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Hyper Production and Eco-Friendly Bleaching of Kraft Pulp by Xylanase From Bacillus pumilus SV-205 Using Agro Waste Material


Hyper production of an extra cellular, alkali stable, cellulase free and thermostable xylanase have been produced from Bacillus pumilus SV-205 using wheat bran as a substrate under solid state fermentation. Enzyme production under optimized conditions enhanced the production level from 4510 to 65,130 ± 1000 IU/g dry substrate, which was 14.4 fold as compared to production under un-optimized conditions. The application of crude xylanase was investigated in pulp bio bleaching. The bio-bleaching of kraft pulp with xylanase was the most effective at an enzyme dose of 12.5 IU/g oven dried pulp, pH 10.0 and 120 min incubation at 60 °C. Under the optimized conditions, xylanase pre-treatment reduced Kappa number by 0.8 points and increased brightness by 1.08 points as compared with the control. The pre-treatment of pulp with xylanase resulted in 19.01% reduction in chlorine consumption by maintaining the same brightness as in control. These results clearly demonstrated that the B. pumilus SV-85S xylanase was effective as a pulp bio-bleaching agent. The decrease in chlorine consumption by pre-treatment of pulp with xylanase apparently made the bio-bleaching process not only economical but also eco-friendly. The xylanase from B. pumilus SV-205 exhibit remarkable properties which are suitable for application in paper pulp bleaching and an elevated production of xylanase by B. pumilus SV-205 under solid state fermentation over wheat bran, a cheap and easily available agro-residue would apparently reduce the enzyme cost substantially.

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  1. 1.

    Alokika Singh, B.: Production, characteristics, and biotechnological applications of microbial xylanases. App. Microbiol. Biotechnol 103, 8763–8784 (2019).

    Article  Google Scholar 

  2. 2.

    Nagar, S., Gupta, V.K., Kumar, D., Kumar, L., Kuhad, R.C.: Production and optimization of cellulase-free, alkali-stable xylanase by Bacillus pumilus SV-85S in submerged fermentation. J. Ind. Microbiol. Biotechnol. 37, 71–83 (2010).

    Article  Google Scholar 

  3. 3.

    Bhardwaj, N., Kumar, B., Verma, P.: A detailed overiview of xylanases: an emerging biomolecule for current and future prospective. Bioprocess. Bioresor. (2019).

    Article  Google Scholar 

  4. 4.

    Kumara, D., Nagar, S., Bhushan, I., Kumara, L., Parshad, R., Gupta, V.K.: Covalent immobilization of organic solvent tolerant lipase on aluminum oxide pellets and its potential application in esterification reaction. J. Mol. Cata. B: Enz. 87, 51–61 (2013).

    Article  Google Scholar 

  5. 5.

    Bala, A., Singh, B.: Concomitant production of cellulase and xylanase by thermophilic mould Sporotrichum thermophile in solid state fermentation and their applicability in bread making. World J. Microbiol. Biotechnol. (2017).

    Article  Google Scholar 

  6. 6.

    Walia, A., Guleria, S., Mehta, P., Chauhan, A., Parkash, J.: Microbial xylanases and their industrial application in pulp and paper biobleaching: a review. 3 Biotech 7, 1–12 (2017).

    Article  Google Scholar 

  7. 7.

    Nagar, S., Mittal, A., Kumar, D., Kumar, L., Gupta, V.K.: Immobilization of xylanase on glutaraldehyde activated aluminum oxide pellets for increasing digestibility of poultry feed. Proc. Biochem. 47, 1402–1410 (2012).

    Article  Google Scholar 

  8. 8.

    Katsimpouras, C., Dedes, G., Thomaidis, N.S., Topakas, E.: A novel fungal GH30 xylanase with xylobiohydrolase auxiliary activity. Biotechnol. Biofuels 12, 1–14 (2019).

    Article  Google Scholar 

  9. 9.

    Karlsson, E.N., Schmitz, E., Linares-Pastén, J.A., Adlercreutz, P.: Endo-xylanases as tools for production of substituted xylooligosaccharides with prebiotic properties. Appl. Microbol. Biotechnol. 102, 9081–9088 (2018).

    Article  Google Scholar 

  10. 10.

    Nagar, S., Mittal, A., Gupta, V.K.: Enzymatic clarification of fruit juices (Apple, Pineapple, and Tomato) Using Purified Bacillus pumilus SV-85S xylanase. Biotechnol. Bioproc. Engg. 17, 1165–1175 (2012).

    Article  Google Scholar 

  11. 11.

    Kumar, L., Kumar, D., Nagar, S., Gupta, R., Kuhad, R.C., Gupta, V.K.: Modulation of xylanase production from alkaliphilic Bacillus pumilus VLK-1 through process optimization and temperature shift operation. 3 Biotech 4, 345–356 (2014).

    Article  Google Scholar 

  12. 12.

    Gautam, A., Kumar, A., Bharti, A.K., Dutt, D.: Rice straw fermentation by Schizophyllum commune ARC-11 to produce high level of xylanase for its application in pre-bleaching. J. Genet. Eng. Biotechnol. 16, 693–701 (2018).

    Article  Google Scholar 

  13. 13.

    Nagar, S., Jain, R.K., Thakur, V.V., Gupta, V.K., : Biobleaching application of cellulase poor and alkali stable xylanase from Bacillus pumilus SV-85S. 3 Biotech (2013).

    Article  Google Scholar 

  14. 14.

    Álvarez-Cervantes, J., Domínguez-Hernández, E.M., Mercado-Flores, Y., Díaz-Godínez, G.: Mycosphere essay: properties and characteristics of microbial xylanases. Mycosphere 7, 1600–1619 (2016).

    Article  Google Scholar 

  15. 15.

    Nagar, S., Mittal, A., Gupta, V.K.: Two way strategy for utilizing agricultural waste ‘wheat bran’ for production and immobilization of xylanase. J. Innov. Biol. 1, 035–044 (2014)

    Google Scholar 

  16. 16.

    Harris, A.D., Ramalingam, C.: Xylanases and its application in food industry: a review. J. Exp. Sci. 1, 1–11 (2010)

    Google Scholar 

  17. 17.

    Nagar, S., Mittal, A., Kumar, D., Gupta, V.K.: Production of alkali tolerant cellulase free xylanase in high levels by Bacillus pumilus SV-205. Int. J. Biological. Macro. 50, 414–420 (2012).

    Article  Google Scholar 

  18. 18.

    Nagar, S., Gupta, V.K., Kumar, D., Kumar, L., Kuhad, R.C.: Production and optimization of cellulase-free, alkali-stable xylanase by Bacillus Pumilus SV-85S in submerged fermentation. J. Ind. Microbiol. Biotechnol. 37, 71–83 (2010).

    Article  Google Scholar 

  19. 19.

    Yadav, P., Maharjan, J., Korpole, S.: Production, purification, and characterization of thermostable alkaline xylanase from Anoxybacillus kamchatkensis NASTPD13. Front. Bioeng. Biotechnol. 6, 6–65 (2018).

    Article  Google Scholar 

  20. 20.

    Azeri, C., Tamer, U.A., Oskay, M.: Thermoactive cellulase-free xylanase production from alkaliphilic Bacillus strains using various agro-residues and their potential in biobleaching of kraft pulp. Afr. J. Biotechnol. 9, 63 (2010)

    Google Scholar 

  21. 21.

    Nagar, S., Mittal, A., Kumar, D., Kumar, L., Gupta, V.K.: Hyper production of alkali stable xylanase in lesser duration by Bacillus pumilus SV-85S using wheat bran under solid state fermentation. New Biotechnol. (2011).

    Article  Google Scholar 

  22. 22.

    Sharma, P.K.: Xylanases current and future perspectives : a review. New. Biol. Rep. 6, 122 (2017)

    Google Scholar 

  23. 23.

    Gomes, A.F.S., Santos, B.S.L., Ransciscon, E.G., Baffi, M.A.: Substrate and temperature effect on xylanase production by Aspergillus fumigatus using low cost agricultural wastes. Biosci. J. 32, 915–921 (2016).

    Article  Google Scholar 

  24. 24.

    Sanghi, A., Garg, N., Sharma, J., Kuhar, K., Kuhad, R.C., Gupta, V.K.: Optimization of xylanase production using inexpensive agroresidues by alkalophilic Bacillus subtilis ASH in solid-state fermentation. World J. Microb. Biotechnol. 24, 633–640 (2008).

    Article  Google Scholar 

  25. 25.

    Sindhu, I., Chhibber, S., Caplash, N., Sharma, P.: Production of cellulase free xylanase from Bacillus megaterium by solid state fermentation for biobleaching of pulp. Curr. Microbiol. 53, 167–172 (2006).

    Article  Google Scholar 

  26. 26.

    Battan, B., Sharma, J., Kuhad, R.C.: High level xylanase production by Bacillus pumilus ASH under solid-state fermentation. World J. Microbiol. Biotechnol. 22, 1281–1287 (2006).

    Article  Google Scholar 

  27. 27.

    Kumar, V., Shukla, P.: Extracellular xylanase production from T. lanuginosus VAPS24 at pilot scale and thermostability enhancement by immobilization. Process Biochem. 71, 53–60 (2018).

    Article  Google Scholar 

  28. 28.

    Kshirsagar, S.D., Saratale, G.D., Saratale, R.G., Govindwar, S.P., Oh, M.K.: An isolated Amycolatopsis sp GDS for cellulase and xylanase production using agricultural waste biomass. J. Appl. Microbiol. 120, 112–125 (2015).

    Article  Google Scholar 

  29. 29.

    Archana, A., Satyanarayana, T.: Purification and characterization of a cellulase-free xylanase of a moderate thermophile Bacillus licheniformis A99. World J. Microbiol. Biotechnol. 19, 12–17 (2003).

    Article  Google Scholar 

  30. 30.

    Dhiman, S.S., Sharma, J., Battan, B.: Industrial applications and future prospects of microbial xylanases: a review. BioResources 3, 1377–1402 (2008)

    Google Scholar 

  31. 31.

    Xiong, L., et al.: The ACEII recombinant Trichoderma reesei QM9414 strains with enhanced xylanase production and its applications in production of xylitol from tree barks. Microb. Cell. Fact. (2016).

    Article  Google Scholar 

  32. 32.

    Mittal, A., Nagar, S., Gupta, V.K.: Production and purification of high levels of cellulase-free bacterial xylanase by Bacillus sp SV-34S using agro-residue. Annal. Microbiol. 63, 1157–1167 (2013).

    Article  Google Scholar 

  33. 33.

    Khandeparkar, R.D.S., Bhosle, N.B.: Isolation, purification and characterization of the xylanase produced by Arthrobacter sp MTCC 5214 when grown in solid-state fermentation. Enzyme Microb. Technol. 39, 732–742 (2006).

    Article  Google Scholar 

  34. 34.

    Manimaran, A., Santhosh, K.K., Permaul, K., Singh, S.: Hyper production of cellulase-free xylanase by Thermomyces lanuginosus SSBP on bagasse pulp and its application in biobleaching. Appl. Microbiol. Biotechnol. 81, 887–893 (2019)

    Article  Google Scholar 

  35. 35.

    Heck, J., Flores, S., Hertzm, P., Ayub, M.: Optimization of cellulase free xylanase activity by Bacillus coagulans BL69 in solid state cultivation. Proc. Biochem. 40, 107–112 (2005)

    Article  Google Scholar 

  36. 36.

    Oliveira, D.S., Meherb-Dini, C., Franco, C.M.L., Gomes, E., Da-Silva, R.: Production of crude xylanase from Thermoascus Aurantiacus CBMAI 756 aiming the baking process. J. Food. Sci. (2010).

    Article  Google Scholar 

  37. 37.

    Poorna, C.A., Prema, P.: Production and partial characterization of endoxylanase by Bacillus pumilus using agro industrial residues. Biochem. Eng. J. (2006).

    Article  Google Scholar 

  38. 38.

    Battan, B., Sharma, J., Dhiman, S.S., Kuhad, R.C.: Enhanced production of cellulase-free thermostable by Bacillus pumilus ASH and its potential application in paper industry. Enzyme Microb. Technol. 41, 733–739 (2007).

    Article  Google Scholar 

  39. 39.

    Sonia, K.G., Chadha, B.S., Saini, H.S.: Sorghum straw for xylanase hyper-production by Thermomyces lanuginosus (D2W3) under solid state fermentation. Biores. Technol. (2005).

    Article  Google Scholar 

  40. 40.

    Masui, D.C., Zimbardi, A.L.R.L., Souza, F.H.M., Guimarães, L.H.S., Furriel, R.P.M., Jorge, J.A.: Production of a xylose-stimulated β-glucosidase and a cellulase-free thermostable xylanase by the thermophilic fungus Humicola brevis var. thermoidea under solid state fermentation. World J. Microbiol. Biotechnol. (2012).

    Article  Google Scholar 

  41. 41.

    Yang, S., Yang, B., Duan, C., Puller, D.A., Wang, X.: Applications of enzymatic technologies to the production of high-quality dissolving pulp: a review. Bioresour. Technol. 228, 440–448 (2019).

    Article  Google Scholar 

  42. 42.

    Zhao, L., Yuan, Z., Kapu, N.S., Chang, X.F.: Increasing efficiency of enzymatic hemicellulose removal from bamboo for production of high-grade dissolving pulp. Bioresour. Technol. 223, 40–46 (2017).

    Article  Google Scholar 

  43. 43.

    Garg, G., Mahajan, R., Kaur, A., Sharma, J.: Xylanase production using agro-residue in solid-state fermentation from Bacilluspumilus ASH for biodelignification of wheat straw pulp. Biodegradation (2011).

    Article  Google Scholar 

  44. 44.

    Motta, F.L., Andrade, C.C.P., Santana, M.H.A.: A review of xylanase production by the fermentation of xylan: classification, characterization and applications, in Sustainable degradation of lignocellulosic biomass-techniques, applications and commercialization. InTechOpen, New York (2013)

    Google Scholar 

  45. 45.

    Lin, X., Wu, Z., Zhang, C., Liu, S., Nie, S.: Enzymatic pulping of lignocellulosic biomass. Ind. Crop. Prod. (2018).

    Article  Google Scholar 

  46. 46.

    Zhang, K., Zhang, Y., Yan, D., Zhang, C., Nie, S.: Enzyme-assisted mechanical production of cellulose nanofibrils: thermal stability. Cellulose (2018).

    Article  Google Scholar 

  47. 47.

    Nie, S., et al.: Enzymatic pretreatment for the improvement of dispersion and film properties of cellulose nanofibrils. Carbohyd. Polym. (2018).

    Article  Google Scholar 

  48. 48.

    Ko, C.H., et al.: Identification of Paenibacillus sp. 2S–6 and application of its xylanase on biobleaching. Int. Biodeter. Biodegr. (2011).

    Article  Google Scholar 

  49. 49.

    Mittal, A., Nagar, S., Kirti, K.S.J., Gupta, V.K.: Isolation, purification and characterization of alkali and thermostable xylanase from Bacillus sp KS09. Inter. J. Res. Develop. Pharm. Life Sci. 2, 63–68 (2012)

    Google Scholar 

  50. 50.

    Raj, A., Kumar, S., Singh, S.K., Prakash, J.: Production and purification of xylanase from alkaliphilic Bacillus licheniformis and its pretreatment of eucalyptus kraft pulp. Biocatal. Agric. Biotechnol. (2018).

    Article  Google Scholar 

  51. 51.

    Kiddinamoorthy, J., Anceno, A.J., Haki, G.D., Rakshit, S.K.: Production, purification and characterization of Bacillus sp. GRE7 xylanase and its application in eucalyptus kraft pulp biobleaching. World J. Microbiol. Biotechnol. (2008).

    Article  Google Scholar 

  52. 52.

    Thomas, L., Sindhu, R., Binod, P., Pandey, A.: Production of an alkaline xylanase from recombinant Kluyveromyces lactis (KY1) by submerged fermentation and its application in bio-bleaching. Biochem. Eng. J. (2015).

    Article  Google Scholar 

  53. 53.

    Li, Q., Sun, B., Jia, H., Hou, J., Yang, R., Xiong, K., Xu, Y.: Engineering a xylanase from Streptomyce rochei L10904 by mutation to improve its catalytic characteristics. Int. J. Biol. Macromol. 101, 366–372 (2017).

    Article  Google Scholar 

  54. 54.

    Dhiman, S.S., Garg, G., Mahajan, R., Garg, N., Sharma, J.: Single lay out and mixed lay out enzymatic processes for biobleaching of kraft pulp. Bioresour. Technol. (2009).

    Article  Google Scholar 

  55. 55.

    Gupta, S., Bhushan, B., Hoondal, G.S.: Isolation, purification and characterization of xylanasefrom Staphylococcus sp. SG-13 and its application in biobleaching of kraft pulp. J. Appl. Microbiol. 88 (2):325–334 (2000)

    Article  Google Scholar 

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Dr. Sushil Nagar greatly acknowledges the help received from Electron Microscope Facility (SAIF), All India Institute of Medical Sciences, New Delhi for SEM studies. The authors are thankful to Dr. R. Malhotra, Scientist (Statistics), National Dairy Research Institute (NDRI), Karnal for his help in statistical analysis of the data.

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Nagar, S., Gupta, V.K. Hyper Production and Eco-Friendly Bleaching of Kraft Pulp by Xylanase From Bacillus pumilus SV-205 Using Agro Waste Material. Waste Biomass Valor 12, 4019–4031 (2021).

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  • Xylanase
  • Xylan
  • Solid state fermentation
  • Agro-residues
  • Wheat bran