International Journal of Technology 10(8): 1523-1532

ISSN 2086-9614 © IJTech 2019

DELIGNIFICATION OF OIL PALM EMPTY FRUIT BUNCH USING PERACETIC

ACID AND ALKALINE PEROXIDE COMBINED WITH THE ULTRASOUND

Heri Hermansyah1*, Dwini Normayulisa Putri1, Andiko Prasetyanto1, Zhofran Bintang

Chairuddin1, Meka Saima Perdani1, Muhamad Sahlan1, Masafumi Yohda2
1
Department of Chemical Engineering, Faculty of Engineering, Universitas Indonesia, Kampus UI
Depok, Depok 16424, Indonesia
2
Department of Biotechnology and Life Sciences, Faculty of Engineering, Tokyo University of
Agriculture and Technology, 2-24-16 Naka-cho, Koganei, Tokyo 184-8588, Japan

(Received: April 2019 / Revised: September 2019 / Accepted: November 2019)

ABSTRACT
Lignocellulosic biomass has great potential as a low-cost source of fermentable sugar for the
production of biofuels and high value organic acids. One potential biomass is oil palm empty fruit
bunch, since it has high cellulose and hemicellulose content. However, its lignin content can
hinder the access of cellulose and hemicellulose during the hydrolysis process. Therefore, an
effective pretreatment for the delignification of lignocellulose biomass should be considered to
reduce the lignin content. In this study, delignification of oil palm empty fruit bunch using
peracetic acid and alkaline peroxide solution combined with the ultrasound method is investigated
as a novel combination method of biomass pretreatment. The effect of pretreatment time was
observed by using a peracetic acid solution for 1, 3, 5, 7 and 9 hours, followed by an alkaline
peroxide solution for 4, 6, 8 and 10 hours. Based on the results, the best delignification was
achieved by pretreatment using peracetic acid pretreatment for 3 hours, followed by alkaline
peroxide pretreatment for 10 hours. Both pretreatments were assisted by the ultrasound method.
The results show hemicellulose, cellulose and lignin content of 14.13%, 77.27% and 8.6%
respectively. The lignin content was reduced by 68.73% and the cellulose content increased by
121.85%, relative to the untreated EFB. This result was considered as the best pretreatment, since
the pretreatment time was shorter and high cellulose content together with low lignin content was
achieved, which will improve the hydrolysis process.

Keywords: Delignification; Pretreatment; Oil palm empty fruit bunch; Ultrasound

1. INTRODUCTION
Lignocellulosic biomass has great potential as a low price raw material for the production of
biofuels and high value products, such as bioethanol, enzyme and organic acid (Hermansyah et
al., 2015; Hermansyah et al., 2018). One potential lignocellulose biomass is oil palm empty fruit
bunch (EFB), since annually it is widely generated worldwide, especially in Indonesia, but
unfortunately it has limited uses, only as an organic fertilizer or boiler fuel to generate electricity
(Palamae et al., 2014).
Typically, EFB is composed of 2465% cellulose, 2134% hemicellulose, and 1431% lignin
(Chang, 2014). Based on its composition, EFB can potentially be used as a raw material for
biofuels and organic acid production by utilizing the cellulose and hemicellulose content.

*
Corresponding author’s email: [email protected], Tel. +62-813-85399048, Fax. +62-21-7863515
Permalink/DOI: https://doi.org/10.14716/ijtech.v10i8.3464
1524 Delignification of Oil Palm Empty Fruit Bunch using Peracetic Acid and
Alkaline Peroxide Combined with the Ultrasound

Hemicellulose and cellulose can be hydrolyzed into simple sugars for fermentation into biofuels
and other products via microbial processes (Pattanamanee et al., 2012; Kim & Kim, 2013;
Sklavounos et al., 2013).
Fortunately, EFB is a great source of cellulose and hemicellulose, since the lignin content can
be removed, while the loss of hemicellulose and cellulose is kept to a minimum. Therefore,
effective pretreatment for the delignification of EFB should be considered in order to improve
the removal of lignin content.
In recent years, there have been many studies on the pretreatment of lignocellulosic biomass for
the delignification process, either chemically or physically. Chemical pretreatment has been
widely reported as a potential method for delignification, including acid and alkaline
pretreatments (Mosier et al., 2005). In acid treatment, the bonds between cellulose,
hemicellulose and lignin are broken down by H+ ions. The main objective of acid treatment is
to solubilize the hemicellulose fraction of the biomass (Alvira et al., 2010) and change the
structure of the lignin (Mosier et al., 2005) by solubilizing the acid soluble lignin. Otherwise,
the main objective of alkali treatments is to remove nearly all the lignin and some of the
hemicellulose. This treatment will have a great affect on the enzymatic hydrolysis of cellulose
to sugars (Taherzadeh & Karimi, 2008). Furthermore, in some chemical treatments, the agents
mentioned above are combined with oxidizing agents such as hydrogen peroxide and sodium
hypochlorite water (Nazir et al., 2013) to assist the delignification and depolymerization
processes.
Several researchers have succeeded in achieving the delignification of EFB by using chemical
treatment. It has been delignified simultaneously and consecutively by using NaOH and H2O2,
with lignin removal of 72% and 99% (Misson et al., 2009). Another chemical treatment has been
attempted using peracetic acid solution. In this approach, approximately 53% of the lignin was
removed, but nearly all the hemicellulose was retained (Palamae et al., 2014). On the other
hand, a recent study has employed a sequential treatment using peracetic acid (PA) solution and
alkaline peroxide (AP) solution, with around 92% of the lignin content from the EFB fiber
removed (Palamae et al., 2017).
Besides using chemical treatment, physical treatment has also been studied to improve the results
of lignocellulose biomass pretreatment. Previously, EFB has been successfully pretreated by
using a sequential dilute acid and microwave alkali pretreatment, resulting in high
delignification and a large amount of cellulose (Akhtar & Idris, 2017). In addition, ultrasound-
assisted dilute aqueous ammonia pretreatment has also been investigated for the intensification
of enzyme hydrolysis for corn cob, corn stover and sorghum stalk. It has been found that a
combination of ultrasonic pretreatment can increase the accessibility of cellulose in the biomass
and increase the enzymatic hydrolysis sugar yield (Xu et al., 2017). However, the combination
of ultrasound and chemical pretreatment for EFB is still limited. Therefore, this study will
modify the pretreatment method conducted by previous researcher which only using PA and AP
solutions (Palamae et al., 2017). The aim of this study is to observe the performance of the
delignification of EFB by employing a combination of chemical and physical treatments using
PA and AP solutions and assisted by the ultrasound method. The study will focus on the time
arrangement of the pretreatment in order to obtain the best delignification process.

2. METHODOLOGY
2.1. Materials
The EFB fiber was purchased from a local supplier (Palembang, Indonesia). Prior to use, it was
washed and sun-dried and then dried in an oven at 70°C for 24 hours. The samples were then
Hermansyah et al. 1525

ground and sieved using a sieve with a mesh number of 30 and 0.6 mm aperture before being
stored in a container at room temperature.
The following chemicals were used: glacial acetic acid 100%, sodium hydroxide, and
hydrochloric acid 37%, purchased from Merck KGaA (Darmstadt, Germany); sulfuric acid and
acetone, purchased from Mallinckrodt (Phillipsburg, NJ, USA); and hydrogen peroxide 35%,
purchased from PT. Wiloso Yasa Pratama (Jakarta, Indonesia).
2.2. EFB Pretreatment
The pretreatment process to perform the EFB delignification consisted of peracetic acid and
alkaline peroxide pretreatment; both pretreatments were assisted by the ultrasound method. A
schematic diagram of the pretreatment process is shown in Figure 1.
Oil Palm Empty Fruit Bunch
(EFB)
washed, dried, ground and sieved

EFB Pretreatment

Peracetic Acid Pretreatment Alkaline Peroxide Pretreatment

assisted by an ultrasonic processor assisted by an ultrasonic processor

Filtered Filtered

Neutralized Neutralized

Washed Washed

Dried Dried

Pretreated EFB

Figure 1 Schematic diagram of pretreatment process

2.2.1. Peracetic acid pretreatment
The PA solution was prepared by mixing glacial acetic acid with 30% w/w of hydrogen peroxide,
at volumes of 600 mL and 400 mL respectively, and adding 15 mL of sulfuric acid as a catalyst.
The solution was prepared at a room temperature of 35 ± 3°C and mixed for 72 h (Palamae et
al., 2014). Delignification of the EFB fiber was conducted in a 2 L Duran glass bottle held in an
ultrasonic processor (53 kHz, 90W). 75 g of the fiber was mixed with 1500 mL PA solution (20
mL of PA solution per gram of EFB fiber) and placed inside the ultrasonic processor set to 35°C,
for 1, 3, 5, 7 and 9 hours. The resulting slurry was filtered through a muslin cloth. The pretreated
EFB was then washed with distilled water, neutralized with 6 M NaOH, and then further washed
with distilled water. The solids obtained were dried in an oven at 80°C for 48 h. The PA-
pretreated solids, referred to as “delignified EFB fiber”, were stored at room temperature.
2.2.2. Alkaline peroxide pretreatment
AP pretreatment was conducted in a 500 mL Duran glass bottle inside a 35°C ultrasonic
processor (53 kHz, 90W). 10 g of delignified EFB fiber was mixed with 174 mL of sodium
hydroxide (40% w/w) and 26 mL of hydrogen peroxide (35% w/w). The temperature was
controlled at 35°C for 4, 6, 8 and 10 hours. The resulting slurry was then filtered through a
1526 Delignification of Oil Palm Empty Fruit Bunch using Peracetic Acid and
Alkaline Peroxide Combined with the Ultrasound

muslin cloth. Pretreated EFB was washed with distilled water, neutralized to a final pH of 5.5–
6.0 with 6 M hydrochloric acid, and washed again with distilled water. The final EFB solids
fraction was dried in an oven at 80°C for 48 h.
2.3. EFB Analysis
The content of the EFB fiber was determined by measuring the acetone extractable material,
hemicellulose content, and acid insoluble lignin contents. The methods used were based on
National Renewable Energy Laboratory (NREL), version 08-03-2012.
To measure the acetone extractives, 5 gr of EFB fiber (W0) was extracted using acetone (80 mL)
in a Soxhlet extractor. The extraction was performed at 90°C for 1 h; subsequently, fresh acetone
was used to wash the EFB fibers for 30 min, which were then air dried at room temperature. The
fibers were then oven dried at 105°C to a constant weight. The dried EFB fibers were then cooled
in a desiccator and weighed (W1). The acetone extractable contents E (%) of the EFB fibers were
calculated using the Equation 1 (Li et al., 2004):
W0  W1
E (%)   100 % (1)
W0
In order to measure the hemicellulose, 0.3 g acetone extracted dry sample (W 2) was put into a
15 ml test tube and 3 ml of NaOH (0.5 mol/L) added. The mixture was then heated at 80°C for
3.5 h. Subsequently, the mixture was cooled to room temperature and filtered under vacuum.
The solid residue was washed with distilled water until a pH level of 7 was reached. The residue
was then oven dried at 105°C to a constant weight, and the, dried residue cooled in a desiccator
and weighed (W3). The hemicellulose content H (%) of the EFB fibers was calculated using
Equation 2:
W 2  W3
H (%)   100 % (2)
W2

To determine the acid insoluble lignin content, 0.2 g acetone extracted dry sample (W 4) was
placed in a Erlenmeyer flask, and sulfuric acid (200 ml, 720 g/L) then poured gently into the
flask. The flask was then reacted at 30°C for 1 h. Next, 56 mL of distilled water was added to
the flask and then autoclaved at 121°C for 1 h. After cooling, the residues were filtered using
Whatman no. 1 filter paper and washed with distilled water. The washed residue was then oven
dried to a constant weight at 105°C, and the dried residue cooled in a desiccator and weighed
(W5). The contents of acid insoluble lignin L (%) in the original sample were calculated using
Equation 3:
W5
L (%)   100 % (3)
W4

As acetone extractives, hemicellulose, acid insoluble lignin and cellulose are the only
components found in EFB fiber (Di Blasi et al., 1999; Lin et al., 2010; Menon & Rao, 2012),
the cellulose content C (%) was generally calculated from the other measured values, as follows:
C (%)  100  H (%)  L (%) (4)
On the other hand, the EFB morphology was analyzed using a transmission electron microscope
(TEM) for untreated and pretreated EFB.
2.4. Calculation
The percentages of lignin removal, the decrease in hemicellulose and increase in cellulose were
calculated using Equations 57:
Hermansyah et al. 1527

L in E1  L in E1 i
Lignin removal (%)   100 (5)
L in E1
H in E1  H in E1 i
Decrease in hemicellulose (%)   100 (6)
H in E1
C in E1 i  C in E1
Increase in cellulose (%)   100 (7)
C in E1

3. RESULTS AND DISCUSSION

3.1. Untreated EFB Content
Prior to the pretreatment, the EFB content was analyzed in order to measure the composition of
cellulose, hemicellulose and lignin. Based on the results of the content analysis, as seen in Table
1, the untreated EFB contained 37.67% hemicellulose, 34.83% cellulose and 27.5% lignin.
These levels are comparable with the general range of EFB composition, which are 2465%
cellulose, 2134% hemicellulose and 1431% lignin (Chang, 2014). According to the results,
the cellulose and lignin content is in line with values previously reported, while the
hemicellulose content is 3.67% higher (Chang, 2014).
3.2. Pretreated EFB Content
After PA pretreatment, followed by AP pretreatment assisted by ultrasound, the EFB content
was again analyzed, the results of which are presented in Table 1.
Table 1 Pretreated EFB content
Composition Lignin Decrease in Increase in
Sample Pretreatment Condition (% w/w) Removal Hemicellulose Cellulose
H C L (%) (%) (%)
E1 None 37.67 34.83 27.50
E2 PA (35°C, 1 h) AP (35°C, 4 h) 37.53 43.07 19.40 29.45% 0.37% 23.66%
E3 PA (35°C, 3 h) AP (35°C, 4 h) 27.07 53.73 19.20 30.18% 28.14% 54.26%
E4 PA (35°C, 5 h) AP (35°C, 4 h) 29.33 62.82 7.85 71.45% 22.14% 80.36%
E5 PA (35°C, 7 h) AP (35°C, 4 h) 6.20 71.70 22.10 19.64% 83.54% 105.86%
E6 PA (35°C, 9 h) AP (35°C, 4 h) 7.50 71.45 21.05 23.45% 80.09% 105.14%
E7 PA (35°C, 1 h) AP (35°C, 6 h) 23.83 59.92 16.25 40.91% 36.74% 72.04%
E8 PA (35°C, 3 h) AP (35°C, 6 h) 23.20 62.45 14.35 47.82% 38.41% 79.30%
E9 PA (35°C, 5 h) AP (35°C, 6 h) 19.10 65.00 15.90 42.18% 49.30% 86.62%
E10 PA (35°C, 7 h) AP (35°C, 6 h) 7.33 72.77 19.90 27.64% 80.54% 108.93%
E11 PA (35°C, 9 h) AP (35°C, 6 h) 9.83 74.77 15.40 44.00% 73.90% 114.67%
E12 PA (35°C, 1 h) AP (35°C, 8 h) 15.60 61.50 22.90 16.73% 58.59% 76.57%
E13 PA (35°C, 3 h) AP (35°C, 8 h) 22.33 65.47 12.20 55.64% 40.72% 87.97%
E14 PA (35°C, 5 h) AP (35°C, 8 h) 18.97 69.28 11.75 57.27% 49.64% 98.91%
E15 PA (35°C, 7 h) AP (35°C, 8 h) 18.60 65.80 15.60 43.27% 50.62% 88.92%
E16 PA (35°C, 9 h) AP (35°C, 8 h) 27.00 61.75 11.25 59.09% 28.32% 77.29%
E17 PA (35°C, 1 h) AP (35°C, 10 h) 17.07 62.88 20.05 27.09% 54.69% 80.53%
E18 PA (35°C, 3 h) AP (35°C, 10 h) 14.13 77.27 8.60 68.73% 62.49% 121.85%
E19 PA (35°C, 5 h) AP (35°C, 10 h) 4.00 77.60 18.40 33.09% 89.38% 122.80%
E20 PA (35°C, 7 h) AP (35°C, 10 h) 14.93 69.77 15.30 44.36% 60.37% 100.32%
E21 PA (35°C, 9 h) AP (35°C, 10 h) 25.13 64.47 10.40 62.18% 33.29% 85.10%
E1 - Untreated EFB; E2-21 - Pretreated EFB with different PA and AP pretreatment times, assisted by the ultrasonic
processor (53 kHz, 90 W)

Based on these results, after pretreatment the lignin and hemicellulose content decreased in all
the samples, whereas the cellulose content increased. The decrease in lignin content varied
between 27.09% (sample E2) and 68.73% (sample E3), relative to the composition of untreated
EFB. The same trend was observed in the hemicellulose content, which decreased between
33.29% (sample E6) and 89.38% (sample E4), again relative to the composition of untreated
1528 Delignification of Oil Palm Empty Fruit Bunch using Peracetic Acid and
Alkaline Peroxide Combined with the Ultrasound

EFB. On the other hand, the increase in cellulose content varied between 80.53% (sample E2)
and 122.80% (sample E4).
These results mean that the modified method proposed in the study has successfully delignified
the EFB, as proven by the reduction in lignin content and the increase in cellulose content. This
is because pretreatment by the PA solution is highly selective in removing lignin, through several
mechanisms including hydroxylation of aromatic rings, oxidative demethylation, oxidative ring
opening, and epoxidation (Sundara, 1998; Song et al., 2013). In addition, pretreatment by AP
solution plays a role in removing hemicellulose (Palamae et al., 2014). Moreover, the
ultrasound-assisted method facilitates the disintegration and disruption of the EFB structure,
meaning a larger surface area is exposed by the solutions (Xu et al., 2017). The results of a
previous study also proved that the combination of chemical and mechanical/ physical methods
was able to improve the performance of biomass pretreatment and also enzymatic hydrolysis
(Aditiya et al., 2015).
3.3. Effect of Pretreatment Time on EFB Content
In the study, the effect of pretreatment time on EFB content was determined. A previous study
by Palamae et al. (2017) established that the optimum conditions for EFB delignification were
PA pretreatment for 9 hours at 35°C, followed by 12 hours of treatment with AP at 20°C, which
achieved 92.02% lignin content removal. However, the pretreatment time in the study is
relatively long and determination of the optimum conditions related to the pretreatment time
have not been made. Therefore, in this study the effect of PA pretreatment time on EFB content,
especially to the removal of lignin, will be considered.
PA pretreatment time varied between 1, 3, 5, 7 and 9 hours, while AP pretreatment time varied
between 4, 6, 8 and 10 hours. In order to enhance the EFB delignification process, PA and AP
pretreatment were combined with physical treatment using ultrasound. The results of EFB
content after the sequential PA-AP pretreatment assisted with ultrasound are presented in Figure
2.
The results in Figure 2(d) show that after 3 hours of PA pretreatment, followed by 10 hours of
AP pretreatment (E18), the hemicellulose, cellulose and lignin content were 14.13 %, 77.27 %
and 8.6 % respectively. These are considered to be the best conditions for the delignification
process, according to the criteria of high cellulose and hemicellulose content, and low lignin
content. This is because the low lignin and high cellulose content will be preferable for achieving
high sugar concentration in the enzymatic hydrolysis process, since the existence of lignin will
prevent the enzymes from accessing cellulose and hemicellulose (Hendriks & Zeeman, 2009).
Moreover, the overall pretreatment time is also reduced when compared to the previous study
by Palamae et al. (2017).
According to the results, a reduction in pretreatment time has been achieved compared to
previous research, with PA pretreatment for 9 hours, followed by AP pretreatment for 12 hours,
yielding 2.8% lignin content (Palamae et al., 2017). This is because the combination of PA and
AP pretreatment with ultrasound can help the destruction of the cell wall structure (Kumakura
& Kaetsu, 1983). In this way, the delignification process can be improved. Besides, ultrasonic
pretreatment will improve the specific surface area, reduce the degree of polymerization, and
increase the biodegradability of lignocellulosic biomass (Mason & Peters, 1991). Therefore, the
pretreatment time can be reduced with the assistance of ultrasound.
Hermansyah et al. 1529

Hemicellulose Cellulose Lignin Hemicellulose Cellulose Lignin

100 100

74.77
72.77
71.45
71.7
80 80

62.82

62.45
59.92

65
53.73
EFB Content

EFB Content
60 60
43.07
37.53
(%)

(%)
29.33
27.07

40 40

23.83
21.05

23.2
22.1

19.9
19.1
16.25
19.4

19.2

14.35

15.9

15.4
9.83
20 20

7.33
7.85

7.5
6.2

0 0
PA PA PA PA PA PA PA PA PA PA
1 hour 3 hours 5 hours 7 hours 9 hours 1 hour 3 hours 5 hours 7 hours 9 hours
(a) (b)
Hemicellulose Cellulose Lignin
Hemicellulose Cellulose Lignin
100
100

77.27

77.6

69.77
69.28

64.47
80

62.88
65.47

80
65.8

61.75
61.5

EFB Content
EFB Content

60 60
(%)
(%)

40

25.13
40
22.33

20.05
22.9

27
18.97

17.07

18.4
18.6

14.93
14.13

15.3
15.6

15.6
11.75

11.25
12.2

10.4
20 20
8.6
4

0 0
PA PA PA PA PA PA PA PA PA PA
1 hour 3 hours 5 hours 7 hours 9 hours 1 hour 3 hours 5 hours 7 hours 9 hours
(c) (d)
Figure 2 Effect of pretreatment time on EFB content: (a) 4 hours; (b) 6 hours; (c) 8 hours; (d) 10 hours
of AP pretreatment
3.4. Comparison of Results from the Previous Study
A comparison of the results from the previous study by Palamae et al. (2017) is presented in the
Table 2.

Table 2 Comparison of untreated and pretreated EFB content

Composition (% w/w)
Pretreatment Condition Ref.
Hemicellulose Cellulose Lignin
None 36.60 28.30 35.10 (Palamae et al., 2017)
PA (35°C, 9 h) AP (20°C, 12 h)* 11.20 81.90 2.80 (Palamae et al., 2017)
None 37.67 34.83 27.50 This study
PA (35°C, 3 h) AP (35°C, 10 h)** 14.13 77.27 8.60 This study
*
without ultrasound-assistance; **with ultrasound-assistance
Based on the table, the results of this study show 68.73% lignin removal compared to the
composition of untreated EFB. On the other hand, the cellulose content is 77.27%, which is an
increase of 121.85% relative to the composition of untreated EFB; however, the loss of
hemicellulose is 62.49%. Compared with the previous study, after the sequential PA and AP
pretreatment, the lignin content removed was 92.02%, while the cellulose content increased by
1530 Delignification of Oil Palm Empty Fruit Bunch using Peracetic Acid and
Alkaline Peroxide Combined with the Ultrasound

189.4% and that of hemicellulose decreased by 69.39%, again relative to the composition of
untreated EFB (Palamae et al., 2017).
3.5. Morphology Analysis
To ensure a thorough experiment, TEM analysis was conducted to further understand the effect
of pretreatment on the morphology of EFB. The morphology of untreated and pretreated EFB is
shown in Figure 3.

(a) (b)
Figure 3 Morphology of: (a) untreated EFB; (b) pretreated EFB (E18)
Based on the results of the TEM imaging, there is a visual difference between the untreated and
pretreated EFB (E18: 3 hours PA pretreatment and 10 hours AP pretreatment). The reduced
contrast of the cell walls by pretreatment is a result of lignin removal, while the decreased
staining density indicates reduced lignin content in the cell wall following the pretreatment
process. Lignin enriched regions are black to dark gray in color, while hemicellulose and
cellulose enriched regions are lighter gray. The disruption of the biomass structure is caused by
the ultrasound. This imaging observation result signifies the effect of ultrasonic assistance on
the PA and AP pretreatment.

4. CONCLUSION
Delignification of EFB has been successfully achieved in this study. The best delignification
was obtained by pretreatment using PA for 3 hours, followed by AP pretreatment for 10 hours
and assisted by ultrasound. Hemicellulose, cellulose and lignin content of 14.13%, 77.27% and
8.6%, respectively was achieved. After pretreatment, lignin content was reduced by 68.37%,
while that of cellulose increased by 121.82%, relative to the composition of untreated EFB.
Besides, the overall pretreatment time was also reduced if compared to the previous study by
Palamae et al. (2017).

5. ACKNOWLEDGEMENT
The authors are grateful for the research support provided by Universitas Indonesia and the
Ministry of Research, Technology and Higher Education Republic of Indonesia through
International Research Collaboration Grant with Grant Number NKB-
1778/UN2.R3.1/HKP.05.00/2019.
Hermansyah et al. 1531

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