, Volume 21, Issue 6, pp 993–1004 | Cite as

Delignification and detoxification of peanut shell bio-waste using an extremely halophilic laccase from an Aquisalibacillus elongatus isolate

  • Rezvan Rezaie
  • Shahla Rezaei
  • Nasrin Jafari
  • Hamid Forootanfar
  • Mohammad Reza Khoshayand
  • Mohammad Ali Faramarzi
Original Paper


Lignocellulose bioconversion is a harsh process requiring the use of surfactants and organic solvents. Consequently, the incorporation of laccases in this bioconversion requires the bioprospecting of enzymes that can remain stable under extreme conditions. An extracellular, highly stable laccase was produced by the halophilic isolate Aquisalibacillus elongatus in submerged liquid culture fermentation. Statistical and non-statistical strategies gave the highest enzymatic activity (8.02 U mL−1) following addition of glucose (1.7 g L−1), copper sulfate (0.8 g L−1), urea (15 g L−1), and CaCl2 (0.8 g L−1). The enzyme, after purification using a synthetic affinity support, delignified a peanut shell substrate by 45%. A pH of 8.0 and a temperature of 35 °C were optimal for delignification of this bio-waste material. Addition of [Bmim][PF6], 1,4-dioxane, acetone, and HBT promoted this bio-waste delignification. Bio-treatment in the presence of 50% [Bmim][PF6] gave a maximal lignin removal of 87%. The surfactants tested had no significant effects on the delignification yield. The laccase also detoxified the toxic phenols found in peanut shell waste. The high catalytic efficiency of this enzyme against a lignocellulosic sample under extreme conditions suggests the suitability of this laccase for industrial applications.


Laccase Halophile Optimization Aquisalibacillus elongatus Delignification Peanut shell 



Research reported in this publication was supported by Elite Researcher Grant Committee under Award Number 943687 from the National Institutes for Medical Research Development (NIMAD), Tehran, Iran to M.A.F.

Supplementary material

792_2017_958_MOESM1_ESM.docx (175 kb)
Supplementary material 1 (DOCX 176 kb)


  1. Addleman K, Archibald F (1993) Kraft pulp bleaching and delignification by dikaryons and monokaryons of Trametes versicolor. Appl Environ Microbiol 59:266–273PubMedPubMedCentralGoogle Scholar
  2. Aghaie-Khouzani M, Forootanfar H, Moshfegh M, Khoshayand MR, Faramarzi MA (2012) Decolorization of some synthetic dyes using optimized culture broth of laccase producing ascomycete Paraconiothyrium variabile. Biochem Eng J 60:9–15CrossRefGoogle Scholar
  3. Anike FN, Yusif M, Isikhuemhen OS (2016) Co-substrating of peanut shells with cornstalks enhances biodegradation by Pleurotus ostreatus. J Biorem Biodegrad 7:327–333Google Scholar
  4. Asina F, Brzonova I, Voeller K, Kozliak E, Kubátová A, Yao B, Ji Y (2016) Biodegradation of lignin by fungi, bacteria and laccases. Bioresour Technol 220:414–424CrossRefPubMedGoogle Scholar
  5. Bharthare P, Shrivastava P, Singh P, Ttiwari A (2014) Peanut shell as renewable energy source and their utility in production of ethanol. Int J Adv Res 2:1–12Google Scholar
  6. Bradford MM (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72:248–254CrossRefPubMedGoogle Scholar
  7. Brander S, Mikkelsen JD, Kepp KP (2014) Characterization of an alkali-and halide-resistant laccase expressed in E. coli: CotA from Bacillus clausii. PLoS One 9:1–11CrossRefGoogle Scholar
  8. Camarero S, Ibarra D, Martínez ÁT, Romero J, Gutiérrez A, del Río JC (2007) Paper pulp delignification using laccase and natural mediators. Enzyme Microb Technol 40:1264–1271CrossRefGoogle Scholar
  9. Chandel AK, Kapoor RK, Singh A, Kuhad RC (2007) Detoxification of sugarcane bagasse hydrolysate improves ethanol production by Candida shehatae NCIM3501. Bioresour Technol 98:1947–1950CrossRefPubMedGoogle Scholar
  10. de Lourdes Moreno M, Pérez D, García MT, Mellado F (2013) Halophilic bacteria as a source of novel hydrolytic enzymes. Life 3:38–51CrossRefPubMedPubMedCentralGoogle Scholar
  11. do Valle JS, de Souza Vandenberghe LP, Santana TT, Linde GA, Colauto NB, Soccol CR (2014) Optimization of Agaricus blazei laccase production by submerged cultivation with sugarcane molasses. Afr J Microbiol Res 8:939–946CrossRefGoogle Scholar
  12. Elsayed MA, Hassan MM, Elshafei AM, Haroun BM, Othman AM (2012) Optimization of cultural and nutritional parameters for the production of laccase by Pleurotus ostreatus ARC280. Br Biotechnol J 2:115–132CrossRefGoogle Scholar
  13. Fang Z, Li T, Wang Q, Zhang X, Peng H, Fang W, Hong Y, Ge H, Xiao Y (2011) A bacterial laccase from marine microbial metagenome exhibiting chloride tolerance and dye decolorization ability. Appl Microbiol Biotechnol 89:1103–1110CrossRefPubMedGoogle Scholar
  14. Forootanfar H, Faramarzi MA (2015) Insights into laccase producing organisms, fermentation states, purification strategies, and biotechnological applications. Biotechnol Prog 31:1443–1463CrossRefPubMedGoogle Scholar
  15. Ghorbani F, Karimi M, Biria D, Kariminia HR, Jeihanipoura A (2015) Enhancement of fungal delignification of rice straw by Trichoderma viride sp. to improve its saccharification. Biochem Eng J 101:77–84CrossRefGoogle Scholar
  16. Gutiérrez A, Rencoret J, Cadena EM, Rico A, Barth D, del Río JC, Martínez ÁT (2012) Demonstration of laccase-based removal of lignin from wood and non-wood plant feedstocks. Bioresour Technol 119:114–122CrossRefPubMedGoogle Scholar
  17. Jafari N, Rezaei S, Rezaie R, Dilmaghani H, Khoshayand MR, Faramarzi MA (2017) Improved production and characterization of a highly stable laccase from the halophilic bacterium Chromohalobacter salexigens for the efficient delignification of almond shell bio-waste. Int J Biol Macromol. doi: 10.1016/j.ijbiomac.2017.07.055 Google Scholar
  18. Jurado M, Prieto A, Martínez-Alcalá A, Martínez AT, Martínez MJ (2009) Laccase detoxification of steam-exploded wheat straw for second generation bioethanol. Bioresour Technol 100:6378–6384CrossRefPubMedGoogle Scholar
  19. Karp SG, Faraco V, Amore A, Letti LAJ, Soccol VT, Soccol CR (2015) Statistical optimization of laccase production and delignification of sugarcane bagasse by Pleurotus ostreatus in solid-state fermentation. Biomed Res Int 2015:1–8CrossRefGoogle Scholar
  20. Knežević A, Milovanović I, Stajić M, Lončar N, Brčeski I, Vukojević J, Ćilerdžić J (2013) Lignin degradation by selected fungal species. Bioresour Technol 138:117–123CrossRefPubMedGoogle Scholar
  21. Lu C, Wang H, Luo Y, Guo L (2010) An efficient system for pre-delignification of gramineous biofuel feedstock in vitro: application of a laccase from Pycnoporus sanguineus H275. Process Biochem 45:1141–1147CrossRefGoogle Scholar
  22. Lu L, Wang TN, Xu TF, Wang JY, Wang CL, Zhao M (2013) Cloning and expression of thermo-alkali-stable laccase of Bacillus licheniformis in Pichia pastoris and its characterization. Bioresour Technol 134:81–86CrossRefPubMedGoogle Scholar
  23. Moreno AD, Ibarra D, Fernández JL, Ballesteros M (2012) Different laccase detoxification strategies for ethanol from lignocellulosic biomass by the thermotolerant yeast Kluyveromyces marxianus CECT 10875. Bioresour Technol 106:101–109CrossRefPubMedGoogle Scholar
  24. Moreno AD, Ibarra D, Ballesteros I, Fernández JL, Ballesteros M (2013) Ethanol from laccase-detoxified lignocellulose by the thermotolerant yeast Kluyveromyces—effects of steam pretreatment conditions, process configurations and substrate loadings. Biochem Eng J 79:94–103CrossRefGoogle Scholar
  25. Moshfegh M, Shahverdi AR, Zarrini G, Faramarzi MA (2013) Biochemical characterization of an extracellular polyextremophilic α-amylase from the halophilic archaeon Halorubrum xinjiangense. Extremophiles 17:677–687CrossRefPubMedGoogle Scholar
  26. Niladevi KN, Prema P (2008) Effect of inducers and process parameters on laccase production by Streptomyces psammoticus and its application in dye decolourization. Bioresour Technol 99:4583–4589CrossRefPubMedGoogle Scholar
  27. Oren A (2010) Industrial and environmental applications of halophilic microorganisms. Environ Technol 31:825–834CrossRefPubMedGoogle Scholar
  28. Periasamy R, Palvannan T (2010) Optimization of laccase production by Pleurotus ostreatus IMI 395545 using the Taguchi DOE methodology. J Basic Microbiol 50:548–556CrossRefPubMedGoogle Scholar
  29. Poojary H, Mugeraya G (2012) Laccase production by Phellinus noxius hp F17: optimization of submerged culture conditions by response surface methodology. Res Biotechnol 3:9–20Google Scholar
  30. Raddadi N, Cherif A, Daffonchio D, Neifar M, Fava F (2015) Biotechnological applications of extremophiles, extremozymes and extremolytes. Appl Microbiol Biotechnol 99:7907–7913CrossRefPubMedGoogle Scholar
  31. Rezaei S, Tahmasbi H, Mogharabi M, Firuzyar S, Ameri A, Khoshayand MR, Faramarzi MA (2015) Efficient decolorization and detoxification of reactive orange 7 using laccase isolated from Paraconiothyrium variabile, kinetics and energetics. J Taiwan Inst Chem Eng 56:113–121CrossRefGoogle Scholar
  32. Rezaei S, Shahverdi AR, Faramarzi MA (2017) Isolation, one-step affinity purification, and characterization of a polyextremotolerant laccase from the halophilic bacterium Aquisalibacillus elongatus and its application in the delignification of sugar beet pulp. Bioresour Technol 230:67–75CrossRefPubMedGoogle Scholar
  33. Samaei-Nouroozi A, Rezaei S, Khoshnevis N, Doosti M, Hajihoseini R, Khoshayand MR, Faramarzi MA (2015) Medium-based optimization of an organic solvent-tolerant extracellular lipase from the isolated halophilic Alkalibacillus salilacus. Extremophiles 19:933–947CrossRefPubMedGoogle Scholar
  34. Sindhu R, Binod P, Pandey A (2016) Biological pretreatment of lignocellulosic biomass-An overview. Bioresour Technol 199:76–82CrossRefPubMedGoogle Scholar
  35. Sivakumar R (2010) Isolation, screening and optimization of production medium for thermostable laccase production from Ganoderma sp. Int J Eng Sci Technol 2:7133–7141Google Scholar
  36. Soden DM, Dobson ADW (2001) Differential regulation of laccase gene expression in Pleurotus sajor-caju. Microbiology 147:1755–1763CrossRefPubMedGoogle Scholar
  37. Sondhi S, Sharma P, George N, Chauhan PS, Puri N, Gupta N (2015) An extracellular thermo-alkali-stable laccase from Bacillus tequilensis SN4, with a potential to biobleach softwood pulp. 3 Biotech 5:175–185CrossRefPubMedGoogle Scholar
  38. Tappi Standard Procedures (2012) Tappi Press, Atlanta, Ga, USAGoogle Scholar
  39. Uthandi S, Saad B, Humbard MA, Maupin-Furlow JA (2010) LccA, an archaeal laccase secreted as a highly stable glycoprotein into the extracellular medium by Haloferax volcanii. Appl Environ Microbiol 76:733–743CrossRefPubMedGoogle Scholar
  40. Ventosa A, Oren A, Ma Y (2004) Halophiles and hypersaline environments: current research and future trends, 1st edn. Springer, Berlin, p 27Google Scholar
  41. Virk AP, Sharma P, Capalash N (2012) Use of laccase in pulp and paper industry. Biotechnol Prog 28:21–32CrossRefPubMedGoogle Scholar
  42. Vithanage LNG, Barbosa AM, Borsato D, Dekker RFH (2015) Value adding of poplar hemicellulosic prehydrolyzates: laccase production by Botryosphaeria rhodina MAMB-05 and its application in the detoxification of prehydrolyzates. BioEnergy Res 8:657–674CrossRefGoogle Scholar
  43. Woolridge EM (2014) Mixed enzyme systems for delignification of lignocellulosic biomass. Catalysts 4:1–35CrossRefGoogle Scholar
  44. Yamoum C, Magaraphan R (2017) Effect of peanut shell content on mechanical, thermal, and biodegradable properties of peanut shell/polylactic acid biocomposites. Polym Compos 38:682–690CrossRefGoogle Scholar

Copyright information

© Springer Japan KK 2017

Authors and Affiliations

  • Rezvan Rezaie
    • 1
  • Shahla Rezaei
    • 1
  • Nasrin Jafari
    • 1
  • Hamid Forootanfar
    • 2
  • Mohammad Reza Khoshayand
    • 3
  • Mohammad Ali Faramarzi
    • 1
  1. 1.Department of Pharmaceutical Biotechnology, Faculty of Pharmacy and Biotechnology Research CenterTehran University of Medical SciencesTehranIran
  2. 2.Department of Pharmaceutical Biotechnology, Faculty of PharmacyKerman University of Medical SciencesKermanIran
  3. 3.Department of Drug and Food Control, Faculty of PharmacyTehran University of Medical SciencesTehranIran

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