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This article isprotected bycopyright. Allrights reserved. Accepted Article -al akmrni@bs [email protected] E-mail: 334354003 Fax: +46 4355903 33 Tel: +46 *Corresponding author Univer Recovery, CentreforResource Swedish J.Taherzadeh Mohammad B. Nair*,MagnusLundin,PatrikLennartsson, Ramkumar R. straw Short title using and a demonstrationin plant for and edibleethanol fungal biomass production laboratory the in of wheat straw pretreatment phosphoric dilute Optimizing

article as doi: 10.1002/ differencesbetween version to this may been through the copyediting, typesetting, pagination andproofreading process, which This article hasbeen accepted for publication andundergone full peer review but has not Neurospora intermedia Neurospora intermedia : and edible filamentous fungal biomass from pretreated wheat from pretreated fungalbiomass ediblefilamentous Ethanoland : jctb.5119

sity of Borås, SE 50190 Borås, Sweden Borås,Sweden ofBorås, 50190 SE sity

and the Versionof Record. Please cite this This article isprotected bycopyright. Allrights reserved. Accepted Article Fermentation Fermentation Keywords plants. wheat-to-ethanol existing in fungaledible biomass and ethanol of for theproduction straw of wheat use for theefficient strategy analternative opens up study This CONCLUSION: of the maximum. acid straw) theoretical dilute phosphoric pretreated 94% (with intermedia maximum. Ethanol fermentation enzymaticallyof hydrolyzed wheat straw using of polysaccharefficiency with the subsequent acid. Scaleup of the pretreatment at a biorefin and86% forhydrolysis was 36% untreatedstraw minimuminhibitorreleas withthe digestibility (w/v)min,maximum at1.75% enzymatic a temperature190°Cfor of15onthe based pretreatment conditions Theoptimal RESULTS: (5–20 min). w/ (0.5–3.0% phosphoric acidconcentrations forthe acid conditions dilute such include factors as pretreatment first time.Thevarious a laboratory scale, and the results were validated in ademonstration biorefinery plant for the intermedia Neurospora andfunga andethanol pretreatment subsequent method acid thatuseswheatstraw dilute We phosphoric for report a BACKGROUND: Abstract : Dilute phosphoric acid; Pretreatment;: Dilute Op phosphoric showed showed an improvement in the ethanol yield from 29%(withuntreated straw) to . Dilute phosphoricacidpretreatment of . wheat wasoptimized straw at v), temperature (150–210°C), temperature andreactiontime v), ery demonstration improved the ery plant process, e. Theefficiencyofenzymatic polysaccharide ide hydrolysis beingoftheide hydrolysis theoretical 95% l biomass productionedible with fungus the were determined as an acid concentration were asof an acidconcentration determined for straw pretreated with dilute phosphoric dilutefor straw phosphoric pretreated with timization; Ethanol; Filamentous fungi; N. This article isprotected bycopyright. Allrights reserved. Accepted Article acid treatment is the most widely used pretreatment method. However, the use of method. theuseof However, most usedpretreatment the widely is acidtreatment underscores the for aneed pretreatment prior to process its sugar hydrolysis hemicellulosea cellulose, 20%–30% 35%–45% alsomaintain thefacilities current should DD processes base ethanolproduction to introducing asanimal feed thatissoldseparately solubles), abyproduct grains with generation) (first production based bioethanol Th applications. forvarious substrate abundant ratio of 1.3 (for most wheat va 2014/15 (Dec/Nov) year the in 720 million tons was approximately The Internationalof the worldwheat Council plant. production annual Grainthe reported that approach forim a promising facilitiescan be wheat-to-ethanol generation first existing at straw wheat of use Integrated decades. several for an ethanolmaterials feedstockas considered has waste been lignocellulosic The of use substrates such as corn such stover substrates aThere arefewstudies only have usedthat these problems. overcoming acid wouldbeaviablefor option of dilute phosphoric form ofterrestrial,driven eutrophication marine fresh-use ofa Hence,the waterand ecosystems. acetone as step, whichusesorganicsolvents such recovery additional an 85.9%)necessitates acid (83– phosphoric of concentrated of andimproves the DDGS the quality as a source nutrient serves residue the problemoffeedfor overcoming contamina oftheDDGS the quality suchas of the reactor acid hydrochloric as such , concentrated 1.

INTRODUCTION 5 . The use of other acids, such as phosphoric acid such asphosphoric acids, other . Theuseof 9 , sugarcane bagasse bagasse , sugarcane 4 or high sulfur contamination of feed residue, thus affecting affecting thus contaminationhigh sulfur of feedresidue, or rieties), thus highlighting theavailability highlighting ofstraw asan rieties), thus GS Wheatquality. contains strawapproximately proving the overall ethanol production capacity production overall ethanol capacity the proving dilute phosphoricacid for pretreatment, for or , poses several sulfuricacid, disadvantages, or depends greatly on DDGS (distillers on greatly dried depends tion. Moreover, the residual in the in the Moreover,the residual phosphate tion. nd 8%–15% ligninand itsrecalcitrant nature d on wheat straw at existing wheat-to-ethanol d onwheatstrawatexisting wheat-to-ethanol e statusofcurrentwheat- economic overall 8 and could potentially potentially results inphosphorus and could 10 , softwood 6 and wheat bran bran wheat and 1 , with a straw to grain grain to a straw , with 2 . Hence, any approach 7 6 . However, the use , is a viable option option a viable , is 3 . In this. In regard, 11 . This article isprotected bycopyright. Allrights reserved. Accepted Article studies ontheuseof stillage using thin andbiomass fungal production to ethanol cake) (peanut press fermentation inand werestudied for their recently Indonesia contribution as Edible fungi,such quality. inthe DDGS content protein the enriches plants the ethanol utilizing hemicellulosicsugars andfungal attention fortheiruse inethanol fermentationlignocellulose iscrit with DDGSfeed Therefore, quality. the useofnaturally existing microorganisms for for this strains modified of genetically The use substrates. fromlignocellulosic that areliberated sugars the hemicellulose toutilize unable such used as fermentation, conventionally forethanol are for exam pretreatment, the otherthan processes various by isalsoinfluenced production ethanol oflignocellulosic commercialization The wheat-to-ethanol plants. existing edible fungalat ethanoland biomass production of for the acid-pretreated phosphate-rich, use strawas for of possibilities a feedstock the conditions were validated de ina biorefinery the optimized and scale, laboratory at wereoptimized hydrolysis and pretreatment affect the theedible processusingfungus, fermentation novel subsequent a acid, with of wheatphosphoric straw withdilute pretreatment involving the wastoaprocess develop study present primary objectiveofthe the Hence, substrates. lignocellulosic N. intermedia N. intermedia Neurospora intermedia Neurospora 12, 13 ical. Several ediblefilamentous . The presence of fungal biomass in the feed residue from feed the fungalbiomass in of . Thepresence for ethanoland fungal biomass production from N. ple the fermentation. The ple fermentation. microorganismsthe that biomass arecapable production asthey of

process oftenleadsproblemsassociated to monstration plant. This study widens the monstration widens the study plant. This intermedia. intermedia. , have historically been used for oncom for oncom beenused historically , have Saccharomyces cerevisiae Saccharomyces 13 Various processthat parameters , thusimproving itsnutritional 14 . However, there are limited are limited there . However, fungi have recently gained gained recently fungihave , are often , areoften This article isprotected bycopyright. Allrights reserved. Accepted Article FPU/mL activity Technology GmbH, Germany). Cellulase Cellic CTec2 (Novozymes, Denmark)with 94 Retsch 100, mill (SM rotorbeater laboratory a using mmsize) to 0.25 (0.2 milled was 0.24±0.08, xylan and mannan 0.0047±0.0011, glucan0.315±0.061, galactan 0.0053±0.0015, mean)0.048±0.013, arabinanSE of of basiswith dry composition (g/g, the with straw, experiments was Agroetan supplied by Lantmännen demonstration and content) both laboratory for dry (92.4% wheatstraw Commercial 2.1.Materials gently agitating the mycelium wi agitating the gently fungal wereplates flooded with 20mL sterile di extractcontaining (ing/L)potato 4; dextrose Schimmelcultures ascomycete, edible An 2. suspension (with a spore concentration of 3.1 of a sporeconcentration (with suspension × 10 optimization. A optimization. full-factorial experimentalmodel wasdesigned using MINITAB de experimental developed The statistically fordiluteacid 2.3.Experimental design pretreatment overnight. biomass at105°C calculated the drying weight by withthedry as the andused inoculum, cultivation the was incubated for48h and at 35°C125 rpm. The fungalbiomass harvestedwas atthe endof mL100 brothcontaining extractYPD 20; 20;culture (ing/L) dextrose peptone 10.The yeast into inoculated were spores the inoculum, fungal biomass preparing For cultivations. for the 2.2. Fungal strain2.2. Fungal (Minitab USA) soft Inc., PA, College, State

MATERIALS AND METHODS METHODS AND MATERIALS 15 , Netherlands), was maintained on potato dextrose agar (PDA) plates agar (PDA) dextrose onpotato maintained was , Netherlands), was used forthe hydrolysis. Neurospora intermedia th aAn inoculumof 50 cellspreader. disposable mL spore ware. Thephosphoricacid concentration (0.5%, sign was usedforthe pretreatment-conditions sign 20; agar 15. For agar 15.For preparing 20; spore suspension, stilled water and the spores were released by stilled waterand wereby the released spores 5 spores/mL) literper medium of was used ol Sweden).ol (Norrköping, Priorto its use, CBS 131.92 CBS131.92 (Centraalbureau voor ® 17 This article isprotected bycopyright. Allrights reserved. Accepted Article optimize determinin asetof overall responses by valuewas (D) calculated.Composit desirability to of 1 a in scale the significancearesponse) of minimumtheirinhibitors weresetto produced individual theand produced level maximum their to was set polysaccharides hydrolyzed total of the individual desirability limit forthe upper function. The desirability individual valuedefining wasspecifiedby desirability (HMF)) and acetic acid werethe selectedre as i glucan), arabinan, (xylan, polysaccharides The responses. the on factor each effect of the mixture and forfactorial, The responses optimization surface, plot shows response designs. howdifferent shows tool optimizer response using carried out was and fermentation) Thepr withanotherfactor. eachinteract level whether datathe means for one factor (such as ex yi inhibitor example sugaror as (such response interaction plots. Themain effect examines how factors various between and the responses were ofvarian (analysis anANOVA with analyzed variedfro parameters,although acidconcentration Eachof making 16groups. model full-factorial from generated the design later testwas individual interaction runs.Asplit developed 96 plot Thefull-factorialdesign content). weight dry straw wheat (based on w/v 15% was loading minu the pretreatment20 process(5,10,15,and and 3%w/v),thetemperature 1.75% reaction (1 an ANOVA responsean ANOVA optimizer. The Minitab ocess optimization (for pretreatment, hydrolysis, an upper and lower limit value to calculate the calculate the limit to lower value upper and an 5 was assigned to each response. A composite A each response. to was assigned 5 eld) differently.examines plot The interaction nhibitors (furfural, hydroxymethyl furfural nhibitorshydroxymethyl (furfural, level. A weightage point of 5(whichdefines point of Aweightage level. of duration the and 210°C), and 190, 170, 50, modeled, generating the main effectsand main modeled, generatingthe the the group had constanttemperatureand time e desirability (D) eval sponse variables. Forindividual sponse variables. response,a ce) general linear model. The relationship model. relationship The linear ce) general g the combined effect of individual response individual response effect of g thecombined experimental settings affect the predicted predicted the affect settings experimental tes) were the factors examined. Initial solid examined.Initial the factors tes) were the differentlevels of ample, time or temperature or acid conc) at conc) acid or temperature or time ample, yields (g/g substrate) of ethanol, released released of ethanol, substrate) (g/g yields m testrun toone another. The results were uates how the settings the factors affect the This article isprotected bycopyright. Allrights reserved. Accepted Article 20 min). Acirculating oil bath provided temperat time andtemperature 1.75 and3.0%), (5,10,15 170,190 210°C) and holding (150, (0.5, Pretreatment combinations ofacidconcentration carriedofconditions outatvarious was mL, scalewith15%solidloading. usedat phosphoricwere bench acidfor dilute pretreatment mL)100 of steelwithaworkingvolume Tubular (150 stainless (Swagelok, reactorsU.S.A.), op Minitab’s response variables. control. as a straw was used Untreated conditions. identical carried under out was plant demonstration inthe pretreated was that straw ofthewheat hydrolysis Enzymatic underFPU/g dry substrateinitiated of35°C,the hydrolysis conditions 125rpm for48h. of10 loading 5.5±0.1. Enzyme at pH mL) 100 volume of 7.5%(working loading a solid sl ofthepretreated hydrolysis carried for out all the pretreated samples from 96 test runs.Enzymatic the individual immersing reactions by thereactor were quenched 2.4. scale Experimental setup forbench functions. in results as the desirability specified individual favorable achieve to appear settings the that 1indicates to close fairly is value that desirability morezero indicatesor one responses that ar case; ideal the to onerepresents ofzero a range with value desirability Acomposite response. a single optimize thesettings how evaluates that value desirability theindividual dis where desirability function(also called desirability hydrolysis hydrolysis urry (with the solid and liquid unseparated) was carried outwith wascarried unseparated) andliquid (withthe solid urry timizer calculates composite de calculatescomposite timizer utility transferfunction). D= (d e outside their acceptable limits.composite acceptable A their outside e dilute acid pretreatment and enzymatic ure controlfor theure reactorsandpretreatment in an ice bath. Enzymatic hydrolysis was was hydrolysis bath. Enzymatic inanice sirability using a composite usinga sirability 1 ×d 2 ×d 3 × … × d ×… m ) 1/m , This article isprotected bycopyright. Allrights reserved. Accepted Article pretreated and enzymatically hydrolyzed (48 (48 hydrolyzed andenzymatically pretreated andfermentationSeparate (SHF hydrolysis experiments 2.6.Fermentation conditions. thefermentationpretreated of were slurry subsequent and hydrolysis Enzymatic run. produced foreach being material pretreated Lof test 1,000 ofpretreatment Two reaching runs with the performed the reactor. were (30 reactor L) with in flow continuous theacidcontinuously added feed the to prior screw verticalplug-reactions ina performed thetestruns.The maintained were for were 190±2°C) resi 1.75%, of concentration conditions (acid results, pretreatment thelaboratory optimum on the Sweden.Based in Örnsköldsvik, Sweden) of Institute Research (Technical SP by operated (BDP) Demo Plant Biorefinery the pretreatmentacid Dilute ofstraw was scaledup th pretreatment in acid 2.5. Dilute compared. also were processes sterilized and Non-sterilized a time. at excluded component being with oneindividual medium fungal growth, on components effect of the modified al. from etas Sues 3.5, CaCl was strawhydrolysate s control. The a as used strawwas Untreated wheat 7.5%). (2.5,5, concentrations loading (substrate) solid Addition of M NaOH. 5.5using1 to adjusted was ofthe pre-washing). slurry pH The subjecting ittoany 2 ·2H N. intermedia 2 O 1.0 andMgSO O spores (3.1 ×10 (3.1 spores 16 . Various sets of minimal. Varioussetsmedia of 4 ·7H e biorefinery demonstration plant plant e biorefinerydemonstration upplemented (inwith salts g/L) (NH4) 2 O 0.75; trace metals (10 mL/L); and vitamin (1 mL/L),mL/L);metals vitamin(10 and (1 0.75;trace O 5 initiatedspores/mL) atvarying the fermentation conducted at laboratory at laboratory scaleconducted under controlled h) straw from the demonstration plant (without h) from(without the demonstration plant straw ) experiments wereperformed usingacid- dence timeof of mindence 10 andtemperature using the previously optimized conditions at optimizedconditions previously using the wereformulated todetermine 2 SO 4 7.5, KH 2 PO 4

This article isprotected bycopyright. Allrights reserved. Accepted Article

Structural changes in the pretreated straw were investigated using a FourierTransform using straw were investigated pretreated inthe Structural changes Micro-Guard guard column (Bio-Rad)Carbo-P wa a column, by two Micro-Guard De-ashing followed HPX-87P, Bio-Rad) was Asetof used. mannose, galactose, and cellobiose,xylose, ethanol, acetic acid, furfural, and 5-hydroxymet (Bio-Rad) and0.6 mL/min 5 mMH guardcolumn cation-H aMicro-Guard 60°C with at U.S.A.) CA, Bio-RadHercules, 87H, fr allliquid used toanalyze methodsLaboratory (NREL) measured were straw phosphoric acid pretreated 1/250 of volume a in counted were spores the and measurement, the ten-foldbefore was diluted The spore Germany). Axiostarplus, (Carl Zeiss mm) microscope 0.1 alight under measured was using Initial sporeconcentration methods 2.7. Analytical 3. two standard deviations. are repo and results out in were carried duplicate analyses experiments and AB,Sweden).All Instruments (Chemical DV-E viscometer-model cm (CI) was calculated as the ratio 1429 atcm of wavenumbers the absorbances index Thecrystallinity U.S.A.). eFTIR FTIR, (Essential by Corp.)andanalyzed Instrument generate data were Thespectral U.S.A.). (ImpactInfrared (FTIR)spectrometer 410iS10

− RESULTS RESULTS 1

19 μ . The . The viscosity of the wasdeterminedslurry pretreated a using Brookfield digital L each. The total solid, suspended solid and sugar contents in the untreated and untreated the in contents and sugar solid suspended solid, total each. The L actions. A -based ion-exchange columnactions.A hydrogen-based(Aminex HPX- ion-exchange 17, 18 . HPLC (Waters 2695, Waters Corporation, U.S.A.) wasCorporation, U.S.A.) Waters (Waters 2695, . HPLC 2 SO 4, used as eluent, wasusedfortheofglucose, analyses d by Nicolet OMNIC 4.1 software (Nicolet software 4.1 OMNIC Nicolet d by arabinose, alead(II) based column (Aminex rted with error barsan rted with a Bürker counting chamber (with depthof a (with chamber a Bürkercounting according totheRenewable National Energy hyl-furfural. For thehyl-furfural. For separation ofglucose, , Nicolet WI, Instrument Madison, Corp., s coupled with the lead (II) based column. based column. lead (II) the s coupledwith d intervals representing − 1 ad 893 and This article isprotected bycopyright. Allrights reserved. Accepted Article conditions, a model was developed withthe model range conditions, a developed was acidconcentration (3%) (Fig.2). Hence, for th ( yield inhibitor in increase considerable wasa there addition, ( min range of 10to20 thetime for were found degradation ofpolysaccharide values the highest andinhibitors degradation range ofpolysaccharides release.For all combinations, treatment awide in resulted pretreatment conditions 2(c)). Thevarious (Fig. its yield in increase slight conditions,change in acid conc pretreatment a Although the acetic acid increasedyield steadily conditions for theacid,210 low (Fig.2(a))except (3% pretreatment highest 20 °C, min). thetemperature increasing Unlike furfural, range fromthe 190210°C. HMF to yield remains from considerable 150to170°C,inits a however, decrease wasyield observed while showedaninitialinhibitors. Furfural yield in Efforts weremade to determine the pretreatmentconditions that would generateminimum min) 20 0.00-0.016) and temperatures range: (p-value (190,210°C)( (15, time pretreatment at higher in yield there1(c)), was adecrease andarabinan (Fig. 1(b)) (Fig. temperature 1(a)).However,forxylan timeand (Fig. interaction pretreatment between yield glucan trend for increasing glucan. The 41%for maxima)and study xylan,arabinan, (theoretical the present for for 68% were 76% yield, hydrolysis in Themaximalfactor, 0.9and0.88). polysaccharide (conversion observed content polysaccharide respective the calculating by followed released, mono-sugar wa process pretreatment duringthe hydrolyzed straw subs gdry per hydrolyzed polysaccharide acid pretreatment ofwheat Dilute phosphoric 3.1. Optimization oftheconditions p -value: andtemperatures to210°C ( 0.00-0.032) 170 - bench scale dilute acid pretreatment pretreatment acid dilute scale - bench crease (Fig. 2(b)) when thetemperature rose straw at bench scale yields 0.441±0.003 g 0.441±0.003 yields scale straw atbench entration from 1.75 to 3% resulted in only aentration resultedinonly from 1.75 to 3% e statistical optimization ofthe pretreatment was observed as a function factorial was observedas ofthe trate. The yield of polysaccharides that are that are of polysaccharides yield The trate. s determined by measuring the individual individual measuring the by s determined of the factorial set limited to 0.5 to 1.75% to 0.5 limited to the factorialset of with an increase in the severity ofthe the severity in withanincrease p -value: 0.001 (Fig1).In -value:– 0.043) 0.001 p -value: 0.016 – 0.033) at a high ata high –0.033) -value: 0.016 p -value: 0.01 -0.023). -0.023). 0.01 -value: This article isprotected bycopyright. Allrights reserved. Accepted Article condition based on the desired responses set condition based onthe desired responses Ahigherand 0.84, respectively. composite desirability value the represents optimum 190 acid, 1.75% °C, 15 min 1),withcompos (Table conditions: acid, a)pretreatment 190°C, 0.5% as the best combinations three model predicted optimizer response The set. response preset usingthe was calculated conditions pretreatment effect ofthe for thecombinational acetic acid Thecomposite 0.014. desirability value (D)that the extent of optimumdefines fit 0.002 and furfural0.004,HMF biomass loadingof dry g/g minimumthevalue, to being set released inhibitors 0.032andthe 0.16,andarabinan xylan loadingofglucan0.08, biomass dry ofthe polysaccharid with theyield out carried conditions). Basedobtained inthe on the results model prediction optimizer considers all po the time to210°C,and1020 a acid, of170 frametemperatures of min. The response- conditions was increased. acidconcenWhen the decreased slurry eachof pretreated when ofthe viscosity of theparameters pretreatment was a decreaseinthe temperature CIvalue(210°C). atthe study, increased In the present of the pretreatment irrespective time (Fig.3( concen acid alower However,at concentration. measuredlowest value (0.19±0.01) for treatment conditions190°C,5min, and 3%acid with the points, percentage 76 CIvalueby inthe a resulted reduction acid pretreatment 1429 cm index(CI)valuemeasuring ofabsorbancesatwavenumbers the (ratio crystallinity by weredetermined instraw changes thestructural on reactions effects ofpretreatment The 3.2. Effect of dilute acid pretreatment on functional groups and viscosity 3.2. Effecton functionalandviscosity acid pretreatment ofdilute groups − 1 and 893 cm 893 and − 1 ). The CI value of untreated straw wasOverall, the diluteCI 0.79±0.25. of untreated ). Thevalue 11 a)). At a higher acid concentration (3%), there . es hydrolyzed set to the maximum value, g/g g/g value, maximum tothe set hydrolyzed es tration (0.5%) the CI value remained constant, constant, remained (0.5%)the CIvalue tration 15 min; b) 0.5% acid, 190°C, 20 min; min;c) b)0.5%acid,15 20 and 190°C, tration wasincreased 0.5 from to 1.75%, the present study, the response optimization was was optimization the response study, present ssible factorial combinations (pretreatment combinations (pretreatment ssible factorial ite (D) factors of0.73, 0.77, desirability This article isprotected bycopyright. Allrights reserved. Accepted Article for wheat straw pretreated acid,with 1.75% min 15 (Fig. and 190°C 4). A maximum total maximal was arabinan and ofxylan hydrolysis min, theother hand, and190°C. On acid,15 hydrolysis after enzymatic glucan hydrolysis loading) 0.31±0.02 glucan,0.18± 0.01 andxylan, 0.023±0.003 arabinan. The maximum scalelaboratory conditions yieldedhydroly at Enzymatic (following hydrolysis acid pretreat pretreatedwheat strawof laboratory scale 3.3.Enzymatic hydrolysis was again observed. in viscosity where adecrease (Fig. 3(b)), andtime withacidconcentration oftemperature interaction the regarding were alsomade enzymati thesubsequent during transfer) heat mixing and (mass aids slurry of viscosity thepretreated Thelow combinations). temperature 60%)whenthetime approximately was increa (by 3(b))showed that and time (Fig. concentration 52%.The ofthe factorial decreasedacid by function interaction viscosity between and 190°C(Table2). acid,min 20 c) 0.5% min190°C and and acid,15 190°C;b)0.5% min,and 15 acid, 1.75% was hydrolysis enzymatic after polysaccharides maximum. theoretical the of arabinan) 36% of (19% gave ofandof the only the untreated wheatstraw 12% thexylan 5% glucan, of hydrolysis enzymatic while maximum theoretical the of 86% gave straw wheat pretreated terms ofoverallefficiency of In acid pretreatment. maximum, following after hydrolysis observed the was theoretical of of0.52±0.03g/g dry hydrolysis polysaccharide polysaccharidehydrol The maximumThe freesugarsfrom release of occurred for wheatwith 0.5% strawpretreated there was a considerable decrease in viscosity in viscosity decrease a considerable there was substrate whichcorresponded loading, to86% c processes.hydrolysis Similar observations ment) ofstraw pretreated underarange of obtained under theobtained pretreatmenta) conditions under zed polysaccharides at (g/g dry substrate dry at (g/g zed polysaccharides sed from15 to 20 min (atdifferent acidand ysis, enzymatic hydrolysis oftheacid This article isprotected bycopyright. Allrights reserved. Accepted Article straw slurry had a viscosity of 82.3±0.6 cP. This is lower than that of the slurry produced in the slurry that of thanlower Thisis of 82.3±0.6cP. viscosity a had slurry straw The wheat total of16%. pretreated content content of24%andasuspendedsolids a flowrateand 1.7L/h, in 0.7resulting a pretreated between 2.25±0.05, a of with apH slurry The straw process. the hydrolysis to contribute heatinlonger the scale as demonstration the at at min the to of15 identified compared optimum min andacid, (Fig.4)at 190°C 15the laborator 1.75% pretreated with maximal straw forthewheat was arabinan and of xylan hydrolysis c acid The optimum plant. the biorefinery of 190±2°C),results, obtainedlaboratory usedforthetestrunsin from was temperature the conditionspretreatment (anacid forthefirsttime. optimum The here plantdemonstration isreported biorefinery atthe straw of fordilute acidwheatThe pretreatment conditions phosphoric validation of the optimum 3.4. ofp dilute phosphoric Validation acid yield by 38% compared to the laboratory-scale pr tothe laboratory-scale compared 38% by yield the demonstration-sc Hence, straw substrate. dry per gof hydrolyzed gpolysaccharide of0.192±0.008 lower yield aconsiderably showed min an Similar (1.75%acid,15 conditions straw substrate. g dry per gpolysaccharides 0.310±0.003 be to calculated was yield hydrolysis (0.63±0.03). Basedcompositionof theon analysis pretreated sample(Table 3), the total experiments fromlaboratory-scale with the the results obtained accordance isin which ahadCI value 0.58±0.01, solidfraction of pretreated scale.The conditionsatthe laboratory non-mixed the to compared scale larger the mixing at and transfer ofheat efficiency (108.5± experiments the laboratory-scale 0.4 cP),whichmight be due improved to the demonstration plant concentration of 1.75%, aresidencetimeconcentration minof 10 and a retreatment of wheat straw at the biorefinery the biorefinery wheat at ofstraw retreatment oncentration of 1.75% was chosen since chosen of 1.75% wasthe oncentration d 190°C) in the laboratory-scale experiments laboratory-scale 190°C) inthe d ale pretreatment improved the polysaccharide polysaccharide the improved ale pretreatment g and cooling periodsofthe also reactor could g slurry was added to the reactor continuously at thereactor was to added slurry laboratory scale in Section 3.4) was chosen chosen scalewas laboratory inSection 3.4) y scale. A reduced residence time (10 min, (10 time reduced residence scale. A y ocess. In addition, the pretreatment process process the ocess. Inpretreatment addition, This article isprotected bycopyright. Allrights reserved. Accepted Article maximumethanol was0.086± production g/g substrate dry there 0.002 However, loading. media, detrimental minimal effectscomplete the fungalgrowth.Withthewould have on mediacomponents to determine whether leaving out individual components of themedium Fermentation wasalsodonewith pretreated su respectively, 5(a)). wereobtained (Fig. wheat of pretreated straw,ethanol yields maximum) theoretical of (29% loading substrate after theenzymatic of untreatedhydrolysis (control)straw gave0.052±0.001 g ethanol/g dry content wheat based ontheglucan the substrate of substrate corre g/gdry loading, 0.127±0.002 of 8.1±0.3mgweight perL) concentration dry at laboratory scale. Fermentationwith afterprocessing substrate loading, g/gdry maximum 0.072±0.004 of a yield showed ethanol hydrolyzed enzymatically ofthe Fermentation 3.5. Fermentation using untreatedwheat of straw). composition on the per substrate g dry whichpolysaccharide straw, was95% maximumof the theoretical (based plant resu in thedemonstration pretreated HMF 0.16±0.02; and acetic (for48acid Theenzymatichydrolysis 1.32±0.01. h) of the straw acid,(1.75% minunder similarconditions 15 a significant). The prehydrolysateliquid from laboratory pretreatment theprocess performed process (with a laboratory the to compared yield resulted inapproximatelydemonstration 4%reductionintheinhibitor at theplant N. intermedia N. intermedia N. intermedia 0.102±0.002 and 0.082±0.005 g/g dry substrate, substrate, dry g/g 0.082±0.005 and 0.102±0.002 p -value in the range 0.00–0.031, proving it to be be it to proving 0.00–0.031, the range in -value sponding to 71% of the theoretical maximum ofthetheoretical sponding to71% pretreated strawfrom demonstrationplant the lted in hydrolysis of 0.578±0.039 0.578±0.039 of hydrolysis lted in g total bstrate supplemented with specific (minimal) nd 190°C) contained (g/L): furfural 4.1±0.1; resulted in an increased amount of ethanol, amountof ethanol, anresulted in increased straw (Fig. 5(a)). In contrast, fermentation contrast,fermentation 5(a)). In (Fig. straw . At solids loadings of 5% and 2.5% of 2.5% and 5% of At solidsloadings . mycelial biomass (with an initial mycelial aninitial biomass (with This article isprotected bycopyright. Allrights reserved. Accepted Article for the firsta time. novelfermentation also describes work theprocess Theusing edible was in thedetermined laboratory scaleandsubs dilutephosphoric foracidpretreatmentwheat straw of conditions In thiswork,theoptimum 4. spores; performed, except that the fermentation was carried out with the inoculation of theinoculation with out carried was fermentation the that except performed, (approx. U.S. $200–400/metric ton for92.5%H $200–400/metric ton (approx. U.S. sulfuricacid grade asindustrial used acids,such than that higher conventionally of other initiated by the inoculation of the mycelium of of the the inoculation initiated by to composition similar inhibitorthe sterilized substrate straw pretreated the non-sterilized substrate (94% g/g loading of thedry maximum) theoretical with the addition of nutrients to to0.171±0.01 vitamins. without The ethanol increased yield loading wasobtained substrate inthe case except of vitamins 5(b)). (Fig. An components, medium the oneof any lacked that samples in growth fungal significant no was as the cost of industrial grade acid (approx. U.S. $600–750/metric ton for 85% forH $600–750/metricton U.S. of industrial gradeacid(approx. cost as the use phosphoricmore acid.However, the of limitationspopular sulfuric acidhassomeof the theuse avoiding acid, hemicellulasemixtures. In thepresent study, we dilute phosphoric used sugars solubilizing hemicellulose convertinalso but technology for pretreatinghas lignocellulosic itonly theadvantage ofnot and biomass filamentous fungus in itsadvantages bi is offset by acid phosphoric

DISCUSSION 21

this gave a lower ethanol concentration of concentration ethanol lower gave a this . The dilute acid pretreatment, thus, eliminates or reduces the need for use of of use for need the reduces or eliminates thus, pretreatment, acid dilute . The N. intermedia . Dilute acid pretreatment has become a art ofthe hasbecome state pretreatment acid Dilute . ethanol concentration of 0.056±0.0024 g/g of dry ethanol concentration 0.056±0.0024 omassin section pretreatment 1). (described substrate) and with the fermentation being being the fermentation and withsubstrate) g solubilized hemicellulose to hemicelluloseg solubilized to fermentable equently equently validated in the demonstration scale (containing initial polysaccharides and initial(containing an polysaccharides 0.083±0.004 g/g dry substrate g/g dry (Fig.5(b)). 0.083±0.004 2 N. intermedia N. SO 4 ) 11 . Nevertheless, the higher cost of . A similar experiment was . Asimilarexperiment was N. intermedia 3 PO 4 ) is

This article isprotected bycopyright. Allrights reserved. Accepted Article maximum yield of sugars was 76% after enzymatic saccharification of 0.5% H 0.5% of saccharification afterenzymatic 76% was ofsugars yield maximum substrate. at 180°C has been reported previously for pretreatmenthas beenreportedwheat using 0.5% H previously straw concentration acid and temperature ahigh at acid acetic furfuraland of levels the in change with pretreated substrates lignocellulosic such as furfural reachedmaxi its higher pretreatment inthe pr temperatures used used, for neutralizing the effect inhibi of acid or fermentability means to the thata detoxificationThis step isnecessary improve usually during pretreatment. the inability of the microorganism fermentative therea criticalis forthefermentationHowever, dilute problem of acid namely hydrolysates, et Sahaexample, al. acids. For dilute other with straw pretreatment onwheat studies to the previous as compared hydrolysis, enzymatic followedby pretreatment acid wasobtained afterdilute yield an In this study, improvedhydrolysis polysaccharide ethanol yield of 0.29 of ethanol yield g/g(86% of wheatstraw recombinant using fermentation while the pilot-scaleRecently, conversionstudying of wheat strawto ethanol, and studiesthe microorganisms. previous that usedgenetically-modified ethanol-fermenting Using study, where a polysaccharide hydrolysis of 95% of the theoretical maximum was observed. maximumtheoreticalof the observed. was of95% hydrolysis a polysaccharide where study, the present in reported that thanmin lower at 180°C. Thisis wheat15 pretreated straw for

N. intermedia N. N. intermedia, N. intermedia,

21

, namely , namely 0.032 g furfural andg 0.027 acetic acid per gram of dry wheat straw

24. In noadditional stepthis study, forpHadjustment)detoxification (expect , tolerated the inhibitors present in the hydrolysate slurry. However, at at However, slurry. hydrolysate the in present inhibitors the tolerated , in the present study, a higher ethanol yield was obtained compared to to compared obtained was yield ethanol ahigher study, the in present mum furfural level.other High levelsalsoobservedfor were Escherichia coli Escherichia dilute phosphoric acid. For example, a similar example, acid.asimilar diluteFor phosphoric tors was necessary since thewas necessarysincefilamentoustors fungus to withstand inhibitory compounds produced inhibitory to withstand esent at study, particularly 190°C,inhibitors of the theoretical ethanolbased yield on FBR5, Saha etal. 3 reported amaximum 2 SO 21 reportedthat the 4 (v/v) for 15 min15 for (v/v) 2 SO 4 (v/v) This article isprotected bycopyright. Allrights reserved. Accepted Article 5. feed. animal as use for itssuitability increasing fermentation, after ethanol solid residue the of value nutritional the enriching ascomycete fungus, an uses edible study the present work, current in the obtained that than higher are ethanol yields these min. Although 9.7 acid, and 161°C, 0.78% strain maximum, from dilute-H theoretical H ligno otherfor reported previously been have cerevisiae. using wheat maximum(based straw), substrate on 88% ofthe theoretical toacid pretreat 1% sulfuric wheat at 100°Cand2 straw of yield ethanol an and obtained h toZhuet Similarly, prior fermentation. al. step (overlimingcalcium ) with detoxification a used also study Their yield). ethanol maximum theoretical the (94% of study present the in reported values the than lower comparatively was which straw), wheat substrate utilization nutrient of wi supplements together the with additionexperiment mycelial biomassfermentation of of a non-sterile in achieved was straw) of content on glucan (based maximum theoretical of substrcomposition onthe (based hydrolysis maximum after enzymatic 95%of theoretical the ofapproximately hydrolysis polysaccharide atotal showed plant demonstration a in biorefinery conditions at theoptimum pretreated Wheat straw loading. substrate straw initial on the maximumbased of thetheoretical 86% enzymatic from 36%to hydrolysis efficiency by the increasing hydrolysis polysaccharide acid The straw. of wheat the pretreatment report descri this ourknowledge, of To thebest 3

PO CONCLUSIONS 4 E. coli . For example, in a recent study, Avci et al. et Avci study, a recent in . For example, However, ethanol yields that are higher than those obtained in the present study in the presentstudy obtained those than higher are that However, ethanol yields were study in their reported conditions pretreatment The optimum FBR5. 3 PO 4 -pretreated corn stover using the recombinant stoverthe using recombinant corn -pretreated th straw hydrolysate. The present work hence hence work present The hydrolysate. straw th bes the first use of dilute phosphoric acid for acidfor dilutephosphoric first the useof bes pretreatment process improved the overall overall improved the process pretreatment ate straw). An ethanol yield of 94%the ethanolyield ate straw).An cellulosic substrates pretreated with dilute dilute with pretreated substrates cellulosic 23 reported an ethanol 96%ofthe ethanol yield reportedanof N. intermedia N. Saccharomyces Saccharomyces , and the , andthe 25 used used This article isprotected bycopyright. Allrights reserved. Accepted Article biorefinery. Bioresource Technology 135: 46-52 (2013). (2013). 46-52 135: Technology Bioresource biorefinery. acid pretreatment of phosphoric forproduction Santos SM, AMP, deVasconcelos 7. 125:76-83(2014). Applied Energy phosphoric-acid steam pretreatment of Euca BandIngram ofdilute-LO, Optimization Tamang DL, Rockwood Z, MT, Tian Mullinnix MT, IU, Castro E,Nieves 6. (2011). 1-3 IBC: Extension, University IowaState Center, Beef Iowa diet? feedlot my in include much grainscanI S, distillers DandHoyer How Loy S, Hansen M, Drewnoski 5. 3713-3729(2009). Engineering Chemistry Research 48: Biomass for Lignocellulosic Efficient and Hydrolysis Biofuel Industrial Production. & Kumar BarrettDM,DelwicheMJand P, 4. 175:17-22(2015). Technology Bioresource via si toethanol of wheatstraw conversion Itenand CottaMA,Pilotscale GJ, LB Kennedy QureshiN, NN, SahaBC,Nichols 3. (2013). 55:251-259 Bioenergy and applications. Biomass anditscoproduct (DDGS) characterizationforbiocomposite corn ethanol of the andMisra water-washing ZarrinbakhshMohanty AK studies M,Fundamental on N, 2. http://wwwigcint/en/grainsupdate anddemand Five-year global supply IGC, 1. REFERENCES discussions. valuable Kilberg, Kristina Laurila and Hägglund Karin express their gratitudeTomas toDr. Brandberg, like to Theauthorswould Agency. Energy was financiallybySwedish This work supported ACKNOWLEDGMENTS feed. asanimal an separately besold could which residue, fermentation straw used. With the useof an edible ascomycete,the also study produced nutritionally enriched eliminating problems associated with sulfur contam efficient asan therebyoutlinesacid pretreatmentagent, using potential the of phosphoric /sdaspx?crop=Wheat (April2015). /sdaspx?crop=Wheat Rocha GJMandSouto-Maior AM,Diluted Sagues WJ, HoffmanSagues RW,Fernández-Sandoval multaneous saccharifica lyptus benthamii for biofuel production. forbiofuel benthamii lyptus Stroeve Methodsfor Pretreatment P, of projections. International Council, Grains fermentable sugarsina sugarcane-based (SEKAB) for their technical assistance and and assistance for theirtechnical (SEKAB) Dr.Jorge Ferreira,Mostafa Jabbari, Marlén ination of feed stock when sulfuric when acidis of feedstock ination tion and fermentation. This article isprotected bycopyright. Allrights reserved. Accepted Article amorphous cellulose. Journal of Applied Polymer Science 8: 1311-1324 (1964). 1311-1324 Science 8: ofApplied Polymer cellulose.amorphous Journal I, II, IIIand of types oflattice PartI.Spectra type. crystal latticed and crystallinity RT,Relation and O'Connor ML Nelson 19. NREL/TP-510-42618(2012).analysis. standardbiomass Analytical Proceduresfor NREL Laboratory lignin inbiomass. and carbohydrates Determination ofstructural andal.TempletonD e, SluiterJ, C, Ruiz R,Scarlata SluiterA,Hames B, 18. TechnicalReport (2008). NREL/TP-510-42621 No Proce Analytical Laboratory NREL Samples. Liquid Process in Solids and TotalDissolved Solids inBiomass ofTotal Determination SluiterA,Hames B,Hyman D,PayneR, ScarlataRuiz C,Cand J, Wolfe 17. (2005). hydrolyzate Mucorindicus.yeast by research 5:669-676 FEMS dilute-acid and pentoses, fromhexoses, production Ethanol MJ, LandTaherzadeh A,Millati Sues R,Edebo 16. (2008). NREL/TP-510-42628. Procedure, Analytical Laboratory Activities. ofCellulase J, Measurement B andBaker Adney 15. (2015). Sciences doi:10.1002/elsc.201400213 Engineering inLife from stillageNeurosporaintermedia: thin by PRandTaherz Lennartsson FerreiraJA, 14. 3872 (2014). Energies 7: and Ascomycetes from Zygomycetes Filamentous ThinStillageUsing Food-Grade Fungi. and Biomass Ethanol of M,Production andTaherzadeh P FerreiraJ,Lennartsson 13. 167: 1501-1512(2012). applications.Biochemistry and Biotechnology Applied characterizationfungi fromand ofzygomycetes and tempe production ethanol biomass for PR Lennartsson R, Millati Wikandari 12. (2015).69: 314-323 andProducts Industrial Crops edibleNeurospora intermedia. productionfungiethanol by acid pretreatment phosphoric ofwheat bran PRand M,Brandberg T,Lennartsson Taherzadeh Nair RB,Lundin MJ,Dilute 11. (2011). 102: 5145-5152 Technology coli. Bioresource Escherichia ethanologenic by bagasse sugarcane pretreated Ingram LO,Effect sulfurcompoof reduced HoffmanMiller RW,FuZ,TongZand MullinnixMT, EN, Geddes CC, IU, Nieves 10. 50:478-484(2013). Products acid stoverforfurfura ofcorn pretreatment phosphoric dilute High temperature andCottaMA, GJ SahaBC,Kennedy Avci A, 9. andBi conditions.Biotechnology McMillan reca LR, Fractionating JRandLynd ME, Himmel RT,Laser M, CuiJ-B,Elander ZhangY-HP,Ding S-Y,Mielenz JR, 8. oengineering 97: 214-223 (2007). oengineering 214-223 97: l and ethanoll andproduction. IndustrialCrops and unds on the fermentation of phosphoric acid ofphosphoric on thefermentation unds for enzymatic hydrolysis and subsequent andsubsequent hydrolysis for enzymatic of certain infrared tocellulose bands adeh MJ, Production of ethanol and biomassand of ethanol adeh MJ,Production , Harmayani E and Taherzadeh MJ, Isolation E andMJ, Isolation , Taherzadeh Harmayani A pilot study for A pilotstudy process diversification. dures for standard biomass analysis. NREL analysis. biomass forstandard dures lcitrant atmodest lignocellulose reaction This article isprotected bycopyright. Allrights reserved. Accepted Article (2015). 185: 234-239 Technology from Bioresource wheat ethanol straw. and lignosulfonate xylose, Coproductionof WuY, ChenQand Huang W,WangK, Zhu S, 25. 18:312-331(1996). Technology and Microbial Enzyme production. ethanol for hydrolysates oflignocellulosic B,Fermentation Hahn-Hägerdal Olsson Land 24. (2013). 130: 603-612 Bioresource Technology production. andethanol hydrolysis acid forenzymatic stover pretreatment usingdilute ofcorn optimization phosphoric surface GJ andCottaMA,Response Kennedy BS, SahaBC,Dien Avci A, 23. fungal cellulases. by acid followed Ingram LO,Optimizingthe saccharification of and MT, Shanmugam KT MullinnixPeterson JJ,RoslanderC,Zacchi Geddes CC,G, 22. (2005). 3693-3700 wheat st of saccharification andfermentation acidYV, Dilute enzymatic pretreatment, Wu and ItenCotta MA LB, 21. Saha BC, (2013).Fuel 112:331-337 wheat saccharificationand fermentationforethanolproduction. of straw simultaneous DandKongH,OptimisationWu Q, WangX, of Zhang ZhangW,Lin Y, 20.

Bioresour Technol 101: 1851 - 1857(2010).Bioresour Technol101:1851

raw to ethanol. Process Biochemistry 40:to ethanol.Biochemistry raw Process sugar cane bagasse using dilute bagasse phosphoric sugarcane using This article isprotected bycopyright. Allrights reserved. Acetic acid acid Acetic HMF Furfural Arabinan Glucan Xylan Accepted Article Optimization Response min) ofwheat strawpretreatment. acid,190 °C,and20 1. Table TABLES Polysaccharide hydrolysis and inhibitor yield at the optimum conditions (0.5% conditions (0.5% at the hydrolysis yield optimum and inhibitor Polysaccharide Minimum Minimum Minimum Minimum Maximum Maximum Maximum 0.014 0.002 0.004 0.032 0.08 0.162 loading) substrate (g/g dry Yield 0.011 0.011 0.001 0.003 0.026 0.009 0.140 Upper Lower Lower Upper limits limits Confidence 95% 0.016 0.002 0.005 0.039 0.026 0.184 0.009 0.009 0.0004 0.001 0.021 0.003 0.125 limits limits Prediction 95% 0.018 0.008 0.024 0.043 0.032 0.200 (% w/v) load Acid conditions Pretreatment 1.75 0.5 0.5

This article isprotected bycopyright. Allrights reserved. Accepted Article (°C) Temp 190 190 190 190 diluteacid pretreatmentenzymatic and ofwheatstraw. hydrolysis 2. Table

(min) (min) Time 15 20 15 Optimum conditions for the maximum Optimum after forthe conditions hydrolysis yield polysaccharide loading substrate initial dry Yield (g/g) Glucan 0.240 0.216 0.280 0.256 0.305 0.281 limits Confidence L 95% owe r Upr Lower Upper Upper 0.264 0.303 0.329 Yield Yield loading) substrate (g/g dry Xylan Xylan 0.189 0.151 0.135

limits 95% Confidence 0.156 0.118 0.102 0.222 0.184 0.168 l i Y substrate substrate (g/g) oading oading nitial dry 0.023 0.013 0.015 Arabinan Arabinan ield 95% Confidence limits limits Confidence 95% L 0.012 0.003 0.005 owe r Upper

0.035 0.023 0.041

This article isprotected bycopyright. Allrights reserved. Accepted Article plant demonstration fromthe substrate 3. Table

HMF Furfural Acetic acid Xylan Glucan Arabinan Component Composition of Compositionthesolid andliquid of fractions the pretreated of straw wheat 0.89± 0.03 0.89± 0.03 2.71± 0.12 1.82± 0.03 16.1± 0.1 11.53± 0.06 15.02± 0.14 fraction (g/L) ofliquidComposition 0.037± 0.006 0.037± 0.006 0.235 ±0.01 0.022± 0.002 substrate) matter fraction (g/gdry ofsolid Composition

Sugarcane Sugarcane Substrate This article isprotected bycopyright. Allrights reserved. bagasse bagasse bagasse Wheat Wheat Wheat stover stover Acceptedstover Article straw straw straw Corn Corn Corn

rteteto incluoi usrts oflignocellulosicpretreatment substrates 4. Table acid 1% (w/w) acid 1% (v/v) acid 1% (v/v) Sulfuric acid acid Sulfuric acid Sulfuric 0.75% (v/v) (v/v) 0.75% acid 1.75% acid 1.75% Phosphoric acid 0.75% Phosphoric acid 0.20% Phosphoric Phosphoric Phosphoric Phosphoric Studies on the acid pretreatment of wheat straw and use of phosphoric acid for use ofphosphoricacid for the strawand Studiesonacidpretreatment ofwheat 1 % (w/w) (w/w) 1 % acid 0.5% 0.5% acid (w/v) (v/v) (v/v) (v/v) (v/v) Acid

186°C;min 24 186°C; 8 min min 8 186°C; Pretreatment Pretreatment (temp; time) time) (temp; conditions 180 °C; °C; 180 °C; 160 °C; 100 190 °C; °C; 190 160°C; 160°C; 180°C; 121°C; 10 min 10 min 10 min 15 min 15 min and and 2 h 2 h 1h 1h

hydrolysis yeild yeild hydrolysis hydrolysis yield yield hydrolysis yield hydrolysis hydrolysis yield yield hydrolysis Polysaccharide Polysaccharide polysaccharide polysaccharide recovery yield yield recovery recovery yield yield recovery 96% and 98% 98% and 96% hemicellulose hemicellulose 86.4% xylose xylose 86.4% 91.4% xylose xylose 91.4% 85% glucose 70.3% total 70.3% 76% xylan xylan 76% sugar yield theoretical 74% total 74% total maximum maximum 95% of yield total

Microorganism – Escherichia coli Escherichia coli Escherichia coli Escherichia coli Saccharomyces Saccharomyces cerevisiae YC- NRRL Y-2034 Recombinant Recombinant Recombinant fermentation S. cerevisiae Neurospora intermedia Ethanol LY160 LY160 FBR5 FBR5 FBR5 097 097 -

theoretical) (94% substrate ethanol/g dry 0.17 g Ethanol yield yield Ethanol ethanol/g dry ethanol/g dry ethanol (89% ethanol (89% wheat straw corn stover corn stover theoretical) theoretical) ethanol /g ethanol/g ethanol/g ethanol/g substrate 16.4 g /L /L g 16.4 0.334 g 0.334 0.24 g 0.13 g sugar sugar sugar 0.49 0.49 -

Geddes et Geddes Reference Vasconc Present Avci et Avci et Avci et Saha et elos et Zhu et study study al. al. al. al. al. al. al. de

23 23 25 21 9 7 22

This article isprotected bycopyright. Allrights reserved. Accepted Article FIGU yield ( Figure R g /g dry subs ES 1. Impact t of various rate loadin g) of a) glu a) g) of

pretreatm e e can, b)xyl nt conditi o a ns onthe ns n, and c) a n, and r r p abinan. abinan. olysaccha r ide hydro ide l ysis

This article isprotected bycopyright. Allrights reserved. Accepted Article loading Figure ) 2. ofa)HM Impac F t of vario of , b) furfur u a

s pretreat lc) ac and m m e e ent condit ent tic acid i ons on th ons on e yield (g/ g g dry subs dry

t rate

This article isprotected bycopyright. Allrights reserved. Accepted Article Figure o value f 3. wheat str wheat Impact a o w cellulosw f pretreat e m

and b) vis andb) ent condi c t ions on thions on osity valu osity e e e ofthe pre a) chang e h in the cry the in ydrolyzate ydrolyzate stallinity i stallinity

n dex

This article isprotected bycopyright. Allrights reserved. Accepted Article

e g F nzymatic lucan ( igure 4. ■

) P P h , and xyla olysaccha ydrolysis n r (

ides hydro ides 48 h) h) 48 ( ■ ) after l dilute pho ysis yield s ( phoric aci phoric g/g dry str g/g dry d d aw substra pretreat m t ent and su ent and e) of arabi of e)

b n sequent an ( ■ ), This article isprotected bycopyright. Allrights reserved. Accepted Article Figure 5. Minimal media with the absence of vitamins ( ofvitamins absence media withthe Minimal sterilized ( b) Complete minimalmedia additionfungal spores- of with sterilized ( control fermentation using acid untreated but enzymatically hydrolyzed straw ( enzymatically aciduntreated but control fermentation using ( ofextract with fungal a) addition substrate biomass with yeast Fermentation profile of Fermentation ▲ ) pretreated slurry; fungal biomass to non-sterilized ( fungal tonon-sterilized biomass ) pretreated slurry; N. intermedia ■ ) indiluteacid pretreated wheatstraw ● ) and spores (

■ ) pretreated slurry; slurry; ) pretreated ∆ ) and non- ) and ♦ ) ○ ); and