Skip to main content
SpringerLink
Log in

Keratinases and sulfide from Bacillus subtilis SLC to recycle feather waste

  • Original Paper
  • Published:
World Journal of Microbiology and Biotechnology Aims and scope Submit manuscript

Abstract

The aim of this study is to investigate the culture conditions of chicken feather degradation and keratinolytic enzyme production by the recently isolated Bacillus subtilis SLC and to evaluate the potential of the SLC strain to recycle feather waste discarded by the poultry industry. The SLC strain was isolated from the agroindustrial waste of a poultry farm in Brazil and was confirmed to belong to Bacillus subtilis by rDNA gene analysis. There was high keratinase production when the medium was at pH 8 (280 U ml−1). Activity was higher using the inoculum propagated for 72 h on 1% whole feathers supplemented with 0.1% yeast extract. In the enzymatic extract, the keratinases were active in the pH range from 2.0 to 12.0 with a maximum activity at pH 10.0 and temperature 60°C. For gelatinase the best pH was 5.0 and the best temperature was 37°C. All keratinases are serine peptidases. The crude enzymatic extract degraded keratin, gelatin, casein, and hemoglobin. Scanning electron microscopy showed Bacillus cells adhered onto feather surfaces after 98 h of culture and degraded feather filaments were observed. MALDI-TOF mass spectrometric analysis showed multiple peaks from 522 to 892 m/z indicating feather degradation. The presence of sulfide was detected on extracellular medium probably participating in the breakdown of sulfide bridges of the feather keratin. External addition of sulfide increased feather degradation.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6
Fig. 7
Fig. 8
Fig. 9
Fig. 10
Fig. 11

Similar content being viewed by others

References

  • Arikawa Y, Ozawa T, Iwasaki I (1967) An improved photometric method for the determination of sulfite with pararosaniline and formaldehyde. Acad J 41(6):1454–1456

    Google Scholar 

  • Barone JR, Schmidt WF (2006) Effect of formic acid exposure on keratin fiber derived from poultry feather biomass. Bioresour Technol 97(2):233–242. doi:10.1016/j.biortech.2005.02.039

    Article  CAS  Google Scholar 

  • Böckle B, Galunsky B, Muller R (1995) Characterization of a keratinolytic serine proteinase from Streptomyces pactum DSM 40530. Appl Environ Microbiol 61:3705–3710

    Google Scholar 

  • Brandelli A (2008) Bacterial keratinases: useful enzymes for bioprocessing agroindustrial byproducts and beyond. Food Bioprocess Technol 1:105–116. doi:10.1007/s11947-007-0025-y

    Article  Google Scholar 

  • Dalev P, Ivanov I, Liubomirova A (1997) Enzymic modification of feather keratin hydrolysates with lysine aimed at increasing the biological value. J Sci Food Agric 73:242–244. doi:10.1002/(SICI)1097-0010(199702)73:2<242:AID-JSFA712>3.0.CO;2-3

    Article  CAS  Google Scholar 

  • Deivasigamani B, Alagappan KM (2008) Industrial application of keratinase and soluble proteins from feather keratins. J Environ Biol 29(6):933–936

    CAS  Google Scholar 

  • Dozie INS, Okeke CN, Unaeze NC (1994) A thermostable, alkaline-active, keratinolytic proteinase from Chrysosporium keratinophilum. World J Microbiol Biotechnol 10(5):563–567. doi:10.1007/BF00367668

    Article  CAS  Google Scholar 

  • Duarte TR, Oliveira SS, Macrae A, Cedrola SML, Mazotto AM, Souza EP, Melo ACN, Vermelho AB (2011) Increased expression of keratinase and other peptidases by Candida parapsilosis mutants. Braz J Med Biol Res 44(3):212–216. doi:10.1590/S0100-879X2011007500011

    Article  CAS  Google Scholar 

  • Farag AM, Hassan AH (2004) Purification, characterization and immobilization of a keratinase from Aspergillus oryzae. Enzyme Microbiol Technol 34:85–93. doi:10.1016/j.enzmictec.2003.09.002

    Article  CAS  Google Scholar 

  • Fujinami S, Fujisawa M (2010) Industrial applications of alkaliphiles and their enzymes—past, present and future. Environ Technol 8(9):845–856. doi:10.1080/09593331003762807

    Article  Google Scholar 

  • Godde C, Sahm K, Brouns SJJ, Kluskens LD, van der Oost J, de Vos WM, Antranikian G (2005) Cloning and expression of islandisin, a new thermostable subtilisin from Fervidobacterium islandicum, in Escherichia coli. Appl Environ Microbiol 71:3951–3958. doi:10.1128/AEM.71.7.3951-3958.2005

    Article  Google Scholar 

  • Gousterova A, Braikova D, Goshev I, Christov P, Tishinov K, Vasileva-Tonkova E, Haertle T, Nedkov P (2005) Degradation of keratin and collagen containing wastes by newly isolated thermoactinomycetes or by alkaline hydrolysis. Lett Appl Microbiol 40(5):335–340. doi:10.1111/j.1472-765X.2005.01692.x

    Article  CAS  Google Scholar 

  • Gregg MM (2002) Wound closure materials in the new millennium. Semin Cutan Med Surg 21(2):166–170. doi:10.1053/sder.2002.33287

    Article  Google Scholar 

  • Grywnowicz G, Lobarzewski J, Wawrzkiewicz K, Wolski T (1989) Comparative characterization of proteolytic enzymes from Trichophyton gallinae and Trychophyton verrucosum. J Med Vet Mycol 27:319–328

    Article  Google Scholar 

  • Gupta R, Ramnani P (2006) Microbial keratinases and their prospective applications: an overview. Appl Microb Biotechnol 70:21–33. doi:10.1007/s00253-005-0239-8

    Article  CAS  Google Scholar 

  • Heussen C, Dowdle EB (1980) Electrophoretic analysis of plasminogen activators in polyacrylamide gels containing sodium dodecyl sulphate and copolymerized substrates. Anal Biochem 102:196–202. doi:10.1016/0003-2697(80)90338-3

    Article  CAS  Google Scholar 

  • Jeong JH, Jeon YD, Lee OM, Kim JD, Lee NR, Park GT, Son HJ (2010) Characterization of a multifunctional feather-degrading Bacillus subtilis isolated from forest soil. Biodegradation 21:1029–1040. doi:10.1007/s10532-010-9363-y

    Article  CAS  Google Scholar 

  • Jones LB, Fontamini D, Jarvinen M, Pekkarinen A (1998) Simplified endoproteinase assays using gelatin or azogelatin. Anal Biochem 263:214–220. doi:10.1006/abio.1998.2819

    Article  CAS  Google Scholar 

  • Khardenavis AA, Kapley A, Purohit HJ (2009) Processing of poultry feathers by alkaline keratin hydrolyzing enzyme from Serratia sp. HPC 1383. Waste Manage 29(4):1409–1415. doi:10.1016/j.wasman.2008.10.009

    Article  CAS  Google Scholar 

  • Kim SS, Kim YJ, Rhee I (2001) Purification and characterization of a novel extracellular protease from Bacillus cereus KCTC 3674. Arch Microbiol 175:458–461. doi:10.1007/s002030100282

    Article  CAS  Google Scholar 

  • Kunert J (1992) Effect of reducing agents on proteolytic and keratinolytic activity of enzymes of Microsporum gypseum. Mycoses 35:343–348. doi:10.1111/j.1439-0507.1992.tb00892.x

    Article  CAS  Google Scholar 

  • Lin X, Lee CG, Casale ES, Shih JCH (1992) Purification and characterization of a keratinase from a feather-degrading Bacillus licheniformis strain. Appl Environ Microbiol 58(10):3271–3275

    CAS  Google Scholar 

  • Lin X, Inglis GD, Yanke LJ, Cheng KJ (1999) Selection and characterization of feather-degrading bacteria from canola meal compost. J Ind Microbiol Biotechnol 23(2):149–153. doi:10.1038/sj.jim.2900706

    Article  CAS  Google Scholar 

  • Lopes BGB, Santos ALS, Bezerra CCF, Wanke B, Lazéra MS, Nishikawa MM, Mazotto AM, Kussumi VM, Haido RMT, Vermelho AB (2008) A 25-kDa serine peptidase with keratinolytic activity secreted by Coccidioides immitis. Mycopathologia 166:35–40. doi:10.1007/s11046-008-9116-1

    Article  CAS  Google Scholar 

  • Lowry OH, Rosembrough NJ, Farr AL, Randall RJ (1951) Protein measurement with the Folin phenol reagent. J Biol Chem 193(1):267–275

    Google Scholar 

  • Manczinger L, Rozs M, Vagvolgyi C, Kevei F (2003) Isolation and characterization of a new keratinolytic Bacillus licheniformis strain. World J Microbiol Biotechnol 19(1):35–39. doi:10.1023/A:1022576826372

    Article  CAS  Google Scholar 

  • Massol-Deya AA, Odelson DA, Hickey RF, Tiedje JM (1995) Bacterial community fingerprinting of amplified 16S and 16–23S ribosomal DNA gene sequences and restriction endonuclease analysis (ARDRA). In: Akkermans ADL (ed) Molecular microbial ecology manual. Kluwer Academic Publishers, Dordrecht, pp 3.3.2.1.1–3.3.2.1.8

  • Matseliukh OV, Levishko AS, Varbanets LD (2010) Proteolytic enzymes of microorganisms. Mikrobiol Z 72(4):56–73

    CAS  Google Scholar 

  • Matsui T, Yamada Y, Mitsuya H, Shigeri Y, Yoshida Y, Saito Y, Matsui H, Watanabe K (2009) Sustainable and practical degradation of intact chicken feathers by cultivating a newly isolated thermophilic Meiothermus ruber H328. Appl Microb Biotechnol 82:941–950. doi:10.1007/s00253-009-1880-4

    Article  CAS  Google Scholar 

  • Mazotto AM, Cedrola SML, Lins U, Rosado AS, Silva KT, Chaves JQ, Rabinovitch L, Zingali RB, Vermelho AB (2010) Keratinolytic activity of Bacillus subtilis AMR using human hair. Lett Appl Microbiol 50:89–96. doi:10.1111/j.1472-765X.2009.02760.x

    Article  CAS  Google Scholar 

  • Mazotto AM, Melo ACN, Macrae A, Rosado AS, Peixoto R, Cedrola SML, Couri S, Zingali RB, Villa ALV, Rabinovitch L, Chaves JQ, Vermelho AB (2011) Biodegradation of feather waste by extracellular keratinases and gelatinases from Bacillus spp. World J Microbiol Biotechnol 27:1355–1365. doi:10.1007/s11274-010-0586-1

    Article  CAS  Google Scholar 

  • Mokrejs P, Svoboda P, Hrncirik J, Janacova D, Vasek V (2010) Processing poultry feathers into keratin hydrolysate through alkaline-enzymatic hydrolysis. Waste Manag Res 29(3):260–267. doi:10.1177/0734242X10370378

    Article  Google Scholar 

  • Nam G, Lee D, Lee H, Lee N, Kim B, Choe E, Hwang J, Suhartono MT, Pyun Y (2002) Native-feather degradation by Fervidobacterium islandicum AW-1, a newly isolated keratinase-producing thermophilic anaerobe. Arch Microbiol 178:538–547. doi:10.1007/s00203-002-0489-0

    Article  CAS  Google Scholar 

  • Odetallah NH, Wang JJ, Garlich JD, Shih JCH (2005) Versazyme supplementation of broiler diets improves market growth performance. Poult Sci 84:858–864

    CAS  Google Scholar 

  • Onifade AA, Al-Sane NA, Al-Musallan AA, Al-Zarban S (1998) A review: potential for biotechnological applications of keratin-degrading microorganisms and their enzymes for nutritional improvement of feathers and other keratins as livestock feed resource. Bioresour Technol 66:1–11. doi:10.1016/S0960-8524(98)00033-9

    Article  CAS  Google Scholar 

  • Radha S, Gunasekaran P (2009) Purification and characterization of keratinase from recombinant Pichia and Bacillus strains. Protein Expr Purif 64(1):24–31. doi:10.1016/j.pep.2008.10.008

    Article  CAS  Google Scholar 

  • Ramnani P, Singh R, Gupta R (2005) Keratinolytic potential of Bacillus licheniformis RG1: structural and biochemical mechanism of feather degradation. Can J Microbiol 51:191–196. doi:10.1139/W04-123

    Article  CAS  Google Scholar 

  • Riffel A, Lucas F, Heeb P, Brandelli A (2003) Characterization of a new keratinolytic bacterium that completely degrades feather keratin. Arch Microbiol 179:258–265. doi:10.1007/s00203-003-0525-8

    CAS  Google Scholar 

  • Riffel A, Brandelli A, Bellaro CM, Souza GHM, Eberlin MN, Tavarez FCA (2007) Purification and characterization of a keratinolytic metalloprotease from Chryseobacterium sp. kR6. J Biotechnol 128:693–703. doi:10.1016/j.jbiotec.2006.11.007

    Article  CAS  Google Scholar 

  • Riffel A, Daroit DJ, Brandelli A (2011) Nutritional regulation of protease production by the feather-degrading bacterium Chryseobacterium sp. kr6. New Biotechnol 28(2):153–157. doi:10.1016/j.nbt.2010.09.008

    Article  CAS  Google Scholar 

  • Rozs M, Macnczinger L, Vágvölgyi CS, Kevei F (2001) Secretion of a trypsin-like thiol proteases by a new keratinolytic strain of Bacillus licheniformis. FEMS Microbiol Lett 205:221–224. doi:10.1016/S0378-1097(01)00462-1

    Article  CAS  Google Scholar 

  • Suh HJ, Lee HK (2001) Characterization of a keratinolytic serine protease from Bacillus subtilis KS-1. J Protein Chem 20:165–169. doi:10.1023/A:1011075707553

    Article  CAS  Google Scholar 

  • Takami H, Nogi Y, Horikoshi K (1999) Reidentification of the keratinase producing facultatively alkaliphilic Bacillus sp. AH-101 as Bacillus halodurans. Extremophil 2(4):293–296

    Article  Google Scholar 

  • Thys RCS, Lucas FS, Riffel A, Heeb P, Brandelli A (2004) Characterization of a protease of a feather-degrading Microbacterium species. Lett Appl Microbiol 39(2):181–186. doi:10.1111/j.1472-765X.2004.01558.x

    Article  CAS  Google Scholar 

  • Vermelho AB, Mazotto AM, Nogueira de Melo AC, Vieira FHC, Duarte TR, Macrae A, Nishikawa MM, Bon EPS (2010) Identification of a Candida parapsilosis strain producing extra cellular serine peptidase with keratinolytic activity. Mycopathologia 169:57–65. doi:10.1007/s11046-009-9231-7

    Article  CAS  Google Scholar 

  • Wang X, Parsons CM (1997) Effect of processing systems on protein quality of feather meal and hog hair meals. Poultry Sci 76(3):491–496

    CAS  Google Scholar 

  • Warzkiewicz K, Lobarewski J, Wolski T (1987) Intracellular keratinase of Trichophyton gallinae. J Med Vet Mycol 25:261–268

    Article  Google Scholar 

  • Williams CM, Richter CS, Mackeinze JM Jr, Shih JCH (1990) Isolation, identification, and characterization of a feather-degrading bacterium. Appl Environ Microbiol 56:1509–1515

    CAS  Google Scholar 

  • Yamamura S, Yasutaka M, Quamrul H, Yokoyama K, Tamiya E (2002) Keratin degradation: a cooperative action of two enzymes from Stenotrophomonas sp. Biochem Biophys Res Commun 294:1138–1143. doi:10.1016/S0006-291X(02)00580-6

    Article  CAS  Google Scholar 

  • Yoshioka M, Miwa T, Horii H, Takata M, Yokoyama T, Nishizawa K, Watanabe M, Shinagawa M, Muruyama Y (2007) Characterization of a proteolytic enzyme derived from a Bacillus strain that effectively degrades prion protein. J Appl Microbiol 102(2):509–515. doi:10.1111/j.1365-2672.2006.03080.x

    Article  CAS  Google Scholar 

  • Zaghloul TI, Embaby AM, Elmahdy AR (2011) Biodegradation of chicken feathers waste directed by Bacillus subtilis recombinant cells: Scaling up in a laboratory scale fermentor. Bioresour Technol 102:2387–2393. doi:10.1016/j.biortech.2010.10.106

    Article  CAS  Google Scholar 

Download references

Acknowledgments

We thank Ms. Deborah Leite for her technical assistance. This study was supported by grants from Coordenação de Aperfeiçoamento Pessoal de Nível Superior (CAPES), Conselho Nacional de Desenvolvimento Científico e Tecnológico (MCT/CNPq), Conselho de Ensino para Graduados e Pesquisas (CEPG/UFRJ) and Fundação Carlos Chagas Filho de Amparo à Pesquisa do Estado do Rio de Janeiro (FAPERJ). The authors declare no conflicts of interest.

Author information

Authors and Affiliations

  1. Departamento de Microbiologia Geral, Instituto de Microbiologia Paulo de Góes (IMPG), Bloco I, Centro de Ciências da Saúde (CCS), Universidade Federal do Rio de Janeiro (UFRJ), Cidade Universitária, Ilha do Fundão, Rio de Janeiro, RJ, 21941-590, Brazil

    Sabrina Martins Lage Cedrola, Ana Cristina Nogueira de Melo, Ana Maria Mazotto, Ulysses Lins, Alexandre Soares Rosado, Raquel S. Peixoto & Alane Beatriz Vermelho

  2. Departamento de Bioquímica, Instituto de Ciências Biomédicas, Bloco H, Centro de Ciências da Saúde (CCS), Universidade Federal do Rio de Janeiro (UFRJ), Cidade Universitária, Ilha do Fundão, Rio de Janeiro, RJ, 21941-590, Brazil

    Russolina Benedeta Zingali

Authors
  1. Sabrina Martins Lage Cedrola
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Ana Cristina Nogueira de Melo
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Ana Maria Mazotto
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Ulysses Lins
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Russolina Benedeta Zingali
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Alexandre Soares Rosado
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Raquel S. Peixoto
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Alane Beatriz Vermelho
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Alane Beatriz Vermelho.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Cedrola, S.M.L., de Melo, A.C.N., Mazotto, A.M. et al. Keratinases and sulfide from Bacillus subtilis SLC to recycle feather waste. World J Microbiol Biotechnol 28, 1259–1269 (2012). https://doi.org/10.1007/s11274-011-0930-0

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s11274-011-0930-0

Keywords

Search

Navigation