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Procedia Chemistry 18 (2016) 147 – 154

Molecular and Cellular Life Sciences: Infectious Diseases, Biochemistry and Structural Biology
2015 Conference, MCLS 2015

Deep Eutectic Solvent (DES) as a Pretreatment for Oil Palm Empty

Abstract

Oil Palm Empty Fruit Bunch (OPEFB) was pretreated using Deep Eutectic Solvent (DES) at different parameters to enable a
highest yield of sugar. DES is a combination of two or more cheap and safe components to form an eutectic mixture through
hydrogen bond interaction, which has a melting point lower than that of each component. DES can be used to replace Ionic
Liquids (ILs), which are more expensive and toxic. In this study, OPEFB was pretreated with DES mixture of choline chloride :
urea in 1:2 molar ratio. The pretreatment was performed at temperature 110ºC and 80ºC for 4 hours and 1 hour. Pretreatment A
(110ºC, 4 hours), B (110ºC, 1 hour), C (80ºC, 4 hours) and D (80ºC, 1 hour). Enzymatic hydrolysis was done by using the
combination of two enzymes, namely, Cellic Ctec2 and Cellic Htec2. Morphology surface of OPEFB is observed under Scanning
Electron Microscopy (SEM). The treated fiber is tested for crystallinity using XRD and functional group analysis using FTIR, to
check the effect of the pretreatment on the fiber and compared it with the untreated fiber. From XRD analysis, DES successfully
gave an effect towards degree of crystallinity of cellulose. Pretreatment A and B successfully reduce the percentage of
crystallinity while pretreatment C and D increased the percentage of crystallinity. From FTIR analysis, DES can expose the
structure of cellulose even though the functional group of lignin and hemicellulose were still present. Upon enzymatic hydrolysis,
DES-treated fiber successfully produced sugar but not significantly when compared with raw. Pretreatment A, B, C and D
produced glucose at the amount of 60.47 mg/ml, 66.33 mg/ml, 61.96 mg/ml and 59.12 mg/ml respectively. However,
pretreatment C gave the highest xylose (70.01 mg/ml) production compared to other DES pretreatments.

©
© 2016
2015TheTheAuthors. Published
Authors. by by
Published Elsevier B.V.B.V.
Elsevier This is an open access article under the CC BY-NC-ND license
(http://creativecommons.org/licenses/by-nc-nd/4.0/).
Peer-review under responsibility of the organizing committee of the Molecular and Cellular Life Sciences: Infectious Diseases,
Peer-review under responsibility of the organizing committee of the Molecular and Cellular Life Sciences: Infectious Diseases,
Biochemistry and Structural Biology 2015 (MCLS 2015).
Biochemistry and Structural Biology 2015 (MCLS 2015)
Keywords: Deep eutectic solvent; oil palm empty fruit bunch; pretreatment; crystallinity

* Corresponding author. Tel.: - ; fax: -.

1876-6196 © 2016 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY-NC-ND license
(http://creativecommons.org/licenses/by-nc-nd/4.0/).
Peer-review under responsibility of the organizing committee of the Molecular and Cellular Life Sciences: Infectious Diseases, Biochemistry and
Structural Biology 2015 (MCLS 2015)
doi:10.1016/j.proche.2016.01.023
148 Nur Atikah Md Nor et al. / Procedia Chemistry 18 (2016) 147 – 154

1. Introduction

Oil Palm Empty Fruit Bunch (OPEFB) is a lignocellulosic biomass. It consists of lignin, hemicellulose and
cellulose1. Researches had been conducted to convert OPEFB biomass into a value-added product. However, for
efficient sugar production, the OPEFB need to be pretreated prior to hydrolysis by suitable enzymes into value-
added product such as bioethanol2. Several pretreatment can be used such as physical pretreatment, chemical
pretreatment and combination of both pretreatments3.
In this study, chemical pretreatment was chosen because of its high efficiency in treating the biomass4. In this
chemical pretreatment, alkaline is preferred compared to acid because alkaline is believed to have less severe effect
to the biomass or fiber after been treated5. Sodium hydroxide (NaOH) pretreatment was chosen in this study as a
benchmark because NaOH has been extensively used as delignification agent where it can cause the swelling of the
fiber, increase the total internal surface area, break the lignin-carbohydrate bonds and also disrupt the lignin
structure6.
Deep eutectic solvents (DES) is a combination of two substances through hydrogen bond interaction, where the
eutectic mixture is having melting point lower than that each individual component7. DES is used to replace ionic
liquid which already known as expensive and toxic liquid. Mixtures of urea with quaternary ammonium salts such as
choline chloride could be a liquid when heated at temperature around 80ºC 8. This liquid formed will have a lower
melting point compared to individual urea and choline chloride. DES used in this study as the medium in treating the
OPEFB is an attempt to produce low-cost, recyclable, environmentally friendly solvent, to extract valuable
components from OPEFB.
Thus, the objective of this study is to determine whether DES created by the combination between urea and
choline chloride at 1:2 molar ratio can be used as a pretreatment solvent for OPEFB, in production of sugar.

2. Methods

2.1. Sample preparation

Oil Palm Empty Fruit Bunch (OPEFB) was collected from Seri Ulu Langat Palm Oil Mills Sdn Bhd, Selangor,
Malaysia. The collected OPEFB were cleaned with tap water to wash away all the dirt and foreign particles. Next,
the cleaned OPEFB were sun-dried until approximately 10-11% moisture content is achieved. The fiber was cut into
smaller pieces ranging from 4cm–5cm. The fiber were kept in refrigerator at temperature of 4ºC prior proceed to
next experiment.

2.2. Chemicals

Choline Chloride ChCl (99% purity), Urea CH4N2O (98% purity), Xylose (99% purity), Cellobiose (98% purity)
and Glucose (99.5% purity) were purchased from Sigma Aldrich, USA while Sodium Hydroxide pellet, NaOH were
purchased from Systerm. Acetonitrile HPLC grade were purchased from Merck, Germany. Enzymes Cellic Ctec2
and Cellic Htec2 were purchased from Novozymes, Denmark.

2.3. Preparation of Deep Eutectic Solvent

Choline Chloride (99% purity) were mixed together with urea (98% purity) at 1:2 molar ratio8. The mixture were
heated at 80ºC with continuous stirring until a homogenous mixture is obtained. The mixture were kept in Schott
bottle once it cooled down prior used.

2.4. Sodium Hydroxide (NaOH) pretreatment

Raw OPEFB were soaked into 5% NaOH solution with solid to liquid ratio is 1:10. The treatment was done at
120ºC for 2 hours in autoclave. After treatment was done, the treated fiber were washed with distilled water to clean
the fiber and air-dried for 24-48 hours until constant weight is achieved. The treated fibers were kept at 4ºC prior
used for next experiment. The black liquor is kept for next analysis.
 Nur Atikah Md Nor et al. / Procedia Chemistry 18 (2016) 147 – 154 149

2.5. DES pretreatment

Raw OPEFB were soaked into DES mixture with solid to liquid ratio is 1:5. The treatments were performed at
110ºC for 4 hours (pretreatment A), 110ºC for 1 hour (pretreatment B), 80ºC for 4 hours (pretreatment C) and 80ºC
for 1 hour (pretreatment D). The treated fibers were then washed with distilled water and oven-dried at 90ºC until
constant weight is achieved. The treated fibers were kept at 4ºC until used. The black liquor is kept for next analysis.

2.6. Enzyme Hydrolysis

The untreated and treated fibers were next underwent enzyme hydrolysis. In enzyme hydrolysis, the fibers were
treated with combination enzymes which are Cellic Ctech2 and Cellic Htech2. Fiber (0.5 g) was put into 10 mL of
citrate buffer (pH 4.8) and the enzyme was added into it at 1:1 ratio. Next, the mixture was incubated at 50ºC for 24
hours at 150 rpm. The hydrolysate is collected and heated in boiling water for 5 minutes. The hydrolysates were
centrifuged at 3000 rpm for 5 minutes to collect the supernatant.

2.7. Sugar Analysis Using High Performance Liquid Chromatography (HPLC)

Glucose, xylose and cellobiose were used as standard with concentrations: a) 2.5 mg/ml, b) 5.0 mg/ml, c) 7.5
mg/ml, and d) 10.0 mg/ml. Sugar content were analyzed using HPLC equipped with ELSD detector. The column
used was NH2 and eluted with mobile phase acetonitrile and deionized water at 80:20 ratio. The samples and
standard were filtered with 0.22 μm nylon membrane filter before proceed with analysis. The data were acquired and
analyzed using Breeze software.

3. Results and discussion

The treated fiber were then analyzed and observed by using Scanning Electron Micrograph (SEM), X-ray
Diffraction (XRD), and Fourier Transform Infra-red Spectroscopy (FTIR). SEM analysis was for observing any
effect of the pretreatments on the surface morphology of the fiber. XRD analysis was conducted to see whether the
pretreatments had any effect towards the crystallinity of cellulose while FTIR analysis was for determining the
presence and absence of important functional groups of the fiber after the fiber being treated.

3.1. Scanning Electron Micrograph (SEM)

Fig. 1. Image of electron micrograph that shows the morphology structure of OPEFB at 200x magnification: (a) raw b) Pretreatment A (110ºC,
4H) c) Pretreatment B (110ºC, 1H) d) pretreatment C (80ºC, 4H) e) Pretreatment D (80ºC, 1H) and f) NaOH pretreatment
150 Nur Atikah Md Nor et al. / Procedia Chemistry 18 (2016) 147 – 154

Fig. 2. Image of electron micrograph that shows the morphology structure of OPEFB at 500x magnification: (a) raw; (b) Pretreatment A (110ºC,
4H); (c) Pretreatment B (110ºC, 1H); (d) pretreatment C (80ºC, 4H); (e) Pretreatment D (80ºC, 1H); (f) NaOH pretreatment. Examples of cavity
spots are shown in a red box.

Fig. 3. Image of electron micrograph that shows the morphology structure of OPEFB at 1000x magnification: (a) raw; (b) Pretreatment A (110ºC,
4H) ; (c) Pretreatment B (110ºC, 1H); (d) pretreatment C (80ºC, 4H); (e) Pretreatment D (80ºC, 1H); (f) NaOH pretreatment. Examples of cavity
and silica spots are shown in red and green boxes, respectively.

From SEM analysis, the result showed that all the pretreatments were successfully even though not completely
remove the silica. Raw or untreated fiber originally is a waxy, smooth and covered with lignin. It also contained lots
of silica on the surface. The fiber had many silica on the surface due to the hardening of soil mineral into the cell
wall during growth9. These silica should be remove during pretreatment so that the enzymes can penetrate or able to
access to the hemicellulose and cellulose to be converted into sugar. This is why pretreatment is a must before
proceed to enzyme hydrolysis process.
From the Fig. 2 and Fig. 3, we can see that all the pretreatments able to remove the silica and left the fiber with
holes known as cavities. All the pretreatments make the surface of the fiber become rough and lignin structure is
 Nur Atikah Md Nor et al. / Procedia Chemistry 18 (2016) 147 – 154 151

breakdown. NaOH pretreatment is the one that able to remove most of the silica. For DES pretreatment, high
temperature which is 110ºC (pretreatment A and B) can remove the silica more compared to low temperature
(pretreatment C and D).
From Fig. 3, NaOH pretreatment showed that the fiber structure was severely disrupted and almost all the silica
were completely removed after being treated. As we can see, NaOH pretreatment also had lots of cavities due to loss
of many silica. From Fig. 3 (labeled b and c), DES pretreatment that performed at 110ºC able to remove silica more
compared to DES pretreatment that performed at 80ºC (labelled d and e). It showed that high temperature worked
more vigorously and gave more severe impact to the morphology of the fiber. Other than that, longer time which is 4
hours gave more effect towards the fiber when more silica were removed and left lots of cavities on the fiber. These
was clearly shown in Fig. 2 and also supported by Fig. 3.

3.2. X-ray Diffraction (XRD)

In the XRD analysis, all the samples were conducted at angle diffraction (2 ) from 10º to 50º. Fig. 4 shows the
diffractogram (XRD) of untreated fiber (raw) and compared it with fiber treated with pretreatments NaOH, A
(110ºC, 4H), B (110ºC, 1H), C (80ºC, 4H) and D (80ºC, 1H). Sigmacell cellulose Type 20 was used as a standard.
There are three diffraction pattern of cellulose. Standard cellulose shows the highest crystallinity due to the purity of
cellulose. Table 1 shows the percentage of crystallinity of cellulose. Pretreatments NaOH, C and D have higher
percentage of crystallinity compared with the untreated fiber. This increment may be due to removal of amorphous
hemicellulose and lignin10. However, pretreatment A and B successfully reduce the percentage of crystallinity when
being compared with untreated fiber. As been shown in Table 1, the reduction in crystallinity of cellulose may be
due to the high temperature. This condition occurred because high temperature will lead to relocation of amorphous
of cellulose and the paracrystalline in the part of crystalline cellulose had been hydrolyzed11. Hence, this will cause
the loss of crystalline cellulose in the treated fiber and directly reduced thecellulose crystallinity. Reduced in
cellulose crystallinity will help in sugar production because reduced crystallinity will make the fiber more accessible
to enzyme reaction for higher sugar production12.

Fig. 4. X-ray Diffractogram for Sigmacell cellulose Type 20, untreated fiber, treated fiber with pretreatments A, B, C, D and NaOH.

Table 1. Crystallinity percentage of Sigmacell cellulose Type 20, untreated fiber and fiber
treated with pretreatment A, B, C, D and NaOH.
Samples Crystallinity Percentage, %
Sigmacell cellulose Type 20 66.42
Untreated Fiber 38.27
Pretreatment A (110ºC, 4H) 37.16
Pretreatment B (110ºC, 1H) 34.99
Pretreatment C (80ºC, 4H) 38.84
Pretreatment D (80ºC, 1H) 39.23
Pretreatment NaOH 39.44
152 Nur Atikah Md Nor et al. / Procedia Chemistry 18 (2016) 147 – 154

3.3. Fourier Transform Infra-red Spectroscopy (FTIR)

At 1732 cm-1, it is a peak that represent C=O acetyl group of hemicellulose ester or a carbonyl ester of ȡ-
coumaric lignin unit13. Pretreatment A,B,C and D have a same trend with raw. However, the peak was lost after
NaOH pretreatment. Peak 1512 cm-1 represent aromatic asymmetric stretching (C=C) from lignin14. There is no
significant effect at this absorption where all the treatments show the same trend with untreated fiber. In addition, the
intensity was reduced at 1243 cm-1 for NaOH pretreatment but for other pretreatments, they have a similar trend with
the raw. At this absorption, it represents the C-O-C functional group of aryl-alkyl ether in lignin10. Thus, NaOH
pretreatment has removed the functional group compared to other pretreatments. At peak 3329 cm-1, it showed that
DES pretreatments (A,B,C and D) gave a broad shape of absorption compared to NaOH pretreatment. This peak
represents hydrogen bonded stretching absorption OH of functional group in cellulose14. Hence, it means that DES
pretreatment is successful in exposing the cellulose of treated EFB. From this FTIR analysis, it shows that the DES
pretreatment using choline chloride : urea may not be the best delignification agent as NaOH pretreatment, because
this DES combination cannot successfully remove the functional group of lignin. However, it is believed that DES
pretreatment can expose the cellulose better than NaOH pretreatment. This can be proved by looking at the presence
of broad peak representing DES pretreatments at 3329 cm-1. It might be that the mechanism of DES pretreatment
worked is not as same as the NaOH pretreatment.

Fig. 5. FTIR spectra of untreated fiber and compared with fiber treated with NaOH, pretreatments A, B, C and D.

3.4. Sugar Analysis by High Performance Liquid Chromatography (HPLC)

The treated fiber undergo sugar analysis by using HPLC. Glucose, xylose and cellobiose are used as standard at
different concentration (as stated above). Fig. 6 showed the yield of sugar been produced from treated fiber and the
result is being compared with sugar production of untreated fiber.
From Fig. 6, NaOH pretreatment produced the highest production of xylose and glucose which are 83.88 mg/ml
and 351.61 mg/ml respectively. Pretreatment A (110ºC, 4 hours), B (110ºC, 1 hour), C (80ºC, 4 hours) and D (80ºC,
1 hour) produced glucose at the amount of 60.47 mg/ml, 66.33 mg/ml, 61.96 mg/ml and 59.12 mg/ml respectively
when the fiber is hydrolyzed with combination of enzymes: Cellic Ctech & Cellic Htech. However, pretreatment C
also gave the highest xylose production compared to other DES pretreatments which is 70.01 mg/ml. Pretreatment
A, B & D gave a low xylose production when using combination of enzyme. It could be that hemicellulose escaped
into the black liquor which will undergo further test and analysis. This sugar analysis shows the DES pretreatment
still cannot produced more sugar when compared with raw fiber. Raw fiber produced xylose at 6.87 mg/ml while
glucose at 47.38 mg/ml. This amount is not significantly different with the sugar production from DES
pretreatments(p>0.05).The low sugar yield could also be due to the limited temperature and time ranges, selected in
 Nur Atikah Md Nor et al. / Procedia Chemistry 18 (2016) 147 – 154 153

this study.From above figure, NaOH pretreatment gave the highest sugar production compared to DES pretreatment
and untreated fiber (p<0.05). It may due to lots of silica were removed and loss of functional group of lignin and also
hemicellulose.

Fig. 6. Xylose and glucose production of untreated fiber, NaOH-treated fiber and fiber treated with different conditions: pretreatment A (110ºC,
4H), B (110ºC, 1H), C (80ºC, 4H) and D (80ºC, 1H).

4. Conclusion

Upon SEM analysis, both pretreatments: DES and NaOH were able to remove silica, left many cavities and
disrupt the structure of OPEFB. Furthermore, combination of high temperature and longer time in DES pretreatment
were the best parameter in giving the severe effect on the morphology of fiber in terms of removal of silica and left
the fiber with lots of cavities. From XRD analysis, DES gave an effect to the degree of crystallinity of cellulose. At
temperature 110ºC, DES successfully reduced the cellulose crystallinity while lower temperature (80ºC), an increase
in cellulose crystallinity was observed, when compared with untreated fiber. From FTIR analysis, DES can not
remove the functional group of lignin and hemicellulose completely or successfully but it is believed that DES can
expose the structure of cellulose. Upon sugar analysis, NaOH pretreatment produce the highest sugar, followed by
DES pretreatment and then untreated fiber (raw). Hence, further investigation need to be done by treating untreated
fiber with different range of temperatures, time and also use other combination of DES which is choline chloride and
glycerol to see whether these parameters can help in increasing sugar production.

Acknowledgements

The authors would like to thank School of Chemical Sciences and Food Technology, Faculty of Science and
Technology, Universiti Kebangsaan Malaysia for providing the working area, Yayasan Sime Darby for providing
financial support which grant no: ST-2014-017 and Centre for Research Instrumentation Management (CRIM),
Universiti Kebangsaan Malaysia for the instruments used in the analysis throughout the study.

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