Comparison of Hydrolysis Conditions to Recover Reducing Sugar from Various Lignocellulosic ...

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384 Chiang Mai J. Sci. 2009; 36(3)

Chiang Mai J. Sci. 2009; 36(3) : 384-394

www.science.cmu.ac.th/journal-science/josci.htmlContributed Paper

Comparison of Hydrolysis Conditions to RecoverReducing Sugar from Various LignocellulosicMaterialsSomkid Deejing* and Wuttichai Ketkorn

Department of Biology, Faculty of Science, Maejo University, Chiang Mai, 50290, Thailand.

*Author for correspondence; e-mail: [email protected]

Received: 10 April 2008Accepted: 1 February 2009

ABSTRACT

Lignocellulosic materials such as rice straw, corn hull, corn stover and vetiver grass

leaves were pretreated with 2.0 M NaOH and hydrolyzed with 1.0, 5.0 and 10.0% (v/v)

sulfuric acid at 100, 111 and 121oC for 15 and 30 min. Among the test conditions, the

maximum reducing sugar concentration from various hydrolysis condition obtained by

hydrolysis with 1.0% (v/v) sulfuric acid at 111oC for 30 min for all materials. In this study,

vetiver grass leaves was selected. The optimal condition of pretreatment and hydrolysis of

vetiver grass leaves were investigated. It was found that the pretreatment with 1.0 M NaOH

and hydrolysis with 2.0% (v/v) sulfuric acid at 111oC for 30 min showed high reducing sugar

concentration.

Keywords : hydrolysis, lignocellulose, ethanol.

1. INTRODUCTION

Lignocelluloses are the most abundant

organic compounds in nature and represent

an important resource for producing valuable

products. Thailand is an agricultural country,

a lot of agricultural wastes are available in

every year. Agricultural residues such as rice

straw, corn hull, corn stover, bagasse and

vetiver grass contain lignocellulose as the majorcomponent. Bioethanol can be produced

from lignocellulosic materials using

pretreatment followed by hydrolysis and

fermentation [1-4]. Pretreatment is one of the

most important steps in the process and high

cost in the production of ethanol from

lingocellulosic materials. The most common

chemical pretreatment methods used for

cellulosic feedstocks are dilute acid, alkaline,

organic solvent, ammonia, sulfur dioxide of

other chemicals to make the biomass more

digestible by enzymes. Lignocellulosic materials

requires a particular combination of the

pretreatment methods to optimize the yields

of the feedstock, minimize the degradation

of substrate, and maximize the sugar yield.One of the most thoroughly investigated

methods is steam pretreatment using an acid

catalyst [5]. Steam pretreatment of corn stover

at 190oC for 5 min using SO2

as acid catalyst

has been shown to give high sugar yields

(almost 90% overall glucose yield and almost

80% overall xylose yield) after 72 h enzymatic

hydrolysis [6]. Alkali pretreatment refers to the


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Chiang Mai J. Sci. 2009; 36(3) 385

application of alkaline solutions to removelignin and various uronic acid substations on

hemicellulose that lower the accessibility of

enzyme to the hemicellulose and cellulose [7].

Generally alkaline pretreatment is more effective

on agricultural residues and herbaceous crops

than wood materials. There are three major

hydrolysis processes for agricultural and wastes

to produce a variety of sugars capable of

making ethanol: dilute acid, concentrated acid,

and enzymatic hydrolysis [8].The main

advantage of the low concentration acid in

the hydrolysis process is that acid recovery may

not be required and there will be no significant

losses of acid. The diluted acid hydrolysis

process uses high temperature (160oC) and

presssure (10 atm).The acid concentration in

the dilute acid hydrolysis process is in the range

of 2-5% [9].

The objective of this study was to compare

the pretreatment and hydrolysis condition

giving high reducing sugar concentration from

various lignocellulosic materials.

2. MATERIALS AND METHODS

2.1 The Hydrolysis Condition of Various

Lignocellulosic Materials

Rice straw, corn hull, corn stover and

vetiver grass leaves were air dried for 1 week

and used as raw materials. The materials were

pretreated with 2.0 M NaOH for 24 hours

at ambient temperature and then washed with

distilled water and dried overnight at 80oC.

Three gram of pretreated materials were

hydrolyzed with 20 ml of 1.0, 5.0 and 10.0%

(v/v) sulfuric acid under autoclaving conditionat 100, 111 and 121oC for 15 and 30 min.

The hydrolysis was performed in tripicate at

each condition. The hydrolysed materials

were centrifuged at 6,000 rpm for 15 min.

The supernatant was determined for total

reducing sugar concentration by using glucose

as standard follow Somogyi method [10]. The

lignocellulosic materials which giving high

reducing sugar concentration was selected.

2.2 Optimization of Pretreatment of

Selected Lignocellulosic Materials with

Sodium Hydroxide

The material giving high reducing sugar

concentration was pretreated with 1.0, 2.0 and

4.0 M NaOH at ambient temperature for 24

hours. After pretreatment, the raw material

was washed by distilled water and dried at

80oC. The pretreated material was hydrolysed

according to the optimal condition. The

hydrolysate was centrifuged at 6,000 rpm for

15 min. The supernatant was determined the

total reducing sugar concentrations by Somogyi

method. The treatment was performed in

tripicate at each condition.

2.3 Optimization of Hydrolysis of

Selected Lignocellulosic Materials with

Sulfuric Acid

After the pretreatment of selected material

under optimal sodium hydroxide concentration,

the samples was hydrolysed with 0.25 0.5, 1.0,1.5, 2.0, 2.5, 3.0, 3.5 and 4.0% (v/v) sulfuric

acid at 111oC for 30 min. After that the

hydrolysate solution was centrifuged at

6,000 rpm for 15 min. The total reducing

sugar concentration in liquid fraction was

determined follow Somogyi method. The

hydrolysis was performed in tripicate at each

condition.

3. RESULTS AND DISCUSSION

3.1 The Hydrolysis Condition of Various

Lignocellulosic MaterialsRice straw which pretreated with 2.0 M

NaOH and hydrolysed with 0, 1.0, 5.0 and

10.0% sulfuric acid at 100, 111 and 121oC

for 15 and 30 min were investigated. The

results of pretreatment and hydrolysis for 15

min were shown in Figure 1a. The hydrolysis

at 100oC obtained reducing sugar 0, 1,341,

511 and 0 g/ml, respectively. The reducing


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386 Chiang Mai J. Sci. 2009; 36(3)

sugar concentration after hydrolysis at 111oCwere 0, 1,233, 468 and 0 g/ml, respectively.

The hydrolysis at 121oC the reducing sugar

concentration were 0, 1,307, 179 and 0 g/ml,

respectively. The results of hydrolysis of rice

straw with 0, 1.0, 5.0 and 10.0% at 100,

111 and 121oC for 30 min were shown in

Figure 1b. The reducing sugar concentration

after hydrolysis at 100oC were 0, 1,639, 208and 307 g/ml, respectively. After hydrolysis

at 111oC obtained the reducing sugar

concentration 0, 1,658, 326 and 0 g/ml,

respectively. The hydrolysis at 121oC obtained

reducing sugar 0, 1,424, 1,002 and 52 g/ml,

respectively.

Figure 1. Reducing sugar concentration of rice straw that pretreated with 2.0 M NaOH

and hydrolyzed with 0, 1.0, 5.0 and 10.0% (v/v) at 100, 111 and 121oC for a) 15 min and b)

30 min (1 2 3

1 2 3) 0% H2SO

4( ) 1.0% H

2SO

4(

1 2 3

1 2 3)5.0% H

2SO

4( ) 10.0% H

2SO

4.

In this study, the results showed maximum

reducing sugar concentration of rice straw at

1,658 g/ml after pretreatment with 2.0 M

NaOH and hydrolysis with 1.0% (v/v) sulfuric

acid at 111oC for 30 min. Hydrolysis of rice

straw was usually carried out under mild

condition, ie., low pressure and long retention

time in connection to the hydrolysis of

hemicellulose. Valdes and Planes [11] studied

the hydrolysis of rice straw using 5-10%

H2SO

4at 80-100oC. They reported the best

sugar yield at 100oC with 10% H2SO4 for 240min. Yin et al. [12] studied the hydrolysis of

hemicellulose of cellulose fraction of rice straw

with 2% H2SO

4at 110-120oC, where they

succeeded to hydrolyze more than 70% of

pentoses. Valkanas et al. [13] presented a

method for hydrolysis of rice straw with dilute

acid at 95oC and different acid type and

concentration of acids (0.5-1% H2SO

4, 2-3%

HCl and 0.5-1% H3PO

3). After 3 h retention

time, rice straw pentosans converted to a

solution of monosaccharides, suitable for

fermentation. Roberto et al. [14] studied the

effects of H2SO

4concentration and retention

time on the production of sugars and the

by-products from rice straw at relative low

temperature (121oC) and long time(10-30

min) in a 350 L batch reactor. The optimum

acid concentration of 1% and retention time

of 27 min was found to attain high yield of

xylose (77%). Acid and water impregnationfollowed by stem explosion of barley straw

was the best pretreatment in terms of resulting

glucose concentration in liquid hydrolysate

[15]. The highest yield of xylose (indicating

efficient hydrolysis of hemicellulsoe) from

Bolivian straw were found at a temperature

of 190oC, and reaction time of 5-10 min,

whereas considerably higher temperature


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Chiang Mai J. Sci. 2009; 36(3) 387

(230oC) were needed for hydrolysis of cellulose[16]. The treatment of rice straw with sulfuric

acid increase digestibility 57.0% [17].

The corn hull was pretreated with 2.0 M

NaOH and hydrolyzed with 0, 1.0, 5.0 and

10.0% (v/v) sulfuric acid at 100, 111 and

121oC for 15 and 30 min. The results of

pretreatment and hydrolysis for 15 min were

shown in Figure 2a. The reducing sugar

concentration after hydrolysis at 100oC were

42, 1,238, 180 and 0 g/ml, respectively. The

hydrolysis at 111oC the reducing sugar

concentration were 50, 1,320, 112 and 0 g/

ml, respectively. After hydrolysis at 121oC the

reducing sugar concentration obtained 0,

1,307, 179 and 0 g/ml, respectively. The

hydrolysis of corn hull with 0, 1.0, 5.0 and

10.0% at 100, 111 and 121oC for 30 min were

shown in Figure 2b. The reducing sugarconcentration after hydrolysis at 100oC were

22, 1,656, 258 and 0 g/ml, respectively.

After hydrolysis at 111oC the reducing sugar

obtained 50, 1,674, 53 and 0 g/ml, respectively.

The reducing sugar concentrat ion af ter

hydrolysis at 121oC were 3, 1,579, 1,121 and

108 g/ml, respectively. In this study, the

results showed maximum reducing sugar

concentration of corn hull 1,674 g/ml after

pretreatment with 2.0 M NaOH and hydrolysis

with 1.0% (v/v) sulfuric acid at 111oC for

30 min. Saha and Bothast [18] found that

the dilute sulfuric acid (0.51.0% (v/v)

pretreatment of corn fiber at 121oC obtained

the monomeric sugar yield 85100% of the

theoretical yield.

Figure 2. Reducing sugar concentration of corn hull that pretreated with 2.0 M NaOH and

hydrolyzed with 0, 1.0, 5.0 and 10.0% (v/v) sulfuric acid at 100, 111 and 121oC for a) 15 min

and b) 30 min (1 2 3

1 2 3) 0% H2SO

4( ) 1.0% H

2SO

4(

1 2 3

1 2 3)5.0% H

2SO

4( ) 10.0% H

2SO

4.

The pretreatment of corn stover with 2.0

M NaOH and hydrolysis with 0, 1.0, 5.0 and


121oC for 15 and 30 min were studied. The

results in Figure 3a showed reducing sugar

concentration after hydrolysis under for 15

min. The hydrolysis at 100oC obtained the

reducing sugar concentration 78, 1,605, 1,123

and 0 g/ml, respectively. The reducing sugar


89, 1,409, 1,246 and 0 g/ml, respectively.

After hydrolysis at 121oC obtained reducing

sugar 12, 1,673, 1,513 and 327 g/ml,

respectively. The hydrolysis with 0, 1.0, 5.0 and


121oC for 30 min were examined. The results


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388 Chiang Mai J. Sci. 2009; 36(3)

were shown in Figure 3b. The reducing sugarconcentration after hydrolysis at 100oC were


The hydrolysis at 111oC obtained reducing

sugar concentration 70, 1,785, 1,367 and 15

g/ml, respectively. The hydrolysis at 121oC

the reducing sugar concentration were 1,

1,672, 1,599 and 591 g/ml, respectively. In

this study, the results showed maximum

reducing sugar concentration of corn stover

1,785 g/ml after pretreatment with 2.0 M

NaOH and hydrolysis with 1% (v/v) sulfuric

acid at 111oC for 30 min. Wyman et al. [19]

reported an increase of the overall sugar yield

from 56.8% (when using water pretreated

corn stover) up to 93% by diluted sulfuric

acid pretreatment. This improvement was

attributed to the increase of xylan solubilization

much faster than xylose degradation, due to

acid action. After hydrolysis of corn stover

with diluted sulfuric acid, glucose yield are

maximized at short time (6 sec) and high

temperature (240oC) [20]. The glucose yield

(96-104%) was obtained from acid catalysedsteam pretreated corn stover and xylose yields

increase the 70-74% yield when pretreatment

severity was reduced by using autocatalysis

instead of acid-catalysed steam pretreament,

xylose yields were increased to 80-86 %. Partialdelignification of pretrerated material was

also evaluated as a way to increase the overall

sugar yield. The overall glucose yield increased

sligthly due to delignification but the overall

xylose yield decreased due to hemicellulose

loss in the delignification step [6]. The maximum

sugar recovery by water extraction of corn

stover is produced by a synergistic effect

treatment of 190oC for 5 min and an acid

loading of 1.5% (w/w) at these conditions

25% of the sugars in the feedstock can be

recovered as monomers or oligomers. The

acid has greatly improved the sugar solubility

at 180oC the sugar recovery has been only

1.5% without acid, while by using 3 (%w/v)

of acid the sugar recovery has increased to

16.8%. Also the cellulose digestibility has been

improved by the acid pre- impregnation, after

48 h of digestion the yield of glucose reached

93% of the theoretical by using the substrate

that was pre-impregnated with 3% w/v of

acid and treated at 190oC. The high acid

loading has also been required to achieve thebest recovery of glucose (85% of the initial

glucan) as sum of water extraction and 48 h

hydrolysis [21].

Figure 3. Reducing sugar concentration of corn stover that pretreated with 2.0 M NaOH

and hydrolyzed with 0, 1.0, 5.0 and 10.0% (v/v) sulfuric acid at 100, 111 and 121oC for a) 15

min and b) 30 min (1 2 3

1 2 3) 0% H2SO

4( ) 1.0% H

2SO

4(

1 2 3

1 2 3

1 2 3)5.0% H

2SO

4( ) 10.0% H

2SO

4.


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Chiang Mai J. Sci. 2009; 36(3) 389

The pretreatment of vetiver grass leaveswith 2.0 M NaOH and hydrolysis with 0, 1.0,

5.0 and 10.0% (v/v) sulfuric acid and at 100,

111 and 121oC for 15 and 30 min were

studied. The results of pretreament and

hydrolysis of vetiver grass leaves for 15 min

were shown in Figure 4a. The reducing sugar


0, 1,591, 865 and 512 mg/ml, respectively.

After hydrolysis at 111oC the reducing sugar

concentration were 0, 1,476, 864 and 0 g/ml,

respectively. The hydrolysis at 121oC obtained

reducing sugar concentration 0, 1,609, 1,524

and 181 g/ml, respectively. For the hydrolysis

of vetiver grass leaves with 0, 1.0, 5.0 and

10.0% (v/v) sulfuric acid at 100, 111 and121oC for 30 min were examined. The results

showed in Figure 4b. The reducing sugar



After hydrolysis at 111oC obtained 0, 1,774,

755 and 19 g/ml, respectively. The reducing

sugar concentration after hydrolysis at 121oC

we re 0, 1, 621, 1,647 and 151 g/ml,

respectively. The results showed maximum

reducing sugar concentration of vetiver grass

leaves 1,774 g/ml after pretreatment with

2.0 M NaOH and hydrolysis with 1.0% (v/v)

sulfuric acid at 111oC for 30 min.

Figure 4. Reducing sugar concentration of vetiver grass leaves that pretreated with 2.0 M

NaOH and hydrolyzed with 0, 1.0, 5.0 and 10.0% (v/v) sulfuric acid at 100, 111 and 121 oC

for a) 15 min and b) 30 min (1 2 3

1 2 3

1 2 3) 0% H2SO

4( ) 1.0% H

2SO

4(

1 2 3

1 2 3)

5.0% H2SO

4( ) 10.0%

H2SO

4.

In this study, rice straw, corn hull, corn

stover and vetiver grass leaves were pretreated

with 2.0 M NaOH and hydrolysed with 0,

1.0, 5.0 and 10.0% sulfuric acid for 15 min

and 30 min. The results of these conditionshowed low reducing sugar concentration at

high concentration of sulfuric acid hydrolysis

for every materials and every condition.

Because of at high concentration of acid might

the cause of the reducing sugar degradation

[22]. The soft hydrolysis conditions led to a

sugar-rich prehydrolysate. When using harsh

pretreatment conditions, sugar recovery in

liquids decreased. The optimization of

pretreatment should be the maximization of

the overall sugar yield, taking into account all

fermentable sugars. The overall sugar yields

obtained at mild pretreament temperature170-190oC increased with acid concentration

(for acid concentrtaions up to 1%). By contrast,

high pretreament temperatures and elevated

acid concentrations produced a decrease in

overall sugar yield because cellulose recovery

in the pretreated solids came down and sugars

content in the prehydrolysates decreased as a

result of degradation processes. The maximum


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390 Chiang Mai J. Sci. 2009; 36(3)

overall sugar yield of olive tree (36.3 g sugar/100 g raw material) was obtained at 180oC

and 1% sulfuric acid concentration,

representing 75% of all sugars in the olive tree

biomass (48.7 g/100 g) [23]. Steam explosion

of lignocellulosic materials can be related to

the significant increases of the crystallinity

index values of cellulose [24].

For this study, the maximum reducing

sugar concentration of rice straw, corn hull,

corn stover and vetiver grass leaves were 1,658,

1,674, 1,785 and 1,774 g/ml, respectively.

In this study, vetiver grass leaves were selected

as raw material for ethanol production.

3.2 Optimization of Pretreatment Con-

dition of Vetiver Grass Leaves with Sodium

Hydroxide

The vetiver grass leaves was pretreated

with 1.0, 2.0 and 4.0 M NaOH and hydrolysed

with 1.0% (v/v) sulfuric acid at 111oC for 30

min. The results in Figure 5 showed that the

optimal concentration of sodium hydroxide

for pretreatment of vetiver grass leaves at 1.0M NaOH. Gould [25] reported the extraction

condition of hemicelluloses from the vetiver

grass with a maximum yield of 35% with

4 M NaOH at ambient temperature for 8 h.

The treatment of dewaxed maize stems, rye

straw and rice straw with 1 M NaOH at

30oC for 18 h resulted in a dissolution of 78.0,68.8 and 82.1% of the original lignin, and 72.1,

72.6 and 84.6% of the original hemicelluloses,

respectively [26]. The optimal condition for

the pretreatment of casava rhizome was 2 M

NaOH [27]. The pretreatment of rice bran

with 5 N NaOH for 10 min could increase

20% of sugar [28]. The pretreatment of

cotton stalk and agricultural straw with 2%

NaOH 90 min, 121oC/15psi resulted in the

highest level of delignification 66% and 75.09-

84.52%, respectively [29, 30] but increase

54.83% cellulose and decreased hemicellulose

61.07% and lignin 36.24% [31]. The pretreat-

ment of corn stover with 10% NaOH for

1 hour in the autoclave reduced 95% lignin

content. An increase in the concentration of

sodium hydroxide significantly improved

delignification at all combinations of

temperature and time [32]. The main effect

of sodium hydroxide pretreatment on

lignocellylosic biomass is delignification by

breaking the ester bonds cross-linking lignin

and xylan, thus increasing the porosity ofbiomass [4,29,33]. Alkali pretreatment also

caused glucan solubilization [29]. Neely

[34] present that effective of alkaline pretreat-

ment depends on the lignin content of the

materials [34].

Figure 5. Reducing sugar concentration of vetiver grass leaves after pretreated with 1.0, 2.0

and 4.0 M NaOH and hydrolyzed with 1% (v/v) sulfuric acid at 111oC for 30 min.

Reducingsugarcon

centration(g/ml)

NaOH concentration (M)


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Chiang Mai J. Sci. 2009; 36(3) 391

3.3 Hydrolysis of Vetiver Grass Leaves withSulfuric Acid

The vetiver grass leaves were pretreated

with 1 M NaOH and hydrolyzed with 0.25,

0.5, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5 and 4.0% sulfuric

acid at 111oC for 30 min. The maximum

reducing sugar concentration was obtained

after hydrolysis of vetiver grass leaves with

2% sulfuric acid at 111oC for 30 min. The

results showed in Figure 6. In this study, dilute

acid (2% sulfuric acid) showed highest

reducing sugar concentration. Hinman et al.

[22] reported that dilute acid hydrolysis has

been successfully developed for pretreatment

of lignocellulosic materials. The dilute sulfurics

acid pretreatment can achieve high reaction

rates and significantly improve cellulose

hydrolysis [32,35,36]. Recently developed

diluted acid hydrolysis processes use less

severe conditions and achieve high xylan to

xylose conversion yields. Achieving high xylan

to xylose conversion yields is neccesary to

achieve favorable overall process ecnomics

because xylan accounts for up to a third ofthe total carbohydrate in many lignocelulosic

materials. Sun and Cheng [3] reported the

glucose concentration in the prehydrolyzate

of rye straw was not significantly influencedby the sulfuric acid concentration and residence

time, but it increased in the prehydrolyzate of

bermudagrass with the increase of pretreatment

severity. The xylose concentration in the

filtrates increased with the increase sulfuric acid

concentration and residence time.

The effect of combined heat treatment

and acid hydrolysis at various concentrations

on lignocellulosic biomass was reported. At

high concentration of sulfuric acid (1.0-5.0 M

H2

SO4

), hydrolysis the casava grate waste

was achieved but with excessive carring or

dehydration reaction. At lower acid concentra-

tions, hydrolysis of casava grate waste

biomass was also achieved with 0.3-0.5 M

H2SO

4, while partial hydrolysis was obtained

below 0.3 M H2SO

4(the lowest acid concen-

tration that hydrolysed casava grate waste

biomass) at 120oC and 1 atm pressure for 30

min. High acid concentration is therefore not

required for casava grate waste biomass

hydrolysis [37]. The dilute sulfuric acid (0.5

1.0% (v/v) pretreatment of corn fiber at121oC found that the monomeric sugar yield

was 85100% of the theoretical yield [18].

Figure 6. Reducing sugar concentration of vetiver grass leaves after pretreated with 1.0 M

NaOH and hydrolyzed with 0.25, 0.5, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5 and 4.0% (v/v) sulfuric acid at

111oC for 30 min.

Reducings

ugarconcentration(g/ml)

Sulfuric acid concentration (% v/v)


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392 Chiang Mai J. Sci. 2009; 36(3)

In this study, we decided to select thevetiver grass leaves as raw material for ethanol

production. The reason of our decision,

because of the vetiver was normal practice

of growing for soil and water conservation.

The vetiver need to cut down the leaves every

few months to encourage tiller growth and

to reduce the danger of fire in the dry season.

For the value added of vetiver grass leaves,

we tried to promote the growing of vetiver

for soil and water conservation for solving

the problem of soil destruction to the farmer.

His Majesty the King of Thailand has

repeatedly summoned that uses other than

ones just mention, as well as the utilization of

harvested material, should not be overempha-

sized to nullify the main uses of vetiver. There

is no question that vetiver has a considerable

ecological benefit in soil and water conservation

as well as environmental protection. Its hidden

economic value of and inexpensive means of

stabilizing backslopes and sideslopes of the

highway, railroad, as well as earthen dams is

also increasingly being recognized. Increasingawareness of the ecological potential of vetiver

in controlling pollution, and in protecting our

environment such as in wastewater treatment,

heavy metal contamination etc. are likely to

be envisaged in the near future. The vetiver

grass leaves could be applied as raw materials

for ethanol production.

4. CONCLUSION

Rice straw, corn hull, corn stover and

vetiver grass leaves were pretreated with 2.0

M NaOH and hydrolyzed with 1.0, 5.0 and10.0% (v/v) sulfuric acid at 100, 111 and

121oC for 15 and 30 min. High reducing sugar

concentration was obtained by hydrolysis

with 1.0% (v/v) sulfuric acid at 111oC for

30 min in all of materials. The vetiver grass

leaves was selected. The optimal condition of

pretreatment and hydrolysis of vetiver grass

leaves was 1.0 M NaOH and 2.0% (v/v)

sulfuric acid at 111oC for 30 min.

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