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Enzymatic Hydrolysis of Lignocellulosic Biomass Using an Optimized Enzymatic Cocktail Prepared from Secretomes of Filamentous Fungi Isolated from Amazonian Biodiversity

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Abstract

The use of lignocellulosic biomass (LCB) has emerged as one of the main strategies for generating renewable biofuels. For the efficient use of such feedstock, pre-treatments are essential. The hydrolysis of cellulose – major component of LCB – demands enzymatic cocktails with improved efficiency to generate fermentable sugars. In this scenario, lignocellulolytic fungi have enormous potential for the development of efficient enzyme platforms. In this study, two enzymatic cocktails were developed for hydrolysis of two lignocellulosic biomasses: industrial cellulose pulp and cassava peel. The solid biomass ratio in relation to the protein content of the enzyme cocktail was performed by experimental design. The optimized cocktail for the hydrolysis of cellulose pulp (AMZ 1) was composed, in protein base, by 43% of Aspergillus sp. LMI03 enzyme extract and 57% of T. reesei QM9414, while the optimal enzyme cocktail for cassava peel hydrolysis (AMZ 2) was composed by 50% of Aspergillus sp. LMI03 enzyme extract, 25% of the extract of P. citrinum LMI01 and 25% of T. reesei. The ratio between solids and protein loading for AMZ 1 cocktail performance was 52 g/L solids and 30 mg protein/g solids, resulting in a hydrolytic efficiency of 93%. For the AMZ 2 cocktail, the hydrolytic efficiency was 78% for an optimized ratio of 78 g/L solids and 19 mg protein/g solids. These results indicate that cocktails formulated with enzymatic extracts of P. citrinum LMI01, Aspergillus sp. LMI03, and T. reesei QM9414 are excellent alternatives for efficient hydrolysis of plant biomass and for other processes that depend on biocatalysis.

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References

  1. Pereira Jr, N., Couto, M. A. P. G., & Santa Anna, L. M. M. (2008). Biomass of lignocellulosic composition for fuel ethanol production within the context of biorefinery. Series on Biotechnology (Vol. 2).

  2. Adsul, M., Sandhu, S. K., Singhania, R. R., Gupta, R., Puri, S. K., & Mathur, A. (2020). Designing a cellulolytic enzyme cocktail for the efficient and economical conversion of lignocellulosic biomass to biofuels. Enzyme and Microbial Technology, 133, 109442. https://doi.org/10.1016/j.enzmictec.2019.109442

    Article  CAS  PubMed  Google Scholar 

  3. Mandels, M., & Reese, E. (1957). Induction of Cellulase in Trichoderma Viride As Influenced By Carbon Sources and Metals. Journal of Bacteriology, 73(2), 269–278. https://doi.org/10.1128/jb.73.2.269-278.1957

  4. Bischof, R. H., & Ramoni, J. (2016). Seiboth B 2016 Cellulases and beyond: The first 70Â years of the enzyme producer Trichoderma reesei. Microbial Cell Factories, 15(1), 106. https://doi.org/10.1186/S12934-016-0507-6

    Article  PubMed  PubMed Central  Google Scholar 

  5. Dashtban, M., Buchkowski, R., & Qin, W. (2011). Effect of different carbon sources on cellulase production by Hypocrea jecorina (Trichoderma reesei) strains. International Journal of Biochemistry and Molecular Biology, 2(3), 274–86. Retrieved from https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3193291/pdf/ijbmb0002-0274.pdf. Accessed 05.28.2019.

  6. Zabed, H., Sahu, J. N., Boyce, A. N., & Faruq, G. (2016). Fuel ethanol production from lignocellulosic biomass: An overview on feedstocks and technological approaches. Renewable and Sustainable Energy Reviews, 66, 751–774. https://doi.org/10.1016/j.rser.2016.08.038

    Article  CAS  Google Scholar 

  7. Sun, F. F., Hong, J., Hu, J., Saddler, J. N., Fang, X., Zhang, Z., & Shen, S. (2015). Accessory enzymes influence cellulase hydrolysis of the model substrate and the realistic lignocellulosic biomass. Enzyme and Microbial Technology, 79–80, 42–48. https://doi.org/10.1016/j.enzmictec.2015.06.020

    Article  CAS  PubMed  Google Scholar 

  8. Valadares, F., Gonçalves, T. A., Gonçalves, D. S. P. O., Segato, F., Romanel, E., Milagres, A. M. F., & Ferraz, A. (2016). Exploring glycoside hydrolases and accessory proteins from wood decay fungi to enhance sugarcane bagasse saccharification. Biotechnology for Biofuels, 9(1), 110. https://doi.org/10.1186/s13068-016-0525-y

  9. Levasseur, A., Drula, E., Lombard, V., Coutinho, P. M., & Henrissat, B. (2013). Expansion of the enzymatic repertoire of the CAZy database to integrate auxiliary redox enzymes. Biotechnology for Biofuels, 6(1), 41. https://doi.org/10.1186/1754-6834-6-41

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  10. Eijsink, V. G. H., Ludwicka, K., Felice, A. K. G., Preims, M., Ludwig, R., Breslmayr, E., & Scheiblbrandner, S. (2016). Extracellular electron transfer systems fuel cellulose oxidative degradation. Science, 352(6289), 1098-1101.https://doi.org/10.1126/science.aaf3165

  11. Saloheimo, M., Paloheimo, M., Hakola, S., Pere, J., Swanson, B., & Nyysso, E. (2002). Swollenin, a Trichoderma reeseiprotein with sequence similarity to the plant expansins, exhibits disruption activity on cellulosic materials. European journal of biochemistry / FEBS, 4211, 4202–4211. https://doi.org/10.1046/j.1432-1033.2002.03095.x

    Article  CAS  Google Scholar 

  12. Obeng, E. M., Adam, S. N. N., Budiman, C., Ongkudon, C. M., Maas, R., & Jose, J. (2017). Lignocellulases: A review of emerging and developing enzymes, systems, and practices. Bioresources and Bioprocessing, 4(1). https://doi.org/10.1186/s40643-017-0146-8

  13. Cesar, F., Maria, B., Silvello, A., & Goldbeck, R. (2020). Cellulase and oxidative enzymes : New approaches, challenges and perspectives on cellulose degradation for bioethanol production. Biotechnology Letters, 42(6), 875–884. https://doi.org/10.1007/s10529-020-02875-4

    Article  CAS  Google Scholar 

  14. Mohanram, S., Amat, D., Choudhary, J., Arora, A., & Nain, L. (2013). Novel perspectives for evolving enzyme cocktails for lignocellulose hydrolysis in biorefineries. Sustainable Chemical Processes, 1(1), 15. https://doi.org/10.1186/2043-7129-1-15

    Article  CAS  Google Scholar 

  15. Borin, G. P., Sanchez, C. C., de Santana, E. S., Zanini, G. K., dos Santos, R. A. C., de Oliveira Pontes, A., & Oliveira, J. V. de C. (2017). Comparative transcriptome analysis reveals different strategies for degradation of steam-exploded sugarcane bagasse by Aspergillus niger and Trichoderma reesei. BMC Genomics, 18(1), 501. https://doi.org/10.1186/s12864-017-3857-5

  16. Lynd, L. R., Weimer, P. J., Zyl, W. H. Van, Isak, S., & Pretorius, I. S. (2002). Microbial cellulose utilization : Fundamentals and biotechnology microbial cellulose utilization . Fundamentals and Biotechnology, 66(3). https://doi.org/10.1128/MMBR.66.3.506

  17. Schuster, A., Schmoll, M., Martins, F. L., Kolling, D., Camassola, M., Jose, A., & Singh, O. V. (2010). Biology and biotechnology of Trichoderma. Journal of Industrial Microbiology & Biotechnology, 87(3), 787-799.https://doi.org/10.1016/S0960-8524(03)00195-0

  18. Seiboth, B., Ivanova, C., & Seidl-seiboth, V. (2011). Trichoderma reesei: A Fungal Enzyme Producer for Cellulosic Biofuels. In Biofuel Production-Recent Developments and Prospects. InTech. https://doi.org/10.5772/16848

  19. Singhania, R. R., Patel, A. K., Sukumaran, R. K., Larroche, C., & Pandey, A. (2013). Role and significance of beta-glucosidases in the hydrolysis of cellulose for bioethanol production. Bioresource Technology, 127, 500–507. https://doi.org/10.1016/j.biortech.2012.09.012

    Article  CAS  PubMed  Google Scholar 

  20. Santa-Rosa, P. S., Souza, A. L., Roque, R. A., Andrade, E. V., Astolfi-Filho, S., Mota, A. J., & Nunes-Silva, C. G. (2018). Production of thermostable β-glucosidase and CMCase by Penicillium sp. LMI01 isolated from the Amazon region. Electronic Journal of Biotechnology, 31, 84–92. https://doi.org/10.1016/j.ejbt.2017.11.005

    Article  CAS  Google Scholar 

  21. Mandels, M., & Weber, J. (1969). The production of cellulases. Advances in Chemistry, 95, 391–414. https://doi.org/10.1021/ba-1969-0095

    Article  CAS  Google Scholar 

  22. Laemmli, U. K. (1970). Cleavage of Structural Proteins during Assembly of Head of Bacteriophage‐T4. Nature, 227, 680–685. https://doi.org/10.1038/227680a0

  23. Schwarz, W. H., Bronnenmeier, K., Staudenbauer, W. L. (1987). Activity staining of cellulases in polyacrylamide gels containing mixed linkage β-glucans. Analytical Biochemistry, 164(1), 72–77. https://doi.org/10.1016/0003-2697(87)90369-1

  24. Barcelos, C. A., Maeda, R. N., Betancur, G. J. V., & Pereira, N. (2013). The essentialness of delignification on enzymatic hydrolysis of sugar cane bagasse cellulignin for second generation ethanol production. Waste and Biomass Valorization, 4(2), 341–346. https://doi.org/10.1007/s12649-012-9137-3

    Article  CAS  Google Scholar 

  25. Ghose, T. K. (1987). Measurement of cellulase activities. Pure & Applied Chemical, 59(2), 257–268.

    Article  CAS  Google Scholar 

  26. Eveleigh, D. E., Mandels, M., Andreotti, R., & Roche, C. (2009). Measurement of saccharifying cellulase. Biotechnology for Biofuels, 2(1), 21. https://doi.org/10.1186/1754-6834-2-21

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  27. Bradford, M. M. (1976). A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Analytical Biochemistry, 72(1–2), 248–254. https://doi.org/10.1016/0003-2697(76)90527-3

    Article  CAS  PubMed  Google Scholar 

  28. Li, J., Zhou, P., Liu, H., Xiong, C., Lin, J., Xiao, W., & Liu, Z. (2014). Synergism of cellulase, xylanase, and pectinase on hydrolyzing sugarcane bagasse resulting from different pretreatment technologies. Bioresource Technology, 155, 258–265. https://doi.org/10.1016/j.biortech.2013.12.113

  29. Zerva, A., Pentari, C., Grisel, S., Berrin, J. G., & Topakas, E. (2020). Biotechnology for biofuels a new synergistic relationship between xylan - active LPMO and xylobiohydrolase to tackle recalcitrant xylan. Biotechnology for Biofuels, 1–13. https://doi.org/10.1186/s13068-020-01777-x

  30. Marjamaa, K., Toth, K., Bromann, P. A., Szakacs, G., & Kruus, K. (2013). Novel Penicillium cellulases for total hydrolysis of lignocellulosics. Enzyme and Microbial Technology, 52(6–7), 358–369. https://doi.org/10.1016/j.enzmictec.2013.03.003

    Article  CAS  PubMed  Google Scholar 

  31. Rocha, V. A. L., Maeda, R. N., Santa Anna, L. M. M., & Pereira, N., Jr. (2013). Sugarcane bagasse as feedstock for cellulase production by Trichoderma harzianum in optimized culture medium. Journal of BElehnologyctroniciotec, 16(5), 1–13. https://doi.org/10.2225/vol16-issue5-fulltext-1

    Article  CAS  Google Scholar 

  32. Nathan, V., Esther Rani, M., Rathinasamy, G., Dhiraviam, K., & Jayavel, S. (2014). Process optimization and production kinetics for cellulase production by Trichoderma viride VKF3. Springerplus, 3(1), 92. https://doi.org/10.1186/2193-1801-3-92

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  33. Benoliel, B., Torres, F. A. G., & de Moraes, L. M. P. (2013). A novel promising Trichoderma harzianum strain for the production of a cellulolytic complex using sugarcane bagasse in natura. Springerplus, 2, 656. https://doi.org/10.1186/2193-1801-2-656

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  34. Pirzadah, T., Garg, S., Singh, J., Vyas, A., Kumar, M., Gaur, N., & Bala, M. (2014). Characterization of actinomycetes and Trichoderma spp. for cellulase production utilizing crude substrates by response surface methodology, SpringerPlus, 3(622), 1–12. https://doi.org/10.1186/2193-1801-3-622

  35. Reyes-Sosa, F. M., López Morales, M., Platero Gómez, A. I., Valbuena Crespo, N., Sánchez Zamorano, L., Rocha-Martín J., & DíezGarcía, B. (2017). Management of enzyme diversity in high-performance cellulolytic cocktails. Biotechnology for Biofuels, 10(1), 1–10.https://doi.org/10.1186/s13068-017-0845-6

  36. de Mojana di Cologna, N., Gómez-Mendoza, D. P., Zanoelo, F. F., Giannesi, G. C., de Alencar Guimarães, N. C., de Souza Moreira, L. R., & Ricart, C. A. O. (2018).Exploring Trichoderma and Aspergillus secretomes: Proteomics approaches for the identification of enzymes of biotechnological interest. Enzyme and Microbial Technology, 109, 1–10. https://doi.org/10.1016/J.ENZMICTEC.2017.08.007

  37. Sajith, S., Sreedevi, S., Priji, P., Unni, K. N., & Benjamin, S. (2015). Production and partial purification of cellulase from a new isolate, Penicillium verruculosum BS3. British Microbiology Research Journal, 9(1), 1–12. https://doi.org/10.9734/BMRJ/2015/17865

    Article  CAS  Google Scholar 

  38. Parambath, J. N., Valsala, G., & Krishnan, S. R. (2016). Purification and characterization of carboxymethyl cellulase ( CMCase ) from P enicillium ochrochloron isolated from forest soil of Neyyar Wild Life Sanctuary, India. International Journal of Biotechnology and Biochemistry, 12(2), 131–144.

    Google Scholar 

  39. Ritter, C. E. T., Camassola, M., Zampieri, D., Silveira, M. M., & Dillon, A. J. P. (2013). Cellulase and xylanase production by Penicillium echinulatum in submerged media containing cellulose amended with sorbitol. Enzyme Research, 2013, 1–9. https://doi.org/10.1155/2013/240219

  40. dos Santos, F. C., de Oliveira, M. A. S., Seixas, F. A. V., & Barbosa-Tessmann, I. P. (2020). A novel cellobiohydrolase I ( CBHI ) from Penicillium digitatum : Production, purification, and characterization. Applied Biochemistry and Biotechmology, 192, 257–282.

    Article  Google Scholar 

  41. Prajapati, B. P., Kumar Suryawanshi, R., Agrawal, S., Ghosh, M., & Kango, N. (2018). Characterization of cellulase from Aspergillus tubingensis NKBP-55 for generation of fermentable sugars from agricultural residues. Bioresource Technology, 250, 733–740. https://doi.org/10.1016/j.biortech.2017.11.099

    Article  CAS  PubMed  Google Scholar 

  42. Liu, D., Zhang, R., Yang, X., Zhang, Z., Song, S., Miao, Y., & Shen, Q. (2012). Characterization of a thermostable b -glucosidase from Aspergillus fumigatus Z5, and its functional expression in Pichia pastoris X33. Microbial Cell Factories, 11(1), 25. https://doi.org/10.1186/1475-2859-11-25

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  43. Grigorevski-Lima, A. L., Da Vinha, F. N. M., Souza, D. T., Bispo, A. S. R., Bon, E. P. S., Coelho, R. R. R., & Nascimento, R. P. (2009). Aspergillus fumigatus thermophilic and acidophilic endoglucanases. Applied Biochemistry and Biotechnology, 155(1–3), 18–26. https://doi.org/10.1007/s12010-008-8482-y

    Article  CAS  Google Scholar 

  44. Tao, Y. M., Zhu, X. Z., Huang, J. Z., Ma, S. J., Wu, X. B., Long, M. N., & Chen, Q. X. (2010). Purification and properties of endoglucanase from a sugar cane bagasse hydrolyzing strain, Aspergillus glaucus XC9. Journal of Agricultural and Food Chemistry, 58(10), 6126–6130. https://doi.org/10.1021/jf1003896

    Article  CAS  PubMed  Google Scholar 

  45. Imran, M., Anwar, Z., Zafar, M., Ali, A., & Arif, M. (2018). Production and Characterization of Commercial Cellulase Produced Through Aspergillus Niger Immis1 after Screening Fungal Species. Pakistan Journal of Botany, 50(27), 1563–1570. Retrieved from http://www.pakbs.org/pjbot/papers/1524266431.pdf. Accessed 22 Sep 2020.

  46. Das, A., Jana, A., Paul, T., Halder, S. K., Ghosh, K., & Maity, C. (2013). Thermodynamics and kinetic properties of halostable endoglucanase from Aspergillus fumigatus ABK9. Journal of Basic Microbiology, 54, 1–10. https://doi.org/10.1002/jobm.201300350

  47. Narra, M., Dixit, G., Divecha, J., Kumar, K., Madamwar, D., & Shah, A. R. (2014). Production, purification and characterization of a novel GH 12 family endoglucanase from Aspergillus terreus and its application in enzymatic degradation of delignified rice straw. International Biodeterioration & Biodegradation, 88, 150–161. https://doi.org/10.1016/j.ibiod.2013.12.016

    Article  CAS  Google Scholar 

  48. Moraes, A. D. O., Modesto, L. F., Isabel, N., Ramirez, B., & Pereira, N., Jr. (2016). Reuse of residual biomass of cellulose industry for second generation bioethanol production. Journal of Advances in Biotechnology, 6(1), 768–772.

    Article  Google Scholar 

  49. Bayitse, R., Hou, X., Bjerre, A.-B., & Saalia, F. K. (2015). Optimisation of enzymatic hydrolysis of cassava peel to produce fermentable sugars. AMB Express, 5(1), 60. https://doi.org/10.1186/s13568-015-0146-z

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  50. Efeovbokhan, V. E., Egwari, L., Alagbe, E. E., Adeyemi, J. T., & Taiwo, O. S. (2019). Production of bioethanol from hybrid cassava pulp and peel using microbial and acid hydrolysis. BioResources, 14(2), 2596–2609. Retrieved from https://bioresources.cnr.ncsu.edu/resources/production-of-bioethanol-from-hybrid-cassava-pulp-and-peel-using-microbial-andacid-hydrolysis. Accessed 22 Sept 2020.

  51. Jun, H., Kieselbach, T., & Jönsson, L. J. (2011). Enzyme production by filamentous fungi: Analysis of the secretome of Trichoderma reesei grown on unconventional carbon source. Microbial Cell Factories. https://doi.org/10.1186/1475-2859-10-68

    Article  PubMed  PubMed Central  Google Scholar 

  52. Chen, C., Duan, C., Li, J., Liu, Y., & Ma, X. (2016). Cellulose (dissolving pulp) manufacturing processes. BioResources, 11(2), 5553–5564. Retrieved from https://ojs.cnr.ncsu.edu/index.php/BioRes/article/view/BioRes_11_2_Review_Chen_Cellulose_Manufacturing_Processes. Accessed 25 Aug 2020.

  53. Lopes, A. M., Filho, E. X. F., & Moreira, L. R. S. (2018). An update on enzymatic cocktails for lignocellulose breakdown. Journal of Applied Microbiology, 125(3), 632–645. https://doi.org/10.1111/jam.13923

  54. Maeda, N. R., Isabel, V., Alves, V., Rocha, L., Aparecida, R., Mesquita, A., & São-carlense, A. T. (2011). Enzymatic hydrolysis of pretreated sugar cane bagasse using Penicillium funiculosum and Trichoderma harzianum cellulases. Process Biochemistry, 46(5), 1196–1201.https://doi.org/10.1016/j.procbio.2011.01.022

  55. Adsul, M., Sharma, B., Singhania, R. R., Saini, J. K., Sharma, A., Mathur, A., & Tuli, DK. (2014) Blending of cellulolytic enzyme preparations from different fungal sources for improved cellulose hydrolysis by increasing synergism. RSC Advances. 4(84), 44726–44732. https://doi.org/10.1039/C4RA08129C

  56. Arias, J. M., Modesto, L. F. A., Polikarpov, I., & Pereira Jr., N. (2016). Design of an Enzyme Cocktail Consisting of Different Fungal Platforms for Efficient Hydrolysis of Sugarcane Bagasse: Optimization and Synergism Studies. Biotechnology Progress, 00, 1–8. https://doi.org/10.1002/btpr.2306

  57. Agrawal, R., Semwal, S., Kumar, R., Mathur, A., & Gupta, R. P. (2018). Synergistic enzyme cocktail to enhance hydrolysis of steam exploded wheat straw at pilot scale 6 November 1 11. https://doi.org/10.3389/fenrg.2018.00122

  58. Teter, S. A., Sutton, K. B., & Emme, B. (2014). Enzymatic processes and enzyme development in biorefining. In Advances in Biorefineries (pp. 199–233). Elsevier. https://doi.org/10.1533/9780857097385.1.199

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Pimentel, P.S.SR., de Oliveira, J.B., Astolfi-Filho, S. et al. Enzymatic Hydrolysis of Lignocellulosic Biomass Using an Optimized Enzymatic Cocktail Prepared from Secretomes of Filamentous Fungi Isolated from Amazonian Biodiversity. Appl Biochem Biotechnol 193, 3915–3935 (2021). https://doi.org/10.1007/s12010-021-03642-5

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