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Biodegradation of Shrimp Biowaste by Marine Exiguobacterium sp. CFR26M and Concomitant Production of Extracellular Protease and Antioxidant Materials: Production and Process Optimization by Response Surface Methodology

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Abstract

Twelve marine bacterial cultures were screened for extracellular protease activity, and the bacterium CFR26M which exhibited the highest activity on caseinate agar plate was identified as an Exiguobacterium sp. Significant amount of extracellular protease (5.9 ± 0.3 U/ml) and antioxidant materials, measured as 2,2′-diphenyl picrylhydrazyl (DPPH) radical scavenging activity (44.4 ± 0.5 %), was produced by CFR26M in submerged fermentation using a shrimp biowaste medium. Response surface methodology (RSM) was employed to optimize the process variables for maximum production of protease and antioxidant materials by CFR26M. Among the seven variables screened by two-level 2**(7–2) fractional factorial design, the concentration of shrimp biowaste, sugar, and phosphate was found to be significant (p ≤ 0.05). The optimum levels of these variables were determined by employing the central composite design (CCD) of RSM. The coefficient of determination (R 2) values of 0.9039 and 0.8924 for protease and antioxidant, respectively, indicates the accuracy of the CCD models. The optimum levels of shrimp biowaste, sugar, and phosphate were 21.2, 10.5, and 2.3 % (w/v) for production of protease and 28.8, 12, and 0.32 % (w/v) for production of antioxidant material, respectively. The concentration of shrimp biowaste, sugar, and phosphate had linear and quadratic effect on both protease and antioxidant productions. RSM optimization yielded 6.3-fold increases in protease activity and 1.6-fold in antioxidant material production. The crude protease of CFR26M had a maximum activity at 32 ± 2 °C with pH 7.6. This is the first report on the use of marine Exiguobacterium sp. for concomitant production of protease and antioxidant materials from shrimp biowaste.

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References

  • Altschul SF, Gish W, Miller W, Myers EW, Lipman DJ (1990) Basic local alignment search tool. J Mol Biol 215:403–410

    Article  CAS  PubMed  Google Scholar 

  • Amoozegar MA, Fatemi AZ, Karbalaei-Heidari HR, Razavi MR (2007) Production of an extracellular alkaline metalloprotease from a newly isolated, moderately halophile, Salinivibrio sp. strain AF-2004. Microbiol Res 162:369–377

    Article  CAS  Google Scholar 

  • Annamalai N, Rajeswari MV, Vijayalakshmi S, Balasubramanian T (2011) Purification and characterization of chitinase from Alcaligenes faecalis AU02 by utilizing marine wastes and its antioxidant activity. Ann Microbiol 61:801–807

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  • AOAC (2000) Official methods of analysis of AOAC International, vols. 1 and 2, 17th edn. AOAC International, Gaithersburg

    Google Scholar 

  • APHA (1998) Standard methods for examination of waste and waste water, 20th edn. American Public Health Association, Washington DC

    Google Scholar 

  • Beg QK, Vikram S, Gupta R (2003) Statistical media optimization and alkaline protease production from Bacillus mojavensis in a bioreactor. Process Biochem 39:203–209

    Article  CAS  Google Scholar 

  • Bhaskar N, Suresh PV, Sakhare PZ, Sachindra NM (2007) Shrimp bio-waste fermentation with Pediococcus acidolactici CFR2182: optimization of fermentation conditions by response surface methodology and effect of optimized conditions on deproteinization/demineralization and carotenoid recovery. Enzymes Microb Technol 40:1427–1434

    Article  CAS  Google Scholar 

  • Chandrasekaran M (1997) Industrial enzymes from marine microorganisms: the Indian scenario. J Mar Biotchnol 5:86–89

    CAS  Google Scholar 

  • Chauhan B, Gupta R (2004) Application of statistical experimental design for optimization of alkaline protease production from Bacillus sp. RGR-14. Process Biochem 39:2115–2122

    Article  CAS  Google Scholar 

  • Claudiana PS, Bianca CA, Rita RC, Irma NGR (2011) The Importance of chitin in the marine environment. Mar Biotechnol 13:823–830

    Article  Google Scholar 

  • Dhanya G, Swetha S, Nampoothiri MK, Rajeev KS, Pandey A (2008) Response surface methodology for the optimization of alpha amylase production by Bacillus amyloliquefaciens. Bioresour Technol 99:4597–4602

    Article  Google Scholar 

  • Duan XJ, Zhang WW, Li XM, Wang BG (2006) Evaluation of antioxidant property of extract and fractions obtained from a red alga, Polysiphonia urceolata. Food Chem 95:37–43

    Article  CAS  Google Scholar 

  • FAO (2010) Yearbook of fishery statistics. Statistics and Information Service, FAO Fisheries and Aquaculture Department. Food and Agriculture Organization of the United Nations, Rome

    Google Scholar 

  • Gooday GW (1990) Physiology of microbial degradation of chitin and chitosan. Biodegradation 1:177–190

    Article  CAS  Google Scholar 

  • Greene RV, Griffin HL, Cotta MA (1996) Utility of alkaline protease from marine shipworm bacterium in industrial cleansing applications. Biotechnol Lett 18:759–764

    Article  CAS  Google Scholar 

  • Gupta R, Beg QK, Lorenz P (2002) Bacterial alkaline proteases: molecular approaches and industrial applications. Appl Microbiol Biotechnol 59:15–32

    Article  CAS  PubMed  Google Scholar 

  • Healy MG, Romo CR, Bustos R (1994) Bioconversion of marine crustacean shell waste. Resour Conserv Recycling 11:139–147

    Article  Google Scholar 

  • Karbalaei-Heidari HR, Abed-Ali Z, Schaller J, Amoozegar MA (2007) Purification and characterization of an extracellular haloalkaline protease produced by the moderately halophilic bacterium Salinivibrio sp. strain AF-2004. Enzyme Microb Technol 40:266–272

    Article  CAS  Google Scholar 

  • Kumar S, Satyanarayana T (2004) Statistical optimization of a thermostable and natural glucoamylase production by thermophilic mold Thermomucor indicae-seudaticae in solid state fermentation. World J Microbiol Biotechnol 20:895–902

    Article  CAS  Google Scholar 

  • Kumar S, Tamura K, Nei M (2004) MEGA3: integrated software for molecular evolutionary genetics analysis and sequence alignment. Brief Bioinform 5:150–163

    Article  CAS  PubMed  Google Scholar 

  • Kunitz M (1947) Crystalline soyabean trypsin inhibitor II: general properties. J Gen Physiol 30(4):291–310

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  • Kuo YH, Liang TW, Liu KC, Hsu YW, Hsu HC, Wang SL (2011) Isolation and identification of a novel antioxidant with antitumour activity from Serratia ureilytica using squid pen as fermentation substrate. Mar Biotechnol 13:451–461

    Article  CAS  PubMed  Google Scholar 

  • Laxman RS, Sonawane AP, More SV, Rao BS, Rele MV, Jogdand VV, Deshpande VV, Rao MB (2005) Optimization and scale up of production of alkaline protease from Conidiobolus coronatus. Process Biochem 40:3152–3158

    Article  CAS  Google Scholar 

  • Mantgomery DC (1984) Design and analysis of experiments, 2nd edn. Wiley, Singapore

    Google Scholar 

  • Mathew P, Nair KGR (2006) Ensilation of shrimp waste by Lactobacillus fermentum. Fish Technol 43:59–62

    Google Scholar 

  • Meera V, Saramma AV (2007) An alkaline protease from Bacillus circulans BM15, newly isolated from a mangrove station: characterization and application in laundry detergent formulations. Ind J Microbiol 47:298–303

    Article  Google Scholar 

  • Mohapatra BR, Bapuji M, Sree A (2003) Production of industrial enzymes (amylase, carboxymethyl cellulase and protease) by bacteria isolated from marine sedentary organisms. Acta Biotechnol 23:75–84

    Article  CAS  Google Scholar 

  • Perk JK, Morita K, Fukumoto I, Yamasaki Y, Nakagawa T, Kawamukai M, Matsuda H (1997) Purification and characterization of the chitinase (ChiA) from Enterobacter sp. G-1. Biosci Biotechnol Biochem 61:684–689

    Article  Google Scholar 

  • Prabhu GN, Chandrasekaran M (1999) l-Glutaminase production by marine Vibrio costicola under solid state fermentation using different substrate. J Mar Biotechnol 4:176–179

    Google Scholar 

  • Priya R, Suresh SK, Rani G (2005) Concomitant production and downstream processing of alkaline protease and biosurfactant from Bacillus licheniformis RG1: bioformulation as detergent additive. Process Biochem 40:3352–3359

    Article  Google Scholar 

  • Puri S, Khali O, Gupta R (2002) Optimization of alkaline protease production from Bacillus sp. by response surface methodology. Curr Microbiol 44:286–290

    Article  CAS  PubMed  Google Scholar 

  • Rao MB, Aparna MT, Mohini SG, Deshpande VV (1998) Molecular and biotechnological aspects of microbial proteases. Microbiol Mol Bio Rev 62:597–635

    CAS  Google Scholar 

  • Rao YK, Lu S, Liu B, Tzeng Y (2006) Enhanced production of an extracellular protease from Beauveria bassiana by optimization of cultivation. Biochem Eng J 28:57–66

    Article  CAS  Google Scholar 

  • Ressing JL, Strominger JL, Leloir LF (1955) A modified colorimetric methods for estimation of N-acetyl amino sugars. J Bio Chem 217:959–962

    Google Scholar 

  • Roberta CS, Thys S, Florencia CO, Adriano B (2006) Optimization of protease production by Microbacterium sp. in feather meal using response surface methodology. Process Biochem 41:67–73

    Google Scholar 

  • Rondle CJ, Morgan WT (1955) The determination of glucosamine and galactosamine. Biochem J 61(4):586–589

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  • Sachindra NM, Bhaskar N, Siddegowda GS, Sathisha AD, Suresh PV (2007) Recovery of carotenoids from ensilaged shrimp waste. Bioresour Technol 98:1642–1646

    Article  CAS  PubMed  Google Scholar 

  • Sandhya C, Sumantha A, Szakacs G, Pandey A (2005) Comparative evaluation of neutral protease production by Aspergillus oryzae in submerged and solid-state fermentation. Process Biochem 40:689–2694

    Google Scholar 

  • Sorokulova I, Krumnow A, Globa L, Vodyanoy V (2009) Efficient decomposition of shrimp shell waste using Bacillus cereus and Exiguobacterium acetylicum. J Ind Microbiol Biotechnol 36:1123–1126

    Article  CAS  PubMed  Google Scholar 

  • Spinelli J, Lehman L, Wieg D (1974) Composition, processing and utilization of red crab (Pleuroncodes planipes) as an aquaculture feed ingredient. J Fish Res Board Canada 31:1025–1030

    Article  CAS  Google Scholar 

  • StatSoft (1999) Statistica for windows. StatSoft, Inc., Tulsa

    Google Scholar 

  • Subramani R, Narayanasamy M (2009) Screening of marine actinomycetes isolated from the Bay of Bengal, India for antimicrobial activity and industrial enzymes. World J Microbiol Biotechnol 25:2103–2111

    Article  Google Scholar 

  • Suresh PV, Anil Kumar PK (2012) Enhanced degradation of α-chitin materials prepared from shrimp processing byproduct and production of N-acetyl-d-glucosamine by thermoactive chitinases from soil mesophilic fungi. Biodegradation. doi:10.1007/s10532-012-9536-y

    PubMed  Google Scholar 

  • Suresh PV, Chandrasekaran M (1998) Utilization of prawn waste for chitinase production by the marine fungus Beauveria bassiana by solid state fermentation. World J Microbiol Biotechnol 14:655–660

    Article  CAS  Google Scholar 

  • Suresh PV, Chandrasekaran M (1999) Impact of process parameters on chitinase production by an alkalophilic marine Beauveria bassiana in solid state fermentation. Process Biochem 43(3):257–267

    Article  Google Scholar 

  • Suresh PV, Anil Kumar PK, Sachindra NM (2011a) Thermoactive β-N-acetylhexosaminidase production by a soil isolate of Penicillium monoverticillium CFR 2 under solid state fermentation: parameter optimization and application for N-acetyl chitooligosaccharides preparation from chitin. World J Microbiol Biotechnol 27:1435–1447

    Article  CAS  PubMed  Google Scholar 

  • Suresh PV, Sachindra NM, Bhaskar N (2011b) Solid state fermentation production of chitin deacetylase by Colletotrichum lindemuthianum ATCC 56676 using different substrates. J Food Sci Technol 48(3):349–356

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  • Synowiecki J, Al-Khateeb NA (2000) The recovery of protein hydrolysate during enzymatic isolation of chitin from shrimp Crangon crangon processing discards. Food Chem 68:147–152

    Article  CAS  Google Scholar 

  • Tari C, Genckal H, Tokatli F (2006) Optimization of growth medium using a statistical approach for the production of alkaline protease from a newly isolated Bacillus sp. L 21. Proc Biochem 41:659–665

    Article  CAS  Google Scholar 

  • Ventosa A, Nieto JJ (1995) Biotechnological applications and potentialities of halophilic microorganisms. World J Microbiol Biotechnol 11:85–94

    Article  CAS  PubMed  Google Scholar 

  • Wang SL, Lin TY, Yen YH, Liao FH, Chen YJ (2006) Bioconversion of shellfish chitin wastes for the production of Bacillus subtilis W-118 chitinase. Carbohydr Res 341:2507–2515

    Article  CAS  PubMed  Google Scholar 

  • Wang SL, Lin HT, Liang TW, Chen YJ, Yen YH, Guo SP (2008) Reclamation of chitinous materials by bromelain for the preparation of antitumor and antifungal materials. Bioresour Technol 99:4386–4393

    Article  CAS  PubMed  Google Scholar 

  • Wang SL, Chao CH, Liang TW, Chen CC (2009) Purification and characterization of protease and chitinase from Bacillus cereus TKU006 and conversion of marine wastes by these enzymes. Mar Biotechnol 11:334–344

    Article  CAS  PubMed  Google Scholar 

  • Wang SL, Tao JC, Tzu WL (2010a) Conversion and degradation of shellfish waste by Serratia sp. TKU016 fermentation for the production of enzyme and bioactive materials. Biodegradation 21:321–333

    Article  CAS  PubMed  Google Scholar 

  • Wang SL, Liu KC, Liang TW, Kuo YH, Wang CY (2010b) In vitro antioxidant activity of liquor and semi purified fraction from fermented squid pen bio-waste by Serratia ureilytica TKU013. Food Chem 119:1380–1385

    Article  CAS  Google Scholar 

  • Wang SL, Liang TW, Yen YH (2011) Bioconversion of chitin-containing wastes for the production of enzymes and bioactive materials. Carbohydr Polym 84:732–742

    Article  CAS  Google Scholar 

  • Zhang L, An R, Wang J, Sun N, Zhang S, Hu J, Kuai J (2005) Exploring novel bioactive compounds from marine microbes. Curr Opin Microbiol 8:276–281

    Article  CAS  PubMed  Google Scholar 

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Acknowledgments

SPV thanks the Council of Scientific and Industrial Research (CSIR), Government of India, for the partial funding under CSIR-EMPOWER Scheme, and APK thanks the CSIR for RA.

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Correspondence to P. V. Suresh.

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Anil Kumar, P.K., Suresh, P.V. Biodegradation of Shrimp Biowaste by Marine Exiguobacterium sp. CFR26M and Concomitant Production of Extracellular Protease and Antioxidant Materials: Production and Process Optimization by Response Surface Methodology. Mar Biotechnol 16, 202–218 (2014). https://doi.org/10.1007/s10126-013-9531-2

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