RESEARCH ARTICLE

Optimization of Chemical Pretreatment and Acid Saccharificationfor Conversion of Sugarcane Bagasse to Ethanol

S. K. Uppal • Ramandeep Kaur • Poonam Sharma

Received: 3 January 2011 / Accepted: 2 August 2011 / Published online: 19 August 2011

� Society for Sugar Research & Promotion 2011

Abstract The production potential of cellulosic ethanol

from sugarcane bagasse was studied. Chemical pretreat-

ments were carried out by shaking bagasse with 1, 2 and

3% H2O2 (pH 10, 11.5 and 13) for 24, 48 and 72 h with

subsequent saccharification of pretreated bagasse with

H2SO4 (0.8 and 1.0 M) for 50 min for optimization of

process. Acid hydrolysates were fermented with Saccha-

romyces cerevisiae var ellipsoideus for ethanol production.

Maximum weight loss in alkaline pretreatment (52.30%),

amount of reducing sugars (520.84 mg/g) and ethanol

produced (27.94 ml/100 g pretreated bagasse) were found

in 2% H2O2 (pH 11.5, 48 h) pretreated bagasse saccharified

with 0.8 M H2SO4 after fermentation for 72 h. Pretreat-

ment followed by acid saccharification decreased the time

interval for ethanol fermentation.

Keywords Sugarcane bagasse � Alkaline pretreatment �Acid saccharification � Fermentation

Introduction

The incessant exhaustion of fossil fuel reserves and con-

sequent upward spiral in their prices has stimulated an

extensive evaluation of alternative technologies and sub-

strates to meet the global energy demand. Ethanol offers

certain attractive features over solid and gaseous fuel

which makes it more imperative to be utilized throughout

the world in overcoming the problems of energy crisis and

hunger. Alcohol fuels burn cleaner than other fossil fuel;

hence overcome the great burden of air pollution. Bioeth-

anol can contribute to a cleaner environment and with the

implementation of environment protection laws in many

countries; demand for this is increasing (Zaldivar et al.

2001). Currently, alcohol fuels have been produced on

industrial scale by fermentation of sugars derived from

wheat, corn, sugar beets, sugarcane juice etc. Presently,

nearly all fuel ethanol is produced by fermentation of corn

glucose in America or sucrose in Brazil, and any countries

advanced in agriculture can use current technology for fuel

ethanol fermentation (Lin and Tanaka 2006). But this

technology consumes a lot of food materials, costs too

much, and under the pressure of world’s food crisis at

present, there is even a serious competition between human

food and fuel ethanol. Thus, there is need to find a better

way to solve this problem, and now it is mostly concen-

trated in the field of utilization of agricultural materials.

Sugarcane bagasse, the major byproduct of the sugar cane

industry, is economically viable for the production of

environmentally friendly ethanol fuel. Sugarcane bagasse

is primarily composed of lignin, cellulose and hemicellu-

loses. Processing of lignocelluloses to ethanol consists of

pretreatment to break the lignin seal and disrupt crystalline

structure of cellulose, saccharification for conversion of

carbohydrate polymers into monomeric sugars followed by

fermentation of sugars to ethanol. So the large scale utili-

zation of byproducts of the sugarcane industry, if effi-

ciently implemented, has the dual and important advantage

of generating reasonable profits, not only for sugar pro-

ducers themselves but also for the national economy at

large, as exemplified by cheap electricity, imports

replacement, the proficient use of local fuels and forest

S. K. Uppal (&) � P. Sharma

Department of Plant Breeding and Genetics,

Punjab Agricultural University, Ludhiana 141004, India

e-mail: satinderuppal11@yahoo.co.in

R. Kaur

Department of Chemistry, Punjab Agricultural University,

Ludhiana 141004, India

123

Sugar Tech (July-Sept 2011) 13(3):214–219

DOI 10.1007/s12355-011-0091-3

preservation. So, the present study was intended with

objectives of pretreating sugarcane bagasse with different

treatment combinations of alkali to increase the accessi-

bility of substrate for production of sugars by acid sac-

charification and subsequent fermentation giving maximum

yields of ethanol.

Materials and Methods

The sugarcane bagasse used in present study was obtained

from sugarcane varieties CoJ 88 and CoS 8436.

Chemical Composition of Sugarcane Bagasse

Fine bagasse samples were prepared in the laboratory by

method standardized by Uppal et al. (2008). Then the

chemical composition of bagasse was determined by

detergent system method (Goering and Soest 1970).

Pretreatment of Sugarcane Bagasse

Fine and dried sugarcane bagasse was treated with 1, 2 and

3% H2O2 solutions each with pH 10, 11.5 and 13 for 24, 48

and 72 h. The pretreatment process was carried out as

reported in Dawson and Boopathy (2008).

Acid Saccharification of Pretreated Sugarcane Bagasse

The bagasse residue obtained from each of pretreatment

combination was treated with 0.8 and 1.0 M H2SO4 with

solid: liquid ratio of 1: 12 and the mixture was autoclaved for

50 min at 110�C temperature and 1.05 kg/cm2 steam pres-

sure. The mixture was filtered and analyzed for estimation of

reducing sugars by the method given by Nelson (1944).

Fermentation of Bagasse Hydrolysate to Ethanol

Acid hydrolysates of sugarcane bagasse were neutralized to

pH 5.0 and fermented with Saccharomyces cerevisiae var

ellipsoideus in glucose yeast extract (GYE) broth. Fer-

mentation was progressed at 28�C. The samples were taken

after 24, 48 and 72 h and were analyzed for estimation of

ethanol by the method of Caputi et al. (1968).

Results and Discussion

Chemical Composition of Sugarcane Bagasse

The Chemical composition of sugarcane bagasse of two

varieties is given in Table 1. It was found that cellulose,

hemicellulose and lignin, structural components of cell

wall of sugarcane ranged between 45.77–47.33%,

31.70–36.17% and 8.33–13.56% respectively. Similar

results have been reported by Saha (2003) reported that

lignocellulosic biomass had cellulose as its major compo-

nent (35–50%) followed by hemicellulose (20–35%) and

lignin (10–25%). Peng et al. (2009) also reported that

sugarcane bagasse was primarily composed of cellulose

(40–45%), hemicellulose (30–35%) and lignin (20–30%).

Pretreatment of Sugarcane Bagasse

The hydrolysis of cellulose is restricted by crystalline

structure of cellulose microfibrils that are aggregated and

embedded within the lignified cell wall matrix. Due to this

complex structure, lignocellulosics are less susceptible to

acid attack. Therefore to increase the susceptibility of

cellulosic materials to acids, it is essential to expose and

separate elementary cellulose microfibrils by various pre-

treatment methods. Chemical pretreatments have received

more attention because physical pretreatments are rela-

tively inefficient, biological pretreatments are time con-

suming and the combined treatments rarely have improved

digestibility when compared with simple treatments.

Alkaline pretreatment was selected as it is expected to

cause less sugar degradation than acid process. Hydrogen

peroxide, which is a well-known reagent in paper and

cellulose industry, has the great advantage of not leaving

residues in the biomass, as it degrades into oxygen and

water. Moreover, the formation of secondary products with

H2O2 is practically inexistent. Krishna and Chowdary

(2000) also concluded that alkaline peroxide pretreatments

were effective in providing fractionation of hemicellulose

and lignin components and resulted in efficient hydrolysis

in linn leaves. So, alkaline H2O2 was chosen for occurring

under mild conditions i.e. temperature, pressure and

absence of acids (Gould 1984). The per cent weight losses

reported by alkaline pretreatments performed by soaking

sugarcane bagasse of different varieties in various con-

centrations (1, 2 and 3%) of hydrogen peroxide at different

pH (10, 11.5 and 13) and shaking for various time intervals

(24, 48 and 72 h) are shown in Fig. 1.

The alkaline pretreatments 1% (pH 13, 72 h), 2% and

3% H2O2 (pH 11.5, 48 h) removed the most lignin and

hemicellulose in bagasse compared to other treatment

Table 1 Chemical composition of different varieties of sugarcane

bagasse

Parameter (%) CoJ 88 CoS 8436

Bagasse 48.21 46.26

Cellulose 45.77 47.33

Hemicellulose 31.70 36.17

Lignin 13.56 8.33

Sugar Tech (July-Sept 2011) 13(3):214–219 215

123

combinations indicating the maximum delignification and

hemicellulose solubilization. Further increase in pH and

time interval for 2 and 3% H2O2 did not alter much total

amount of lignin and hemicellulose solubilized. Dawson

and Boopathy (2008) also reported that sugarcane bagasse

residue pretreated with 2% H2O2 at pH of 11.5 and soaked

for 48 h removed more lignin than any other pretreatment

option evaluated.

As lignin is partly covalently associated with hemicel-

luloses and celluloses, less lignin content in bagasse of CoS

8436 led to more exposure of hemicellulose and cellulose

to alkali. Loss of weight in alkaline pretreatment was found

to be more from CoS 8436 samples as compared to that in

CoJ 88. This is because along with loss of lignin, hemi-

celluloses were also dissolved by alkali. More hemicellu-

lose and less lignin content in CoS 8436 than in CoJ 88 led

to more weight loss in CoS 8436. During the course of

pretreatment, the integrity of the individual residue parti-

cles was completely lost as the residue disintegrated into

small, highly water absorbent fibers with a pulp-like con-

sistency. This suggested that the degree of crystallinity

within the cellulose fibers had been reduced, yielding a

more hydrated, open structure. Zhao et al. (2008) reported

that enhancement of enzymatic digestibility of sugarcane

bagasse by per acetic acid pretreatment can be achieved

mainly by delignification and an increase in the surface

area and exposure of cellulose fibers. Beardmore et al.

(1980) used Gamma rays irradiation as pretreatment for the

enzymatic hydrolysis of cellulose. Surface area of cellulose

and rate of hydrolysis were drastically increased at higher

doses but the crystallinity remained relatively unaffected.

Acid Saccharification of Pretreated Bagasse

The main purpose of saccharification is hydrolysis of b,

1–4 linkages of cellulose into individual monomer units

which can then be fermented to yield ethanol. The reducing

sugars produced by treating pretreated bagasse with acid

i.e. 0.8 and 1.0 M H2SO4 for 50 min at pressure of 15 psi

have been shown in Fig. 2.

Maximum acid saccharification occurred in samples in

which maximum weight was lost in pretreatment experiment.

With increase in pH of H2O2 solution from 10 to 11.5, pro-

duction of reducing sugars increased significantly and

remained nearly constant with pH 13. From CoS 8436 having

more cellulose content as compared to CoJ 88, more reducing

sugars were produced. The small decrease in rate of hydrolysis

with increase in concentration of sulfuric acid from 0.8 to

10

5

0

15

20

25

30

35

40

45

50

% W

eigh

t Los

s%

Wei

ght L

oss

10

20

30

40

50

60

A B C

Treatments

Treatments

% W

eigh

t Los

s

Treatments

0

A B C

D E F G

D E F G

H I

CoJ 88

CoS 8436

H I

0

10

20

30

40

50

60

A B C D E F G H I

CoJ 88

CoS 8436

CoJ 88

CoS 8436

A

B

C

Fig. 1 Pretreatment of sugarcane bagasse with hydrogen peroxide

solutions of different concentrations and pH values for 24, 48 and

72 h. (A) 1% H2O2, pH 10; (B) 1% H2O2, pH 11.5; (C) 1% H2O2, pH

13; (D) 2% H2O2, pH 10; (E) 2% H2O2, pH 11.5; (F) 2% H2O2, pH

13; (G) 3% H2O2, pH 10; (H) 3% H2O2, pH 11.5; (I) 3% H2O2, pH 13

c

216 Sugar Tech (July-Sept 2011) 13(3):214–219

123

0

100

200

300

400

500

600

A B C D E F G H IR

educ

ing

suga

rs (

mg/

g ba

gass

e)R

educ

ing

suga

rs (

mg/

g ba

gass

e)R

educ

ing

suga

rs (

mg/

g ba

gass

e)Treatments

A B C D E F G H I

Treatments

Treatments

CoJ 88, 0.8M acid

CoJ 88, 1.0M acid

CoS 8436, 0.8M acid

CoS 8436, 1.0M acid

CoJ 88, 0.8M acid

CoJ 88, 1.0M acid

CoS 8436, 0.8M acid

CoS 8436, 1.0M acid

CoJ 88, 0.8M acid

CoJ 88, 1.0M acid

CoS 8436, 0.8M acid

CoS 8436, 1.0M acid

0

100

200

300

400

500

600

A

B

C

0

100

200

300

400

500

600

A B C D E F G H I

Fig. 2 Production of reducing

sugars by acid saccharification

of sugarcane bagasse pretreated

with different combinations for

24, 48 and 72 h. (A) 1% H2O2,

pH 10; (B) 1% H2O2, pH 11.5;

(C) 1% H2O2, pH 13; (D) 2%

H2O2, pH 10; (E) 2% H2O2, pH

11.5; (F) 2% H2O2, pH 13;

(G) 3% H2O2, pH 10; (H) 3%

H2O2, pH 11.5; (I) 3% H2O2,

pH 13

Sugar Tech (July-Sept 2011) 13(3):214–219 217

123

1.0 M may be due to breakdown of sugars and formation of

hydroxymethylfurfural (Smith et al. 1982). The enzymatic

hydrolysis of waste sugarcane bagasse in water media was

carried out by Zheng et al. (2002). It was concluded that

industrialization of the enzymatic hydrolysis in water medium

is feasible. Acid hydrolysis of cellulosic pyrolysate to glucose

and its fermentation to ethanol were investigated by Yu and

Zhang (2004). The maximum glucose yield (17.4%) was

obtained by the hydrolysis with 0.2 mol/l sulfuric acid using

autoclaving at 121�C for 20 min.

Fermentation of Bagasse Hydrolysate to Ethanol

Hydrolysate samples prepared by three pretreatment com-

binations i.e. 1% (pH 13, 72 h), 2% and 3% H2O2 (pH

11.5, 48 h) and saccharification with 0.8 M H2SO4 were

chosen for further fermentation experiments as maximum

delignification, hemicellulose solubilization and maximum

saccharification were achieved with these treatments. The

yields of ethanol produced by S. cerevisiae var ellipsoideus

are presented in Figs. 3 and 4.

Maximum ethanol production was obtained from 2%

H2O2 pretreated bagasse of CoS 8436 as maximum lignin

and hemicellulose loss and hence maximum reducing

sugars were produced by this treatment. Ethanol production

increased from 24 h of fermentation to 48 h and the

increase after 72 h was less. Similar results were obtained

by Dhillon et al. (1988) by fermenting saccharified

hydrolysate from rice straw containing 7.68% reducing

sugars with S. cerevisiae to produce 2.89% alcohol in 36 h

at 27�C.

Dawson and Boopathy (2008) reported that maximum

ethanol production was achieved on day 18 and 21 of

fermentation from alkali and acid pretreated bagasse

respectively without saccharification. Our studies revealed

that chemical pretreatment followed by acid saccharifica-

tion led to higher ethanol production after 48 h and there

was slight increase in ethanol production after 72 h. These

results suggested that acid saccharification after pretreat-

ment of bagasse reduces fermentation time and hence

hastens the process of ethanol production.

Conclusions

Chemical pretreatments of sugarcane bagasse with 1%

H2O2 (pH 13, 72 h), 2 and 3% H2O2 (pH 11.5, 48 h)

removed the most lignin and hemicellulose. The reducing

sugars produced by acid saccharification (0.8M H2SO4)

were also found to be maximum with above combinations.

The maximum ethanol was produced from 2% H2O2 (pH

11.5, 48 h) pretreated and 0.8 M saccharified bagasse. It

was found that maximum ethanol production by fermen-

tation can be achieved in shorter time intervals from

bagasse which was firstly pretreated followed by sacchar-

ification than directly from pretreated bagasse.

References

Beardmore, D.H., L.T. Fan, and Y.H. Lee. 1980. Gamma ray

irradiation as a pretreatment for enzymatic hydrolysis of

cellulose. Biotechnology Letters 2: 435–438.

10

15

20

25

Eth

anol

(m

l/100

g pr

etre

ated

bag

asse

)

0

5

AB

Treatments C

24 hrs

48 hrs

72 hrs

Fig. 3 Ethanol production from 0.8 M acid hydrolysed bagasse of

CoJ 88 by fermentation with yeast S. cerevisiae var ellipsoideus for

24, 48 and 72 h. Acid hydrolyzed bagasse obtained from three best

pretreatments viz. (A) 1% H2O2, pH 13, 72 h; (B) 2% H2O2, pH 11.5,

48 h; (C) 3% H2O2, pH 11.5, 48 h selected for further fermentation

0

5

10

15

20

25

30

AB

C

Eth

anol

(m

l/100

g pr

etre

ated

bag

asse

)

Treatments

24 hrs

48 hrs

72 hrs

Fig. 4 Ethanol production from 0.8 M acid hydrolysed bagasse of

CoS 8436 by fermentation with yeast S. cerevisiae var ellipsoideus for

24, 48 and 72 h. Acid hydrolyzed bagasse obtained from three best

pretreatments viz. (A) 1% H2O2, pH 13, 72 h; (B) 2% H2O2, pH 11.5,

48 h; (C) 3% H2O2, pH 11.5, 48 h selected for further fermentation

218 Sugar Tech (July-Sept 2011) 13(3):214–219

123

Caputi, A.J., M. Yeda, and T. Brown. 1968. Spectrophotometric

determination of ethanol in wine. American Journal of Enologyand Viticulture 19: 60.

Dawson, L., and R. Boopathy. 2008. Cellulosic ethanol production

from sugarcane bagasse without enzymatic saccharification.

Bioresources 3: 452–460.

Dhillon, G.S., S.K. Grewal, A. Singh, and M.S. Kalra. 1988.

Production of sugars from rice straw. Acta MicrobiologicaPolonica 37: 167–173.

Goering, H.K., and P.J. Van Soest. 1970. Forage fibre analysis

(apparatus, reagents, procedures and some applications). USDA

Agricultural handbook no. 379.

Gould, J.M. 1984. Alkaline peroxide delignification of agricultural

residues to enhance enzymatic saccharification. Biotechnologyand Bioengineering 26: 46–52.

Krishna, S.H., and G.V. Chowdary. 2000. Optimization of simulta-

neous saccharification and fermentation for the production of

ethanol from lignocellulosic biomass. Journal of AgriculturalFood Chemistry 48: 1971–1976.

Lin, Y., and S. Tanaka. 2006. Ethanol fermentation from biomass

resources: Current state and prospects-mini review. AppliedMicrobiology and Biotechnology 69: 627–642.

Nelson, N. 1944. A photometric adaptation of the somogyi method for

determination of glucose. Journal of Biological Chemistry 153:

375–380.

Peng, F., Ren, J.L., Xu, F., Bian, J., Peng, P., and R.C. Sun. 2009.

Comparative study of hemicelluloses obtained by graded ethanol

precipitation from sugarcane bagasse. Journal of AgriculturalFood Chemistry. doi:10.1021/jf900986b.

Saha, B.C. 2003. Hemicellulose bioconversion. Journal of IndianMicrobiology and Biotechnology 30: 279–291.

Smith, C.P., H.E. Grethlein, and A.O. Converse. 1982. Glucose

decomposition at high temperature, mild acid and short residence

time. Solar Energy 28: 41–82.

Uppal, S.K., R. Gupta, R.S. Dhillon, and S. Bhatia. 2008. Potential of

sugarcane bagasse for production of furfural and its derivatives.

Sugar Tech 10: 298–301.

Yu, Z., and H. Zhang. 2004. Ethanol fermentation of acid-hydrolyzed

cellulosic pyrolysate with Saccharomyces cerevisiae. Biore-source Technology 93: 199–204.

Zaldivar, J., J. Nielsen, and L. Olsson. 2001. Fuel ethanol production

from lignocelluloses: A challenge for metabolic engineering and

process integration. Applied Microbiology and Biotechnology56: 17–34.

Zhao, X.B., L. Wang, and D.H. Liu. 2008. Peracetic acid pretreatment

of sugarcane bagasse for enzymatic hydrolysis: A continued

work. Journal of Chemical Technology and Biotechnology 83:

950–956.

Zheng, C., Y. Lei, Q. Yu, X. Lui, and K. Huan. 2002. Enzymatic

hydrolysis of waste sugarcane bagasse in water media. Environ-mental Technology 23: 1009–1016.

Sugar Tech (July-Sept 2011) 13(3):214–219 219

123