Enzymes Involved in Lignocellulose Decomposition and Effects of Fungal Pretreatment During Solid-state Fermentation of Lignocellulosic Agricultural Residues: The Case of Trametes versicolor
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Abstract
Ligninolytic fungi are considered useful for different biotechnological uses such as lignocellulose pretreatment for biorefinery applications, composting, mushroom production, or bioremediation purposes. Enzymes contributing to lignocellulose breakdown represent valuable biocatalysts. Trametes versicolor is a well-known ligninolytic white-rot basidiomycete capable of growing on different lignocellulosic materials. In the present study, the use of T. versicolor, which secretes several enzymes capable of degrading lignocellulosic biomass, was evaluated from two biotechnological aspects including fungal crude extracts and effects on enzymatic saccharification of wheat straw. We used 20-day-old wheat straw cultures of this fungus to characterize its lignocellulose-decomposing multi-enzyme complex. At the end of the cultivation period, losses of approximately 41% in lignin and 30% in total dry mass were observed relative to their initial values. A total of 17 enzyme activities, each acting differently on the polysaccharide components of the lignocellulosic substrate, and lignin-modifying enzyme activities (laccase, peroxidases) were investigated in aqueous extracts of fungal cultures. The activities of carboxylesterase (substrate: 4-nitrophenyl valerate), endo-1,4-β-D-glucanase (substrate: 2-hydroxyethylcellulose), and laccase were found to predominate. In summary, fungal crude extracts containing different enzyme activities may be promising for various biotechnological purposes. Trametes versicolor effectively pretreated wheat straw, significantly increasing reducing sugar levels compared to controls.
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References
Artola, A., Font, X., Moral-Vico, J., & Sánchez, A. (2024). The role of solid-state fermentation to transform existing waste treatment plants based on composting and anaerobic digestion into modern organic waste-based biorefineries, in the framework of circular bioeconomy. Frontiers in Chemical Engineering, 6, Article 1463785. https://doi.org/10.3389/fceng.2024.1463785
Asgher, M., Wahab, A., Bilal, M., & Iqbal, H. M. N. (2018). Delignification of lignocellulose biomasses by alginate–chitosan immobilized laccase produced from Trametes versicolor IBL-04. Waste and Biomass Valorization, 9(11), 2071-2079. https://doi.org/10.1007/s12649-017-9991-0
Bailey, M. J. (1988). A note on the use of dinitrosalicylic acid for determining the products of enzymatic reactions. Applied Microbiology and Biotechnology, 29(5), 494-496. https://doi.org/10.1007/BF00269074
Baral, N. R., & Shah, A. (2017). Comparative techno-economic analysis of steam explosion, dilute sulfuric acid, ammonia fiber explosion and biological pretreatments of corn stover. Bioresource Technology, 232, 331-343. https://doi.org/10.1016/j.biortech.2017.02.068
Baruah, J., Nath, B. K., Sharma, R., Kumar, S., Deka, R. C., Baruah, D. C., & Kalita, E. (2018). Recent trends in the pretreatment of lignocellulosic biomass for value-added products. Frontiers in Energy Research, 6, Article 141. https://doi.org/10.3389/fenrg.2018.00141
Bhat, M. K. (2000). Cellulases and related enzymes in biotechnology. Biotechnology Advances, 18(5), 355-383. https://doi.org/10.1016/S0734-9750(00)00041-0
Bhatia, S. K., Kim, S. H., Yoon, J. J., & Yang, Y. H. (2017). Current status and strategies for second generation biofuel production using microbial systems. Energy Conversion and Management, 148, 1142-1156. https://doi.org/10.1016/j.enconman.2017.06.073
Bornscheuer, U. T. (2002). Microbial carboxyl esterases: classification, properties and application in biocatalysis. FEMS Microbiology Reviews, 26(1), 73-81. https://doi.org/10.1016/S0168-6445(01)00075-4
Bucić-Kojić, A., Šelo, G., Zelić, B., Planinić, M., & Tišma, M. (2017). Recovery of phenolic acid and enzyme production from corn silage biologically treated by Trametes versicolor. Applied Biochemistry and Biotechnology, 181(3), 948-960. https://doi.org/10.1007/s12010-016-2261-y
Canam, T., Town, J. R., Tsang, A., McAllister, T. A., & Dumonceaux, T. J. (2011). Biological pretreatment with a cellobiose dehydrogenase-deficient strain of Trametes versicolor enhances the biofuel potential of canola straw. Bioresource Technology, 102(21), 10020–10027. https://doi.org/10.1016/j.biortech.2011.08.045
Cardoso, W. S., Queiroz, P. V., Tavares, G. P., Santos, F. A., Soares, F. E. D. F., Kasuya, M. C. M., & de Queiroz, J. H. (2018a). Multi-enzyme complex of white rot fungi in saccharification of lignocellulosic material. Brazilian Journal of Microbiology, 49(4), 879-884. https://doi.org/10.1016/j.bjm.2018.05.006
Cardoso, W. S., Soares, F. E. D. F., Queiroz, P. V., Tavares, G. P., Santos, F. A., Sufiate, B. L., Kasuya, M. C. M., & de Queiroz, J. H. (2018b). Minimum cocktail of cellulolytic multi-enzyme complexes obtained from white rot fungi via solid-state fermentation. 3 Biotech, 8(1), Article 46. https://doi.org/10.1007/s13205-017-1073-2
Cerda, A., Artola, A., Barrena, R., Font, X., Gea, T., & Sánchez, A. (2019). Innovative production of bioproducts from organic waste through solid-state fermentation. Frontiers in Sustainable Food Systems, 3, Article 63. https://doi.org/10.3389/fsufs.2019.00063
Daâssi, D., Prieto, A., Zouari-Mechichi, H., Martínez, M. J., Nasri, M., & Mechichi, T. (2016). Degradation of bisphenol A by different fungal laccases and identification of its degradation products. International Biodeterioration and Biodegradation, 110, 181-188. https://doi.org/10.1016/j.ibiod.2016.03.017
Dauda, M. Y., & Erkurt, E. A. (2020). Investigation of reactive Blue 19 biodegradation and byproducts toxicity assessment using crude laccase extract from Trametes versicolor. Journal of Hazardous Materials, 393, Article 121555. https://doi.org/10.1016/j.jhazmat.2019.121555
Dubois, M., Gilles, K., Hamilton, J., Rebers, P., & Smith, F. (1956). Colorimetric method for determination of sugars and related substances. Analytical Chemistry, 28(3), 350-356. https://doi.org/10.1021/ac60111a017
Duong, H. L., Paufler, S., Harms, H., Maskow, T., & Schlosser, D. (2022a). Applicability and information value of biocalorimetry for the monitoring of fungal solid-state fermentation of lignocellulosic agricultural by-products. New Biotechnology, 66, 97-106. https://doi.org/10.1016/j.nbt.2021.11.001
Duong, H. L., Paufler, S., Harms, H., Schlosser, D., & Maskow, T. (2022b). Fungal lignocellulose utilisation strategies from a bioenergetic perspective: quantification of related functional traits using biocalorimetry. Microorganisms, 10(8), Article 1675. https://doi.org/10.3390/microorganisms10081675
Duong, H. L., Paufler, S., Harms, H., Maskow, T., & Schlosser, D. (2024). Biocalorimetry-aided monitoring of fungal pretreatment of lignocellulosic agricultural residues. Applied Microbiology and Biotechnology, 108(1), Article 394. https://doi.org/10.1007/s00253-024-13234-y
Falkoski, D. L., Guimarães, V. M., de Almeida, M. N., Alfenas, A. C., Colodette, J. L., & de Rezende, S. T. (2013). Chrysoporthe cubensis: a new source of cellulases and hemicellulases to application in biomass saccharification processes. Bioresource Technology, 130, 296-305. https://doi.org/10.1016/j.biortech.2012.11.140
Franklin, M. R. (2007). Phase I biotransformation reactions-esterases, and amidases. In S. J. Enna & D. B. Bylund (Eds.). xPharm: The comprehensive pharmacology reference (pp. 1-3). Elsevier. https://doi.org/10.1016/B978-008055232-3.60279-X
Giacobbe, S., Pezzella, C., Della Ventura, B., Giacobelli, V. G., Rossi, M., Fontanarosa, C., Amoresano, A., Sannia, G., Velotta, R., & Piscitelli, A. (2019). Green synthesis of conductive polyaniline by Trametes versicolor laccase using a DNA template. Engineering in Life Sciences, 19(9), 631-642. https://doi.org/10.1002/elsc.201900078
Gonçalves, C., Rodriguez-Jasso, R. M., Gomes, N., Teixeira, J. A., & Belo, I. (2010). Adaptation of dinitrosalicylic acid method to microtiter plates. Analytical Methods, 2(12), 2046-2048. https://doi.org/10.1039/c0ay00525h
Gong, S., Chen, C., Zhu, J., Qi, G., & Jiang, S. (2018). Effects of wine-cap Stropharia cultivation on soil nutrients and bacterial communities in forestlands of northern China. PeerJ, 6, Article e5741. https://doi.org/10.7717/peerj.5741
Gottschalk, L. M. F., Oliveira, R. A., & Bon, E. P. D. S. (2010). Cellulases, xylanases, β-glucosidase and ferulic acid esterase produced by Trichoderma and Aspergillus act synergistically in the hydrolysis of sugarcane bagasse. Biochemical Engineering Journal, 51(1), 72-78. https://doi.org/10.1016/j.bej.2010.05.003
Hermosilla, E., Rubilar, O., Schalchli, H., da Silva, A. S. A., Ferreira-Leitao, V., & Diez, M. C. (2018). Sequential white-rot and brown-rot fungal pretreatment of wheat straw as a promising alternative for complementary mild treatments. Waste Management, 79, 240-250. https://doi.org/10.1016/j.wasman.2018.07.044
Hölker, U., Höfer, M., & Lenz, J. (2004). Biotechnological advantages of laboratory-scale solid-state fermentation with fungi. Applied Microbiology and Biotechnology, 64, 175-186. https://doi.org/10.1007/s00253-003-1504-3
Hu, J., Arantes, V., & Saddler, J. N. (2011). The enhancement of enzymatic hydrolysis of lignocellulosic substrates by the addition of accessory enzymes such as xylanase: is it an additive or synergistic effect? Biotechnology for Biofuels, 4(1), Article 36. https://doi.org/10.1186/1754-6834-4-36
Johan, U. U. M., Rahman, R. N. Z. R. A., Kamarudin, N. H. A., & Ali, M. S. M. (2021). An integrated overview of bacterial carboxylesterase: structure, function and biocatalytic applications. Colloids and Surfaces B: Biointerfaces, 205, Article 111882. https://doi.org/10.1016/j.colsurfb.2021.111882
Kademi, A., Aït-Abdelkader, N., Fakhreddine, L., & Baratti, J. C. (2000). Characterization of a new thermostable esterase from the moderate thermophilic bacterium Bacillus circulans. Journal of Molecular Catalysis - B Enzymatic, 10, 395-401. https://doi.org/10.1016/S1381-1177(99)00111-3
Kandhola, G., Rajan, K., Labbé, N., Chmely, S., Heringer, N., Kim, J.-W., Hood, E. E., & Carrier, D. J. (2017). Beneficial effects of Trametes versicolor pretreatment on saccharification and lignin enrichment of organosolv-pretreated pinewood. RSC Advances, 7(72), 45652-45661. https://doi.org/10.1039/C7RA09188E
Kapoor, R. K., Rajan, K., & Carrier, D. J. (2015). Applications of Trametes versicolor crude culture filtrates in detoxification of biomass pretreatment hydrolyzates. Bioresource Technology, 189, 99-106. https://doi.org/10.1016/j.biortech.2015.03.100
Kostylev, M., & Wilson, D. (2012). Synergistic interactions in cellulose hydrolysis. Biofuels, 3(1), 61-70. https://doi.org/10.4155/bfs.11.150
Kovacs, K., Macrelli, S., Szakacs, G., & Zacchi, G. (2009). Enzymatic hydrolysis of steam-pretreated lignocellulosic materials with Trichoderma atroviride enzymes produced in-house. Biotechnology for Biofuels, 2(1), Article 14. https://doi.org/10.1186/1754-6834-2-14
Kračun, S. K., Schückel, J., Westereng, B., Thygesen, L. G., Monrad, R. N., Eijsink, V. G. H., & Willats, W. G. T. (2015). A new generation of versatile chromogenic substrates for high-throughput analysis of biomass-degrading enzymes. Biotechnology for Biofuels, 8(70), Article 70. https://doi.org/10.1186/s13068-015-0250-y
Kumla, J., Suwannarach, N., Sujarit, K., Penkhrue, W., Kakumyan, P., Jatuwong, K., Vadthanarat, S., & Lumyong, S. (2020). Cultivation of mushrooms and their lignocellulolytic enzyme production through the utilization of agro-industrial waste. Molecules, 25(12), Article 2811. https://doi.org/10.3390/molecules25122811
Lee, J., Gwak, K., Park, J., Park, M., Choi, D., Kwon, M., & Choi, I. (2007). Biological pretreatment of softwood Pinus densiflora by three white rot fungi. The Journal of Microbiology, 45(6), 485-491.
Lizardi-Jiménez, M. A., & Hernández-Martínez, R. (2017). Solid state fermentation (SSF): diversity of applications to valorize waste and biomass. 3 Biotech, 7, Article 44. https://doi.org/10.1007/s13205-017-0692-y
López-Abelairas, M., Álvarez Pallín, M., Salvachúa, D., Lú-Chau, T., Martínez, M. J., & Lema, J. M. (2013). Optimisation of the biological pretreatment of wheat straw with white-rot fungi for ethanol production. Bioprocess and Biosystems Engineering, 36(9), 1251-1260. https://doi.org/10.1007/s00449-012-0869-z
López-Gutiérrez, I., Razo-Flores, E., Méndez-Acosta, H. O., Amaya-Delgado, L., & Alatriste-Mondragón, F. (2021). Optimization by response surface methodology of the enzymatic hydrolysis of non-pretreated agave bagasse with binary mixtures of commercial enzymatic preparations. Biomass Conversion and Biorefinery, 11(6), 2923-2935. https://doi.org/10.1007/s13399-020-00698-x
McIlvaine, T. C. (1921). A buffer solution for colorimetric comparison. Journal of Biological Chemistry, 49(1), 183-186. https://doi.org/10.1016/s0021-9258(18)86000-8
Meyer, V., Basenko, E. Y., Benz, J. P., Braus, G. H., Caddick, M. X., Csukai, M., De Vries, R. P., Endy, D., Frisvad, J. C., Gunde-Cimerman, N., Haarmann, T., Hadar, Y., Hansen, K., Johnson, R. I., Keller, N. P., Kraševec, N., Mortensen, U. H., Perez, R., Ram, A. F. J., Record, Ross, P., … Wösten, H. A. B. (2020). Growing a circular economy with fungal biotechnology: a white paper. Fungal Biology and Biotechnology, 7(5), Article 5. https://doi.org/10.1186/s40694-020-00095-z
Mosier, N., Wyman, C., Dale, B., Elander, R., Lee, Y. Y., Holtzapple, M., & Ladisch, M. (2005). Features of promising technologies for pretreatment of lignocellulosic biomass. Bioresource Technology, 96(6), 673-686. https://doi.org/10.1016/j.biortech.2004.06.025
Olughu, O. O., Tabil, L. G., Dumonceaux, T., Mupondwa, E., Cree, D., & Li, X. (2023). Technoeconomic analysis of a fungal pretreatment-based cellulosic ethanol production. Results in Engineering, 19, Article 101259. https://doi.org/10.1016/j.rineng.2023.101259
Pallín, M. Á., González-Rodríguez, S., Eibes, G., López-Abelairas, M., Moreira, M. T., Lema, J. M., & Lú-Chau, T. A. (2024). Towards industrial application of fungal pretreatment in 2G biorefinery: scale-up of solid-state fermentation of wheat straw. Biomass Conversion and Biorefinery, 14(1), 593-605. https://doi.org/10.1007/s13399-022-02319-1
Salvachúa, D., Prieto, A., López-Abelairas, M., Lu-Chau, T., Martínez, Á. T., & Martínez, M. J. (2011). Fungal pretreatment: an alternative in second-generation ethanol from wheat straw. Bioresource Technology, 102(16), 7500-7506. https://doi.org/10.1016/j.biortech.2011.05.027
Sánchez, A. (2024). A perspective of solid-state fermentation as emergent technology for organic waste management in the framework of circular bioeconomy. ACS Sustainable Resource Management, 1(8), 1630-1638. https://doi.org/10.1021/acssusresmgt.4c00062
Shirkavand, E., Baroutian, S., Gapes, D. J., & Young, B. R. (2016). Combination of fungal and physicochemical processes for lignocellulosic biomass pretreatment - a review. Renewable and Sustainable Energy Reviews, 54, 217-234. https://doi.org/10.1016/j.rser.2015.10.003
Shirkavand, E., Baroutian, S., Gapes, D. J., & Young, B. R. (2017). Pretreatment of radiata pine using two white rot fungal strains Stereum hirsutum and Trametes versicolor. Energy Conversion and Management, 142, 13-19. https://doi.org/10.1016/j.enconman.2017.03.021
Sindhu, R., Binod, P., & Pandey, A. (2016). Biological pretreatment of lignocellulosic biomass - an overview. Bioresource Technology, 199, 76-82. https://doi.org/10.1016/j.biortech.2015.08.030
Singh, J., Kumar, P., Saharan, V., & Kapoor, R. K. (2019). Simultaneous laccase production and transformation of bisphenol-A and triclosan using Trametes versicolor. 3 Biotech, 9(4), Article 129. https://doi.org/10.1007/s13205-019-1648-1
Singh, S., Harms, H., & Schlosser, D. (2014). Screening of ecologically diverse fungi for their potential to pretreat lignocellulosic bioenergy feedstock. Applied Microbiology and Biotechnology, 98(7), 3355-3370. https://doi.org/10.1007/s00253-014-5563-4
Soccol, C. R., da Costa, E. S. F., Letti, L. A. J., Karp, S. G., Woiciechowski, A. L., & Vandenberghe, L. P. D. S. (2017). Recent developments and innovations in solid state fermentation. Biotechnology Research and Innovation, 1(1), 52-71. https://doi.org/10.1016/j.biori.2017.01.002
Tišma, M., Jurić, A., Bucić-Kojić, A., Panjičko, M., & Planinić, M. (2018). Biovalorization of brewers’ spent grain for the production of laccase and polyphenols. Journal of the Institute of Brewing, 124(2), 182-186. https://doi.org/10.1002/jib.479
Tišma, M., Šalić, A., Planinić, M., Zelić, B., Potočnik, M., Šelo, G., & Bucić-Kojić, A. (2020). Production, characterisation and immobilization of laccase for an efficient aniline-based dye decolourization. Journal of Water Process Engineering, 36, Article 101327. https://doi.org/10.1016/j.jwpe.2020.101327
Tišma, M., Žnidaršič-Plazl, P., Šelo, G., Tolj, I., Šperanda, M., Bucić-Kojić, A., & Planinić, M. (2021). Trametes versicolor in lignocellulose-based bioeconomy: state of the art, challenges and opportunities. Bioresource Technology, 330, Article 124997. https://doi.org/10.1016/j.biortech.2021.124997
Vasco-Correa, J., & Shah, A. (2019). Techno-economic bottlenecks of the fungal pretreatment of lignocellulosic biomass. Fermentation, 5(2), Article 30. https://doi.org/10.3390/fermentation5020030
Visser, E. M., Falkoski, D. L., de Almeida, M. N., Maitan-Alfenas, G. P., & Guimarães, V. M. (2013). Production and application of an enzyme blend from Chrysoporthe cubensis and Penicillium pinophilum with potential for hydrolysis of sugarcane bagasse. Bioresource Technology, 144, 587-594. https://doi.org/10.1016/j.biortech.2013.07.015
Wan, C., & Li, Y. (2012). Fungal pretreatment of lignocellulosic biomass. Biotechnology Advances, 30(6), 1447-1457. https://doi.org/10.1016/j.biotechadv.2012.03.003
Wang, F., Terry, N., Xu, L., Zhao, L., Ding, Z., & Ma, H. (2019). Fungal laccase production from lignocellulosic agricultural wastes by solid-state fermentation: a review. Microorganisms, 7(12), Article 665. https://doi.org/10.3390/microorganisms7120665
Xu, L., Sun, K., Wang, F., Zhao, L., Hu, J., Ma, H., & Ding, Z. (2020). Laccase production by Trametes versicolor in solid-state fermentation using tea residues as substrate and its application in dye decolorization. Journal of Environmental Management, 270, Article 110904. https://doi.org/10.1016/j.jenvman.2020.110904