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Industrial Crops and Products 44 (2013) 367– 372

Contents lists available at SciVerse ScienceDirect

Industrial Crops and Products

journa l h o me page: www.elsev ier .com/ locate / indcrop

ydrothermal pretreatment and enzymatic saccharification of corn stover forfficient ethanol production�

adal C. Sahaa,∗, Tsuyoshi Yoshidaa,b, Michael A. Cottaa, Kenji Sonomotob,c

Bioenergy Research Unit, National Center for Agricultural Utilization Research, Agricultural Research Service, U.S. Department of Agriculture, 1815N, University Street, Peoria, IL1604, USADepartment of Bioscience and Biotechnology, Faculty of Agriculture, Graduate School, Kyushu University, 6-10-1 Hakozaki, Higashi-ku, Fukuoka 812-8581, JapanBio-Architecture Center, Kyushu University, 6-10-1 Hakozaki, Higashi-ku, Fukuoka 812-8581, Japan

r t i c l e i n f o

rticle history:eceived 4 September 2012eceived in revised form 6 November 2012ccepted 16 November 2012

eywords:

a b s t r a c t

Corn stover used in this study contained 37.0 ± 0.4% cellulose, 31.3 ± 0.6% hemicellulose and 17.8 ± 0.2%lignin on dry basis. Hydrothermal pretreatment and enzymatic saccharification were evaluated for con-version of corn stover cellulose and hemicellulose to fermentable sugars. Under the optimum conditionsof hydrothermal pretreatment of corn stover (10%, w/v; 200 ◦C; 5 min) and enzymatic saccharification(45 ◦C, pH 5.0, 72 h), a total of 550 ± 5 mg of fermentable sugars was obtained per g corn stover which

thanolorn stoverydrothermal pretreatmentnzymatic hydrolysisecombinant ethanologenic Escherichia coli

is equivalent to 72% of theoretical sugar yield. The corn stover hydrolyzate was fermented withoutany detoxification by recombinant Escherichia coli strain FBR 5 at pH 6.5 and 37 ◦C for 74 h to produce20.9 ± 0.5 g ethanol from 42.8 ± 1.7 g sugars per L with a yield of 0.49 g ethanol per g available sugarsand 0.27 g ethanol per g corn stover which is equivalent to 68.7% of theoretical ethanol yield from cornstover. This is the first report on the production of ethanol from hydrothermally pretreated corn stover

rium

ixed sugar fermentation by the recombinant bacte

. Introduction

Ethanol is the most widely used liquid renewable biofuel alter-ative to fossil fuel. Lignocellulosic biomass represents the World’sost abundant carbohydrate reserve and has the potential to

ecome a major source of fermentable sugars for production ofecond generation fuel ethanol. The conversion of native lignocel-ulosic biomass, in which three polymeric components – cellulose,emicellulose and lignin are interpenetrated, to fermentable sugars

nvolves two steps – feedstock pretreatment and enzymatic saccha-ification. Due to the heterogeneous complexity and recalcitrancef lignocellulosic feedstocks, pretreatment is required to break theignin seal and/or disrupt the structure of crystalline cellulose toncrease the accessibility of cellulase enzymes (Saha, 2004). Severalretreatment technologies using acid or alkali at elevated tempera-ures are available to pretreat lignocellulosic biomass. Dilute H2SO4

typically below 2%) at high temperature (120–200 ◦C) is effectiven breaking down hemicellulose and providing improved acces-ibility for cellulose hydrolysis (Saha, 2003). A major drawback

� Mention of trade names or commercial products in this article is solely for theurpose of providing specific information and does not imply recommendation orndorsement by the U.S. Department of Agriculture.∗ Corresponding author. Tel.: +1 309 681 6276; fax: +1 309 681 6427.

E-mail address: Badal.Saha@ars.usda.gov (B.C. Saha).

926-6690/$ – see front matter. Published by Elsevier B.V.ttp://dx.doi.org/10.1016/j.indcrop.2012.11.025

.Published by Elsevier B.V.

of dilute acid pretreatment is the generation of sugar degrada-tion products such as furfural and hydroxymethyl furfural (HMF)which are toxic to the fermentative microorganisms. The gen-eration of these fermentation inhibitors depends on a numberof factors such as acid dose, temperature and duration of pre-treatment. After acid pretreatment, the pH is to be adjusted towithin the optimal activity range of the hydrolytic enzymes. As aresult, the hydrolyzate contains salts which can be detrimental tofermentation.

Hydrothermal pretreatment (also known as hydrothermolysis,autohydrolysis, uncatalyzed solvolysis, aquasolv, hot-compressedwater treatment, or liquid hot water pretreatment) can be con-sidered as an eco-friendly green processing technology, sincethe medium contains lignocellulosic feedstock and water only,avoiding corrosion problems, acid recycling and the formation ofneutralization sludges (Garrote et al., 1999; Gullon et al., 2012). Itresults in lower fermentation inhibiting hydrolyzates than steamexplosion pretreatment (Laser et al., 2002). Perez et al. (2008)reported the optimal liquid hot water pretreatment conditions onwheat straw as 188 ◦C and 40 min which lead to sugar yield of79.8% of theoretical after enzymatic hydrolysis of pretreated wheatstraw. Diaz et al. (2010) showed that enzymatic hydrolysis of solids

obtained after hydrothermal pretreatment of rapeseed at 217.7 ◦Cfor 42.2 min released about 70% of glucose present in raw material.da Cruz et al. (2012) reported that the optimal hydrothermal pre-treatment condition of sugarcane bagasse that gave highest yield

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50.1% of maximum theoretical ethanol yield) of ethanol by recom-inant Saccharomyces cerevisiae YRH400, while minimizing furfuralroduction, was at 190 ◦C for 17.2 min.

The main objective of this work is to optimize hydrothermalretreatment and enzymatic saccharification conditions to releaseermentable sugars using corn stover as feedstock and evaluate theermentability of the corn stover hydrolyzate by a mixed sugartilizing ethanologenic recombinant Escherichia coli strain. Theecombinant bacterium (strain FBR5) has the ability to utilize allugars such as glucose, xylose, arabinose, galactose and mannoseypically present in lignocellulosic materials and produce ethanolDien et al., 2000). It is stable without antibiotics and can toleratep to 50 g ethanol per L (Saha and Cotta, 2012).

. Materials and methods

.1. Materials

Corn stover (∼20% moisture) was collected from a corn fieldn greater Peoria area. It was air-dried to about 8% moisture con-ent at room temperature for 2 days, chopped and milled to passhrough a 2.36 mm screen. The milled corn stover was storedt room temperature. Celluclast 1.5 L (cellulase) and Novozym88 (�-glucosidase) were purchased from Brenntag Great Lakes,ilwaukee, WI, USA. Fiberzyme (hemicellulase) was supplied byyadic Corp., Jupiter, FL, USA. Membrane Filter Unit (0.2 mm)as purchased from Nalge Company, Rochester, NY, USA. AminexPX 87P column (300 × 7.8 mm), Aminex HPX 87H column

300 × 7.8 mm), De-ashing cartridge (30 × 4.6 mm), Carbo-P micro-uard cartridge (30 × 4.6 mm), and Cation H micro-guard cartridge30 × 4.6 mm) were purchased from Bio-Rad Laboratories, Inc., Her-ules, CA, USA. All other chemicals used were of standard analyticalrades.

.2. Hydrothermal pretreatment

Milled corn stover (10 g/100 mL) was slurried in water and pre-reated in a rotating 200 mL stainless steel reactor with infraredeating (Labomat BFA-12, Mathis USA, Inc., Concord, NC, USA) at00 ◦C for 5 min, unless otherwise specified. Twelve reactors (each00 mL capacity) were used at one time. The heating and coolingimes of the reactors were not considered part of the reported pre-reatment time, even though the heat-up ramp took about 50 mino reach the final temperature at 200 ◦C. The reactors were water-ooled using tap water following the pretreatment. The coolingime was about 25 min. The heating temperature was varied from20 to 200 ◦C and the pretreatment time was varied from 5 mino 1 h. The reactors were routinely rotated at 50 rpm with 60-

clockwise followed by 60-s counter clockwise rotations duringretreatment for proper mixing.

The severity factor (SF) was determined by the following equa-ion (Garrote et al., 1999).

F = log[R] = log10

(t × exp

[T − 10014.75

])

here t is the reaction time in min and T is the temperature ofretreatment in ◦C. R or Ro is commonly used to represent SF. Itas originally designated by Overend and Chornet (1987) as reac-

ion ordinate or severity parameter. It is being used to represent SF

ince then. In practice, log10 R or log10 Ro is often used instead of

or Ro. Pretreatment conditions can be compared combining bothretreatment time and temperature into a single reaction ordinate,hrough severity factor (R or Ro) (Overend and Chornet, 1987).

Products 44 (2013) 367– 372

2.3. Enzyme assays

The cellulase activity in terms of filter paper activity was assayedand expressed as filter paper unit (FPU) by the procedure describedby Ghose (1987). Carboxymethyl cellulase (CMCase), �-glucosidaseand xylanase activities were assayed by the procedures describedpreviously (Saha et al., 2005). All enzyme assays were performedat pH 5.0 and 45 ◦C and the activities were expressed in terms ofinternational units (IU, �mol product formed per min).

2.4. Enzymatic saccharification

The enzymatic saccharification of the pretreated corn stoverwas performed by shaking slowly (100 rpm) at 45 ◦C for 72 h afterdiluting the pretreated material to 5% solid level (unless otherwisespecified), adjusting the pH to 5.0 with 10 M NaOH and adding afilter sterilized enzyme cocktail of 3 commercial enzyme prepa-rations. The enzyme cocktail contained 150 �L Celluclast, 30 �LNovozym and 150 �L Fiberzyme per g of corn stover (unless oth-erwise specified). The residual solids were separated from theliquid by centrifugation (12,000 × g; 10 min) before using the liquidportion as enzymatically saccharified corn stover hydrolyzate. Thecocktail of commercial enzyme preparations used for enzymaticsaccharification contained small quantities of glucose. For simpli-fication purpose, the quantity of glucose present in the enzymecocktail was subtracted from the measured glucose in each case.

2.5. Recombinant ethanologenic Escherichia coli strain FBR5 andpreparation of inoculum

Recombinant E. coli strain FBR5 (provided by Dr. Bruce S. Dien,USDA-ARS, NCAUR, Peoria, IL, USA) was maintained in glycerol vialsat −80 ◦C for use as a working stock. It was plated on Luria broth(LB; 10 g tryptone, 5 g yeast extract, and 5 g NaCl per L) containing4 g xylose and 20 mg tetracycline solidified with 15 g agar per L (pH6.5). Plates were incubated at 35 ◦C. Cells from a single well-isolatedcolony were inoculated into a 125 mL Erlenmeyer flask containing100 mL of LB with 2 g xylose and 2 mg tetracycline. The flask wasincubated at 35 ◦C and 100 rpm for 24 h. This grown culture wasused as seed culture for fermentation experiments.

2.6. Fermentation experiments

The fermentation experiments were carried out in pH-controlled 500 mL fleakers with a working volume of 350 mL atpH 6.5 and 35 ◦C essentially as described previously (Bothast et al.,1994) except that 4 M NaOH was used instead of 4 M KOH forpH control. A diagram of the fleaker system was presented pre-viously by Beall et al. (1991). Rubber fleaker caps were drilled toallow the insertion of a pH probe, CO2 vent, sampling needle, and aport for addition of base. A magnetic stirrer was located beneaththe water bath and maintained at 130 rpm during the experi-ments. No attempts were made to exclude oxygen. Corn stover(10%, w/v) hydrolyzate was prepared by hydrothermal pretreat-ment at 200 ◦C for 5 min and then enzymatically saccharifying thepretreated materials without dilution at pH 5.0 and 45 ◦C for 72 h.The solid residues were removed by centrifugation. The mediumwas prepared by dissolving 10 g tryptone and 5 g yeast extract in theliquid portion of the hydrolyzate (per L) and filter-sterilizing usinga membrane filter unit. The inoculum size was 5% (v/v). Sampleswere withdrawn periodically and the optical densities (A660 nm) ofthe samples were monitored immediately after withdrawal. The

withdrawn fermentation broth was centrifuged to remove cells andkept at −20 ◦C prior to high pressure liquid chromatography (HPLC)analysis. Reactor performance was monitored by quantifying unuti-lized sugars (glucose, xylose, arabinose, galactose and mannose)

B.C. Saha et al. / Industrial Crops and Products 44 (2013) 367– 372 369

Table 1Composition of corn stover used in this study.

Component wt%

Cellulose 37.0 ± 0.4Hemicellulose 31.3 ± 0.6Xylan 25.2 ± 0.1Arabinan 3.3 ± 0.2Galactan 1.7 ± 0.3Mannan 1.1 ± 0.0Lignin 17.8 ± 0.2Ash 7.3 ± 0.1

Data expressed as weight percent on dry basis are averages of two separate deter-m

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Fig. 1. Effect of temperature (120–200 ◦C) of hydrothermal pretreatment (15 min)on enzymatic saccharification (pH 5.0, 45 ◦C, 72 h) of pretreated corn stover(10 g/100 mL). The enzyme cocktail contained three commercial enzyme (cellulase,

consequence of hydrolysis of acetyl groups from the hemicellulose

inations.

nd fermentation product (ethanol). Base consumption and pHere recorded. Duplicate parallel fermentation experiments were

un for reproducibility and comparison. For simplification purpose,he quantity of ethanol produced from the additional sugars (glu-ose and fructose) present in the enzyme cocktail was subtractedrom the measured ethanol yield.

.7. Analytical procedures

The composition of corn stover with respect to cellulose, hemi-ellulose, total lignins and ash contents was determined using thetandard laboratory analytical procedures for biomass analysis pro-ided by National Renewable Energy Laboratory (NREL) (Sluitert al., 2008a,b). Moisture content was determined using a mois-ure analyzer (Mark 2, Sartorius Mechatronics Corp., Bohemia, NY,SA). Sugars, ethanol, acetic acid, furfural, and HMF were analyzedy HPLC (Saha and Bothast, 1999). The separation system consistedf a fully integrated solvent delivery system (LC-20AD Prominence,himadzu America, Inc., Columbia, MD, USA) equipped with anIL-20AC autosampler, RID-10A refractive index detector, a SDP-0A UV detector, a CTO-10AS VP column heater and a computeroftware based integration system (LC solutions 1.23 SP1, Shi-adzu). Two ion moderated partition chromatography columns

Aminex HPX-87P with De-ashing and Carbo-P micro-guard car-ridges, Aminex HPX 87H with Cation H micro-guard cartridge)ere used. The Aminex HPX-87P column was maintained at 85 ◦C,

nd the sugars, furfural and HMF were eluted with Milli-Q filteredater at a flow rate of 0.6 mL/min. The Aminex HPX-87H columnas maintained at 65 ◦C, and the sugars, acetic acid, and ethanolere eluted with 10 mM HNO3 prepared using Milli-Q filteredater at a flow rate of 0.6 mL/min. Peaks were detected by refractive

ndex or UV absorption (277 nm) and were identified and quantifiedy comparison to retention times of authentic standards (glucose,ylose, arabinose, galactose, mannose, ethanol, acetic acid, furfuralnd HMF). The yield of total sugars was calculated by the procedurerovided by NREL (Sluiter et al., 2008a). The value is expressed asercentage of maximum theoretical sugar yield.

. Results and discussion

.1. Composition of corn stover

Corn stover, collected from a local corn field, was used with-ut washing. The composition of the stover on dry basis is given inable 1. Cellulose was the most abundant fraction (37.0 ± 0.4%), fol-owed by hemicellulose (31.3 ± 0.6%) and lignin (17.8 ± 0.2%). These

alues are consistent with those previously reported for corn stoverrom other sources (Aden and Foust, 2009; Bura et al., 2009; Chent al., 2009; Kim and Holtzapple, 2005).

�-glucosidase, and hemicellulase) preparations. The data presented are averagesof two separate experiments. Galactose and mannose data were not shown. Glu,glucose; Xyl, xylose; Ara, arabinose.

3.2. Hydrothermal pretreatment and enzymatic saccharificationof corn stover

3.2.1. Effect of temperature of pretreatmentThree commercial enzyme preparations (cellulase, �-

glucosidase and hemicellulase) were used for saccharificationof pretreated corn stover. The activity levels of cellulase (filterpaper activity), CMCase, �-glucosidase and xylanase are given inTable 2. Hydrothermal pretreatment of corn stover (10 g/100 mL)was carried out at 120 to 200 ◦C for 15 min. The result of effec-tiveness of temperature on the yield of sugars from pretreatedcorn stover after enzymatic saccharification at pH 5.0 and 45 ◦Cfor 72 h is presented in Fig. 1. We have used pH 5.0 and 45 ◦Cfor enzymatic hydrolysis. Typically, the enzyme assays wereperformed at pH 5.0 and 50 ◦C for a short period of time (30 minor less). In our previous studies, we reported that a temperatureof 45 ◦C works better than 50 ◦C for longer (72 h) reaction time(Saha and Cotta, 2007, 2008). The total sugar yield decreasedto 87% at 50 ◦C in comparison to that at 45 ◦C for 72 h reaction.The reaction time for enzymatic hydrolysis was chosen at 72 hbecause there was a very slow increase of each sugar yield after48 h incubation period. Each individual sugar released after enzy-matic hydrolysis increased slowly with increasing pretreatmenttemperature up to 160 ◦C but at 180 and 200 ◦C, the yields oftotal sugars released were 352 ± 13 (glucose, 210 ± 4; xylose120 ± 6; arabinose 12 ± 2) mg and 505 ± 2 (glucose, 342 ± 5; xylose141 ± 6; arabinose, 12 ± 1) mg per g of stover, respectively. Thisdata shows that temperature of pretreatment is very importantfor hydrothermal pretreatment of corn stover as the yield of totalsugars increased by 55.0% at 180 ◦C in comparison to that at 160 ◦Cand by 43.5% at 200 ◦C in comparison to 180 ◦C. Negligible amountsof furfural (0.31 ± 0.00 mg/g) and HMF (0.11 ± 0.01 mg/g) wereproduced at temperatures 140–160 ◦C. These values increased to0.63 ± 0.03 mg and 0.11 ± 0.01 mg per g, respectively, at 180 ◦C.At 200 ◦C, 12.00 ± 2.00 mg furfural and 1.00 ± 0.10 mg HMF weregenerated per g stover. The release of acetic acid by hydrothermalpretreatment followed similar trend. At 200 ◦C, 32 ± 0.0 mg aceticacid was generated per g corn stover. Acetic acid was formed as a

fraction of corn stover while furfural and HMF were pentose andhexose sugar degradation products, respectively (Saha, 2003).Based on the data on the release of total sugars, it was decided

370 B.C. Saha et al. / Industrial Crops and Products 44 (2013) 367– 372

Table 2Activity level of three commercial enzyme preparations used in enzymatic hydrolysis of hydrothermally pretreated corn stover.

Enzyme Filter paper CMCase �-Glucosidase XylanasePreparation (U/mL) (U/mL) (U/mL) (U/mL)

Celluclast (cellulase) 44 ± 2 788 ± 51 17 ± 1 858 ± 22Novozym 188 (�-glucosidase) 0 ± 0 – 420 ± 25 –

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Fa(h

ing into account of the amount of HMF (0.42 ± 0.02 mg g stover)formed from it using the enzyme combination # 4 or 5 underthe conditions used which is very attractive. The hemicelluloseconversion to sugars was only 58% of the theoretical yield not

Fiberzyme (hemicellulase) 8 ± 1

t pH 5.0 and 45 ◦C. The data presented are means ± standard deviation for triplicat

o use 200 ◦C for hydrothermal pretreatment. The SF values forydrothermal pretreatment for 15 min at 120, 140, 160, 180 and00 ◦C were 1.76, 2.35, 2.94, 3.53 and 4.12, respectively calculatedsing the formula described in the Materials and Methods sectionGarrote et al., 1999).

.2.2. Effect of duration of pretreatmentThe effects of duration (5–60 min) of hydrothermal pretreat-

ent of corn stover (10 g/100 mL) at 200 ◦C were investigated. TheF of the pretreatment for 5, 15, 25, 35 and 60 min were 3.63, 4.12,.34, 4.49 and 4.72, respectively. The relationships between SF andurfural, and SF and HMF are presented in Fig. 2. Both furfural andMF contents in the pretreated corn stover increased with the

ncrease of duration of pretreatment except that at 60 min pre-reatment, the furfural level in the hydrolyzate did not increase.he reason is unknown. The results of enzymatic saccharificationf pretreated corn stover are presented in Fig. 3. The glucoseelease increased up to 25 min pretreatment (412 ± 21 mg/g) afterhich it decreased slowly to 396 ± 21 g/g after 35 min pretreat-ent and 385 ± 18 g/g stover after 60 min pretreatment. The xylose

elease upon enzymatic hydrolysis, on the other hand, decreasedramatically from 5 min pretreatment (184 ± 5 mg/g stover) to0 min pretreatment (17 ± 1 mg/g stover). As a result, the totalugar released was maximum (553 ± 8 mg/g corn stover) at 5 minretreatment and it decreased to 402 ± 13 mg/g stover at 60 minretreatment. The glucose yield decreased 6.5% from 25 to 60 minretreatment time (Fig. 3). The HMF level increased to 6.7 ± 0.6 mgrom 3.5 ± 0.3 mg per g of stover (Fig. 2). The xylose yield decreased3.7% (Fig. 3) while the furfural level increased from 8.3 ± 2.2o 25.5 ± 4.6 mg per g stover from 5 to 15 min pretreatmentime (Fig. 2). The furfural and HMF levels in the corn stoverydrolyzate after 5 min pretreatment were 3.89 ± 1.00 mg and

.42 ± 0.02 mg per g corn stover, respectively which are quite low.ased on these results, it was decided to use 5 min duration forretreatment.

Severi ty fac tor

3.6 3.8 4.0 4.2 4.4 4.6 4.8

Fu

rfu

ral (m

g/g

)

0

5

10

15

20

25

HM

F (

mg

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0.0

0.5

1.0

1.5

2.0

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Furfural

HMF

ig. 2. Effect of severity factor of hydrothermal pretreatment at 200 ◦C on gener-tion of fermentation inhibitory compounds (furfural and HMF) from corn stover10 g/100 mL). The data presented are averages of two separate experiments. HMF,ydroxymethyl furfural.

95 ± 25 45 ± 3 10,254 ± 250

ys.

3.2.3. Effect of enzyme loading on hydrolysisThere was only negligible amount of sugar released [Total sug-

ars, 22 ± 2 (glucose, 2 ± 0; xylose, 14 ± 0; arabinose, 6 ± 2) mg/gstover] during hydrothermal pretreatment of corn stover (10%,w/v) at 200 ◦C for 5 min. The composition analysis of the solidresidues left after pretreatment indicate that 5.2 ± 0.0% cellulose,14.7 ± 0.0% hemicellulose and 3.4 ± 0.0% lignin of the original cornstover material were solubilized. In other words, 14.1 ± 0.0% oforiginal cellulose content, 47.0 ± 0.0% of original hemicellulose con-tent and 19.1 ± 0.0% of original lignin content were solubilized.That is why, enzymatic saccharification using both cellulase andhemicellulase enzymes is important to generate free sugars fromhydrothermally pretreated corn stover. The effects of 9 differentcombinations of commercial cellulase and hemicellulase enzymepreparations on the enzymatic hydrolysis of hydrothermally pre-treated (200 ◦C, 5 min) corn stover (10 g/100 mL) were studied.The �-glucosidase concentration was kept at 30 �L per g cornstover as at this concentration, no cellobiose and higher cello-oligosaccharides were detected in the enzymatic hydrolyzatesby HPLC analysis. The results of the release of glucose, xylose,arabinose and total sugars which include galactose and mannosegeneration at each enzyme combination after enzymatic hydroly-sis at pH 5.0 and 45 ◦C for 72 h are given in Table 3. It is evidentfrom the data that combination # 4 or 5 was able to generate maxi-mum amount of sugars from the pretreated corn stover using 9 FPUof cellulase enzyme per g of corn stover (equivalent to 24 FPU/gglucan content of corn stover). Based on theoretical glucose yieldfrom corn stover, 86% cellulose was converted to glucose not tak-

Fig. 3. Effect of duration (5–60 min) of hydrothermal pretreatment at 200 ◦Con enzymatic saccharification (pH 5.0, 45 ◦C, 72 h) of pretreated corn stover(10 g/100 mL). The enzyme cocktail contained three commercial enzyme (cellulase,�-glucosidase, and hemicellulase) preparations. The data presented are averagesof two separate experiments. Galactose and mannose data were not shown butincluded in total sugars calculations. Glu, glucose; Xyl, xylose; Ara, arabinose; TS,total sugars.

B.C. Saha et al. / Industrial Crops and Products 44 (2013) 367– 372 371

Table 3Effect of enzyme dose on saccharification of hydrothermally pretreated (200 ◦C, 5 min) corn stover.

# Celluclast Fiberzyme Glucose Xylose Arabinose Galactose Total sugars(�L/g) (�L/g) (mg/g) (mg/g) (mg/g) (mg/g) (mg/g)

1 150 150 334 ± 13 166 ± 10 13 ± 1 6 ± 2 524 ± 222 225 150 337 ± 4 174 ± 1 15 ± 1 6 ± 2 536 ± 33 75 150 314 ± 5 170 ± 1 13 ± 1 6 ± 2 507 ± 34 150 225 350 ± 10 177 ± 2 13 ± 1 6 ± 2 550 ± 55 225 225 350 ± 10 177 ± .2 13 ± 1 6 ± 2 550 ± 56 75 225 327 ± 6 173 ± 6 13 ± 1 6 ± 2 521 ± 47 150 75 336 ± 0 175 ± 2 11 ± 1 6 ± 2 532 ± 18 225 75 340 ± 11 176 ± 9 12 ± 1 6 ± 2 538 ± 229 75 75 318 ± 16 167 ± 0.0 12 ± 1 6 ± 2 507 ± 13

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-Glucosidase used was 30 �L/g corn stover in all cases. Enzymatic saccharificationxperiments. Mannose data was not shown but included in total sugars calculation

aking into account the amount of furfural (3.89 ± 1.00 mg/g stover)ormed from pentose sugars during pretreatment. This indicateshat the hemicellulase preparation used in this study may note effective for hydrolyzing the hemicellulose from corn stover.he exact reason is unknown. Previous studies indicate that cornber hemicellulose was also not effectively hydrolyzed by com-ercial hemicellulase preparations (Saha and Bothast, 1999). In

omparison to hydrothermal pretreatment of corn stover, Pangt al. (2013) reported that a combination of steam explosionnd microwave irradiation pretreatment method under opti-ized condition (200 ◦C, 5 min) gave a maximum glucose, xylose

nd total sugar yield 57.4, 17.8 and 75.2%, respectively (cor-esponding to 100% maximum total sugars in feedstock) afternzymatic hydrolysis. Li et al. (2012) compared different alkali-ased pretreatments of corn stover for improving enzymaticaccharification. They obtained a maximum total sugar yield of8.2% on the basis of glucose and xylose released from corntover pretreated with alkaline sodium sulfite (Na2SO3: NaOH,:1) at 140 ◦C for 30 min. The yield was 59.4% with alkaliretreatment under the same conditions. The time course ofnzymatic hydrolysis of hydrothermally pretreated corn stoversing the enzyme preparation (combination # 4) is shown inig. 4. After 6 h, the yield of total sugars was 62% of that at 72 h.ost of the sugars (∼88% of total sugars) were released within

4 h of enzymatic reaction. But the sugar release continued toncrease slowly and at 48 h the sugar yield was 96% of that at2 h.

Time (h)

0 12 24 36 48 60 72

Su

gar

(mg

/g)

0

100

200

300

400

500

600 Glu

Xyl

Ara

TS

ig. 4. Time course of enzymatic hydrolysis at pH 5.0 and 45 ◦C of hydrothermallyretreated (200 ◦C, 5 min) corn stover (10 g/100 mL). The enzyme cocktail containedhree commercial enzyme (cellulase, �-glucosidase, and hemicellulase) prepara-ions. The data presented are averages of two separate experiments. Galactose and

annose data were not shown but included in total sugars calculations. Glu, glucose;yl, xylose; Ara, arabinose; TS, total sugars.

erformed at pH 5.0 and 45 ◦C for 72 h. Data presented are averages of two separate

3.3. Fermentability of hydrothermally pretreated andenzymatically hydrolyzed corn stover

The conversion of the sugars generated after enzymatically sac-charified hydrolyzate of hydrothermally pretreated corn stoverto ethanol without detoxification was investigated using a mixedsugar utilizing ethanologenic recombinant E. coli strain FBR5. Theresult is presented in Fig. 5. The data indicates that the bacteriumwas able to utilize all sugars completely and produce ethanol. How-ever, the bacterium started to utilize glucose first and then xyloseand arabinose were slowly utilized after a considerable amountof glucose (27%) was depleted from the fermentation broth. Thispattern of mixed sugar utilization was observed with other stud-ies reported previously (Saha et al., 2005; Saha and Cotta, 2007,2008). The cell density (OD 660 nm) increased with the increase offermentation time up to 48 h (9.1 ± 0.3) and then it decreased to6.1 ± 0.3 at 72 h which is 32.7% less. We had observed such phe-nomenon in previous studies also (Saha et al., 2005; Saha andCotta, 2007, 2008). One reason may be that most of the sugarswere utilized by this time (Fig. 5). The fermentation was completewithin 72 h with an ethanol productivity of 0.29 g L−1 h−1. The bac-terium produced 20.9 ± 0.5 g ethanol from 42.8 ± 1.7 g sugars per Lwhich is equivalent to 0.49 g ethanol per g available sugars. Thetheoretical maximum yield of ethanol from glucose, xylose andarabinose is 0.51 g/g sugar (McMillan, 1993). This given a yield of

0.27 g ethanol per g corn stover which is 68.7% of the theoreti-cal ethanol yield from corn stover based on its total carbohydratecontent.

Time (h)

0 12 24 36 48 60 72 84 96

Su

gar

or

Eth

an

ol (g

/L)

0

10

20

30

40

50

60C

ell d

en

sit

y (

A6

60

nm

)

0

2

4

6

8

10

Glu

Xyl

Ara

TS

EtOH

CD

Fig. 5. Time course of utilization of sugars and production of ethanol by mixedsugar utilizing ethanologenic recombinant E. coli strain FBR5 from hydrothermallypretreated (200 ◦C, 5 min) and enzymatically saccharified (pH 5.0, 45 ◦C, 72 h) cornstover (10 g/100 mL) hydrolyzate at pH 6.5 and 37 ◦C. The data presented are aver-ages of two separate experiments. Galactose and mannose utilization data were notshown. Glu, glucose; Xyl, xylose; Ara, arabinose; TS, total sugars; CD, cell density.

3 ps and

xv(a2s3(bdb

4

ctcttwwoci

A

R

A

B

B

B

B

B

C

biomass. Technical Report NREL/TO-510-42618. http://www.nrel.gov/

72 B.C. Saha et al. / Industrial Cro

In comparison, a newly developed a newly developed glucose,ylose and arabinose fermenting recombinant Saccharomyces cere-isiae strain 424A(LNH-ST) produced ethanol with a yield of 72.5%0.37 g/g sugar) from a mixture of sugars containing glucose, xylose,rabinose, galactose and mannose – each at a concentration of0 g/L (Bera et al., 2010). An improved recombinant S. cerevisiaetrain 424A(LNH-ST) containing overexpressed glyceraldehyde--phosphate dehydrogenase enzyme produced 82.2% ethanol0.42 g/g xylose) from xylose (70 g/L) (Bera et al., 2011). The recom-inant yeast strain 424A(LNH-ST) produced 0.19 g ethanol per g ofry corn stover pretreated with ammonia fiber expansion (AFEX)y separate hydrolysis and fermentation (Lau and Dale, 2009).

. Conclusions

Based on the results obtained, hydrothermal pretreatment oforn stover has proved to be very simple and effective pretreatmentechnique. The efficiency of the enzymatic hydrolysis of pretreatedorn stover is dependent on the temperature and duration of pre-reatment. Longer duration of pretreatment was highly detrimentalo hemicellulose hydrolysis. The cellulose hydrolysis increasedith the increase of duration of pretreatment up to 25 min afterhich it slowly decreased monitored up to 60 min. The hydrolyzate

btained after enzymatic hydrolysis of hydrothermally pretreatedorn stover was readily fermentable as there was no need to detox-fy the hydrolyzate before its fermentation.

cknowledgement

The authors thank Gregory J. Kennedy for technical assistance.

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