Keratinase Production by Bacillus sp. MD24 in Sub-merge and Solid State Fermentation


  • Suharti Suharti Department of Chemistry, Faculty of Mathematics and Science, Universitas Negeri Malang, Jl. Semarang No. 5 Malang, East Java, Indonesia
  • Hanif Nur Rozaq Department of Chemistry, Faculty of Mathematics and Science, Universitas Negeri Malang, Jl. Semarang No. 5 Malang, East Java, Indonesia
  • Aulia Qisti Department of Chemistry, Faculty of Mathematics and Science, Universitas Negeri Malang, Jl. Semarang No. 5 Malang, East Java, Indonesia
  • Mieke Alvionita Department of Chemistry, Faculty of Mathematics and Science, Universitas Negeri Malang, Jl. Semarang No. 5 Malang, East Java, Indonesia
  • Surjani Wonorahardjo Department of Chemistry, Faculty of Mathematics and Science, Universitas Negeri Malang, Jl. Semarang No. 5 Malang, East Java, Indonesia



Keratinase, Bacillus, chicken feathers, fermentation, amino acids


Chicken feathers are valuable solid waste that mainly contains insoluble proteins called keratin. Naturally, keratin is degraded by microbes that produce a specific protease called keratinase.  Many reports have been addressed to study Bacillus species as keratin-degrading microbes. However, most reported works were using pathogenic soil Bacillus. Our work has been concentrated on keratinase produced by a soil bacterium, Bacillus sp. MD24 and we confirmed it as a non-pathogenic bacterium. This report describes the different profiles of keratinase fermentation between sub-merged fermentation (SmF) and solid-state fermentation (SSF) using chicken feathers as a sole carbon and nitrogen source.  Keratinase production and weight loss were followed for 10 consecutive days, the keratinase molecular weight was investigated, the type and concentration of 18 amino acids were determined using LC-MS/MS after 10 days to seek its potential as the amino acid source, and protein by-product concentration was measured. The results showed that keratinase production as well as weight loss in SmF was better compared to SSF with different optimum production times. Surprisingly, L-arginine was a dominant amino acid in SmF hydrolysate which contained 42.06% of total amino acids and made Bacillus sp. MD24 is a potential organism for L-arginine production. As protein by-product concentration was relatively low in both conditions showing effective utilization of chicken feathers as matter and energy source, consequently, cell-free keratinase should be applied to degrade chicken feathers into valuable materials.


Abdel-Fattah, A., A. Hashem, M. El-Gamal, M. Emran, and S. Ismail. (2018). Biodegradation of feather waste by keratinase produced from newly isolated Bacillus Licheniformis ALW1. Journal of Genetic Engineering and Biotechnology, 16(2), 311-18.

Adelina, Adelina, Feli Feliatra, Yusni Ikhwan Siregar, Iskandar Putra, and Indra Suharman. (2021). Use of chicken feather meal fermented with Bacillus Subtilis in Diets to increase the digestive enzymes activity and nutrient digestibility of silver pompano Trachinotus Blochii (Lacepede, 1801). F1000Research 10:25.

Andriyani, Andriyani, Fernanda Tabetha Claudia Wongkar, and Suharti Suharti. (2021). Production of keratinase under solid-state fermentation (SSF) by Bacillus Sp. MD24 and potential of its liquid by-product as organic fertilizers. AIP Conference Proceedings, 2353(1), 030103.

Bokveld, Amahle, Nonso E. Nnolim, and Uchechukwu U. Nwodo. (2021). Chryseobacterium Aquifrigidense FANN1 produced detergent-stable metallokeratinase and amino acids through the abasement of chicken. Front. Bioeng. Biotechnol., 9, 720176.

Catalán, Eva, and Antoni Sánchez. (2020). Solid-state fermentation (SSF) versus submerged fermentation (SmF) for the recovery of cellulases from coffee husks: A life cycle assessment (LCA) based comparison. Energies, 13(11).

Chen, Jinyang, Siyuan Ding, Yimei Ji, Junying Ding, Xuanyu Yang, Mihua Zou, and Zhilian Li. (2015). Microwave-enhanced hydrolysis of poultry feather to produce amino acid. Chemical Engineering and Processing: Process Intensification, 87, 104-9.

Eaksuree, Warintorn, Akkharadet Prachayakitti, Tewa Upathanpreecha, Rutjawate Taharnklaew, Sunee Nitisinprasert, and Suttipun Keawsompong. (2016). In vitro and in vivo evaluation of protein quality of enzymatic treated feather meals. SpringerPlus, 5(1), 2-7.

Habbeche, Amina, Boudjema Saoudi, Bassem Jaouadi, Soumaya Haberra, Bilal Kerouaz, Mokhtar Boudelaa, Abdelmalek Badis, and Ali Ladjama. (2014). Purification and biochemical characterization of a detergent-stable keratinase from a newly thermophilic actinomycete actinomadura keratinilytica strain Cpt29 isolated from poultry compost. Journal of Bioscience and Bioengineering, 117(4), 413-21.

Hardiman, Jhon, and Manouchehr Katanbaf. (2012). Good feather cover for optimising energy utilisation - Poultry World. Poultry World. Accessed on March 22nd, 2023.

Hashemi, Maryam, Seyyed Mohammad Mousavi, Seyed Hadi Razavi, and Seyed Abbas Shojaosadati. (2013). Comparison of submerged and solid state fermentation systems effects on the catalytic activity of Bacillus sp. KR-8104 α-amylase at different PH and temperatures. Industrial Crops and Products, 43(1), 661-67.

Jiang, Zehui, Daochun Qin, Chung Yun Hse, Monlin Kuo, Zhaohui Luo, Ge Wang, and Yan Yu. (2008). Preliminary study on chicken feather protein-based wood adhesives. Journal of Wood Chemistry and Technology, 28(3), 240-46.

Kani, T., K. Subha, and P. Madhanraj. (2012). Degradation of chicken feathers by Leuconostoc sp. and Pseudomonas Microphilus. Eur. J. Exp. Biol., 2(2), 358-62.

Kim, Hyang Yeon, Do Yeon Heo, Hye Min Park, Digar Singh, and Choong Hwan Lee. (2016). Metabolomic and transcriptomic comparison of solid-state and submerged fermentation of Penicillium Expansum KACC 40815. PLoS ONE, 11(2).

Kushibiki, Toshihiro, Yoshine Mayumi, Eiko Nakayama, Ryuichi Azuma, Kenichiro Ojima, Akio Horiguchi, and Miya Ishihara. (2021). Photocrosslinked gelatin hydrogel improves wound healing and skin flap survival by the sustained release of basic fibroblast growth factor. Scientific Reports, 11(1).

Machado, Geruza Silveira, Ana Paula Folmer Correa, Paula Gabriela da Silva Pires, Letícia Marconatto, Adriano Brandelli, Alexandre de Mello Kessler, and Luciano Trevizan. (2021). Determination of the nutritional value of diet containing Bacillus Subtilis hydrolyzed feather meal in adult dogs.” Animals An Open Access Journal from MDPI, 11(12).

Mazotto, Ana Maria, Rosalie Reed Rodrigues Coelho, Sabrina Martins Lage Cedrola, Marcos Fábio De Lima, Sonia Couri, Edilma Paraguai De Souza, and Alane Beatriz Vermelho. (2011). Keratinase production by three Bacillus Spp. using feather meal and whole feather as substrate in a submerged fermentation. Enzyme Research.

Mazotto, Ana Maria, Sonia Couri, MÔnica C. T. Damaso, and Alane Beatriz Vermelho. (2013). Degradation of feather waste by Aspergillus Niger Keratinases: Comparison of submerged and solid-state fermentation. International Biodeterioration and Biodegradation, 85,189-95.

Mendelson, Neil H., Adrienne Bourque, Kathryn Wilkening, Kevin R. Anderson, and Joseph C. Watkins. (1999). Organized cell swimming motions in Bacillus Subtilis colonies: Patterns of short-lived whirls and jets. Journal of Bacteriology, 181(2), 600-609.

Nagal, Swetlana, and P. C. Jain. (2010). Feather degradation by strains of Bacillus isolated from decomposing feathers. Brazilian Journal of Microbiology, 41(1), 196-200.

Nkemnaso, Clifford. (2019). Solid state fermentation: Substrates uses and applications in biomass and metabolites production-a review. South Asian Research Journal of Biology and Applied Biosciences, 1(1):20-29.

Nurkhasanah, U., E. Susanti, A. M. Idris, and S. Suharti. (2020). keratin biofilm from chicken feathers. IOP Conference Series: Earth Environ.l Sci., 475, 012073.

Nurkhasanah, Uswatun, and Suharti. (2019). Preliminary study on keratinase fermentation by Bacillus Sp. MD24 under solid state fermentation. IOP Conference Series: Earth Environ.l Sci., 276, 012 276.

Pernicova, Iva, Vojtech Enev, Ivana Marova, and Stanislav Obruca. (2019). Interconnection of waste chicken feather biodegradation and keratinase and Mcl-PHA production employing Pseudomonas Putida KT2440. Applied Food Biotechnology, 2(1), 83-93.

Qiu, Jingwen, Casper Wilkens, Kristian Barrett, and Anne S. Meyer. (2020). Microbial enzymes catalyzing keratin degradation: Classification, structure, function. Biotechnology Advances, 44, 107607.

Rai, Sudhir K., and Ashis Kumar Mukherjee. (2015). Optimization for production of liquid nitrogen fertilizer from the degradation of chicken feather by iron-oxide (Fe3O4) magnetic nanoparticles coupled β-keratinase. Biocatalysis and Agricultural Biotechnology, 4(4), 632-44.

Saenz, Jerson Anders Cuellar. (2022). Global Poultry Market Dynamics and Current Trends. Veterinaria Digital 13 May 2022. accessed on 12 January 2025.

Suharti Suharti, Maurilla Trisna Riesmi, Arina Hidayati, Umi Faridatuz Zuhriyah, Surjani, and Evi Susanti Wonorahardjo. (2018). Enzymatic dehairing of goat skin using keratinase from Bacillus sp. MD24, A newly isolated soil bacterium. Journal of Tropical Agricultural Science, 41(3), 1449-1461.

Suharti, Suharti, Evi Susanti, Uswatun Nurkhasanah, and Yuniar Avia Mulyadewi. (2019). Fibrous keratin powder as by-product of keratinase fermentation under solid state fermentation using chicken feathers as substrate fibrous keratin powder as by-product of keratinase fermentation under solid state fermentation using chicken feathers as sub. Iop Conf. ser.: Mater. Sci. Eng., 515, 012030

Tesfaye, Tamrat, Bruce Sithole, and Deresh Ramjugernath. (2018). Preparation, characterization and application of keratin based green biofilms from waste chicken feathers. 16(3), 1-16.

Tiwary, Ekta, and Rani Gupta. (2010). Medium optimization for a novel 58 KDa dimeric keratinase from Bacillus Licheniformis ER-15: Biochemical characterization and application in feather degradation and dehairing of hides. Bioresource Technology, 101(15), 6103-10.

Tuly, Jamila A., Hossain M. Zabed, Abdul Sattar Nizami, Md Mehedi Hassan, S. M. Roknul Azam, Mukesh Kumar Awasthi, Quaisie Janet, Gu Chen, Nelson Dzidzorgbe Kwaku Akpabli-Tsigbe, and Haile Ma. (2022). Bioconversion of agro-food industrial wastes into value-added peptides by a Bacillus Sp. Mutant through solid-state fermentation. Bioresource Technology, 364, 126513.

Wang, Ju, Shilei Hao, Tiantian Luo, Zhongjun Cheng, Wenfeng Li, Feiyan Gao, Tingwang Guo, Yuhua Gong, and Bochu Wang. (2017). Feather Keratin Hydrogel for Wound Repair: Preparation, Healing Effect and Biocompatibility Evaluation. Colloids and Surfaces B: Biointerfaces, 149, 341-50.

Yao, Dawei, Jiao Qu, Peiwei Chang, Yanhua Tao, and Deji Yang. (2012). Production and characterization of alkaline protease from hemoglobin-degrading Bacillus Pumilus NJM4 to produce fermented blood meal. Journal of Food Science and Technology, 49(5), 626-31.