review. Current Enzyme Inhibition.
Cupp-Enyard C. (2008). Sigma's non-specific protease activity assay-casein as a substrate. Journal of Visualized Experiments, JoVE. 899. https://doi.org/10.3791/899
Gautam M & Agrawal M. (2020). Greenhouse gas emissions from municipal solid waste management: a review of global scenario. Carbon footprint case studies: Municipal solid waste management, Sustainableroad transport and carbon sequestration. 123-160. https://doi.org/10.1007/978-981-15-9577-6_5
Gregg K & Rogers GE. (1986). Feather keratin: composition, structure and biogenesis. In: Biology of the Integument: 2 Vertebrates. Springer. p. 666-694.
He H, Zhang Y & Shi G, Li Y. (2023). Recent biotechnological advances and future prospective of Bacillus licheniformis as microbial cell factories. Systems Microbiology and Biomanufacturing, 3, 521-532. https://doi.org/10.1007/s43393-023-00162-7
Hodgson P & Fane A. (1992). Crossflow microfiltration of biomass with inorganic membranes: The influence of membrane surface and fluid dynamics. Key Engineering Materials, 61, 167-174. https://doi.org/10.4028/www.scientific.net/KEM.61-62.167
Ignatova Z, Gousterova A, Spassov G & Nedkov P. (1999). Isolation and partial characterisation of extracellular keratinase from a wool degrading thermophilic actinomycete strain Thermoactinomyces candidus. Canadian Journal of Microbiology, 45, 217-222. https://doi.org/10.1139/w98-230
Ilmiah SN, Mubarik NR & Wahyuntari B. (2018). Characterization of protease from Bacillus licheniformis F11.1 as a bio-detergent agent. Makara Journal of Science, 105-112. https://doi: 10.7454/mss.v22i3.8809
Jana A, Halder SK, Dasgupta D, Hazra S, Mondal P, Bhaskar T & Ghosh D. (2020). Keratinase biosynthesis from waste poultry feathers for proteinaceous stain removal. ACS Sustainable Chemistry & Engineering, 8, 17651-17663. https://doi.org/10.1021/acssuschemen g.0c04378
Kittler S, Müller F, Elshazly M, Wandrey GB, Klein T, Daub A, Spadiut O & Kopp J. (2025). Transferability of bioprocessing modes for recombinant protease production: from fed-batch to continuous cultivation with Bacillus licheniformis. BMC Biotechnology, 25, 13.
Kumar P, Dubey R & Maheshwari D. (2012). Bacillus strains isolated from rhizosphere showed plant growth promoting and antagonistic activity against phytopathogens. Microbiological Research, 167, 493-499. https://doi.org/10.1016/j.micres.2012.05.002
Lange L, Huang Y & Busk PK. (2016). Microbial decomposition of keratin in nature—a new hypothesis of industrial relevance. Applied Microbiology and Biotechnology, 100, 2083-2096. https://doi.org/10.1007/s00253-015-7262-1
Li Q. (2019). Progress in microbial degradation of feather waste. Frontiers in Microbiology, 10, 2717. https://doi.org/10.3389/fmicb.2019.02717
Manczinger L, Rozs M, Vágvölgyi C & Kevei F. (2003). Isolation and characterization of a new keratinolytic Bacillus licheniformis strain. World Journal of Microbiology and Biotechnology, 19, 35-39. https://doi.org/10.1023/A:1022576826372
Masi C, Gemechu G & Tafesse M. (2021). Isolation, screening, characterization, and identification of alkaline protease-producing bacteria from leather industry effluent. Annals of Microbiology, 71, 24. https://doi.org/10.1186/s13213-021-01631-x
Mattiello S, Guzzini A, Del Giudice A, Santulli C, Antonini M, Lupidi G & Gunnella R. (2022). Physico-chemical characterization of keratin from wool and chicken feathers extracted using refined chemical methods. Polymers, 15, 181. https://doi.org/10.3390/polym15010181
Maxton A & Masih SA. (2025). Potential of industrial waste to transfer microbial electron in microbial fuel cell using dye reduction assay. Water, Air, & Soil Pollution, 236, 126.
Mottet A & Tempio G. (2017). Global poultry production: current state and future outlook and challenges. World's Poultry Science Journal, 73, 245-256. https://doi.org/10.1017/S0043933917000071
Muras A, Romero M, Mayer C & Otero A. (2021). Biotechnological applications of Bacillus licheniformis. Critical Reviews in Biotechnology, 41, 609-627. https://doi.org/10.1080/07388551.2021.1873239
Mushtaq H, Ganai SA, Jehangir A, Ganai BA & Dar R. (2023). Molecular and functional characterization of protease from psychrotrophic Bacillus sp. HM49 in North-western Himalaya. PLoS One, 18, e0283677. https://doi.org/10.1371/journal.pone.0283677
Nguyen V-M-L, Ndao A, Peterson EC, Blais J-F & Adjallé K. (2025). Bacillus species: evolving roles in bio-based detergents. Processes, 13, 1885.
Okoroma EA, Garelick H, Abiola OO & Purchase D. (2012). Identification and characterisation of a Bacillus licheniformis strain with profound keratinase activity for degradation of melanised feather. International Biodeterioration & Biodegradation, 74, 54-60. https://doi.org/10.1016/j.ibiod.2012.07.013
Olajuyigbe FM. (2013). Optimized production and properties of thermostable alkaline protease from Bacillus subtilis SHS-04 grown on groundnut (Arachis hypogaea) meal. Advances in Enzyme Research, 1, 112.
Olajuyigbe FM, Ajele JO. (2005). Production dynamics of extracellular protease from Bacillus species. African Journal of Biotechnology, 4, 776-779.
Park G-T & Son H-J. (2009). Keratinolytic activity of Bacillus megaterium F7-1, a feather-degrading mesophilic bacterium. Microbiological Research, 164, 478-485. https://doi.org/10.1016/j.micres.2007.02.004
Peydayesh M, Bagnani M, Soon WL & Mezzenga R. (2022). Turning food protein waste into sustainable technologies. Chemical Reviews, 123, 2112-2154. https://doi.org/10.1021/acs.chemrev.2c00236
Rao Y, Ishaq AR, Zhang Y, Zhang R, Cai D, He P & Chen S. (2025). Cell membrane engineering of Bacillus licheniformis for the enhancement of heterologous protein production. International Journal of Biological Macromolecules, 305, 141178.
Sellami-Kamoun A, Haddar A, Ali NE-H, Ghorbel-Frikha B, Kanoun S & Nasri M. (2008). Stability of thermostable alkaline protease from Bacillus licheniformis RP1 in commercial solid laundry detergent formulations. Microbiological Research, 163, 299-306. https://doi.org/10.1016/j.micres.2006.06.001
Sharma S & Gupta A. (2016). Sustainable management of keratin waste biomass: applications and future perspectives. Brazilian Archives of Biology and Technology, 59, e16150684. https://doi.org/10.1590/1678-4324-2016150684
Tyndall JD, Nall T & Fairlie DP. (2005). Proteases universally recognize beta strands in their active sites. Chemical Reviews, 105, 973-1000. https://doi.org/10.1021/cr040669e
Waterborg JH. (2009). The lowry method for protein quantitation. The protein protocols handbook. 7-10. https://doi.org/10.1007/978-1-59745-198-7_2
Zhang Y, Liu Z, Hu J, Yao Q, Xu H, Zhang Q, Chen S & Wang Y. (2025). Elucidating metabolic mechanisms underlying the influence of specific growth rate on alkaline protease synthesis in Bacillus licheniformis through combined omics and computational modeling analysis. Bioresource Technology, 132762.
Zilda DS, Harmayani E, Widada J, Asmara W, Irianto HE, Patantis G & Fawzya YN. (2012). Screening of thermostable protease producing microorganisms isolated from Indonesian hotspring. Squalen Bulletin of Marine and Fisheries Postharvest and Biotechnology, 7, 105-114.
Zubair M, Sultan HM, Ghazanfar S, Khalid S, Rehman MZu, Islam A & Ashique S. (2025). Advances in the production of recombinant protease through expression systems: an updated review. Current Enzyme Inhibition.
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Sustainable bioconversion of poultry feather waste into industrial protease by Bacillus licheniformis through optimised fermentation and functional application. (2026). Journal of Applied and Natural Science, 18(2), 1039-1049. https://doi.org/10.31018/jans.v18i2.7568
