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Optimizing the biodegradation of two keratinous wastes through a Bacillus subtilis recombinant strain using a response surface methodology

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Abstract

Statistical optimization of the biodegradation of two keratinous wastes directed by Bacillus subtilis recombinant cells was carried out by means of a response surface methodology. A Box–Behnken design was employed to predict the optimal levels of three variables namely, keratin percent, incubation time and inoculum size. Analysis of variance revealed that, only keratin percent had the highest significant effect. Canonical analysis and ridge max analysis were used to get the optimal levels of the three predictors along with the optimum levels of the responses. The optimal sets of predicted and validated levels of the three variables were [7.69% (w/v) feathers, 96.58 h and 1.28% (v/v) inoculum size] and [8% (w/v) feathers, 98.45 h, 3.9% (v/v) inoculum size] to achieve the highest levels of soluble proteins (1.25–1.7 mg/ml) and NH2-free amino groups (245.82–270.0 μmol leucine/ml), respectively upon using three optimized feathers-based media. These values represented 83.67–100% and 100% adequacy for the models of soluble proteins and NH2-free amino groups, respectively. While, [8.23% (w/v) sheep wool, 5.52% (v/v) inoculum size and 46.58 h] and [8.33% (w/v) sheep wool, 5.89% (v/v) inoculum size and 63.46 h] were the optimal sets of predicted and validated levels of the above variables to achieve the highest yields of soluble proteins (3.4–4.6 mg/ml) and NH2-free amino groups (290.9–302.0 μmol leucine/ml), respectively upon using three optimized sheep wool-based media. These values represented 100% adequacy for the models of soluble proteins and NH2-free amino groups. By the end of the optimization strategy, a fold enhancement (2.14–2.43 and 1.78–2.12) in the levels of released soluble proteins and NH2-free amino groups, respectively was obtained upon using three optimized feathers-based media. However, a fold enhancement (4.25–5.75 and 2.42–2.5) in the levels of soluble proteins and NH2-free amino groups, respectively was obtained upon using three optimized sheep wool-based media. Data would encourage pilot scale optimization of the biodegradation of these wastes.

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References

  • Adinarayana K, Ellaiah P (2002) Response surface optimization of the critical medium components for the production of alkaline protease by a newly isolated Bacillus sp. J Pharm Pharmceut Sci 5(3):272–278

    CAS  Google Scholar 

  • Anbu P, Gopinath SCB, Hilda A, Lakshmipriya T, Annadurai G (2007) Optimization of extracellular keratinase production by poultry farm isolate Scopulariopsis brevicaulis. Bioresour Technol 98:1298–1303

    Article  CAS  PubMed  Google Scholar 

  • Bernal C, Diaz I, Coello N (2006) Response surface methodology for the optimization of keratinase production in culture medium containing feathers produced by Kocuria rosea. Can J Microbiol 52:445–450

    Article  CAS  PubMed  Google Scholar 

  • Bernhardt K, Schrempf H, Goebel W (1978) Bacteriocin and antibiotic resistance plasmids in Bacillus cereus and Bacillus subtilis. J Bacteriol 133:897–903

    Google Scholar 

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

    PubMed  Google Scholar 

  • Box GEP, Behnken DW (1960) Some new three level design for study of quantitative variables. Technometrics 2:455–475

    Article  Google Scholar 

  • Bradford MM (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye bindings. Anal Biochem 72:248–255

    Article  CAS  PubMed  Google Scholar 

  • Cai C-gang, Lou B-gan, Zheng X-dong (2008) Keratinase production and keratinase degradation by a mutant strain of Bacillus subtilis. J. Zhejiang Univ Sci B 9(1):60–67

    Article  CAS  PubMed  Google Scholar 

  • Cai C, Zheng X (2009) Medium characterization for keratinase production in hair substrate by a new Bacillus subtilis KD-N2 using response surface methodology. J Ind Microbiol Biotechnol 36(7):875–883

    Article  CAS  PubMed  Google Scholar 

  • Cao ZJ, Zhang Q, Wei DK, Chen L, Wang XQ, Zhou MH (2009) Characterization of a novel Stenotrophomomnas isolate with high keratinase activity and purification of the enzyme. J Ind Microbiol Biotechnol 36(2):181–188

    Article  CAS  PubMed  Google Scholar 

  • Chang YN, Huang JC, Lee CC, Shih IL, Tzeng YM (2002) Use of response surface methodology to optimize culture medium for production of lovastatin by Monascus ruber. Enzyme Microbiol Technol 30:889–894

    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  PubMed  Google Scholar 

  • Douglas CM (2001) Design and analysis experiments, 5th edn. Wiley, Arizona, USA

    Google Scholar 

  • Draper NR (1963) Ridge analysis’ of response surfaces. Technometrics 5:469–479

    Article  Google Scholar 

  • El-Refai HA, AbdelNaby MA, Gaballa A, El-Araby MH, Abdel Fattah AF (2005) Improvement of the newly isolated Bacillus pumilus FH9 keratinolytic activity. Process Biochem 40:2325–2332

    Article  CAS  Google Scholar 

  • Gheshlaghi R, Scharer JM, Moo-Young M, Douglas PL (2005) Medium optimization for the hen egg white lysozyme production by recombinant Aspergillus niger using statistical methods. Biotechnol Bioeng 90(6):754–760

    Article  CAS  PubMed  Google Scholar 

  • Gokhade DV, Patil SG, Batawde KB (1991) Optimization of cellulose production by Aspergillus niger NCIM. Appl Biochem Biotechnol 30(2):99–109

    Article  Google Scholar 

  • Gousterova A, Braikova D, Geshov I, Christov P, Tishinov K, Tonkova TE, Haertle T, Nedkov P (2005) Degradation of keratin and collagen wastes by newly isolated Thermoactinomycetes or by alkaline hydrolysis. Lett Appl Microbiol 40:335–340

    Article  CAS  PubMed  Google Scholar 

  • Grazziotin A, Pimentel FA, Sangali S, de Jong EV, Brandelli A (2006) Nutritional improvement of feather protein by treatment with microbial keratinases. Animal Feed Sci Technol 126:135–144

    Article  CAS  Google Scholar 

  • Grazziotin A, Pimentel FA, Sangali S, de Jong EV, Brandelli A (2007) Production of feather protein hydrolysate by keratinolytic bacterium Vibrio sp.kr2. Bioresour Technol 98:3172–3175

    Article  CAS  PubMed  Google Scholar 

  • Gupta R, Rammani P (2006) Microbial keratinases and their prospective applications: an overview. Appl Microbiol Biotechnol 70:21–33

    Article  CAS  PubMed  Google Scholar 

  • Heck JX, De Barros S, Ayub M (2005) Optimization of xylanase and mannanase production by Bacillus circulans strain BL53 on solid state cultivation. Enzyme Microb Technol 37:417–423

    Article  CAS  Google Scholar 

  • Hoerl AE (1959) Optimum solution of many variables equations. Chem Eng Prog 55:67–78

    Google Scholar 

  • Jain Z, Nian-fa G (2007) Application of response surface methodology in medium optimization for pyruvic acid production of Torulopsis glabrata TP19 in batch fermentation. J Zhejiang Univ Sci B 8(2):98–104

    Article  Google Scholar 

  • Kalil SJ, Maugeri F, Roderigues MI (2000) Response surface analysis and simulation as a tool for bioprocess design and optimization. Process Biochem 35:539–550

    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 Manag 29:1409–1415

    Article  CAS  PubMed  Google Scholar 

  • Kim JM, Lim WJ, Suh HJ (2001) Feather-degrading Bacillus species from poultry waste. Process Biochem 37:287–291

    Article  CAS  Google Scholar 

  • Kluskens LD, Voorhorst WGG, Siezen RJ, Schwerdtfeger RM, Antranikian G, van der Oost J, de Vos WM (2002) Molecular characterization of fervidolysin, a subtilisin-like serine protease from the thermophilic bacterium Fervidobacterium pennavorans. Extermophiles 6:185–194

    Article  CAS  Google Scholar 

  • Lin X, Kelemen DW, Miller ES, Shih JCH (1995) Nucleotide sequence and expression of ker A; the gene encoding for a keratinolytic protease of Bacillus licheniformis PWD-1. Appl Environ Microbiol 61:1469–1474

    CAS  PubMed  Google Scholar 

  • Lin HH, Yin LJ, Jiang ST (2009a) Cloning, expression and purification of Pseudomonas aeruginosa keratinase in Escherichia coli AD949 (DE3) pLys S expression system. J Agric Food Chem 57(9):3506–3511

    Article  CAS  PubMed  Google Scholar 

  • Lin HH, Yin LJ, Jiang ST (2009b) Expression and purification of Pseudomonas aeruginosa keratinase in Bacillus subtilis DB104 expression system. J Agric Food Chem 57(17):7779–7784

    Article  CAS  PubMed  Google Scholar 

  • Lohomme B, Roux JC (1991) Utilization of experimental design for optimization of Rhizopus arrhizus culture. Bioresour Technol 35(3):301–312

    Article  Google Scholar 

  • Ionata E, Canganrella F, Bianconi G, Benno Y, Sakamoto M, Capasso A, Rossi M, La Cara F (2008) A novel keratinase from Clostridium sporogenes bv Pennavorans from solfataric muds. Microbiol Res 163:105–112

    Article  CAS  PubMed  Google Scholar 

  • Mitsuiki S, Ichikawa M, Oka T, Sakai M, Moriyama Y, Sameshima Y, Goto M, Furukawa K (2004) Molecular characterization of a keratinolytic enzyme from an alkalophilc Nocardiopsis sp. TOA-1. Enzyme Microb Technol 34:482–489

    Article  CAS  Google Scholar 

  • Myers RH (1976) Response surface methodology. Edwards Brothers, Ann Arbor, MI

    Google Scholar 

  • Nam GW, Lee DW, Lee HS, Lee NJ, Kim BC, Choe EA, Hwang JK, Suhartono MT, Pyum YR (2002) Native-feather degradation by Fervidobacterium islandicum AW-1, a newly isolated keratinase-producing thermophilic anaerobe. Arch Microbiol 178:538–547

    Article  CAS  PubMed  Google Scholar 

  • Onifade AA, Al-Sane NA, Al-Mussalam AA, Al-Zarban S (1998) A review: potentials for biotechnological applications of keratin-degrading microorganisms and their enzymes for nutritional improvement of feathers and other keratins as livestock feed resources. Bioresour Technol 66:1–11

    Article  CAS  Google Scholar 

  • Oulad Haddar H, Zaghloul TI, Saeed HM (2009) Biodegradation of native feather keratin by Bacillus subtilis recombinant strains. Biodegradation 20:687–694

    Article  Google Scholar 

  • Pearce KN, Karahalios D, Friedman M (1988) Ninhydrin assay for proteolysis in ripening cheese. J Food Sci 53(2):432–435

    Article  CAS  Google Scholar 

  • Prakash P, Jayalakshmi SK, Sreeramulu K (2010) Production of keratinase by free and immobilized cells of Bacillus halodurans strain PPKS-2: partial characterization and its application in feather degradation and dehairing of the goat skin. Appl Biochem Biotechnol 160(7):1909–1920

    Article  CAS  PubMed  Google Scholar 

  • Radha S, Gunasekaran P (2008) Sustained expression of keratinase gene under PxylA and Pamyl promoters in the recombinant Bacillus megaterium MS 941. Bioresour Technol 99:5528–5537

    Article  CAS  PubMed  Google Scholar 

  • R Development Core Team (2009). R: a language and environment for statistical computing. R Foundation for Statistical Computing, Vienna, Austria. ISBN 3-900051-07-0, http://www.R-project.org/

  • Rucka M, Lamer-Zarawska E, Maliszewska I, Turkiewicz B (1998) Optimization of growth and hydrolytic enzymes production by Fusarium culmorum using response surface methodology. Bioprocess Eng 19:229–232

    CAS  Google Scholar 

  • Sangali S, Brandelli A (2000) Feather keratin hydrolysis by a Vibrio sp. strain kr2. J Appl Microbiol 89:735–743

    Article  CAS  PubMed  Google Scholar 

  • Santos RMDB, Firmino AAP, de Sa CM, Felix CR (1996) Keratinolytic activity of Aspergillus fumigatus Fresenius. Curr Microbiol 33:364–370

    Article  CAS  Google Scholar 

  • Sharma A, Bardhan D, Patel R (2009) Optimization of physical parameters for lipase production from Arthrobacter sp. BGCC#490. Ind J Biochem Biophys 46(2):178–183

    CAS  Google Scholar 

  • Syed DG, Lee JC, Li WJ, Kim CJ, Agasar D (2009) Production, characterization and application of keratinase from Streptomyces gulbargensis. Bioresour Technol 100:1868–1871

    Article  CAS  PubMed  Google Scholar 

  • Wang JJ, Swaisgood HE, Shih JCH (2003) Bioimobilization of keratinase using Bacillus subtilis and Escherichia coli systems. Biotechnol Bioeng 81:21–429

    Article  Google Scholar 

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

    CAS  PubMed  Google Scholar 

  • Xie F, Chao Y, Yang X, Yang J, Xue Z, Luo Y, Qian S (2010) Purification and characterization of four keratinases produced by Streptomyces sp. strain 16 in native human foot skin medium. Biores Technol 101(1):344–500

    Article  Google Scholar 

  • Zaghloul TI, Abdelaziz A, Moustafa MH (1994) High level of expression and stability of the cloned alkaline protease (aprA) gene in Bacillus subtilis. Enzyme Microbiol Technol 16:534–537

    Article  CAS  Google Scholar 

  • Zhang J, Marcin C, Shifflet MA, Salmon P, Brix T, Greasham R, Boukland B, Chartrain M (1996) Development of fermentation process for physotigmine production by Streptomyces griseofuscus. Appl Microbiol Biotechnol 44(5):568–575

    Article  CAS  PubMed  Google Scholar 

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Acknowledgements

The authors are very grateful to Chemist/Heba-Allah S. Marey, Assistant lecturer of the Institute of Graduate Studies and Research (IGSR), Environmental Studies Department, Alexandria University, Egypt and Dr. Nadia A. Soliman, Assistant professor of Mubarak City for Scientific Research and Technology, Bioprocess Department, Alexandria, Egypt for their great help concerning modeling.

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Authors and Affiliations

  1. Department of Biotechnology, Institute of Graduate Studies and Research, Alexandria University, 163 El Horreya Aven., EL Chatby, P.O. Box 830, Egypt

    Amira M. Embaby & Taha I. Zaghloul

  2. Department of Food Science and Technology, Faculty of Agriculture, Alexandria University, Alexandria, Egypt

    Ahmed R. Elmahdy

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  1. Amira M. Embaby
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  2. Taha I. Zaghloul
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  3. Ahmed R. Elmahdy
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Correspondence to Amira M. Embaby.

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Embaby, A.M., Zaghloul, T.I. & Elmahdy, A.R. Optimizing the biodegradation of two keratinous wastes through a Bacillus subtilis recombinant strain using a response surface methodology. Biodegradation 21, 1077–1092 (2010). https://doi.org/10.1007/s10532-010-9368-6

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