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Copyright © 2006 by Humana Press Inc. All rights of any nature whatsoever reserved. 0273-2289/06/129–132/496–508/$30.00 Ethanol Production From Steam-Explosion Pretreated Wheat Straw IGNACIO BALLESTEROS, Ma JOSÉ NEGRO, JOSÉ MIGUEL OLIVA, ARACELI CABAÑAS, PALOMA MANZANARES,* AND MERCEDES BALLESTEROS CIEMAT-Renewable Energies Division, Av. Complutense, 22, 28040-Madrid-Spain; E-mail: Abstract Bioconversion of cereal straw to bioethanol is becoming an attractive alternative
  Ethanol Production From Steam-ExplosionPretreated Wheat Straw I GNACIO B ALLESTEROS , M a J OSÉ N EGRO , J OSÉ M IGUEL O LIVA ,A RACELI C ABAÑAS , P ALOMA M ANZANARES ,* AND M ERCEDES B ALLESTEROS CIEMAT-Renewable Energies Division, Av. Complutense, 22,28040-Madrid-Spain; E-mail:  Abstract Bioconversion of cereal straw to bioethanol is becoming an attractivealternative to conventional fuel ethanol production from grains. In thiswork, the best operational conditions for steam-explosion pretreatment of wheat straw for ethanol production by a simultaneous saccharification andfermentation process were studied, using diluted acid [H 2 SO 4 0.9 % (w/w)]and water as preimpregnation agents. Acid- or water-impregnated biomasswas steam-exploded at different temperatures (160–200°C) and residencetimes (5, 10, and 20 min). Composition of solid and filtrate obtained afterpretreatment, enzymatic digestibility and ethanol production of pretreatedwheat straw at different experimental conditions was analyzed. The bestpretreatment conditions to obtain high conversion yield to ethanol (approx80% of theoretical) of cellulose-rich residue after steam-explosion were190°C and 10 min or 200°C and 5 min, in acid-impregnated straw. However,180°C for 10 min in acid-impregnated biomass provided the highest ethanolyield referred to raw material (140 L/t wheat straw), and sugars recoveryyield in the filtrate (300 g/kg wheat straw). Index Entries: Wheat straw; ethanol; diluted acid pretreatment; steam-explosion. Introduction During the past century, world energy consumption has mostlydepended on the utilization of fossil fuels, which has led to harmfulchanges in our climate and increased the amount of greenhouse gases inthe atmosphere. According to the World energy, technology and climatepolicy outlook report published in 2003 by the European Union (1) , giventhe continued dominance of fossil fuels, world CO 2 emissions are expectedto increase more rapidly than the energy consumption (2.1%/yr on anaverage). In 2030, world CO 2 emissions are expected to be more than twicethe level of 1990. This scenario is a clear encouragement for developing Applied Bioc hemistry and Biotechnology  496  Vol. 129–132, 2006  Copyright © 2006 by Humana Press Inc.All rights of any nature whatsoever reserved.0273-2289/06/129–132/496–508/$30.00 *Author to whom all correspondence and reprint requests should be addressed.  Ethanol Production 497  Applied Biochemistry and BiotechnologyVol. 129–132, 2006  alternative sources that mitigate the detrimental environmental effects of fossil fuels use.With the search for alternative renewable energy sources, bioethanol isfast becoming a viable solution, as it is a nonfossil fuel from a renewablesource that may result in a cost-efficient way to reduce greenhouse gases andgasoline use in transport provided that it is produced in an efficient conver-sion process. Although conventional fuel ethanol is derived from grains suchas corn and wheat competing as a food source for humans, the cereal indus-try produces vast amounts of residue that has little use today. According toKim and Dale (2) , under the 60% ground cover practice, about 354 millionsof tons of wheat straw could be available globally and could produce 104 GLof bioethanol. Europe production would account for about 38% of this world bioethanol capacity. In Spain grain industry generates important amounts of wheat straw, a part of which is used as bedding straw and the remainder is burned or left on the land to fertilize the soil. Bioconversion of this residue tofuel ethanol would provide an attractive possibility to boost the developmentof biofuels in our country in a sustainable way.The lignocellulosic nature of wheat straw makes the pretreatment anessential step because the physical and chemical barriers caused by the closeassociation of main components greatly limits the suceptibility to biopro-cesses such as simultaneous saccharification and fermentation (SSF). Amongprocesses developed to pretreat lignocellulosic biomass, steam-explosion(SE) has been extensively studied and claimed as one of the most successfultechniques for fractionating biomass and enhancing the accessibility of cellu-lose to enzymes. SE has been proved to be effective in a great variety of lig-nocellulosic biomass, including hardwoods (3,4) , softwoods (5,6), andherbaceous residues such as corn stover (7) , sugarcane bagasse (8), and wheatstraw (9,10) .As a way to further improve the effectiveness of SE pretreat-ment, the addition of an impregnation agent before pretreatment has beenshown to be an effective method to increase cellulose digestibility of pre-treated substrates and solubilize a significant portion of the hemicellulosiccomponent. Preimpregnation of biomass with acid catalyst such as diluteSO 2 and H 2 SO 4 has been shown to decrease both temperature and timerequirements whereas achieving optima fractionation, sugar recovery, andenzymatic hydrolysis (EH) of steam-pretreated samples (11) . The ef ficiency of dilute H 2 SO 4 pretreatment in some agricultural residues as corn stover has been already demonstrated (12,13) . Neverthless, few references are foundabout the effect of acid addition in agricultural residues as wheat straw.In this work, the best operational conditions for SE pretreatment of wheat straw for ethanol production by a SSF process were studied, usingdiluted acid (H 2 SO 4 0.9 % [w/w]) or water as impregnation agents beforepretreatment. Acid- or water-impregnated biomass was steam-explodedat different temperatures (160–200°C) and residence times (5, 10, and20 min). The effectiveness of SE was evaluated in terms of cellulose recoveryin the water-insoluble solids (WIS) fraction, hemicellulose-derived sugar  (HS) recovery in the filtrate, and EH yield of WIS fraction. Finally, pre-treated wheat straw was tested in SSF process with the thermotolerantyeast strain Kluyveromyces marxianus CECT 10875. Materials and Methods Raw Material  Wheat straw (6% moisture content) was provided by Ecocarburantesde Castilla y León (Salamanca, Spain). Biomass was coarsely crushedusing a laboratory hammer mill (Retsch GmbH & Co. KG, Germany),homogenised and stored until used.The chemical composition of raw material and WIS fraction was deter-mined using the standard laboratory analytical procedures for biomass anal-ysis provided by the National Renewable Energy Laboratory (Colorado) (14) .The chemical analysis of raw material showed the following compostion(% dry weight): cellulose, 30.2; hemicellulose, 22.3 (xylan, 18.7; arabinan, 2.8;and galactan, 0.8); acid insoluble lignin, 15.3; acid soluble lignin, 1.7; acetylgroups, 2.6; ash, 4.7; and extractives, 14.7 (total, 91.5%). SE Pretreatment  Pretreatment assays were performed by applying Masonite technol-ogy in a 2L-SE pilot unit as described in a previous work (3) . Before pre-treatment, wheat straw was soaked for 18 h at 45°C in 0.9% w/w dilutedsulphuric acid solution or water (solid–liquid ratio: 1/10). The soakedmaterial was vacuum filtered to approx 20% solids content and thensteam-exploded. Temperature pretreatment ranged from 160°C to 200°Cand time from 5 to 20 min, depending on temperature. Pretreatment exper-iments on wheat straw preimpregnated with water were performed at alltempertaures and selected times, except for the lowest temperature of 160°C where only acid impregnated biomass was tested. Asummary of theoperation conditions assayed are shown in Table 1. After pretreatment, the material was recovered in a cyclone, cooled to approx 40°C and filtered torecover two fractions: (1) the WIS fraction and (2) the filtrate or prehy-drolyzate. After separating the filtrate, WIS fraction was throughlywashed with water, weighted and dried at 45°C for storage. Solid recoveryyield was then calculated as dry weight of WIS remaining after pretreat-ment referred to 100 g of raw material.WIS fraction was analyzed for carbohydrates and acid-insoluble lignincontent, and used as substrate in EH and SSF tests. Sugars, furfural, andhydroxymethylfurfural (HMF) content of the filtrate were also analyzed. Enzymatic Hydrolysis Tests  The washed WIS fraction after pretreatment was used as substrate forEH experiments. EH tests were performed in 100-mLErlenmeyer flasks, 498 Ballesteros et al. Applied Biochemistry and BiotechnologyVol. 129–132, 2006   Ethanol Production 499  Applied Biochemistry and BiotechnologyVol. 129–132, 2006  each containing 25 mLof 0.1  M sodium acetate buf fer (pH 4.8), 10% (w/v)dry WIS loading, at 50°C for 72 h. Enzyme loading of 15 FPU/g dry WISof Celluclast 1.5 Land 12.6 IU/g dry WIS of  β -glucosidase Novozyme 188was employed. Enzymes were a gift from Novozymes A/S (Bagsvaerd,Denmark). After EH assays completion, glucose was analyzed by HPLC asdecribed earlier. Experiments were performed in duplicate. EH yield wascalculated as the ratio of g glucose in the EH/100 g potential glucose inWIS. Glucose yield in the EH referred to untreated initial material was alsocalculated by taking into account the solid recovery yield attained in eachexperiment. Microorganisms and Gro wth Conditions  K. marxianus CECT 10875, purchased in the Spanish collection of typecultures, was used in SSF experiments. Active cultures for inoculationwere prepared by growing the organism on a rotary shaker at 150 rpm and42°C for 16 h, in a growth medium containing (g/L): yeast extract, 5;NH 4 Cl, 2; KH 2 PO 4 , 1; MgSO 4 ⋅ 7H 2 O, 0.3; and glucose, 30. All chemicalswere from Sigma (Sigma-Aldrich Inc., St. Louis, MO). SSF Tests  SSF experiments were carried out under no sterile conditions in 100-mLErlenmeyer flasks, each containing 50 mLof the fermentation medium(without glucose) as described earlier, and were incubated at 150 rpm and42°C for 72 h. No contamination was detected at the end of the experi-ments. The WIS fraction obtained after pretreatment was used as substrate Table 1Conditions for SE Pretreatment of Wheat StrawTemperature (°C)TimeImpregnation agent16020Catalyst a 17020H 2 O5Catalyst10Catalyst18010H 2 O5Catalyst10Catalyst19010H 2 O5Catalyst10Catalyst20010H 2 O5Catalyst a 0.9% (w/w) H 2 SO 4 .
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