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Nanocellulose isolation using a thermostable endoglucanase-rich cocktail from Myceliophthora thermophila cultivated in a multilayer packed-bed bioreactor

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Abstract

The isolation of nanocellulose materials by means of enzymatic hydrolysis offers advantages in terms of their properties and operational conditions, but there is still a lack of enzymatic cocktails specifically designed for this application. The present work investigates the use of a thermostable endoglucanase-rich enzymatic cocktail from Myceliophthora thermophila for nanocellulose production. An initial set of experiments was conducted to determine the optimal conditions for enzyme production under solid-state cultivation in small-scale polypropylene plastic bags. A full factorial experimental design was used as a statistical tool to evaluate the effects of the solid substrate and moisture content on the production of endoglucanase and β-glucosidase. Solid-state cultivations in a multilayer packed-bed bioreactor were then carried out under the selected conditions to obtain a larger volume of an enzymatic extract with high endoglucanase selectivity, given by the ratio of endoglucanase (203 ± 6 U/g-substrate) to β-glucosidase activity (1.6 ± 0.1 U/g-substrate). The enzymatic hydrolysis of eucalyptus cellulose pulp at different temperatures, with this endoglucanase-rich cocktail at a loading of 10 mg-protein/g-pulp, resulted in higher glucose release (11 ± 2 g-glucose/L) at 60 °C, with 61 ± 9% cellulose conversion and 16.2% nanocellulose yield. The isolated nanomaterial with average diameter of 36.65 nm and length of 156.63 nm, presented a crystallinity index of 77.4% and thermal stability temperature of 316 °C. These results show the potential to isolate nanocellulose using a thermostable endoglucanase-rich enzymatic cocktail produced locally by solid-state cultivation in a packed-bed bioreactor, in accordance to the biorefinery concept.

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References

  1. Bondancia TJ, Mattoso LHC, Marconcini JM, Farinas CS (2017) A new approach to obtain cellulose nanocrystals and ethanol from eucalyptus cellulose pulp via the biochemical pathway. Biotechnol Prog [Internet] 33(4):1085–95. https://doi.org/10.1002/btpr.2486

    Article  Google Scholar 

  2. Squinca P, Bilatto S, Badino AC, Farinas CS (2020) Nanocellulose production in future biorefineries: an integrated approach using tailor-made enzymes. ACS Sustain Chem Eng [Internet] 8(5):2277–86. https://doi.org/10.1177/004051755902901003

    Article  Google Scholar 

  3. Bilatto S, Marconcini JM, Mattoso LHC, Farinas CS (2020) Lignocellulose nanocrystals from sugarcane straw. Ind Crops Prod [Internet] 157:1–10. https://doi.org/10.1016/j.indcrop.2020.112938

    Article  Google Scholar 

  4. Finny AS, Popoola O, Andreescu S (2021) 3d-printable nanocellulose-based functional materials: fundamentals and applications. Nanomaterials [Internet]. 11(9). Available from: https://doi.org/10.3390/nano11092358

  5. Mateo S, Peinado S, Morillas-Gutiérrez F, La Rubia MD, Moya AJ (2021) Nanocellulose from agricultural wastes: products and applications—a review. Processes [Internet]. 9(9). Available from: https://doi.org/10.3390/pr9091594

  6. Trache D, Tarchoun AF, Derradji M, Hamidon TS, Masruchin N, Brosse N, et al (2020) Nanocellulose: from fundamentals to advanced applications [Internet]. Vol. 8, Frontiers in Chemistry. Available from: https://doi.org/10.3389/fchem.2020.00392

  7. Espino-Pérez E, Domenek S, Belgacem N, Sillard C, Bras J (2014) Green process for chemical functionalization of nanocellulose with carboxylic acids. Biomacromolecules [Internet] 15(12):4551–60. https://doi.org/10.1021/bm5013458

    Article  Google Scholar 

  8. Berto GL, Mattos BD, Rojas OJ, Arantes V (2021) Single-step fiber pretreatment with monocomponent endoglucanase: defibrillation energy and cellulose nanofibril quality. ACS Sustain Chem Eng [Internet] 9(5):2260–70. https://doi.org/10.1021/acssuschemeng.0c08162

    Article  Google Scholar 

  9. Faradilla RHF, Lee G, Rawal A, Hutomo T, Stenzel MH, Arcot J (2016) Nanocellulose characteristics from the inner and outer layer of banana pseudo-stem prepared by TEMPO-mediated oxidation. Cellulose [Internet] 23(5):3023–37. https://doi.org/10.1007/s10570-016-1025-8

    Article  Google Scholar 

  10. Yamato K, Yoshida Y, Kumamoto Y, Isogai A (2021) Surface modification of TEMPO-oxidized cellulose nanofibers, and properties of their acrylate and epoxy resin composite films. Cellulose [Internet]. Available from: https://doi.org/10.1007/s10570-021-04131-y

  11. Krontiras P, Gatenholm P, Hagg DA (2015) Adipogenic differentiation of stem cells in three-dimensional porous bacterial nanocellulose scaffolds. J Biomed Mater Res - Part B Appl Biomater 103(1):195–203

    Article  Google Scholar 

  12. Yuan H, Chen L, Hong FF (2021) Homogeneous and efficient production of a bacterial nanocellulose-lactoferrin-collagen composite under an electric field as a matrix to promote wound healing. Biomater Sci [Internet] 9(3):930–41. https://doi.org/10.1039/D0BM01553A%0A

    Article  Google Scholar 

  13. Ribeiro RSA, Pohlmann BC, Calado V, Bojorge N, Pereira N (2019) Production of nanocellulose by enzymatic hydrolysis: trends and challenges. Eng Life Sci [Internet] 19(4):279–91. https://doi.org/10.1002/elsc.201800158

    Article  Google Scholar 

  14. Bondancia TJ, Corrêa LJ, Cruz AJG, Badino AC, Mattoso LHC, Marconcini JM et al (2018) Enzymatic production of cellulose nanofibers and sugars in a stirred-tank reactor: determination of impeller speed, power consumption, and rheological behavior. Cellulose [Internet] 25(8):4499–511. https://doi.org/10.1007/s10570-018-1876-2

    Article  Google Scholar 

  15. Rosales‐Calderon O, Pereira B, Arantes V (2021) Economic assessment of the conversion of bleached eucalyptus Kraft pulp into cellulose nanocrystals in a stand‐alone facility via acid and enzymatic hydrolysis. Biofuels, Bioprod Biorefining [Internet]. 1–14. Available from: https://doi.org/10.1002/bbb.2277

  16. Bhati N, Shreya, Sharma AK (2021) Cost-effective cellulase production, improvement strategies, and future challenges. J Food Process Eng [Internet]. 44(2). Available from: https://doi.org/10.1111/jfpe.13623

  17. Siqueira GA, Dias IKR, Arantes V (2019) Exploring the action of endoglucanases on bleached eucalyptus kraft pulp as potential catalyst for isolation of cellulose nanocrystals. Int J Biol Macromol [Internet] 133:1249–59. https://doi.org/10.1016/j.ijbiomac.2019.04.162

    Article  Google Scholar 

  18. Casciatori PA, Casciatori FP, Thomeo JC, da Silva R (2015) Phe Temperatura Ótimos Para Atividade Endoglucanase De Fungos Em Cultivo Sólido. In: Anais do XX Congresso Brasileiro de Engenharia Química - COBEQ 2014. Blucher Chemical Engineering Proceedings, vol 1, no 2. Blucher, São Paulo, pp 2202-2209. Available from: https://doi.org/10.5151/chemeng-cobeq2014-1577-18593-170726

  19. de Oliveira SP, Alvarez Rodrigues N, Casciatori-Frassatto PA, Casciatori FP (2020) Solid-liquid extraction of cellulases from fungal solid-state cultivation in a packed bed bioreactor. Korean J Chem Eng [Internet] 37(9):1530–40. https://doi.org/10.1007/s11814-020-0579-1

    Article  Google Scholar 

  20. Perez CL, Casciatori FP, Thoméo JC (2018) Strategies for scaling-up packed-bed bioreactors for solid-state fermentation: the case of cellulolytic enzymes production by a thermophilic fungus. Chem Eng J [Internet] 2019(361):1142–51. https://doi.org/10.1016/j.cej.2018.12.169

    Article  Google Scholar 

  21. Zanelato AI, Shiota VM, Gomes E, da Silva R, Thoméo JC (2012) Endoglucanase production with the newly isolated Myceliophtora sp. I-1D3b in a packed bed solid state fermentor. Brazilian J Microbiol [Internet] 43(4):1536–44. https://doi.org/10.1590/S1517-83822012000400038

    Article  Google Scholar 

  22. Bruins ME, Janssen AEM, Boom RM (2001) Thermozymes and their applications: a review of recent literature and patents. Appl Biochem Biotechnol - Part A Enzym Eng Biotechnol [Internet] 90(2):155–86. https://doi.org/10.1385/abab:90:2:155

    Article  Google Scholar 

  23. Pandey A (2003) Solid-state fermentation. Biochem Eng J [Internet] 13(2–3):81–4. https://doi.org/10.1016/s1369-703x(02)00121-3

    Article  Google Scholar 

  24. Farinas CS (2015) Developments in solid-state fermentation for the production of biomass-degrading enzymes for the bioenergy sector. Renew Sustain Energy Rev [Internet] 52:179–88. https://doi.org/10.1016/j.rser.2015.07.092

    Article  Google Scholar 

  25. Soccol CR, da Costa ESF, Letti LAJ, Karp SG, Woiciechowski AL, de Vandenberghe S LP (2017) Recent developments and innovations in solid state fermentation. Biotechnol Res Innov [Internet] 1(1):52–71. https://doi.org/10.1016/j.biori.2017.01.002

    Article  Google Scholar 

  26. Mitchell DA, Cunha LEN, Machado AVL, de Lima Luz LF, Krieger N (2010) A model-based investigation of the potential advantages of multi-layer packed beds in solid-state fermentation. Biochem Eng J [Internet]. 48(2):195–203. https://doi.org/10.1016/j.bej.2009.10.008

    Article  Google Scholar 

  27. CasciatoriFrassatto PA, Casciatori FP, Thoméo JC, Gomes E, Boscolo M, da Silva R (2020) Fungal cellulases: production by solid-state cultivation in packed-bed bioreactor using solid agro-industrial by-products as substrates and application for hydrolysis of sugarcane bagasse [Internet]. Ciencias Agrarias Semina 41:2097–116. https://doi.org/10.5433/1679-0359.2020v41n5Supl1p2097

    Article  Google Scholar 

  28. Leang YH, Saw HY (2011) Proximate and functional properties of sugarcane bagasse. Agro Food Ind Hitech 22(2):5–8

    Google Scholar 

  29. Szczerbowski D, Pitarelo AP, Zandoná Filho A, Ramos LP (2014) Sugarcane biomass for biorefineries: comparative composition of carbohydrate and non-carbohydrate components of bagasse and straw. Carbohydr Polym [Internet] 114:95–101. https://doi.org/10.1016/j.carbpol.2014.07.052

    Article  Google Scholar 

  30. Philippini RR, Martiniano SE, Chandel AK, de Carvalho W, da Silva SS (2019) Pretreatment of sugarcane bagasse from cane hybrids: effects on chemical composition and 2G sugars recovery. Waste and Biomass Valorization [Internet] 10(6):1561–70. https://doi.org/10.1007/s12649-017-0162-0

    Article  Google Scholar 

  31. Casciatori FP, Laurentino CL, Taboga SR, Casciatori PA, Thoméo JC (2014) Structural properties of beds packed with agro-industrial solid by-products applicable for solid-state fermentation: experimental data and effects on process performance. Chem Eng J [Internet] 255:214–24. https://doi.org/10.1016/j.cej.2014.06.040

    Article  Google Scholar 

  32. Ramaraj B (2007) Mechanical and thermal properties of polypropylene/sugarcane bagasse composites. J Appl Polym Sci [Internet] 103(6):3827–32. https://doi.org/10.1002/app.25333

    Article  Google Scholar 

  33. Bradford MM (1976) A rapid and sensitive method for the quantitation microgram quantities of protein utilizing the principle of protein-dye. Anal Biochem [Internet] 72:248–54. https://doi.org/10.1016/0003-2697(76)90527-3

    Article  Google Scholar 

  34. Ghose TK (1987) Measurement of cellulase activities. Pure Appl Chem [Internet] 59(2):257–68. https://doi.org/10.1351/pac198759020257

    Article  Google Scholar 

  35. Ghose TK, Bisaria VS (1987) Measurement of hemicellulase activities Part 1: Xylanases. Pure Appl Chem [Internet] 59(12):1739–52. https://doi.org/10.1351/pac198759020257

    Article  Google Scholar 

  36. Segal L, Creely JJ, Martin AE, Conrad CM (1959) An empirical method for estimating the degree of crystallinity of native cellulose using the X-ray diffractometer. Text Res J [Internet] 29(10):786–94. https://doi.org/10.1177/004051755902901003

    Article  Google Scholar 

  37. Massarente VS, de Araujo Zanoni J, Gomes E, Bonilla-Rodriguez GO (2020) Biochemical characterization of endoglucanases produced by Myceliophthora thermophila M.7.7 in solid-state culture. Biocatal Agric Biotechnol [Internet]. 27(July). Available from: https://doi.org/10.1016/j.bcab.2020.101684

  38. de Almeida MN, Falkoski DL, Guimarães VM, de Rezende ST (2019) Study of gamba grass as carbon source for cellulase production by Fusarium verticillioides and its application on sugarcane bagasse saccharification. Ind Crops Prod [Internet] 133(March):33–43. https://doi.org/10.1016/j.indcrop.2019.03.008

    Article  Google Scholar 

  39. de Albuquerque MFG, Guimarães VM, de Rezende ST (2020) Use of sugar beet flour and wheat bran as carbon source improves the efficiency of chrysoporthe cubensis enzymes in sugarcane bagasse saccharification. Bioenergy Res [Internet]. Available from: https://doi.org/10.1007/s12155-020-10224-6

  40. Benocci T, Aguilar-Pontes MV, Zhou M, Seiboth B, Vries RP, de (2017) Regulators of plant biomass degradation in ascomycetous fungi. Biotechnol Biofuels [Internet] 10(1):152. https://doi.org/10.1186/s13068-017-0841-x

    Article  Google Scholar 

  41. Kadowaki MAS, Várnai A, Jameson J-K, T. Leite AE, Costa-Filho AJ, Kumagai PS et al (2018) Functional characterization of a lytic polysaccharide monooxygenase from the thermophilic fungus Myceliophthora thermophila. Berrin J-G, editor. PLoS One. 13(8):1–16. https://doi.org/10.1371/journal.pone.0202148

    Article  Google Scholar 

  42. Meesupthong R, Yingkamhaeng N, Nimchua T, Pinmanee P, Mussatto SI, Li B et al (2021) Xylanase pretreatment of energy cane enables facile cellulose nanocrystal isolation. Cellulose [Internet] 28(2):799–812. https://doi.org/10.1007/s10570-020-03559-y

    Article  Google Scholar 

  43. Hu J, Tian D, Renneckar S, Saddler JN (2018) Enzyme mediated nanofibrillation of cellulose by the synergistic actions of an endoglucanase, lytic polysaccharide monooxygenase (LPMO) and xylanase. Sci Rep [Internet] 8(3195):1–8. https://doi.org/10.1038/s41598-018-21016-6

    Article  Google Scholar 

  44. Bala A, Singh B (2019) Cellulolytic and xylanolytic enzymes of thermophiles for the production of renewable biofuels. Renew Energy [Internet] 136:1231–44. https://doi.org/10.1016/j.renene.2018.09.100

    Article  Google Scholar 

  45. Paës G, O’Donohue MJ (2006) Engineering increased thermostability in the thermostable GH-11 xylanase from Thermobacillus xylanilyticus. J Biotechnol [Internet] 125(3):338–50. https://doi.org/10.1016/j.jbiotec.2006.03.025

    Article  Google Scholar 

  46. Cowan DA (1997) Thermophilic proteins: Stability and function in aqueous and organic solvents. Comp Biochem Physiol - A Physiol [Internet] 118(3):429–38. https://doi.org/10.1016/S0300-9629(97)00004-2

    Article  Google Scholar 

  47. Zhang Y, Chen J, Zhang L, Zhan P, Liu N, Wu Z (2020) Preparation of nanocellulose from steam exploded poplar wood by enzymolysis assisted sonication. Mater Res Express [Internet]. 7(3). Available from: https://doi.org/10.1088/2053-1591/ab7b28

  48. Chen L, Wang Q, Hirth K, Baez C, Agarwal UP, Zhu JY (2015) Tailoring the yield and characteristics of wood cellulose nanocrystals (CNC) using concentrated acid hydrolysis. Cellulose [Internet] 22(3):1753–62. https://doi.org/10.1007/s10570-015-0615-1

    Article  Google Scholar 

  49. Martelli-Tosi M, Torricillas MDS, Martins MA, Assis OBG De, Tapia-Blácido DR (2016) Using commercial enzymes to produce cellulose nanofibers from soybean straw. J Nanomater [Internet]. 2016. Available from: https://doi.org/10.1155/2016/8106814

  50. JT Xu CXQ (2019) Preparation characterization of spherical cellulose nanocrystals with high purity by the composite enzymolysis of pulp fibers. Bioresour Technol [Internet] 291:1–6. https://doi.org/10.1016/j.biortech.2019.121842

    Article  Google Scholar 

  51. Foster EJ, Moon RJ, Agarwal UP, Bortner MJ, Bras J, Camarero-Espinosa S et al (2018) Current characterization methods for cellulose nanomaterials. Chem Soc Rev [Internet] 47(8):2609–79. https://doi.org/10.1039/c6cs00895j

    Article  Google Scholar 

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Acknowledgements

This study was financed in part by the National Council for Scientific and Technological Development (CNPq, grant #30786/2018-2), SISNANO (MCTI), FINEP, the Embrapa AgroNano research network, Coordenação de Aperfeiçoamento de Pessoal de Nível Superior—Brazil (CAPES, Finance Code 001), and the São Paulo State Research Foundation (FAPESP, grants #2019/25261-8, #2016/10636-8, #2018/00996-2, and #2018/10899-4). The AFM measurements were supported by LNNano (Brazilian Nanotechnology National Laboratory) and CNPEM/MCTIC (Brazilian Center for Research in Energy and Materials).

Funding

This study was financed in part by the National Council for Scientific and Technological Development (CNPq, grant #30786/2018–2), SISNANO (MCTI), FINEP, the Embrapa AgroNano research network, Coordenação de Aperfeiçoamento de Pessoal de Nível Superior—Brazil (CAPES, Finance Code 001), and the São Paulo State Research Foundation (FAPESP, grants #2019/25261–8, #2016/10636–8, #2018/00996–2, and #2018/10899–4).

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Authors and Affiliations

  1. National Nanotechnology Laboratory for Agribusiness (LNNA), Embrapa Instrumentation, São Carlos, SP, Brazil

    Eric Katayama, Stanley Bilatto & Cristiane S. Farinas

  2. Graduate Program of Chemical Engineering, Federal University of São Carlos, São Carlos, SP, Brazil

    Eric Katayama, Natalia A. Rodrigues, Fernanda P. Casciatori & Cristiane S. Farinas

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  1. Eric Katayama
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  2. Natalia A. Rodrigues
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Contributions

Eric Katayama: methodology, data curation, formal analysis, investigation, writing—original draft; Natalia A. Rodrigues: methodology, data curation, formal analysis, writing—review and editing; Stanley Bilatto: methodology, data curation, Formal analysis, writing—review and editing; Fernanda P. Casciatori: writing—review and editing, Supervision; Cristiane S. Farinas: conceptualization, funding acquisition, supervision, writing—review and editing.

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Correspondence to Cristiane S. Farinas.

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Katayama, E., Rodrigues, N.A., Bilatto, S. et al. Nanocellulose isolation using a thermostable endoglucanase-rich cocktail from Myceliophthora thermophila cultivated in a multilayer packed-bed bioreactor. Biomass Conv. Bioref. 14, 9121–9136 (2024). https://doi.org/10.1007/s13399-022-02977-1

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