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International Biodeterioration & Biodegradation 59 (2007) 85–89

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Enzymatic hydrolysis of corncob and ethanol productionfrom cellulosic hydrolysate

Ming Chena, Liming Xiaa,�, Peijian Xueb

aDepartment of Chemical Engineering and Bioengineering, Zhejiang University, Hangzhou 310027, ChinabNational Engineering Research Center For Fermentation Technology, Bengbu 233010, China

Received 26 April 2006; accepted 23 July 2006

Available online 27 September 2006

Abstract

Enzymatic hydrolysis of corncob and ethanol fermentation from cellulosic hydrolysate were investigated. After corncob was

pretreated by 1% H2SO4 at 108 1C for 3 h, the cellulosic residue was hydrolyzed by cellulase from Trichoderma reesei ZU-02 and the

hydrolysis yield was 67.5%. Poor cellobiase activity in T. reesei cellulase restricted the conversion of cellobiose to glucose, and the

accumulation of cellobiose caused severe feedback inhibition to the activities of b-1,4-endoglucanase and b-1,4-exoglucanase in cellulase

system. Supplementing cellobiase from Aspergillus niger ZU-07 greatly reduced the inhibitory effect caused by cellobiose, and the

hydrolysis yield was improved to 83.9% with enhanced cellobiase activity of 6.5CBUg�1 substrate. Fed-batch hydrolysis process was

started with a batch hydrolysis containing 100 g l�1 substrate, with cellulosic residue added at 6 and 12 h twice to get a final substrate

concentration of 200 g l�1. After 60 h of reaction, the reducing sugar concentration reached 116.3 g l�1 with a hydrolysis yield of 79.5%.

Further fermentation of cellulosic hydrolysate containing 95.3 g l�1 glucose was performed using Saccharomyces cerevisiae 316, and

45.7 g l�1 ethanol was obtained within 18 h. The research results are meaningful in fuel ethanol production from agricultural residue

instead of grain starch.

r 2006 Elsevier Ltd. All rights reserved.

Keywords: Corncob; Hydrolysis; Cellulase; Cellobiase; Ethanol fermentation

1. Introduction

The importance of ethanol as a clean and safetransportation fuel has increased with the anticipatedshortage of fossil fuel reserves and increased air pollution(Moniruzzaman et al., 1997; Sun and Cheng, 2002).However a dramatic increase in ethanol production usingthe current starch-based technology may not be practical inChina because it will compete for the limited agriculturalland needed for food and feed production. Lignocellulosicbiomass is a cheap, renewable, abundantly availableresource (Ho et al., 1998), and its conversion to glucoseand other fermentable sugars has been considered, in thelast few decades, to be an attractive route for ethanolproduction (Curreli et al., 1997; Gaspar et al., 2005).

e front matter r 2006 Elsevier Ltd. All rights reserved.

iod.2006.07.011

ing author. Tel.: +86571 8795 1840;

95 1358.

ess: xialm@zju.edu.cn (L. Xia).

The hydrolysis of natural cellulose to glucose dependson the synergism of three enzymes in cellulase system, i.e.,b-1,4-endoglucanase (EC 3.2.1.4), b-1,4-exoglucanase(EC 3.2.1.91) and cellobiase (EC 3.2.1.21) (Tolan andFoody, 1999). In previous publication, we have reportedcellulase production by Trichoderma reesei ZU-02 in solid-state fermentation (Xia and Cen, 1999). However the mostwidely used cellulase from T. reesei is poor in cellobiase,and thus restricts the conversion of cellobiose to glucose(Shen and Xia, 2004). The accumulation of cellobiose willcause severe feedback inhibition to the cellulase reaction, asthe enzyme is more susceptible to end-product inhibitioncaused by cellobiose than glucose (Duff and Murray, 1996;Wen et al., 2004).In China, large-amounted, concentrated agricultural

waste corncob is produced annually, and often causesenvironmental pollution due to the lack of effectiveutilization. In this work, hydrolysis of corncob withcellulase from T. reesei ZU-02 and cellobiase from

ARTICLE IN PRESSM. Chen et al. / International Biodeterioration & Biodegradation 59 (2007) 85–8986

Aspergillus niger ZU-07 was studied. Further fermentationof the enzymatic hydrolysate for ethanol production wasalso tested using Saccharomyces cerevisiae 316.

2. Materials and methods

2.1. Microorganism

T. reesei ZU-02 (originally from ATCC 56764) was used for cellulase

production. A. niger ZU-07 (obtained from the Laboratory of Renewable

Resource Engineering, Purdue University) was used for cellobiase

production. The strain of S. cerevisiae 316 (stored by the Laboratory of

Biochemical Engineering, Zhejiang University) was used for ethanol

fermentation.

2.2. Lignocellulosic material and pretreatment

Corncob obtained locally was air-dried and ground to particles of

about 2mm in diameter. One percent H2SO4 was added at a solid-to-liquid

ratio of 1:6. The mixture was pretreated at 108 1C for 3 h and then filtered.

The cellulosic residue was washed to pH 4.8 with water and dried, and had

the following composition: cellulose 59.4%, hemicellulose 6.5%, lignin

22.2%, and others 11.9%.

2.3. Cellulase production

Cellulase was produced by solid-state fermentation according to the

method of Xia (Xia and Cen, 1999). Spores of T. reesei ZU-02 were

inoculated into the seed medium and incubated at 30 1C for 48 h under

aerobic conditions. For solid-state fermentation, 10% liquid seeds were

inoculated into the solid medium (5 cm thick) in a 1000ml Erlenmeyer

flask and cultured in an incubator at 28–30 1C for 7 d. Each gram of dry

koji contained 146 IU of filter paper activity (FPU) and 12 IU of

cellobiase activity (CBU).

2.4. Cellobiase production

Cellobiase was produced according to the method of Shen (Shen and

Xia, 2004). The solid medium was inoculated with spores suspension of

A. niger ZU-07 and cultured at 30 1C for 3 d. The koji produced by solid-

state fermentation contained 376CBUg�1 dry koji and no detectable filter

paper activity.

2.5. Enzymatic hydrolysis of cellulosic residue

2.5.1. Batch enzymatic hydrolysis

Batch enzymatic hydrolysis of corncob cellulosic residue was performed

in 250ml Erlenmeyer flasks, containing a 100ml mixture of tap water and

solid substrate. Cellulase from T. reesei ZU-02 and cellobiase from A.

niger ZU-07 were utilized for enzymatic hydrolysis. The pH and

temperature were adjusted to 4.8 and 50 1C, respectively. The flasks were

incubated in an orbital shaker with speed of 160 rpm.Unless specified, the

reaction time was set 48 h.

2.5.2. Fed-batch enzymatic hydrolysis

Fed-batch enzymatic hydrolysis was carried out at pH 4.8 and 50 1C in

a 5 l reactor with a working volume of 3 l. Experiments were started with

100 g l�1 substrate and enzyme loadings of 20FPUg�1 substrate and

6.5CBUg�1 substrate. Cellulosic residue was then added twice at 6 and

12 h to get a final substrate concentration of 200 g l�1, simultaneously

adding certain amount of cellulase and cellobiase (enzyme loadings of

10FPUg�1 fed substrate and 6.5CBUg�1 fed substrate). The total

hydrolysis time was set 60 h.

2.6. Ethanol fermentation of cellulosic hydrolysate

Inoculum was prepared by transferring a loop of cells of S. cerevisiae

316 into a 250ml flask containing 50ml of culture medium

(30 g l�1 glucose, 5 g l�1 peptone, 3 g l�1 yeast extract), and incubating at

30 1C for 24 h. Cells were harvested by centrifugation (4800 rpm, 5min),

suspended in sterilized water and used to inoculate the fermentation

medium.

Cellulosic hydrolysate obtained from fed-batch hydrolysis, supplemen-

ted with 3 g l�1 yeast extract and 0.25 g l�1 (NH4)2HPO4, was utilized as a

fermentation medium. Ethanol fermentations were carried out at 30 1C

under anaerobic conditions, with 0.5ml of cells suspension inoculated into

a 100ml flask with a working volume of 50ml.

2.7. Analysis methods

Enzyme from koji was extracted with 50 volumes of water at room

temperature for 6 h and filtered. The enzyme extract was centrifuged at

4000 rpm for 10min, and the clear supernatant was used for enzyme assay.

Filter paper activity and cellobiase activity were determined according to

standard International Union of Pure and Applied Chemistry (IUPAC)

procedures (Ghose, 1987). Filter paper activity was assayed by incubating

a reaction mixture containing a strip of Whatman no. 1 filter paper

(1� 6 cm) immersed in 1ml of 0.05M citrate buffer and 0.5ml of

appropriately diluted enzyme solution at 50 1C for 30min. One unit of

filter paper activity (FPU) is defined as the amount of enzyme that forms

1mmol glucose (reducing sugar as glucose) per minute under the assay

conditions. Cellobiase activity was assayed in a reaction mixture contain-

ing 1ml of 15 mM cellobiose solution (prepared in 0.05M citrate buffer,

pH 4.8) and 1ml of appropriately diluted enzyme solution at 50 1C for

30min. One unit of cellobiase activity (CBU) is the amount of enzyme that

forms 2mmol glucose per minute from cellobiose.

The reducing sugar was determined using the 3,5-dinitrosalicylic acid

(DNS) method (Ghose, 1987). Glucose, xylose, cellobiose, arabinose,

ethanol and glycerol were analyzed by HPLC (Syltech model 500 pump,

USA) with an organic acid column (TRANSGENOMIC ICSep ICE-

COREGEL 87H3 Column). Purified water was used as the mobile phase

at a flow rate of 0.5ml/min. The column temperature was fixed at 60 1C.

The eluate was detected by a refractive index detector (Spectra-Physics

6040 XR RI detector).

The yield of enzymatic hydrolysis was calculated as follows:

Hydrolysis yield (%) ¼ reducing sugar� 0.9� 100/polysaccharide in

substrate.

At least three parallel samples were used in all analytical determina-

tions, and data are presented as the mean of three replicates.

3. Results and discussion

3.1. Batch enzymatic hydrolysis of cellulosic residue

3.1.1. Effects of substrate concentration

The effects of substrate concentration on enzymatichydrolysis were investigated at a fixed ratio of cellulasefrom T. reesei ZU-02 to substrate (20FPUg�1 substrate;1.64CBUg�1 substrate). As shown in Fig. 1, reducingsugar concentration and hydrolysis yield showed anopposite variation trend, i.e., with substrate concentrationincreasing, the reducing sugar concentration increased butthe hydrolysis yield decreased. This may be due to the end-product feedback inhibition caused by high reducing sugarconcentration (Wen et al., 2004). Taking reducing sugarconcentration and hydrolysis yield into account, theoptimal substrate concentration was 100 g l�1.

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100

80

60

40

20

050 75 100 125 150

Red

ucin

g su

gar

(g l-1

)

Substrate concentration (g l-1)

80

70

60

50

40

30

20

10

0

Hyd

roly

sis

yiel

d (%

)

Reducing sugarHydrolysis yield

Fig. 1. Effects of substrate concentration on the enzymatic hydrolysis at

fixed ratio of T. reesei ZU-02 cellulase to substrate (20FPUg�1 substrate;

1.64CBUg�1 substrate).

100

80

60

40

20

050 75 100 125 150

Red

ucin

g su

gar

(g l-1

)

Cellulase dosage (FPU g l-1 substrate)

80

70

60

50

40

30

20

10

0

Hyd

roly

sis

yiel

d (%

)

Reducing sugarHydrolysis yield

Fig. 2. Effects of T. reesei ZU-02 cellulase dosage (presented as filter

paper activities per gram of substrate, FPUg�1 substrate) on the

enzymatic hydrolysis.

M. Chen et al. / International Biodeterioration & Biodegradation 59 (2007) 85–89 87

3.1.2. Effects of cellulase dosage

As the cost of cullulase contributes significantly to thetotal cost of biomass conversion process (Cao et al., 1996),the cellulase dosage should be minimized as much aspossible. Hydrolysis experiments were performed with100 g l�1 substrate and different dosages of T. reesei ZU-02 cellulase (presented as filter paper activities per gram ofsubstrate, FPUg�1 substrate) at pH 4.8 and 50 1C, and theresults are shown in Fig. 2. Reducing sugar concentrationand hydrolysis yield had a similar variation trend, that is,both increased sharply with cellulase dosage varying from10 to 20FPUg�1 substrate, and basically leveled off from20 to 30FPUg�1 substrate.

3.1.3. Time course of hydrolysis by cellulase from T. reesei

ZU-02

100 g l�1 of cellulosic residue was hydrolyzed bycellulase from T. reesei ZU-02 (20FPUg�1 substrate;1.64CBUg�1 substrate) at pH 4.8 and 50 1C for 60 h.Glucose, xylose, arabinose and cellobiose produced duringhydrolysis were analyzed by HPLC at the interval of12 h (Table 1). The reducing sugar concentrationreached 49.4 g l�1 and the hydrolysis yield was 67.5% after48 h of hydrolysis, and prolonged hydrolysis time beyond48 h helped little in increasing the hydrolysis yield. Xyloseand arabinose were detected out in the hydrolysate,showing the presence of xylanase in T. reesei ZU-02cellulase.

b-1,4-endoglucanase and b-1,4-exoglucanase in cellulasehydrolyze cellulose chains and result in the forma-tion of cellobiose, which can be further cleavedinto glucose by cellobiase. It was found that a highamount of cellobiose existed in the cellulosic hydrolysate,indicating poor cellobiase activity in T. reesei ZU-02cellulase. The accumulation of cellobiose caused severefeedback inhibition to the activities of b-1,4-endoglucanaseand b-1,4-exoglucanase in cellulase, resulting in lowhydrolysis yield.

3.1.4. Synergistic hydrolysis by T. reesei ZU-02 cellulase

and A. niger ZU07 Cellobiase

To weaken the feedback inhibition caused by cellobioseaccumulation, cellobiase produced by A. niger ZU-07 wassupplemented to the hydrolysis system to enhance the totalactivity of cellobiase. For the given cellulase dosage of20FPUg�1 substrate, reducing sugar concentration andhydrolysis yield both increased with increasing the cello-biase activity till 6.5CBUg�1 substrate. Further additionof cellobiase failed to improve the hydrolysis (data notshown). From the time course of synergistic hydrolysiswith T. reesei ZU-02 cellulase and A. niger ZU-07cellobiase listed in Table 2, it was easily found that thecellobiose concentration was maintained at a low levelduring the whole hydrolysis process. A conclusion could bedrawn that cellobiose formed during hydrolysis processwas quickly hydrolyzed to glucose owing to the improve-ment of cellobiase activity in hydrolysis system. Therebythe feedback inhibition caused by the cellobiose accumula-tion was greatly reduced, which resulted in higher reducingsugar concentration and hydrolysis yield. At 48 h, thehydrolysis yield was improved to 83.9% and the reducingsugar concentration reached 61.4 g l�1.

3.2. Fed-batch hydrolysis with cellulase and cellobiase

In ethanol production from lignocellulosic materials,ethanol concentration in fermentation broth should be ashigh as possible in order to minimize the energy consump-tion in evaporation and distillation (Wingren et al., 2003),which requires a relatively high initial sugar concentrationin hydrolysate. Raising the substrate concentration inbatch hydrolysis helps to obtain higher sugar concentra-tion, but also often causes mixing and heat transferproblems due to the rheological properties of a very densefibrous suspension (Rudolf et al., 2005). While in fed-batchhydrolysis process, such problems could be effectivelyavoided. Since substrate added is gradually degraded, the

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Table 1

Time course of hydrolysis by cellulase from T. reesei ZU-02 (20FPUg�1 substrate; 1.64CBUg�1 substrate)

Time (h) Sugar concentration in hydrolysate (g l�1) Reducing sugar (g l�1) Hydrolysis yield (%)

Cellobiose Glucose Xylose Arabinose

12 3.7 9.0 1.7 0.5 16.9 23.1

24 7.4 16.2 3.9 0.7 30.8 42.1

36 8.6 24.0 5.4 0.7 41.2 56.3

48 11.7 26.9 6.7 0.9 49.4 67.5

60 12.2 28.1 6.9 0.9 51.1 69.8

Table 2

Time course of synergistic hydrolysis by cellulase from T. reesei ZU-02 and cellobiase from A. niger ZU-07 (20FPUg�1 substrate; 6.5CBUg�1 substrate)

Time (h) Sugar concentration in hydrolysate (g l�1) Reducing sugar (g l�1) Hydrolysis yield (%)

Cellobiose Glucose Xylose Arabinose

12 0.4 18.8 2.3 0.7 24.3 33.2

24 0.6 34.1 4.7 0.8 42.3 57.8

36 0.7 43.7 6.2 0.8 53.7 73.4

48 0.9 50.1 6.9 1.0 61.4 83.9

60 0.9 51.2 6.8 1.0 62.2 84.9

120

100

80

60

40

20

012 24 36 48 60

Time (h)

Con

cent

ratio

n of

glu

cose

& r

educ

ing

suga

r (g

l-1)

Reducing sugarGlucose

Fig. 3. Time course of fed-batch synergetic hydrolysis by cellulase from T.

reesei ZU-02 and cellobiase from A. niger ZU-07.

100

80

60

40

20

00 6 12 18 24 30

Time (h)

Con

cent

ratio

n (g

I-1)

GlucoseXyloseEthanolGlycerol

Fig. 4. Fermentation of cellulosic hydrolysate from fed-batch hydrolysis

using S. cerevisiae 316.

M. Chen et al. / International Biodeterioration & Biodegradation 59 (2007) 85–8988

viscosity of reaction mixture can be kept at a low level. Theresults showed that the substrate concentration wasincreased to 200 g l�1 in fed-batch process, and that thereducing sugar concentration reached 116.3 g l�1 with ahydrolysis yield of 79.5% after 60 h of reaction (Fig. 3).The glucose concentration in hydrolysate reached95.3 g l�1, suitable for further ethanol fermentation byS. cerevisiae. In addition, the cellulase dosage was reducedto 15FPUg�1 substrate in fed-batch process. For degrada-tion of equivalent substrate, fed-batch hydrolysis shortenedthe reaction time and therefore enhanced the productivitygreatly compared with batch hydrolysis.

3.3. Fermentation of cellulosic hydrolysate

The glucose-rich cellulosic hydrolysate obtained fromfed-batch hydrolysis, supplemented with yeast extract and(NH4)2HPO4, was utilized as the fermentation medium forethanol fermentation. Results indicated that S. cerevisiae

316 could readily ferment glucose in hydrolysate to ethanolbut could not metabolize xylose due to the lack of xylosereductase and xylitol dehydrogenase (Fig. 4). Within18 h, 95.3 g l�1 glucose was fermented to 45.7 g l�1 ethanol,equivalent to 94% of the theoretical yield (based on thetheoretical yield of 0.51 g ethanol/g glucose).

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Acknowledgment

Financial support from National Engineering ResearchCenter for Fermentation Technology of China for thiswork is gratefully acknowledged.

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