SACCHARIFICATION WITH Phanerochaete Chrysosporium and ETHANOL PRODUCTION WITH Saccharomyces Cerevisiae | Lignin | Cellulose

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Journal of Atoms and Molecules is an international peer reviewed online journal, publish articles in all fields of science. email: editor@jamonline.in visit: www.jamonline.in
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  Jamonline / 2(4); 2012 / 321-331 Ajeet Kumar Srivastava & Pushpa Agrawal All rights reserved© 2011 www.jamonline.in 321 Research Article Journal of Atoms and Molecules   An International Online Journal ISSN  –  2277  –  1247   SACCHARIFICATION WITH  Phanerochaete chrysosporium AND ETHANOLPRODUCTION WITH Saccharomyces cerevisiae  Ajeet Kumar Srivastava * and Pushpa Agrawal Department of Biotechnology, R V College of Engineering, R.V. Vidyaniketan Post,Bangalore,India Received on: 28-07-2012 Revised on: 09-08-2012 Accepted on: 27-08-2012Abstract: Lignocellulosic biomass can be utilized to produce ethanol, a promising alternative energy sourcefor the limited crude oil. Efficient conversion of lignocellulosic materials into fuel ethanol hasbecome a world priority for producing environmentally friendly renewable energy at reasonableprice for the transportation sector. Fuel ethanol can be utilized as oxygenate of gasoline elevating itsoxygen content, allowing a better oxidation of hydrocarbons and reducing the amounts of greenhouse gas emissions into the atmosphere. There are mainly two processes involved in theconversion such as hydrolysis of cellulose in the lignocellulosic biomass to produce reducingsugars, and fermentation of the sugars to ethanol. The cost of ethanol production fromlignocellulosic materials is relatively high based on current technologies, and the main challengesare the low yield and high cost of the hydrolysis process. Considerable research efforts have beenmade to improve the hydrolysis of lignocellulosic materials. Pretreatment of lignocellulosicmaterials to remove lignin and hemicellulose can significantly enhance the hydrolysis of cellulose.The fungi like Phanerochaete chrysosporium have unique oxidative systems which together withligninolytic enzymes are responsible for lignocellulose degradation as well as selective lignindegradation and further ethanol formation done by Saccharomyces cerevisiae through fermentativepathways . Among the three raw materials studied the ethanol yield was observed to be the highestin bagasse (4.5 g/l). Keywords:   Saccharification, lignocellulosic biomass, microbial pretreatment, fermentable sugars,hydrolysis, fermentation, ethanol production.  Jamonline / 2(4); 2012 / 321-331 Ajeet Kumar Srivastava & Pushpa Agrawal All rights reserved© 2011 www.jamonline.in 322 1 Introduction  For some years now, growing efforts hasbeen devoted to the bio-conversion of agricultural biomass (especially energycrops) in order to produce bio-fuels(ethanol, biogas, etc) as alternative to fossilenergy sources. Lignocellulosic biomassconsists mainly of cellulose, hemicelluloseand lignin. Cellulose is the most abundantrenewable biopolymer [1,2,3]. As mostlignocellulosic substrates also containhemicellulose, a system of enzymesomewhat analogue to the cellulase complexis required for the hydrolysis of xylan, themajor constituent of hemicellulose [4, 8].Hydrolysis of agricultural biomass tofermentable sugars (glucose,xylose,arabinose etc.) is required prior to energyconversion process [4,5].Cellulosecrystallinity, accessible surface, as well asthe presence of lignin and hemicellulose,determine the resistance of biomass toenzymatic hydrolysis [3].Hydrolysis can beperformed chemically or enzymatically,following the appropriate preteatment steps[6,7]. The hydrolysis processes used in thepast were essentially chemical, but the costsand the formation of toxic by-products madethem noncompetitive [5,2] and sometimesunsuitable. Enzymatic processes, whichhold several advantages, are nowsubstituting the chemical ones. Theefficiency of enzymatic process is quite highand the mild process conditions requireneither expensive materials nor high processenergy [2,3,7]. The use of cellulase complex(containing endoglucanase, exoglucanaseand β -glucosidase) from differentmicroorganisms or a mixture of cellulaseswith other enzymes (xylanases, amylases,laccase, pectinases etc.) for hydrolysis of lignocellulosic substrates into fermentablesugars gains more and more interest [8, 9, 1,10, 11]. Since the extraction of relevantamounts of fermentable sugars fromlignocellulosic materials requires intensivetreatement using several enzymes to break down recalcitrant lignin and hemicellulosematrix. An alternative approach for biofuelproduction is the use of biomass with a lowlignocellulosic content (e.g. corn cob mix).In this way, high hydrolysis rates could bereached at lower cost. The objective of thepresent research was to achieve high yieldsof fermentable sugar from cellulosic components by Phanerochaete chrysosporium   2 Pretreatment of lignocellulosicmaterials Pretreatment of lignocelluloses is intendedto disorganize the crystalline structure of * Corresponding authorAjeet Kumar SrivastavaEmail: ajeeth@rvce.edu.in  Phone: +91-9916830878  Jamonline / 2(4); 2012 / 321-331 Ajeet Kumar Srivastava & Pushpa Agrawal All rights reserved© 2011 www.jamonline.in 323 macro- and microfibrils, in order to releasethe polymer chains of cellulose andhemicellulose, and/or modify the pores inthe material to allow the enzymes topenetrate into the fibers to render themamenable to enzymatic hydrolysis.Pretreatment is one of the most importantsteps in the process of converting renewablelignocellulosic biomass into useful products.If the pretreatment is not efficient enoughthe resultant residue is not easilyhydrolyzable by cellulase enzyme and if it ismore severe, result is the production of toxiccompounds which inhibit the microbialmetabolism. The yeast S. cerevisiae is themost commonly used microorganism intraditional industrial fermentations,including current sucrose-,starch- andcellulose-based bioethanol production.S.cerevisiae is also generally recognized assafe (GRAS) and can ferment efficientlysimple hexose sugars, such as D-glucose, D-mannose and D-galactose, and disaccharideslike sucrose and maltose,reaching ethanolconcentrations as high as 20% (v/v).Moreover,S. cerevisiae has a relatively goodtolerance to lignocellulose-derivedinhibitors and high osmotic pressure. Also,the fermentation rate is not significantlyreduced at ethanol concentrations below10% (v/v) [12]. The major inconvenience tothe use of S. cerevisiae for lignocellulosicfermentation is its lack of natural ability toutilize the pentose sugars D-xylose and L-arabinose. 2.1   Biological pretreatment  Biological pretreatment offers someconceptually important advantages such aslow chemical and energy use, but acontrollable and sufficiently rapid systemhas not yet been found. Chemicalpretreatments have serious disadvantages interms of the requirement for specializedcorrosion resistant equipment, extensivewashing, and proper disposal of chemicalwastes. Biological pretreatment is a safe andenvironmentally-friendly method for ligninremoval from lignocellulose. The mostpromising microorganisms for biologicalpretreatment are white-rot fungi that belongto class Basidiomycetes [13]. The effects of biological pretreatment of rice straw usingfour white-rot fungi ( Phanerochaetechrysosporium, Trametes versicolor,Ceriporiopsis subvermispora, and Pleurotusostreatus ) were evaluated on the basis of quantitative and structural changes in thecomponents of the pre-treated rice straw aswell as susceptibility to enzymatichydrolysis [13]. Of these white-rot fungi, P.ostreatus selectively degraded the ligninfraction of rice straw rather than theholocellulose component. When rice strawwas pre-treated with P. ostreatus for 60 d,the total weight loss and the degree of Klason lignin degraded were 25% and 41%,  Jamonline / 2(4); 2012 / 321-331 Ajeet Kumar Srivastava & Pushpa Agrawal All rights reserved© 2011 www.jamonline.in 324 respectively. Lignin is an aromatic polymerwith the substituents connected by bothether and carbon-carbon linkages. It iscomposed of three principal building blocks:  p -coumaryl alcohol (  p -hydroxyphenylpropanol), coniferyl alcohol (guaiacylpropanol), and sinapyl alcohol (syringylpropanol) (Fig.1). In softwoods, coniferylalcohol is the principal constituent, thelignin of hardwoods is composed of guaiacyl and syringyl units. Grass ligninscontain guiacyl-, syringyl-, and  p -hydroxyphenyl-units.After the pretreatment,the residual amounts of cellulose andhemicellulose were 83% and 52% of thosein untreated rice straw, respectively. Byenzymatic hydrolysis with a commercialcellulase preparation for 48 h, 52%holocellulose and 44% cellulose in the pre-treated rice straw were solubilized. The netsugar yields based on the amounts of holocellulose and cellulose of untreated ricestraw were 33% for total soluble sugar fromholocellulose and 32% for glucose fromcellulose [13]. The biological pretreatmentinduces structural loosening of cells with asimultaneous increase in porosity. TheScanning Electron Microscopic (SEM)observations show that the pretreatmentwith P. ostreatus resulted in an increase insusceptibility of rice straw to enzymatichydrolysis due to partial degradation of thelignin that is responsible for preventingpenetration of cellulase in the rice straw asdescribed above.Patel et al. (2007)did apreliminary study on the microbialpretreatment and fermentation of theagricultural residues like Lignocellulosicmaterial [14]. Fig.1.   Lignin building blocks. Lignin inter-monomer linkages are similarin softwood, hardwood, and grass lignins.Biosynthesis of lignin proceeds through afree radical coupling mechanism that waspolymerization. Freudenberg synthesizeddehydrogenative polymers (DHP) in thelaboratory by reacting coniferyl alcohol witha fungal laccase. In softwoods, the mostabundant lignin substructure is the b-arylether, which accounts for approximately40% of the interphenylpropane linkages[15]. 3 Material and methods3.1 Raw materials Procured rice straw & wheat straw from localmill. Bagasse from local sugar factory. Each raw
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