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Response surface optimization of corn stover pretreatment using dilutephosphoric acid for enzymatic hydrolysis and ethanol production

Ayse Avci a,b,⇑, Badal C. Saha a, Bruce S. Dien a, Gregory J. Kennedy a, Michael A. Cotta a

a Bioenergy Research Unit, National Center for Agricultural Utilization Research, Agricultural Research Service, U.S. Department of Agriculture1, 1815 N. University Street, Peoria,IL 61604, USAb Department of Food Engineering, Faculty of Engineering, Sakarya University, Sakarya 54187, Turkey

h i g h l i g h t s

" Dilute phosphoric acid pretreatment of corn stover was optimized by RSM." Temperature, acid dose and time were independent variables for pretreatment." Total sugar yield from pretreated corn stover was 78% after enzymatic hydrolysis." Recombinant Escherichia coli FBR5 produced 0.49 ethanol/g sugar from undetoxified hydrolysate.

a r t i c l e i n f o

Article history:Received 3 August 2012Received in revised form 2 October 2012Accepted 14 December 2012Available online 22 December 2012

Keywords:Corn stoverDilute phosphoric acid pretreatmentResponse surface methodologyRecombinant Escherichia coli FBR5Ethanol fermentation

a b s t r a c t

Dilute H3PO4 (0.0–2.0%, v/v) was used to pretreat corn stover (10%, w/w) for conversion to ethanol. Pre-treatment conditions were optimized for temperature, acid loading, and time using central compositedesign. Optimal pretreatment conditions were chosen to promote sugar yields following enzymaticdigestion while minimizing formation of furans, which are potent inhibitors of fermentation. The maxi-mum glucose yield (85%) was obtained after enzymatic hydrolysis of corn stover pretreated with 0.5% (v/v) acid at 180 �C for 15 min while highest yield for xylose (91.4%) was observed from corn stover pre-treated with 1% (v/v) acid at 160 �C for 10 min. About 26.4 ± 0.1 g ethanol was produced per L by recom-binant Escherichia coli strain FBR5 from 55.1 ± 1.0 g sugars generated from enzymatically hydrolyzed cornstover (10%, w/w) pretreated under a balanced optimized condition (161.81 �C, 0.78% acid, 9.78 min)where only 0.4 ± 0.0 g furfural and 0.1 ± 0.0 hydroxylmethyl furfural were produced.

� 2012 Elsevier Ltd. All rights reserved.

1. Introduction

Developing sustainable transportation fuels is a global chal-lenge (Savvanidou et al., 2010). Bioethanol is a renewable, biobasedoxygenated fuel (Saha, 2004) and the current production of bioeth-anol is based on first generation conversion of starch and sugarbased crops (Ajanovic, 2011; Fischer et al., 2010). In particular,the United States and Brazil have expanded corn and sugarcanecultivation, respectively, for production of fuel ethanol (Rathmann

et al., 2010; Hoekman, 2009). Further growth in production willneed to rely on exploiting other non-food and non-animal feedassociated biomass (Hoekman, 2009; Uncu and Cekmecelioglu,2011). Lignocellulosic biomass is the most abundant renewablecarbohydrate source in the world and it is proposed to dominatethe biofuel production in the future (Chen et al., 2012; Fischeret al., 2008). Low cost agricultural residues (corn stover, wheatstraw, rice straw), agricultural processing byproducts (corn fiber,rice hulls, sugar cane bagasse) and energy crops (switchgrass,miscanthus) can be used for ethanol production (Saha and Cotta,2010). One significant source of biomass is corn stover, which isespecially abundant in the United States (Um et al., 2003).

Biological production of ethanol from lignocellulose requiresthat the biomass be pretreated prior to enzymatic hydrolysis forproduction of fermentable sugars (Saha et al., 2011a). Pretreatmentis needed to provide accessibility to enzymes for the hydrolysis ofcellulose and hemicellulose (Öhgren et al., 2005; Saha et al.,2011b). Various physical, chemical, and physicochemical methods

0960-8524/$ - see front matter � 2012 Elsevier Ltd. All rights reserved.http://dx.doi.org/10.1016/j.biortech.2012.12.104

⇑ Corresponding author at: Department of Food Engineering, Faculty of Engi-neering, Sakarya University, Sakarya 54187, Turkey. Tel.: +90 264 295 5464; fax:+90 264 295 5608.

E-mail address: aysea@sakarya.edu.tr (A. Avci).1 Mention of trade names or commercial products in this article is solely for the

purpose of providing specific information and does not imply recommendation orendorsement by the U.S. Department of Agriculture.

Bioresource Technology 130 (2013) 603–612

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for pretreatment have been described (Tian et al., 2011). DiluteH2SO4 is typically used for pretreatment of lignocellulosic biomass.While H3PO4 is more expensive than H2SO4, it has several advanta-ges that merit its use. The H3PO4 is environmentally friendly be-cause phosphorus containing solid residues can be used asfertilizers (Geddes et al., 2010; Lee and Jeffries, 2011). Moreover,phosphorus is an essential nutrient for various fermentation media(Romero et al., 2007). Only a few studies are available on the diluteH3PO4 pretreatment of corn stover. Um et al. (2003) carried out di-lute H3PO4 pretreatment of corn stover at 121 �C for 30–120 min ata solid loading of 5% (w/v). Lin et al. (2010) studied ball milling pre-treatment of corn stover (7.5%, w/v) at room temperature by add-ing six different dilute acids including H3PO4. However, none ofthese two studies are aimed at maximizing the sugar yield afterenzymatic saccharification of pretreated corn stover while mini-mizing the formation of fermentation inhibitory compounds suchas furfural and hydroxyl methyl furfural (HMF) during pretreat-ment. Typical detoxification of the inhibitory compounds involveadditional process step such as over-liming and bio-detoxificationusing an inhibitor utilizing fungal strain prior to fermentation ofthe hydrolysate which adds cost to the process of ethanol produc-tion from lignocellulosic biomass (Saha, 2004). Our aim is to opti-mize the dilute H3PO4 pretreatment of corn stover using a highersolid loading and temperature while minimizing the formation ofthe inhibitory compounds.

Herbaceous biomass yields glucose, xylose, arabinose, and gal-actose upon enzymatic saccharification (Saha, 2003). Current com-mercial strains such as Saccharomyces cerevisiae and Zymomonasmobilis used for ethanol production are unable to ferment pentosesugars such as xylose and arabinose. For this reason, various re-combinant yeasts and bacteria have been developed during the lastfew decades that can ferment these sugars (Saha et al., 2011a).Mixed sugar utilizing ethanologenic recombinant Escherichia colistrain FBR5 is one of the more promising strains in terms of ethanolproductivity, plasmid stability, antibiotic dependence, and broadsubstrate range (Dien et al., 2000; Saha and Cotta, 2011; Sahaand Cotta, 2012). This particular strain utilized a novel metabolicengineering strategy designed to enhance phenotypic stability ofhigh ethanol yields (Dien et al., 2000).

In the present paper, dilute H3PO4 was used as a pretreatmentstrategy for conversion of corn stover to ethanol. Pretreatmentconditions, including temperature, acid loading, and duration wereoptimized for yields of glucose, xylose, arabinose, galactose andtotal sugars following enzymatic digestion. The optimized pre-treatment conditions with respect to maximum yield of totalsugars and tolerable levels of furans were subsequently used forits conversion to ethanol using recombinant E. coli strain FBR5.

2. Methods

2.1. Materials

Corn stover (92.6 ± 0.2% dry matter) was harvested in the fall of2011 (Peoria, IL). It was ground in a hammer mill so as to passthrough a 1.27 mm screen and stored at ambient temperature intightly closed plastic bags. Celluclast 1.5 L (cellulase) and Novozym188 (b-glucosidase) were purchased from Brentag Great Lakes(Milwaukee, WI, USA). Fiberzyme (hemicellulase) was providedby Dyadic Corp., Jupiter, FL. Aminex HPX 87P column(300 � 78 mm), Aminex HPX 87H column, De-ashing cartridge(30 � 4.6 mm), Carbo-P micro-guard cartridge (30 � 4.6 mm) andCation H micro-guard cartridge (30 � 4.6 mm) were purchasedfrom Bio-Rad Laboratories Inc. (Hercules, CA, USA). Sterile filterunits were purchased from Nalgene Co. (Rochester, NY, USA). Allchemicals used were of standard analytical grades.

2.2. Enzyme assays

The cellulase activity in terms of filter paper activity was as-sayed and expressed as filter paper unit (FPU) by the procedure de-scribed by Ghose (1987). The b-glucosidase and xylanase activitieswere assayed by the procedures described previously (Saha andCotta, 2008). All enzyme assays were performed at pH 5.0 and45 �C and the activities were expressed in terms of internationalunits (IU, lmol product formed per min).

2.3. Pretreatment and enzymatic digestion of corn stover

Pretreatments of corn stover (10%, w/w, 100 mL) were carriedout in a multi-vessel rotating reactor system equipped with twelve200-mL stainless steel reactors (Labomat BFA-12, Mathis USA Inc.,Concord, NC). The system is heated with infrared heater and watercooled. The reactors were rotated continuously at 50 rpm (60 sclockwise and 60 s counter clockwise) during the reaction. Pre-treatments were conducted under the reaction conditions listedin Table 1. Following pretreatment and subsequent to enzymaticdigestion, the pH was adjusted to 5.0 with aqueous slurry ofCa(OH)2.

For enzymatic saccharification, a filter sterilized enzyme cocktailcontaining 15 FPU cellulase (Celluclast 1.5 L) and 9 U b-glucosidase(Novozym 188) based on per g glucan content, and 1578 U xylanase(Fiberzyme) per g hemicellulose content was added to the pre-treated corn stover hydrolysate at pH 5.0. Reactions were carriedout at 45 �C for 72 h by gentle shaking at 135 rpm. The hydrolysatewas clarified by centrifuging at 25,000g for 10 min and analyzed forsoluble sugars and furans using HPLC.

2.4. Experimental design and statistical analysis

A three variable central composite design was used to optimizethe pretreatment conditions of corn stover with dilute H3PO4. Theindependent variables were temperature (�C), acid dose (%, v/v)and time (min). The ranges and levels of the independent variablesare given in Table 1. Levels of variables were determined accordingto preliminary studies. The percentages (w/w) of glucose, xylose,total sugars and furfural in pretreated corn stover after enzymatichydrolysis were chosen as responses for analyzing. A total of 20experiments with central points were performed in random orderfor minimizing the effect of any unexplained variability in the ob-served responses due to external factors. The central point wasreplicated six times and each of the other points was replicatedthree times. Regression analysis was performed for each responsein order to estimate the effect of combined independent variableson the responses by using the statistical software package Design-Expert 8.0.5 (Stat Ease, Inc., Minneapolis, USA). Each response wastested for possible linear, quadratic and cubic models to find out tobest fitting model. Backward elimination strategy was used toeliminate insignificant terms from the model. Quadratic polyno-mial model was used to calculate regression coefficients. Signifi-cance of each model term was determined with analysis of

Table 1Range of each independent variable in the central composite design.

Variables

Coded levels of factors Temperature (�C) Acid dose (%, v/v) Time (min)

Axial point (�1.68) 126.36 0.16 1.59Low level (�1) 140 0.5 5Central level (0) 160 1.0 10High level (+1) 180 1.5 15Axial point (+1.68) 193.63 1.84 18.41

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variance (ANOVA). The fit of the models were evaluated by com-paring R2 and adjusted-R2. The statistical significance was checkedby F-test. Three-dimensional surface plots were drawn based onthe final equation determined for each response to demonstratethe effects of independent variables on the responses.

2.5. Fermentation experiments

Recombinant E. coli strain FBR5 was maintained in glycerol vialsat �80 �C. It was plated on Luria Bertani (LB) agar containing 10 gtryptone, 5 g yeast extract, 5 g NaCl and 15 g agar per L supple-mented with 4 g xylose and 20 mg tetracycline. Plates were incu-bated at 35 �C overnight and a single colony was picked forinoculation into a 125 mL flask containing 100 mL LB broth. Com-position of the LB broth was the same as LB agar without the agar.Inoculated liquid cultures were incubated at 35 �C for 24 h withgentle shaking at 85 rpm. A 5% (v/v) inoculum having OD600nm of2.0 was used for fermentation.

Corn stover was pretreated under optimized conditions andenzymatic saccharification was carried out as explained inSection 2.3. Tryptone (10 g/L) and yeast extract (5 g/L) weredissolved in the dilute H3PO4 pretreated and enzymatically saccha-rified corn stover hydrolysate. It was filtered through a filtersterilization unit prior to fermentation experiments.

Fermentation experiments were carried out in pH-controlled250 mL flakers with a working volume of 150 mL at pH 6.5 and

35 �C. The pH was controlled at 6.5 during fermentation by adding2 N KOH. Detailed setup of the bioreactors has been reported pre-viously (Saha et al., 2011b). Samples were taken periodically todetermine ethanol production, sugar utilization and cell growthof the bacterium.

2.6. Analyses

The cellulose, hemicellulose, lignin and ash contents of corn sto-ver were determined using the procedure provided by NationalRenewable Energy Laboratory (NREL), Golden, CO (Sluiter et al.,2008a,b). Moisture content was analyzed using a moisture ana-lyzer (Mark 2, Sartorious Mechatronics Corp., Bohemia, NY, USA).Sugars, furfural, 5-hydroxymethyl furfural (HMF) and ethanol wereanalyzed by high performance liquid chromatography (HPLC). Sug-ars and ethanol were separated using an Aminex HPX-87P columnand detected using a refractive index detector (410 differentialrefractometer, Waters). Two guard columns (De-ashing and Car-bo-P micro guard cartridges) were used with Aminex HPX-87P col-umn. Column temperature was maintained at 85 �C and elutionwas made at 0.6 mL/min flow rate by using Milli-Q (MilliporeCorp., Bedford, MA, USA) filtered water. Furfural and HMF wereseparated in Aminex HPX-87H column and detected by using dualk absorbance detector (2487, Waters) at 277 nm. The column tem-perature was 65 �C and 10 mM HNO3 in Milli-Q filtered water was

Table 2Composition of corn stover.

Per 100 g of original material on dry basis

Component Before pretreatment (original material) Solid residuesa (after pretreatment) Solid residuesb (after enzymatic hydrolysis)

Glucan 37.1 ± 0.0 33.6 ± 0.0 14.1 ± 0.0Xylan 21.0 ± 0.1 7.6 ± 0.0 5.0 ± 0.0Arabinan 3.1 ± 0.4 0.0 ± 0.0 0.0 ± 0.0Galactan 2.1 ± 0.0 2.2 ± 0.1 1.2 ± 0.0Total carbohydrates 63.3 ± 0.5 43.4 ± 0.1 20.3 ± 0.0Lignin 18.9 ± 0.0 16.7 ± 0.6 16.6 ± 0.7Ash 5.0 ± 0.0 6.9 ± 0.6 6.9 ± 0.4

Data presented are averages ± standard deviations of triplicate estimations.a 39.5 ± 0.6% of the original material was solubilized after pretreatment of corn stover (10%, w/w) with 0.78% H3PO4 at 161.81 �C for 9.78 min.b 54.3 ± 0.8% of the original material was solubilized after enzymatic saccharification of the pretreated corn stover at pH 5.0 and 45 �C for 72 h.

Table 3Factors (temperature, acid dose and time) and the responses (glucose, xylose, total sugar, and furfural) of the central composite design used for response surface methodology.Coded values of the variables were indicated in parenthesis.

Trial Factors Responses

Temp (�C) Acid (%, v/v) Time (min) Glucose (%) Xylose (%) Total sugar (%) Furfural (%)

1 140 (�1) 0.5 (�1) 5 (�1) 15.81 9.70 28.92 0.012 180 (+1) 0.5 (�1) 5 (�1) 29.97 19.54 56.74 1.673 140 (�1) 1.5 (+1) 5 (�1) 18.84 15.42 39.08 0.024 180 (+1) 1.5 (+1) 5 (�1) 31.73 15.58 54.63 4.065 140 (�1) 0.5 (�1) 15 (+1) 18.37 13.57 36.36 0.026 180 (+1) 0.5 (�1) 15 (+1) 35.35 16.83 58.48 3.747 140 (�1) 1.5 (+1) 15 (+1) 21.49 19.30 47.57 0.028 180 (+1) 1.5 (+1) 15 (+1) 34.80 11.24 51.40 7.209 126.36 (�1.68) 1 (0) 10 (0) 17.11 10.69 31.70 0.0110 193.64 (+1.68) 1 (0) 10 (0) 33.74 6.78 43.43 8.7011 160 (0) 0.16 (�1.68) 10 (0) 19.01 11.75 32.20 0.0312 160 (0) 1.84 (+1.68) 10 (0) 27.42 21.10 55.75 1.0613 160 (0) 1 (0) 1.59 (�1.68) 21.38 18.83 46.43 0.1114 160 (0) 1 (0) 18.41 (+1.68) 26.80 20.45 53.83 1.0815 160 (0) 1 (0) 10 (0) 28.89 21.84 55.96 0.5016 160 (0) 1 (0) 10 (0) 28.59 21.16 54.81 0.6117 160 (0) 1 (0) 10 (0) 27.29 20.68 52.90 0.4818 160 (0) 1 (0) 10 (0) 28.36 21.00 54.43 0.8119 160 (0) 1 (0) 10 (0) 28.37 21.04 54.45 0.5620 160 (0) 1 (0) 10 (0) 28.96 21.32 55.36 0.68

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used at a flow rate of 0.6 mL/min for elution. Growth of the bacte-rium was determined measuring the optical density at 600 nm.

3. Results and discussion

3.1. Composition of corn stover

The corn stover used in this study was analyzed for compositionand determined to contain 37.1 ± 0.03% glucan, 21.0 ± 0.10% xylan,2.1 ± 0.01% galactan, 3.1 ± 0.4% arabinan, 18.9 ± 0.04% lignin(1.8 ± 0.02% acid soluble, 17.1 ± 0.02% acid insoluble) and5.04 ± 0.02% ash on dry basis (Table 2). The results are consistentwith previously reported compositions of corn stover (Perezet al., 2005; Chen et al., 2012). Glucan and xylan account for91.7% of the total carbohydrates in the corn stover.

3.2. Linear model for dilute-phosphoric pretreatment of corn stover

The response of sugar yields to varying pretreatment reactionconditions were modeled using response surface methodology(Pereira et al., 2010; Ferreira et al., 2009). Based upon prior resultsfrom applying dilute H2SO4 to corn stover, the following rangeswere tested: 140–200 �C, 0.0–2.0% (v/v) H3PO4 loading, and 5–25 min reaction time. The complete list of trials and responsesare listed in Table 3. Each response is the average of triplicate inde-pendent runs. Following pretreatment, the pH of the whole hydro-lysate was adjusted to 5.0 and saccharified with commercialcellulases and hemicellulases. The product stream was analyzedfor glucose, xylose, arabinose, galactose and total sugar yields aswell as for furfural and HMF concentrations. Production of HMFwas found to be minimal because little hexoses were released dur-ing pretreatment (data not shown). The chosen range of conditionswas sufficient to describe the reaction space as evidenced by thebroad range of results for glucose (15.81–35.3%), xylose (6.78–21.84%), and furfural (0.01–8.70%).

The resulting sugars and furfural concentrations were success-fully modeled based upon the reaction conditions using linearregression. Furfural was transformed by the square root. All modelswere significant at the P < 0.0001 level. The model surfaces showedexcellent correlation with the data and the adjusted R2s were: glu-cose, 0.979; xylose, 0.990; total sugars, 0.978 and furfural, 0.985.Analysis of variances (ANOVA) of the models for glucose, xylose,total sugar and furfural are given in Table 4. The final equationswere as follows:

Glucose ¼ 55:64þ 0:415� Tempþ 24:10� Phosþ 1:52� Time

� 0:0618� Temp� Phos� 5:78� Phos2 � 0:060

� Time2

Xylose ¼ 408þ 4:84� Tempþ 42:3� Phosþ 3:43� Time

� 0:262� Temp� Phos� 0:0185� Temp� Time

� 0:0136� Temp2 � 0:0209� Time2

Total sugars ¼ 526:8þ 6:07� Tempþ 63:8� Phosþ 5:11� Time

� 0:382� Temp� Phos� 0:0218� Temp� Time

� 0:0158� Temp2 � 0:0209� Time2

ffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi

Furfuralp

¼ 16:1� 0:216� Temp� 2:12� Phos� 0:186� Time

þ 0:0158� Temp� Phosþ 1:38� 10�3 � Temp

� Timeþ 7:21� 10�4 � Temp2

where sugars and furfural appear as concentrations (%) and the vari-ables are Temp (temperature in �C), Phos (H3PO4 concentration in %,v/v), and Time (in min).

3.3. Optimization of reaction conditions for corn stover pretreatmentwith H3PO4

The effect of reaction conditions on glucose yield followingenzymatic digestion are plotted as surfaces in Fig. 1. These surfaceswere generated with the quadratic equation displayed above. Sep-arate surfaces are shown for time versus temperature, temperatureversus acid loading, and acid loading versus time. From comparingthe generated surfaces, glucose yield is most sensitive to reactiontemperature compared to acid loading and time. Kabel et al.(2007) reported that reaction severity varies with the exponentialof temperature for dilute H2SO4 pretreatment of wheat straw. Therelative insensitivity of the reaction to H3PO4 loading suggests thatthe amount added was sufficient to overcome the natural bufferingcapacity of the biomass. To maximize glucose yield (35.35 g/100 gcorn stover or 86.62% of theoretical yield) the recommended con-ditions are to treat corn stover (10%, w/w) in 0.5% (v/v) H3PO4

and hold at 180 �C for 15 min.Xylose yields for varying reaction conditions are shown in Fig. 2.

As observed for glucose yield, xylose was also most sensitive totemperature. But, unlike glucose, xylose concentration achieved amaximum value at 160 �C through the experimental space. Xyloseconcentration was also somewhat sensitive to acid concentrationat the lower reaction temperature. This difference in behavior be-tween the sugars (glucose and xylose) reflects the different mech-anisms for their generation. Glucose was largely generated afterenzymatic digestion, while xylan was directly hydrolyzed byH3PO4 (data not shown). The reaction sequence is xylan to xyloseto furfural. Therefore, at first as temperature increases, xylan con-version to xylose is favored but under harsher conditions xylose

Table 4ANOVA table of the adjusted models from phosphoric acid pretreated and enzymehydrolyzed corn stover.

Source Sum of squares DF F value P-value

GlucoseModel 522.41 6 139.76 <0.0001 SignificantResidual 8.62 12Lack of fit 6.80 7 2.66 0.1493 Not significantR2 0.9859Adj-R2 0.9788Pred R2 0.9520

XyloseModel 349.31 7 239.51 <0.0001 SignificantResidual 2.08 10Lack of fit 1.33 5 1.75 0.2768 Not significantR2 0.9941Adj-R2 0.9899Pred R2 0.9759

Total sugarModel 1075.27 7 100.39 <0.0001 SignificantResidual 13.77 9Lack of fit 8.37 4 1.94 0.2423 Not significantR2 0.9874Adj-R2 0.9775Pred R2 0.9291

FurfuralModel 13.15 6 208.99 <0.0001Residual 0.14 13Lack of fit 0.11 8 2.20 0.2007R2 0.9897Adj-R2 0.9850Pred R2 0.9752

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conversion to furfural begins to dominate. Similar behavior is alsoobserved for dilute H2SO4 pretreatments (Kabel et al., 2007). Max-imum xylose (21.84 g/100 g corn stover) was obtained at 160 �Cusing 1.0% (v/v) H3PO4 with 10 min retention time at which xyloserecovery was 94.5% of the theoretical yield. It decreased drasticallyat 193.63 �C at which it was at its lowest value (6. 78 g/100 g sto-ver, 29.3% of the theoretical yield).

Fig. 3 shows the response surface for the total sugars yield (glu-cose, xylose, galactose, arabinose and cellobiose). Maximum value(58.48 g/100 g corn stover) was obtained at 180 �C with 0.5% (v/v)

H3PO4 and 15 min retention time. These conditions correspondwith the maximum yield for glucose, but xylose content is low(16.83 g/100 g stover). The furfural concentration is also high un-der these conditions (3.74 g/100 g stover). Because furfural is a po-tent inhibitor of fermentative microorganisms, less harshpretreatment conditions are warranted even though glucose yieldwill be sacrificed.

Xylose and glucose generated during pretreatment can undergofurther dehydration reactions to form furfural and HMF, respec-tively (Nichols et al., 2008). As discussed, both of these products

Fig. 1. Response surface plots of glucose yield (g/100 g corn stover) obtained from central composite design with three independent variables for the optimization of cornstover pretreatment with H3PO4. Top, temperature (�C) and acid dose (%, v/v); middle, temperature (�C) and time (min); bottom, acid dose (%, v/v) and time (min).

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are to be avoided because they are potent inhibitors of fermenta-tion. In this study both furans were detected, but HMF concentra-tions were uniformly low (<1%) because very little precursorglucose was produced directly via H3PO4 pretreatment. However,xylose was largely generated via direct acid hydrolysis and furfuralconcentrations were significant. The response surface plots for fur-fural production during H3PO4 pretreatment are displayed in Fig. 4.As observed for glucose and xylose yields, furfural formation is

most sensitive to reaction temperature. Unlike the case for thesetwo products, however, furfural increased in an exponential likemanner with temperature. This curve may reflect the lag time be-tween xylose formation and its transformation into furfural; xyloseis directly produced from xylan. At higher temperatures, whenmore furfural is formed, its appearance is sensitive to both reactiontemperature and duration. The furfural formation has been deter-mined as 8.7 g/100 g stover at 193.6 �C which is the highest axial

Fig. 2. Response surface plots of xylose yield (g/100 g corn stover) obtained from central composite design with three independent variables for the optimization of cornstover pretreatment with H3PO4. Top, temperature (�C) and acid dose (%, v/v); middle, temperature (�C) and time (min); bottom, acid dose (%, v/v) and time (min).

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point of temperature. Higher xylose degradation with H3PO4 over160 �C was also reported with sugarcane bagasse (Geddes et al.,2010).

A number of reports exist on the pretreatment of corn stoverusing various methods such as dilute H2SO4 (Chen et al., 2012;Schell et al., 2003), steam pretreatment (Öhgren et al., 2005) andhot water pretreatment (Mosier et al., 2005). Yields of the sugar re-leased were dependent on the temperature and the time as well asthe method applied. Yields obtained by other researchers varied

from 73–91% for glucose and 70–82% for xylose for corn stover(Chen et al., 2012; Mosier et al., 2005; Öhgren et al., 2005; Schellet al., 2003). To the best of our knowledge, there is only one priorstudy for H3PO4 pretreatment of corn stover and this was con-ducted under non-optimized conditions: 121 �C for 120 min; atwo hour residence time is unlikely to be commercially feasible.As a result the sugar yields were low: 60% for glucose and 58%for hemicellulose. The H3PO4 concentration was also higher (2%,v/v) which would add considerable chemical costs (Um et al.,

Fig. 3. Response surface plots of total sugar yield (g/100 g corn stover) obtained from central composite design with three independent variables for the optimization of cornstover pretreatment with H3PO4. Top, temperature (�C) and acid dose (%, v/v); middle, temperature (�C) and time (min); bottom, acid dose (%, v/v) and time (min).

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2003). Geddes et al. (2010) used H3PO4 for the pretreatment of sug-arcane bagasse. They reported that the optimum conditions forsugarcane bagasse pretreatment with H3PO4 as 160 �C, 10 minand 1% (w/w) acid concentration, which is similar to our findingsfor corn stover.

Further pretreatment experiments were conducted to validatethe pretreatment model for H3PO4 pretreatment of corn stover.The optimal conditions were determined from the model based

upon the following targets: maximum sugar yield with a furfuralconcentration below 0.5% (w/v). Using these constraints, 25 opti-mal conditions were determined of which five are listed in Table 4.Further experiments were conducted for these selected conditionsand the results for sugar yields and furfural concentrationcompared to those predicted by the developed model. As shownin Table 5, experimental values fit very well with the predictedvalues which indicate the accuracy of the model. This is the first

Fig. 4. Response surface plots of furfural yield (g/100 g corn stover) obtained from central composite design with three independent variables for the optimization of cornstover pretreatment with H3PO4. Top, temperature (�C) and acid dose (%, v/v); middle, temperature (�C) and time (min); bottom, acid dose (%, v/v) and time (min).

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report on the optimization of dilute H3PO4 pretreatment of cornstover by RSM in which conditions were established to releasemaximum sugars after enzymatic hydrolysis while minimizingthe formation of fermentation inhibitory compounds so that nodetoxification step is needed for its fermentation by the recombi-nant E. coli FBR5.

3.4. Fermentation

For ethanol fermentation, corn stover (10%, w/w) was pre-treated with 0.78% H3PO4 at 161.81 �C for 9.78 min and subse-quently hydrolyzed with the enzyme cocktail containingcellulase, xylanase and b-glucosidase preparations for 72 h at45 �C and pH 5.0. The compositions of corn stover solid residuesafter acid pretreatment and also after enzymatic saccharificationare presented in Table 2. The enzymatically digested hydrolysatewas used as substrate. It contained 27.9 ± 0.6 g glucose,20.8 ± 0.3 g xylose (total sugars, 55.1 ± 1.0 g) and 0.50 ± 0.04 gfurfural per 100 g of corn stover. The patterns of sugar utilization,ethanol production, furfural and HMF utilization and cell growthduring fermentation by E. coli FBR5 at 35 �C and pH 6.5 are shown

in Fig. 5A and B. Glucose, arabinose and galactose were consumedcompletely within 24 h and 16.5 ± 0.3 g/L ethanol produced whichrepresents 62.5% of the total ethanol produced. Xylose utilizationstarted after all the other sugars were exhausted (Fig. 5A). Furfuraland HMF were completely metabolized in the first 18 h of the fer-mentation (Fig. 5B) which were converted to furfuryl alcohol andHMF alcohol, respectively (data not shown). It was shown that re-combinant E. coli FBR5 could grow in the medium containing 1.2 g/L furfural after 24–30 h of lag period at pH 7.0 and ethanol produc-tion starts after the lag time during which period both furfural andHMF were metabolized (Saha et al., 2011a,b). It is obvious that un-der the optimized conditions targeted to get maximum 0.5 g/L fur-fural, the fermentation time for ethanol production by therecombinant E. coli FBR5 can be shortened. Fermentation was com-pleted in 42 h where no residual sugars were detected. A total26.4 ± 0.1 g ethanol was produced in 48 h from 55.1 ± 1.0 g totalsugars per L which makes 0.49 ± 0.1 g ethanol/g glucose. Theethanol productivity was 0.55 g/L/h. This is the first report on theproduction of ethanol by the recombinant E. coli strain FBR5 fromdilute H3PO4 pretreated corn stover.

4. Conclusions

The effects of temperature, acid dose and time on pretreatmentof corn stover with H3PO4 have been investigated. Different tem-peratures are required in order to get maximum amounts of glu-cose (180 �C) and xylose (160 �C) with H3PO4. As the conditionsof the pretreatment are optimized within the tolerable furfural le-vel for E. coli FBR5, very good ethanol yield can be achieved by therecombinant bacterium without using any detoxification step. Thiswill simplify the process steps for production of ethanol from cornstover.

Acknowledgements

Ayse Avci gratefully acknowledges the fellowship offered by theTurkish Council of Higher Education to conduct this research andSakarya University for study leave.

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Table 5Confirmation of the predicted optimum condition with the experimental results.

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Glucose (%) Xylose (%) Furfural (%) Glucose (%) Xylose (%) Furfural (%)

1 163.92 0.61 9.37 27.90 21.31 0.50 29.6 ± 1.0 21.2 ± 0.3 0.58 ± 0.102 161.81 0.78 9.78 28.26 21.16 0.50 27.9 ± 0.6 20.8 ± 0.3 0.50 ± 0.043 160.40 0.87 10.32 28.37 21.11 0.50 27.7 ± 0.6 20.9 ± 0.3 0.50 ± 0.044 166.52 0.50 8.04 27.40 21.49 0.50 27.5 ± 0.9 20.0 ± 0.4 0.50 ± 0.065 153.61 1.50 12.87 26.84 21.68 0.50 27.0 ± 0.4 20.5 ± 0.4 0.43 ± 0.02

Time (h)0 8 16 24 32 40 48

Suga

r (g/

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0

10

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60GlucoseXyloseArabinoseGalactoseTotal sugars

Time (h)0 8 16 24 32 40 48

Furf

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, HM

F (g

/L)

0.0

0.1

0.2

0.3

0.4

0.5

Etha

nol (

g/L)

, Cel

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(OD 66

0nm

)

0

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30FurfuralHMFEthanolCell density

A

B

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