Abstract
The use of glycerol obtained as an intermediate of the biodiesel manufacturing process as carbon source for microbial growth is a potential alternative strategy for the production of enzymes and other high-value bioproducts. This work evaluates the production of cellulase enzymes using glycerol for high cell density growth of Trichoderma harzianum followed by induction with a cellulosic material. Firstly, the influence of the carbon source used in the pre-culture step was investigated in terms of total protein secretion and fungal morphology. Enzymatic productivity was then determined for cultivation strategies using different types and concentrations of carbon source, as well as different feeding procedures (batch and fed-batch). The best strategy for cellulase production was then further studied on a larger scale using a stirred tank bioreactor. The proposed strategy for cellulase production, using glycerol to achieve high cell density growth followed by induction with pretreated sugarcane bagasse, achieved enzymatic activities up to 2.27 ± 0.37 FPU/mL, 106.40 ± 8.87 IU/mL, and 9.04 ± 0.39 IU/mL of cellulase, xylanase, and β-glucosidase, respectively. These values were 2 times higher when compared to the control experiments using glucose instead of glycerol. This novel strategy proved to be a promising approach for improving cellulolytic enzymes production, and could potentially contribute to adding value to biomass within the biofuels sector.
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References
Ahamed A, Vermette P (2009) Effect of culture medium composition on Trichoderma reesei’s morphology and cellulase production. Bioresour Technol 100:5979–5987
Amanullah A, Christensen L, Hansen K, Nienow A, Thomas C (2002) Dependence of morphology on agitation intensity in fed-batch cultures of Aspergillus oryzae and its implications for recombinant protein production. Biotechnol Bioeng 77:815–826
Amore A, Giacobbe S, Faraco V (2013) Regulation of cellulase and hemicellulase gene expression in fungi. Curr Genom 14:230–249. doi:10.2174/1389202911314040002
Benoliel B, Torres FAG, de Moraes LMP (2013) A novel promising Trichoderma harzianum strain for the production of a cellulolytic complex using sugarcane bagasse in natura. SpringerPlus 2:656
Bigelow M, Wyman CE (2002) Cellulase production on bagasse pretreated with hot water. Appl Biochem Biotechnol 98:921–934
Bradford MM (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72:248–254
Chen Y-H, Walker TH (2011) Biomass and lipid production of heterotrophic microalgae Chlorella protothecoides by using biodiesel-derived crude glycerol. Biotechnol Lett 33:1973–1983
Da Silva GP, Mack M, Contiero J (2009) Glycerol: a promising and abundant carbon source for industrial microbiology. Biotechnol Adv 27:30–39
Delabona Pda S, Farinas CS, da Silva MR, Azzoni SF, Pradella JG (2012) Use of a new Trichoderma harzianum strain isolated from the Amazon rainforest with pretreated sugar cane bagasse for on-site cellulase production. Bioresour Technol 107:517–521. doi:10.1016/j.biortech.2011.12.048
Delabona Pda S, Farinas CS, Lima DJ, Pradella JG (2013) Experimental mixture design as a tool to enhance glycosyl hydrolases production by a new Trichoderma harzianum P49P11 strain cultivated under controlled bioreactor submerged fermentation. Bioresour Technol 132:401–405. doi:10.1016/j.biortech.2012.11.087
Delabona PdS, Pirota RDPB, Codima CA, Tremacoldi CR, Rodrigues A, Farinas CS (2012) Using Amazon forest fungi and agricultural residues as a strategy to produce cellulolytic enzymes. Biomass Bioenergy 37:243–250. doi:10.1016/j.biombioe.2011.12.006
dos Reis L, Fontana RC, Delabona Pda S, da Silva Lima DJ, Camassola M, Pradella JG, Dillon AJ (2013) Increased production of cellulases and xylanases by Penicillium echinulatum S1M29 in batch and fed-batch culture. Bioresour Technol 146:597–603. doi:10.1016/j.biortech.2013.07.124
Fan X, Burton R, Zhou Y (2010) Glycerol (byproduct of biodiesel production) as a source for fuels and chemicals—mini review. Open Fuels Energy Sci J 3:17–22
Florencio C, Cunha F, Badino A, Farinas C (2015) Validation of a novel sequential cultivation method for the production of enzymatic cocktails from trichoderma strains. Appl Biochem Biotechnol 175:1389–1402
Ghose T (1987) Measurement of cellulase activities. Pure Appl Chem 59:257–268
Himmel ME, Ruth MF, Wyman CE (1999) Cellulase for commodity products from cellulosic biomass. Curr Opin Biotechnol 10:358–364
Ilmen M, Saloheimo A, Onnela M-L, Penttilä ME (1997) Regulation of cellulase gene expression in the filamentous fungus Trichoderma reesei. Appl Environ Microbiol 63:1298–1306
Kovács K, Szakacs G, Zacchi G (2009) Comparative enzymatic hydrolysis of pretreated spruce by supernatants, whole fermentation broths and washed mycelia of Trichoderma reesei and Trichoderma atroviride. Bioresour Technol 100:1350–1357
Li C, Lesnik KL, Liu H (2013) Microbial conversion of waste glycerol from biodiesel production into value-added products. Energies 6:4739–4768
Maeda RN, Barcelos CA, Santa Anna LMM, Pereira N (2013) Cellulase production by Penicillium funiculosum and its application in the hydrolysis of sugar cane bagasse for second generation ethanol production by fed batch operation. J Biotechnol 163:38–44
Mandels M, W J (1969) Production of cellulases. Adv Chem Ser 95:391–414
Robl D, da Silva Delabona P, Costa PS, da Silva Lima DJ, Rabelo SC, Pimentel IC, Büchli F, Squina FM, Paddila G, da Cruz Pradella JG (2015) Xylanase production by endophytic Aspergillus niger using pentose-rich hydrothermal liquor from sugarcane bagasse. Biocatal Biotransform 33:175–187. doi:10.3109/10242422.2015.1084296
Naranjo JM, Posada JA, Higuita JC, Cardona CA (2013) Valorization of glycerol through the production of biopolymers: the PHB case using Bacillus megaterium. Bioresour Technol 133:38–44
Peberdy JF (1994) Protein secretion in filamentous fungi—trying to understand a highly productive black box. Trends Biotechnol 12:50–57
Pereira BMP, Alvarez TM, da Silva Delabona P, Dillon AJP, Squina FM, da Cruz Pradella JG (2013) Cellulase on-site production from sugar cane bagasse using Penicillium echinulatum. BioEnergy Res 6:1052–1062
Portnoy T, Margeot A, Linke R, Atanasova L, Fekete E, Sándor E, Hartl L, Karaffa L, Druzhinina IS, Seiboth B (2011) The CRE1 carbon catabolite repressor of the fungus Trichoderma reesei: a master regulator of carbon assimilation. BMC Genom 12:269
Pradella JGC, Rossell CEV, Scandiffio MIG, Cunha MP, Pinho MGO, Bonomi A (2009) Estudo preliminar do custo de produção in house de celulases na biorrefinaria de etanol de segunda geração. Anais do XVII Simpósio Nacional de Bioprocessos
Robl D, Delabona Pda S, Mergel CM, Rojas JD, Costa Pdos S, Pimentel IC, Vicente VA, da Cruz Pradella JG, Padilla G (2013) The capability of endophytic fungi for production of hemicellulases and related enzymes. BMC Biotechnol 13:94. doi:10.1186/1472-6750-13-94
Rocha GJM, Goncalves AR, Oliveira BR, Olivares EG, Rossell CEV (2012) Steam explosion pretreatment reproduction and alkaline delignification reactions performed on a pilot scale with sugarcane bagasse for bioethanol production. Ind Crops Prod 35:274–279. doi:10.1016/j.indcrop.2011.07.010
Santucci BS, Maziero P, Rabelo SC, Curvelo AA, Pimenta MTB (2015) Autohydrolysis of hemicelluloses from sugarcane bagasse during hydrothermal pretreatment: a kinetic assessment. BioEnergy Res 8:1778–1787
Sarma SJ, Brar SK, Sydney EB, Le Bihan Y, Buelna G, Soccol CR (2012) Microbial hydrogen production by bioconversion of crude glycerol: a review. Int J Hydrogen Energy 37:6473–6490
Sluiter A, Hames B, Ruiz R, Scarlata C, Sluiter J, Templeton D (2006) Determination of sugars, byproducts, and degradation products in liquid fraction process samples. Golden, National Renewable Energy Laboratory
Tang S, Boehme L, Lam H, Zhang Z (2009) Pichia pastoris fermentation for phytase production using crude glycerol from biodiesel production as the sole carbon source. Biochem Eng J 43:157–162
Uzbas F, Sezerman U, Hartl L, Kubicek CP, Seiboth B (2012) A homologous production system for Trichoderma reesei secreted proteins in a cellulase-free background. Appl Microbiol Biotechnol 93:1601–1608
Wang L, Ridgway D, Gu T, Moo-Young M (2005) Bioprocessing strategies to improve heterologous protein production in filamentous fungal fermentations. Biotechnol Adv 23:115–129. doi:10.1016/j.biotechadv.2004.11.001
Ward O, Qin W, Dhanjoon J, Ye J, Singh A (2006) Physiology and biotechnology of Aspergillus. Adv Appl Microbiol 58:1–76
Wu H, Karanjikar M, San K-Y (2014) Metabolic engineering of Escherichia coli for efficient free fatty acid production from glycerol. Metab Eng 25:82–91
Yang F, Hanna MA, Sun R (2012) Value-added uses for crude glycerol—a byproduct of biodiesel production. Biotechnol Biofuels 5:1–10
Zhang Y-HP, Himmel ME, Mielenz JR (2006) Outlook for cellulase improvement: screening and selection strategies. Biotechnol Adv 24:452–481
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The authors would like to thank the technical staff of the National Laboratory of Science and Technology of Bioethanol (CTBE), and acknowledge the financial support provided by the Brazilian agencies CNPq, CAPES and FAPESP.
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The financial support provided by the Brazilian agencies CNPq, CAPES and FAPESP and not have any potential Conflicts of interest (financial or non-financial).
This manuscript complies to the Ethical Rules applicable for Journal of Industrial Microbiology and Biotechnology. In this research is not involve human participants and/or animals.
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da Silva Delabona, P., Lima, D.J., Robl, D. et al. Enhanced cellulase production by Trichoderma harzianum by cultivation on glycerol followed by induction on cellulosic substrates. J Ind Microbiol Biotechnol 43, 617–626 (2016). https://doi.org/10.1007/s10295-016-1744-8
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DOI: https://doi.org/10.1007/s10295-016-1744-8