Optimization of β-Glucosidase, β-Xylosidase and Xylanase Production by Colletotrichum graminicola under Solid-State Fermentation and Application in Raw Sugarcane Trash Saccharification

Efficient, low-cost enzymatic hydrolysis of lignocellulosic residues is essential for cost-effective production of bioethanol. The production of β-glucosidase, β-xylosidase and xylanase by Colletotrichum graminicola was optimized using Response Surface Methodology (RSM). Maximal production occurred in wheat bran. Sugarcane trash, peanut hulls and corncob enhanced β-glucosidase, β-xylosidase and xylanase production, respectively. Maximal levels after optimization reached 159.3 ± 12.7 U g−1, 128.1 ± 6.4 U g−1 and 378.1 ± 23.3 U g−1, respectively, but the enzymes were produced simultaneously at good levels under culture conditions optimized for each one of them. Optima of pH and temperature were 5.0 and 65 °C for the three enzymes, which maintained full activity for 72 h at 50 °C and for 120 min at 60 °C (β-glucosidase) or 65 °C (β-xylosidase and xylanase). Mixed with Trichoderma reesei cellulases, C. graminicola crude extract hydrolyzed raw sugarcane trash with glucose yield of 33.1% after 48 h, demonstrating good potential to compose efficient cocktails for lignocellulosic materials hydrolysis.

[1]  Parameswaran Binod,et al.  Dilute acid pretreatment and enzymatic saccharification of sugarcane tops for bioethanol production. , 2011, Bioresource technology.

[2]  Zhengqiang Jiang,et al.  Characterisation of a thermostable xylanase from Chaetomium sp. and its application in Chinese steamed bread , 2010 .

[3]  M. Rajoka,et al.  Characterization of a β-xylosidase produced by a mutant derivative of Humicola lanuginosa in solid state fermentation , 2010, Annals of Microbiology.

[4]  D. Northcote,et al.  Minimization of variation in the response to different proteins of the Coomassie blue G dye-binding assay for protein. , 1981, Analytical biochemistry.

[5]  J. A. Jorge,et al.  Extracellular β‐D‐glucosidase from Chaetomium thermophilum var. coprophilum: production, purification and some biochemical properties , 2002 .

[6]  Ramesh Chander Kuhad,et al.  Optimization of cellulase production by a brown rot fungus Fomitopsis sp. RCK2010 under solid state fermentation. , 2011, Bioresource technology.

[7]  I. S. Pretorius,et al.  Microbial Cellulose Utilization: Fundamentals and Biotechnology , 2002, Microbiology and Molecular Biology Reviews.

[8]  P. Christakopoulos,et al.  Purification, characterization and mass spectrometric identification of two thermophilic xylanases from Sporotrichum thermophile , 2010 .

[9]  Jianmin Gao,et al.  Purification and characterization of a novel endo-β-1,4-glucanase from the thermoacidophilic Aspergillus terreus , 2008, Biotechnology Letters.

[10]  Severino de Albuquerque Lucena-Neto,et al.  Purification and characterization of a new Xylanase from Humicola grisea var. Thermoidea , 2004 .

[11]  U. Hölker,et al.  Biotechnological advantages of laboratory-scale solid-state fermentation with fungi , 2004, Applied Microbiology and Biotechnology.

[12]  E. Bon,et al.  Milling pretreatment of sugarcane bagasse and straw for enzymatic hydrolysis and ethanol fermentation. , 2010, Bioresource technology.

[13]  Amie D. Sluiter,et al.  Determination of Structural Carbohydrates and Lignin in Biomass , 2004 .

[14]  E. Filho,et al.  Purification and partial characterization οf a new β-xylosidase from Humicola grisea var. thermoidea , 2006 .

[15]  E. Ferreira,et al.  Purification and characterization of a beta-glucosidase from solid-state cultures of Humicola grisea var. thermoidea. , 1996, Canadian journal of microbiology.

[16]  N. Laosiripojana,et al.  Optimisation of synergistic biomass-degrading enzyme systems for efficient rice straw hydrolysis using an experimental mixture design. , 2012, Bioresource technology.

[17]  H. Belghith,et al.  Improvement of Highly Thermostable Xylanases Production by Talaromyces thermophilus for the Agro-industrials Residue Hydrolysis , 2010, Applied biochemistry and biotechnology.

[18]  C. A. Codima,et al.  Effect of initial moisture content on two Amazon rainforest Aspergillus strains cultivated on agro-industrial residues: Biomass-degrading enzymes production and characterization , 2013 .

[19]  J. A. Jorge,et al.  Xylanases from fungi: properties and industrial applications , 2005, Applied Microbiology and Biotechnology.

[20]  R. C. Kasana,et al.  Microbial proteases: Detection, production, and genetic improvement , 2011, Critical reviews in microbiology.

[21]  Antonio Di Pietro,et al.  The Top 10 fungal pathogens in molecular plant pathology. , 2012, Molecular plant pathology.

[22]  Venkatesh Balan,et al.  Hemicellulases and auxiliary enzymes for improved conversion of lignocellulosic biomass to monosaccharides , 2011, Biotechnology for biofuels.

[23]  Carmen Sánchez,et al.  Lignocellulosic residues: biodegradation and bioconversion by fungi. , 2009, Biotechnology advances.

[24]  C. Farinas,et al.  Finding stable cellulase and xylanase: evaluation of the synergistic effect of pH and temperature. , 2010, New biotechnology.

[25]  S. W. Kim,et al.  Production of cellulases and hemicellulases by Aspergillus niger KK2 from lignocellulosic biomass. , 2004, Bioresource technology.

[26]  Venkatesh Balan,et al.  Alkali‐based AFEX pretreatment for the conversion of sugarcane bagasse and cane leaf residues to ethanol , 2010, Biotechnology and bioengineering.

[27]  C. R. Terrasan,et al.  β-Xylosidases from filamentous fungi: an overview , 2010 .

[28]  S. Bansal,et al.  Production of Cellulases through Solid State Fermentation Using Kinnow Pulp as a Major Substrate , 2010 .

[29]  P. Christakopoulos,et al.  Purification and characterization of a thermostable intracellular β-xylosidase from the thermophilic fungus Sporotrichum thermophile , 2006 .

[30]  B. Leite,et al.  ULTRASTRUCTURAL ASPECTS OF COLLETOTRICHUM GRAMINICOLA CONIDIUM GERMINATION, APPRESSORIUM FORMATION AND PENETRATION ON CELLOPHANE MEMBRANES : FOCUS ON LIPID RESERVES , 1998 .

[31]  Rubens Maciel Filho,et al.  Integrated versus stand-alone second generation ethanol production from sugarcane bagasse and trash. , 2012, Bioresource technology.

[32]  C. Krishna Solid-State Fermentation Systems—An Overview , 2005, Critical reviews in biotechnology.

[33]  H. Barnett,et al.  Illustrated Genera of Imperfect Fungi , 1972 .

[34]  Ling Wang,et al.  A xylose-tolerant beta-xylosidase from Paecilomyces thermophila: characterization and its co-action with the endogenous xylanase. , 2008, Bioresource technology.

[35]  I. Dogaris,et al.  Induction of cellulases and hemicellulases from Neurospora crassa under solid-state cultivation for bioconversion of sorghum bagasse into ethanol , 2009 .

[36]  C. A. Codima,et al.  Using Amazon forest fungi and agricultural residues as a strategy to produce cellulolytic enzymes , 2012 .

[37]  T. C. McIlvaine,et al.  A BUFFER SOLUTION FOR COLORIMETRIC COMPARISON , 1921 .

[38]  M. Damaso,et al.  Use of corncob for endoxylanase production by thermophilic fungus Thermomyces lanuginosus IOC-4145 , 2000, Applied biochemistry and biotechnology.

[39]  J. A. Jorge,et al.  Purification and biochemical properties of a thermostable xylose-tolerant β-D-xylosidase from Scytalidium thermophilum , 2004, Journal of Industrial Microbiology and Biotechnology.

[40]  J. A. Jorge,et al.  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 , 2012, World Journal of Microbiology and Biotechnology.

[41]  Rajeev K Sukumaran,et al.  Advancement and comparative profiles in the production technologies using solid-state and submerged fermentation for microbial cellulases , 2010 .

[42]  F. A. Leone,et al.  Purification and biochemical properties of a glucose-stimulated β-D-glucosidase produced by Humicola grisea var. thermoidea grown on sugarcane bagasse , 2010, The Journal of Microbiology.

[43]  G. Cornacchia,et al.  Energy Recovery from Sugarcane-Trash in the Light of 2nd Generation Biofuels. Part 1: Current Situation and Environmental Aspects , 2011 .

[44]  Satinder Kaur Brar,et al.  Value-addition of agricultural wastes for augmented cellulase and xylanase production through solid-state tray fermentation employing mixed-culture of fungi. , 2011 .

[45]  Guido Zacchi,et al.  An approach to the utilisation of CO2 as impregnating agent in steam pretreatment of sugar cane bagasse and leaves for ethanol production , 2010, Biotechnology for biofuels.

[46]  J. van den Brink,et al.  Construction of a cellulase hyper-expression system in Trichoderma reesei by promoter and enzyme engineering , 2012, Microbial Cell Factories.

[47]  D. Gokhale,et al.  Development of biocatalysts for production of commodity chemicals from lignocellulosic biomass. , 2011, Bioresource technology.

[48]  J. A. Jorge,et al.  Beta-D-glycosidase activities of Humicola grisea: biochemical and kinetic characterization of a multifunctional enzyme. , 1990, Biochimica et biophysica acta.

[49]  V. Bisaria,et al.  Microbial β-Glucosidases: Cloning, Properties, and Applications , 2002 .

[50]  J. A. Jorge,et al.  Beta-glucosidase activity from the thermophilic fungus Scytalidium thermophilum is stimulated by glucose and xylose. , 2004, FEMS microbiology letters.

[51]  S. Tokuyama,et al.  Purification and characterization of a high-thermostable β-xylanase from newly isolated Thermomyces lanuginosus THKU-49 , 2010, Mycoscience.

[52]  Seth Debolt,et al.  Synthesis, regulation and utilization of lignocellulosic biomass. , 2010, Plant biotechnology journal.

[53]  R. Peralta,et al.  Production of xylanolytic enzymes by Aspergillus tamarii in solid state fermentation , 1999 .

[54]  J. A. Jorge,et al.  Purification and biochemical characterization of β-xylosidase from Humicola grisea var. thermoidea , 1995 .

[55]  R. Kuhad,et al.  Xylanase production from an alkalophilic actinomycete isolate Streptomyces sp. RCK-2010, its characterization and application in saccharification of second generation biomass , 2012 .

[56]  E. Gomes,et al.  Characterization and comparison of thermostability of purified β-glucosidases from a mesophilic Aureobasidium pullulans and a thermophilic Thermoascus aurantiacus , 2007 .

[57]  B. S. Chadha,et al.  Regulation of cellulase production in two thermophilic fungi Melanocarpus sp. MTCC 3922 and Scytalidium thermophilum MTCC 4520 , 2006 .

[58]  H. Chum,et al.  A techno-economic evaluation of the effects of centralized cellulosic ethanol and co-products refinery options with sugarcane mill clustering , 2010 .

[59]  E. Gomes,et al.  Production and characteristics comparison of crude β-glucosidases produced by microorganisms Thermoascus aurantiacus e Aureobasidium pullulans in agricultural wastes , 2008 .

[60]  P. Christakopoulos,et al.  Studies on the solid-state production of thermostable endoxylanases from Thermoascus aurantiacus : Characterization of two isozymes , 1998 .

[61]  Jack N Saddler,et al.  The enhancement of enzymatic hydrolysis of lignocellulosic substrates by the addition of accessory enzymes such as xylanase: is it an additive or synergistic effect? , 2011, Biotechnology for biofuels.

[62]  E. Bon,et al.  Production of Thermophilic Endo-β-1,4-xylanases by Aspergillus fumigatus FBSPE-05 Using Agro-industrial By-products , 2012, Applied Biochemistry and Biotechnology.

[63]  P. Christakopoulos,et al.  Fungal multienzyme production on industrial by-products of the citrus-processing industry. , 2008, Bioresource technology.

[64]  Charles E Wyman,et al.  Xylooligomers are strong inhibitors of cellulose hydrolysis by enzymes. , 2010, Bioresource technology.

[65]  F. A. Leone,et al.  Purification and biochemical characterization of a mycelial glucose- and xylose-stimulated β-glucosidase from the thermophilic fungus Humicola insolens , 2010 .

[66]  O. Singh,et al.  Sugarcane bagasse and leaves: foreseeable biomass of biofuel and bio‐products , 2012 .

[67]  G. L. Miller Use of Dinitrosalicylic Acid Reagent for Determination of Reducing Sugar , 1959 .

[68]  M. F. Jahromi,et al.  Efficiency of rice straw lignocelluloses degradability by Aspergillus terreus ATCC 74135 in solid state fermentation , 2011 .

[69]  Anoop Singh,et al.  Production of liquid biofuels from renewable resources , 2011 .

[70]  Marcos S. Buckeridge,et al.  Scientific challenges of bioethanol production in Brazil , 2011, Applied Microbiology and Biotechnology.