Abstract— This study aims to investigate a novel pretreatment

method of sugarcane bagasse (SCB) as an essential step for

production of second generation bioethanol. Effect of tween 80 (TW)

and polyethylene glycol 4000 (PEG) on SCB pretreatment using 1-

butyl-3-methyl imidazolium chloride ([BMIM]Cl) as an ionic liquid

(IL) was assessed. Various concentrations of TW and PEG were

utilized to determine the optimum concentration of surfactant

resulting in high cellulose conversion. Both of surfactants increased

the sugar yield of enzymatic saccharification compared to IL only

pretreated SCB and untreated SCB but, PEG in 3% w/w exhibited

significantly enhanced enzymatic digestibility with an efficiency of

96.2% after 12 h of hydrolysis, which was 23% higher than the

efficiency observed for the IL pretreated SCB.

Keywords—1-butyl-3-methyl imidazolium chloride,

Pretreatment, Sugarcane bagasse, Surfactant

I. INTRODUCTION

IGNOCELLULOSIC biomass has potential to serve as a

low cost and renewable feedstock for bioconversion into

fermentable sugars, which can be further utilized for biofuel

production [1]. Pretreatment of lignocellulosic biomass prior

to use as feedstock for biofuel and chemical production is

required either to disrupt the crystalline structure of cellulose,

remove hemicellulose, remove or modify the lignin to increase

the exposure of cellulosic substrate to hydrolytic enzymes,

decrease cellulosic substrate recalcitrance for enzyme attack

and thus increase glucose yield [2]. The very complex

structure of lignocellulosic biomass makes it highly resistant to

enzymatic hydrolysis which results in low sugar yields [3].

Pretreatment is the most challenging step in bioethanol

production from lignocelluloses. Pretreatment disrupts the

compact and highly ordered structure of lignocelluloses so

enhances the sugar yield of enzymatic hydrolysis of biomass.

While all of the pretreatment methods result in enhanced

enzymatic saccharification of the residual polysaccharide, an

effective process that is inexpensive with low capital costs and

minimal impact on downstream processing has yet to be

developed. More recently, ionic liquids (IL) has emerged as a

promising method for lignocellulosic pretreatment as they

Niloofar Nasirpour is with the Tarbiat Modares University, Tehran, Iran

(e-mail: Niloofar.nasirpour@gmail.com).

Seyyed Mohammad Mousavi is with the Tarbiat Modares University,

Tehran, Iran (corresponding author: Tel.: +98-21-82884917; fax: +98-21-

82884931 ; e-mail: mousavi_m@modares.ac.ir).

exhibit outstanding physical and chemical properties [4].

Several reports were available on the pretreatment of

lignocellulosic biomass using various ILs [1]-[5], but

combination of surfactants and IL as a pretreatment agent has

not been studied yet. Lignin is widely recognized to be an

obstacle to efficient enzymatic hydrolysis. First, it

unproductively adsorbs a large fraction of the cellulase,

making it unavailable for enzymatic hydrolysis of cellulose

[6]. On top of that, lignin impedes enzyme access to cellulose

and hemicellulose, resulting in extended reaction times to

achieve high conversions. Thus, it would be beneficial to use

synergistic effect of IL and surfactant for delignification of

biomass.

In the present study, the surfactant-assisted ionic liquid

pretreatment of sugarcane bagasse was investigated for

enhanced enzymatic hydrolysis. Tween 80 and polyethylene

glycol 4000 were as additive surfactants, and [BMIM]Cl was

applied as the solvating ionic liquid. Experiments carried out

at 130 °C for 90 minute.

II. MATERIALS AND METHODS

A. Feedstock and materials

Sugarcane bagasse was supplied by the Iranian Research

Organization for Science and Technology (IROST). Samples

were ground in a cutter mill (Moulinex, AR1044) and passed

through sieves of mesh size 30 and 70. Ionic liquid [BMIM]Cl

was purchased from Sigma-Aldrich. Commercial enzymes of

Celluclast 1.5 L (the cellulases from Trichoderma ressei) and

Novozyme 188 (the cellubiase from Aspergillus niger) were

purchased from Sigma- Aldrich.

B. Surfactant assisted ionic liquid pretreatment

The amount of solid biomass subjected to pretreatment was

0.5 g of pre-milled SCB based on dry weight. All samples

were soaked into the surfactant of determined concentration

over-night to assure sufficient penetration of liquid into the

SCB solids. Subsequently, ionic liquid was added to sample

tubes so that the ratio of IL to solid content was 10:1. The

mixture was heated in an oil bath of 130 ºC for 90 min.

Following pretreatment, 5 ml deionized water was added into

the reaction mixture. The solution was mixed and centrifuged

(Vifion, VF550) at 12000 rpm for 10 min. To remove the

residual IL from the regenerated cellulose, biomass was

washed with 10 ml deionized water for three times. Wet

regenerated SCB was then freeze dried for 24 hours, prior to

Impact of Surfactant on the Ionic Liquid

Pretreatment of Sugarcane Bagasse

Niloofar Nasirpour, and Seyyed Mohammad Mousavi

L

3rd International Conference on Chemical, Ecology and Environmental Sciences (ICEES'2014) March 19-20, 2014 Abu Dhabi (UAE)

98

enzymatic hydrolysis, and further analysis.

C. Enzymatic hydrolysis

Enzymatic saccharification was carried out with 2.5% w/v

of pretreated and untreated SCB in 10 mM of citrate buffer in

pH 4.8. Sodium azide was utilized in 0.02% to prevent

bacterial growth. The substrates were hydrolyzed with

Celluclast 1.5 L at 50 FPU /g substrate and Novozyme 188 at

40 CBU/g substrate. Samples were taken at 0, 3, 6, 12, 24, 48

and 72 h. Collected samples were centrifuged (B. Braun A15)

at 13500 rpm. Concentration of reduced sugars was measured

by utilizing DNS (3,5- dinitrosalicylic acid) method [13]. All

experiments were performed in duplicate.

III. RESULTS AND DISCUSSION

TW and PEG were added to SCB at concentration of 1, 3

and 5% w/w before IL pretreatment. Fig. 1 shows the kinetic

of enzymatic hydrolysis reaction of untreated and pretreated

SCB by IL and different TW concentrations. It is observed that

addition of tween 80 have increased the saccharification

efficiency compared to untreated and IL treated SCB, but

addition of 1% TW have minor effect compared to IL

pretreated SCB. TW with 5% concentration results in highest

sugar yield. Enzymatic digestibility of TW assisted IL

pretreatment of 1, 3 and 5% concentration after 24 h were

87.7, 93.6 and 99.7, respectively.

Fig 1 Kinetic of enzymatic hydrolysis reaction of untreated and

pretreated SCB by IL and different TW concentrations

Fig. 2 shows the kinetic of enzymatic hydrolysis of

untreated and pretreated SCB by IL and different PEG

concentrations. It is obvious that addition of PEG has

increased the efficiency of saccharification process, but similar

to the effect of TW, PEG concentration of 1% doesn’t have

substantial effect on the sugar release compared to the IL

pretreatment.

The optimum concentration of PEG was 3%, with an

efficiency of 96.2% after 12 h of hydrolysis, while increasing

the concentration to 5% the enzymatic digestibility decreased.

High concentration of PEG might occupy the hydrogen

bonding capacity of ionic liquid and thus the IL pretreatment

influence have decreased. However, this is not true for 5%

concentration of TW, as the hydrogen bonding capacity of TW

is different from PEG, because each has different chemical

bonds, interacting with lignocelluloses.

Fig. 2 Kinetic of enzymatic hydrolysis reaction of untreated and

pretreated SCB by IL and different TW concentrations

In the present study surfactants have influenced the substrate

structure and the performance of the surfactant is the structural

change of lignocelluloses as the lignin removal was increased

by surfactant addition. Following the pretreatment step the

SCB is completely washed, while it is possible that a little

amount of surfactant may remain in the pores of pretreated

SCB and affecting the enzymatic hydrolysis of substrate.

Irrespective of the mechanistic effect of surfactant on SCB

during the pretreatment or hydrolysis step, the ultimate result

is the higher concentrations of sugar.

IV. CONCLUSION

This study presents a novel and improved pretreatment

method for lignocelluloses. This research demonstrates the

effectiveness of surfactant addition before ionic liquid

pretreatment of a lignocellulosic biomass, sugarcane bagasse

(SCB). Enzymatic saccharification of surfactant-IL pretreated

samples increased 23% in comparison with IL pretreated

samples.

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