Abstract
Chitin extraction from shrimp shell powder (SSP) using protease-producing microbes is an attractive approach for valorizing shrimp shell waste because it is simple and environmentally friendly. In this study, the protease production and chitin extraction from SSP by Bacillus cereus HMRSC30 were simultaneously optimized using statistical approaches. As a result, fermentation in medium composed of 30 g/L SSP, 0.2 g/L MgSO4 · 7H2O, 3 g/L (NH4)2SO4, 0.5 g/L K2HPO4, and 1.5 g/L KH2PO4 (pH 6.5) for 7 days maximized protease production (197.75 ± 0.33 U/mL) to approximately 1.64-fold compared to unoptimized condition (126.8 ± 0.047 U/mL). This level of enzyme production was enough to achieve 97.42 ± 0.28% deproteinization (DP) but low demineralization (DM) of 53.76 ± 0.21%. The high DM of 90% could be easily accomplished with the post-treatment using 0.4 M HCl and acetic acid. In addition, the study evaluated the possible roadmap to maximize the value of generated products and obtain additional profits from this microbial process. The observation showed the possibility of serving crude chitin as a bio-adsorbent with the highest removal capacity against Coomassie brilliant blue (97.99%), followed by methylene blue (74.42%). The recovered protease exhibited the function to remove egg yolk stain, indicating its potential for use as a detergent in de-staining. The results corroborated the benefits of microbial fermentation by B. cereus HMRSC30 as green process for comprehensive utilization of shrimp shell waste as well as minimizing waste generation along the established process.
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The datasets generated and analyzed during the current study are available from the corresponding author on reasonable request.
References
Abdel Wahab WA, Ahmed SA (2018) Response surface methodology for production, characterization and application of solvent, salt and alkali-tolerant alkaline protease from isolated fungal strain Aspergillus niger WA 2017. Int J Biol Macromol 115:447–458. https://doi.org/10.1016/j.ijbiomac.2018.04.041
Abusham RA, Rahman RNZRA, Salleh A, Basri M (2009) Optimization of physical factors affecting the production of thermo-stable organic solvent-tolerant protease from a newly isolated halo tolerant Bacillus subtilis strain Rand. Microb Cell Fact 8:20. https://doi.org/10.1186/1475-2859-8-20
Ahlafi H, Moussout H, Boukhlifi F, Echetna M, Bennani MN, My Slimane S (2013) Kinetics of N-deacetylation of chitin extracted from shrimp shells collected from coastal area of Morocco. Mediterr J Chem 2:503–513. https://doi.org/10.13171/mjc.2.3.2013.22.01.20
Alca Ahing F, Wid N (2016) Extraction and characterization of chitosan from shrimp shell waste in Sabah. Trans Sci Technol 3:227–237
Antonino RSCMDQ, Fook BRPL, Lima VADO, Rached RÍDF, Lima EPN, Lima RJDS, Covas CAP, Fook MVL (2017) Preparation and characterization of chitosan obtained from shells of shrimp (Litopenaeus vannamei Boone). Mar Drugs 15:1–12. https://doi.org/10.3390/md15050141
Aranaz I, Mengibar M, Harris R, Panos I, Miralles B, Acosta N, Galed G, Heras A (2009) Functional characterization of chitin and chitosan. Curr Chem Biol 3:203–230. https://doi.org/10.2174/187231309788166415
Arbia W, Adour L, Amrane A, Lounici H (2013) Optimization of medium composition for enhanced chitin extraction from Parapenaeus longirostris by Lactobacillus helveticus using response surface methodology. Food Hydrocoll 31:392–403. https://doi.org/10.1016/j.foodhyd.2012.10.025
Asha B, Palaniswamy M (2018) Optimization of alkaline protease production by Bacillus cereus FT 1 isolated from soil. J Appl Pharm Sci 8:119–127. https://doi.org/10.7324/JAPS.2018.8219
Bhunia B, Dey A (2012) Statistical approach for optimization of physiochemical requirements on alkaline protease production from Bacillus licheniformis NCIM 2042. Enzyme Res. https://doi.org/10.1155/2012/905804
Cadano JR, Jose M, Lubi AG, Maling JN, Moraga JS, Shi QY, Vegafria HM, VinceCruz-Abeledo CC (2021) A comparative study on the raw chitin and chitosan yields of common bio-waste from Philippine seafood. Environ Sci Pollut Res 28:11954–11961. https://doi.org/10.1007/s11356-020-08380-5
Cahú TB, Santos SD, Mendes A, Córdula CR, Chavante SF, Carvalho LB, Nader HB, Bezerra RS (2012) Recovery of protein, chitin, carotenoids and glycosaminoglycans from Pacific white shrimp (Litopenaeus vannamei) processing waste. Process Biochem 47:570–577. https://doi.org/10.1016/j.procbio.2011.12.012
Cahyaningtyas HAA, Suyotha W, Cheirsilp B, Yano S (2021) Statistical optimization of halophilic chitosanase and protease production by Bacillus cereus HMRSC30 isolated from Terasi simultaneous with chitin extraction from shrimp shell waste. Biocatal Agric Biotechnol 31:101918. https://doi.org/10.1016/j.bcab.2021.101918
Castro R, Guerrero-Legarreta I, Bórquez R (2018) Chitin extraction from Allopetrolisthes punctatus crab using lactic fermentation. Biotechnol Reports 20:e00287. https://doi.org/10.1016/j.btre.2018.e00287
Chopra AK, Mathur DK (2016) Factors affecting protease production by Bacillus stearothermophilus RM-67. J Food Prot 46:1020–1025. https://doi.org/10.4315/0362-028x-46.12.1020
Chuprom J, Bovornreungroj P, Ahmad M, Kantachote D, Dueramae S (2016) Approach toward enhancement of halophilic protease production by Halobacterium sp. strain LBU50301 using statistical design response surface methodology. Biotechnol Reports 10:17–28. https://doi.org/10.1016/j.btre.2016.02.004
Cira LA, Huerta S, Hall GM, Shirai K (2002) Pilot scale lactic acid fermentation of shrimp wastes for chitin recovery. Process Biochem 37:1359–1366. https://doi.org/10.1016/S0032-9592(02)00008-0
Dahmane EM, Taourirte M, Eladlani N, Rhazi M (2014) Extraction and characterization of chitin and chitosan from Parapenaeus longirostris from Moroccan local sources. Int J Polym Anal Charact 19:342–351. https://doi.org/10.1080/1023666X.2014.902577
Das MP, Renuka M, Vijaylakshmi JV, Suguna PR, Karpuramprasad (2018) Removal of methylene blue by adsorption using fish scale chitin. Nat Environ Pollut Technol 17: 993–998.
Demir D, Öfkeli F, Ceylan S, Bölgen N (2016) Extraction and characterization of chitin and chitosan from blue brab and synthesis of chitosan cryogel scaffolds. J Turkish Chem Soc Sect A Chem 3(3):131–144. https://doi.org/10.18596/jotcsa.00634
Dhananasekaran S, Palanivel R, Pappu S (2016) Adsorption of methylene blue, bromophenol blue, and Coomassie brilliant blue by α-chitin nanoparticles. J Adv Res 7:113–124. https://doi.org/10.1016/j.jare.2015.03.003
Doan CT, Tran TN, Nguyen VB, Vo TPK, Nguyen AD, Wang SL (2019a) Chitin extraction from shrimp waste by liquid fermentation using an alkaline protease-producing strain, Brevibacillus parabrevis. Int J Biol Macromol 131:706–715. https://doi.org/10.1016/j.ijbiomac.2019.03.117
Doan CT, Tran TN, Wen IH, Nguyen VB, Nguyen AD, Wang SL (2019b) Conversion of shrimp head waste for production of a thermotolerant, detergent-stable, alkaline protease by Paenibacillus sp. Catalysts 9:798. https://doi.org/10.3390/catal9100798
Ebrahimzadeh MA, Chabra A, Gharaei-Fathabad E, Pourmorad F (2013) Preparation of chitosan from Penicillium spp. and determination of their degree of deacetylation. Indian J Biotechnol 12:231–235
Francisco F, Mae R, Simora C (2015) Deproteinization and demineralization of shrimp waste using lactic bacteria for the production of crude chitin and chitosan. AACL Bioflux 8(1):107–115. http://www.bioflux.com.ro/aacl. Accessed May 2021
Ganesh Kumar A, Swarnalatha S, Sairam B, Sekaran G (2008) Production of alkaline protease by Pseudomonas aeruginosa using proteinaceous solid waste generated from leather manufacturing industries. Bioresour Technol 99:1939–1944. https://doi.org/10.1016/j.biortech.2007.03.025
Gamal RF, El-Tayeb TS, Raffat EI, Ibrahim HMM, Bashandy AS (2016) Optimization of chitin yield from shrimp shell waste by Bacillus subtilis and impact of gamma irradiation on production of low molecular weight chitosan. Int J Biol Macromol 91:598–608. https://doi.org/10.1016/j.ijbiomac.2016.06.008
Gbenebor OP, Adeosun SO, Lawal GI, Jun S, Olaleye SA (2017) Acetylation, crystalline and morphological properties of structural polysaccharide from shrimp exoskeleton. Eng Sci Technol an Int J 20:1155–1165. https://doi.org/10.1016/j.jestch.2017.05.002
Gharieb MM, El-sabbagh SM, Shalaby MA, Darwesh OM (2015) Production of chitosan from different species of zygomycetes and its antimicrobial activity. Int J Sci Eng Res 6:123–130
Ghorbel-Bellaaj O, Hajji S, Younes I, Chaabouni M, Nasri M, Jellouli K (2013) Optimization of chitin extraction from shrimp waste with Bacillus pumilus A1 using response surface methodology. Int J Biol Macromol 61:243–250. https://doi.org/10.1016/j.ijbiomac.2013.07.001
Ghorbel-Bellaaj O, Younes I, Maâlej H, Hajji S, Nasri M (2012) Chitin extraction from shrimp shell waste using Bacillus bacteria. Int J Biol Macromol 51:1196–1201. https://doi.org/10.1016/j.ijbiomac.2012.08.034
Hackman RH (1962) Studies on Chitin V. The action of mineral acids on chitin. Aust J Biol Sci 15:526–537. https://doi.org/10.1071/BI9620526
Haddar A, Hmidet N, Ghorbel-Bellaaj O, Fakhfakh-Zouari N, Sellami-Kamoun A, Nasri M (2011) Alkaline proteases produced by Bacillus licheniformis RP1 grown on shrimp wastes: Application in chitin extraction, chicken feather-degradation and as a dehairing agent. Biotechnol Bioprocess Eng 16:669–678. https://doi.org/10.1007/s12257-010-0410-7
Hamdi M, Hammami A, Hajji S, Jridi M, Nasri M, Nasri R (2017) Chitin extraction from blue crab (Portunus segnis) and shrimp (Penaeus kerathurus) shells using digestive alkaline proteases from P. segnis viscera. Int J Biol Macromol 101:455–463. https://doi.org/10.1016/j.ijbiomac.2017.02.103
Khedr SA, Shouman MA, Attia AA (2012) Adsorption studies on the removal of cationic dye from shrimp shell using chitin. Biointerface Res Appl Chem 3:507–519
Khorrami M, Younesi H, Hosseinpour M, Najafpour G (2012) Production of chitin and chitosan from shrimp shell in batch culture of Lactobacillus plantarum. Chem Biochem Eng Q 26:217–223
Kotlar C, Ponce A, Roura S (2015) Characterization of a novel protease from Bacillus cereus and evaluation of an eco-friendly hydrolysis of a brewery byproduct. J Inst Brew 121:558–565. https://doi.org/10.1002/jib.257
Lertsutthiwong P, How NC, Chandkrachang S, Stevens WF (2002) Effect of chemical treatment on the characteristics of shrimp chitosan. J Met Mater Miner 12:11–18
Liang TW, Hsieh JL, Wang SL (2012) Production and purification of a protease, a chitosanase, and chitin oligosaccharides by Bacillus cereus TKU022 fermentation. Carbohydr Res 362:38–46. https://doi.org/10.1016/j.carres.2012.08.004
Liang TW, Kuo YH, Wu PC, Wang CL, Dzung NA, Wang SL (2010) Purification and characterization of a chitosanase and a protease by conversion of shrimp shell wastes fermented by Serratia Marcescens subsp. sakuensis TKU019. J Chinese Chem Soc 57:857–863. https://doi.org/10.1002/jccs.201000119
Liang TW, Lin JJ, Yen YH, Wang CL, Wang SL (2006) Purification and characterization of a protease extracellularly produced by Monascus purpureus CCRC31499 in a shrimp and crab shell powder medium. Enzyme Microb Technol 38:74–80. https://doi.org/10.1016/j.enzmictec.2005.04.023
Liu Y, Xing R, Yang H, Liu S, Qin Y, Li K, Yu H, Li P (2020) Chitin extraction from shrimp (Litopenaeus vannamei) shells by successive two-step fermentation with Lactobacillus rhamnoides and Bacillus amyloliquefaciens. Int J Biol Macromol 148:424–433. https://doi.org/10.1016/j.ijbiomac.2020.01.124
Lowry OH, Rosebrought NJ, Farr AL, Randall RJ (1951) Protein measurement with the folin phenol reagent. J Biol Chem 193:265–275
Maruthiah T, Palavesam A (2017) Characterization of haloalkalophilic organic solvent tolerant protease for chitin extraction from shrimp shell waste. Int J Biol Macromol 97:552–560. https://doi.org/10.1016/j.ijbiomac.2017.01.021
Mechri S, Sellem I, Bouacem K, Jabeur F, Laribi-Habchi H, Mellouli L, Hacène H, Bouanane-Darenfed A, Jaouadi B (2020) A biological clean processing approach for the valorization of speckled shrimp Metapenaeus monoceros by-product as a source of bioactive compounds. Environ Sci Pollut Res 27:15842–15855. https://doi.org/10.1007/s11356-020-08076-w
Mekahlia S, Bouzid B (2009) Chitosan-Copper (II) complex as antibacterial agent: synthesis, characterization and coordinating bond- activity correlation study. Phys Procedia 2:1045–1053. https://doi.org/10.1016/j.phpro.2009.11.061
Moosa AA, Ridha AM, Kadhim A (2016) Use of biocomposite adsorbents for the removal of methylene blue dye from aqueous solution. Am J Mater Sci 6:135–146. https://doi.org/10.5923/j.materials.20160605.03
Nessa F, Masum SM, Asaduzzaman M, Roy S, Hossain M, Jahan M (2011) A process for the preparation of chitin and chitosan from prawn shell waste. Bangladesh J Sci Ind Res 45:323–330. https://doi.org/10.3329/bjsir.v45i4.7330
Oh YS, Shih IL, Tzeng YM, Wang SL (2000) Protease produced by Pseudomonas aeruginosa K-187 and its application in the deproteinization of shrimp and crab shell wastes. Enzyme Microb Technol 27:3–10. https://doi.org/10.1016/S0141-0229(99)00172-6
Özgür K, Nilüfer C (2011) Isolation of protease producing novel Bacillus cereus and detection of optimal conditions. African J Biotechnol 10:1160–1164. https://doi.org/10.4314/ajb.v10i7
Pachapur VL, Guemiza K, Rouissi T, Sarma SJ, Brar SK (2016) Novel biological and chemical methods of chitin extraction from crustacean waste using saline water. J Chem Technol Biotechnol 91:2331–2339. https://doi.org/10.1002/jctb.4821
Pal K, Rakshit S, Mondal KC, Halder SK (2021) Microbial decomposition of crustacean shell for production of bioactive metabolites and study of its fertilizing potential. Environ Sci Pollut Res. https://doi.org/10.1007/s11356-021-13109-z
Patel AR, Mokashe NU, Chaudhari DS, Jadhav AG, Patil UK (2019) Production optimisation and characterisation of extracellular protease secreted by newly isolated Bacillus subtilis AU-2 strain obtained from Tribolium castaneum gut. Biocatal Agric Biotechnol 19:101122. https://doi.org/10.1016/j.bcab.2019.101122
Rao MS, Muñoz J, Stevens WF (2000) Critical factors in chitin production by fermentation of shrimp biowaste. Appl Microbiol Biotechnol 54:808–813. https://doi.org/10.1007/s002530000449
Santhi R (2014) Microbial production of protease by Bacillus cereus using cassava waste water. Pelagia Res Libr Eur J Exp Biol 4:19–24
Sathish Kumar R, Ananthan G, Selva Prabhu A (2014) Optimization of medium composition for alkaline protease production by Marinobacter sp. GA CAS9 using response surface methodology - a statistical approach. Biocatal Agric Biotechnol 3:191–197. https://doi.org/10.1016/j.bcab.2013.11.005
Sedaghat F, Yousefzadi M, Toiserkani H, Najafipour S (2017) Bioconversion of shrimp waste Penaeus merguiensis using lactic acid fermentation: an alternative procedure for chemical extraction of chitin and chitosan. Int J Biol Macromol 104:883–888. https://doi.org/10.1016/j.ijbiomac.2017.06.099
Sobucki L, Ramos RF, Daroit DJ (2017) Protease production by the keratinolytic Bacillus sp. CL18 through feather bioprocessing. Environ Sci Pollut Res 24:23125–23132. https://doi.org/10.1007/s11356-017-9876-6
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. https://doi.org/10.1007/s10295-009-0587-y
Suyotha W, Cheirsilp B, Yano S, Matsuba S, Konno H (2021) Production of chitosanase by Lentzea sp. OUR-I1 using acid-pretreated shrimp shell in an air-lift bioreactor and the feasibility of utilizing the residual biomass. Waste Biomass Valor 12:2445–2458. https://doi.org/10.1007/s12649-020-01191-2
Tshinyangu KK, Hennebert GL (1996) Protein and chitin nitrogen contents and protein content in Pleurotus ostreatus var. columbinus. Food Chem 57:223–227. https://doi.org/10.1016/0308-8146(95)00202-2
Vakili M, Rafatullah M, Salamatinia B, Abdullah AZ, Ibrahim MH, Tan KB, Gholami Z, Amouzgar P (2014) Application of chitosan and its derivatives as adsorbents for dye removal from water and wastewater: a review. Carbohydr Polym 113:115–130. https://doi.org/10.1016/j.carbpol.2014.07.007
Wu C, Scott J, Shea JE (2012) Binding of Congo red to amyloid protofibrils of the alzheimer Aβ9-40 peptide probed by molecular dynamics simulations. Biophys J 103:550–557. https://doi.org/10.1016/j.bpj.2012.07.008
Xu Y, Bajaj M, Schneider R, Grage SL, Ulrich AS, Winter J, Gallert C (2013) Transformation of the matrix structure of shrimp shells during bacterial deproteination and demineralization. Microb Cell Fact 12:1–12. https://doi.org/10.1186/1475-2859-12-90
Yan N, Chen X (2015) Sustainability: don’t waste seafood waste. Nature 524(7564):155–157. https://doi.org/10.1038/524155a
Yen YH, Li PL, Wang CL, Wang SL (2006) An antifungal protease produced by Pseudomonas aeruginosa M-1001 with shrimp and crab shell powder as a carbon source. Enzyme Microb Technol 39:311–317. https://doi.org/10.1016/j.enzmictec.2005.11.050
Younes I, Ghorbel-Bellaaj O, Nasri R, Chaabouni M, Rinaudo M, Nasri M (2012) Chitin and chitosan preparation from shrimp shells using optimized enzymatic deproteinization. Process Biochem 47:2032–2039. https://doi.org/10.1016/j.procbio.2012.07.017
Zhang H, Fang J, Deng Y, Zhao Y (2014) Optimized production of Serratia marcescens B742 mutants for preparing chitin from shrimp shells powders. Int J Biol Macromol 69:319–328. https://doi.org/10.1016/j.ijbiomac.2014.05.058
Zhang H, Yun S, Song L, Zhang Y, Zhao Y (2017) The preparation and characterization of chitin and chitosan under large-scale submerged fermentation level using shrimp by-products as substrate. Int J Biol Macromol 96:334–339. https://doi.org/10.1016/j.ijbiomac.2016.12.017
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The authors thank the YU-COE(C) program supported by Yamagata University. Thanks are also to PSU Research and Development Office (RDO) and Assoc. Prof. Seppo Karrila for assistance with the English language.
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This work was financially supported by the Higher Education Research Promotion and the Thailand’s Education Hub for Southern Region of ASEAN Countries (TEH-AC) Scholarship from Prince of Songkla University and the Thailand Research Fund under Grant No. RTA6280014.
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WS conceptualized the experimental design and constructed the methodology section. HAAC performed formal analysis, investigation, and original draft preparation. Funding acquisition, manuscript review, and editing were performed by WS and BC. AAP, SY, and MW contributed to comments and advising. All the authors read and approved the final manuscript.
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Cahyaningtyas, H.A.A., Suyotha, W., Cheirsilp, B. et al. Optimization of protease production by Bacillus cereus HMRSC30 for simultaneous extraction of chitin from shrimp shell with value-added recovered products. Environ Sci Pollut Res 29, 22163–22178 (2022). https://doi.org/10.1007/s11356-021-17279-8
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DOI: https://doi.org/10.1007/s11356-021-17279-8