Accepted Manuscript

Title: Cellulose and hemicellulose recovery from oil palm

empty fruit bunch (EFB) fibers and production of sugars from
the fibers

Author: Suriya Palamae Pongsathorn Dechatiwongse Wanna

Choorit Yusuf Chisti Poonsuk Prasertsan

PII: S0144-8617(16)31058-X
DOI: http://dx.doi.org/doi:10.1016/j.carbpol.2016.09.004
Reference: CARP 11531

To appear in:

Received date: 8-7-2016

Revised date: 1-9-2016
Accepted date: 2-9-2016

Please cite this article as: Palamae, Suriya., Dechatiwongse, Pongsathorn., Choorit,
Wanna., Chisti, Yusuf., & Prasertsan, Poonsuk., Cellulose and hemicellulose recovery
from oil palm empty fruit bunch (EFB) fibers and production of sugars from the
fibers.Carbohydrate Polymers http://dx.doi.org/10.1016/j.carbpol.2016.09.004

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 1

Highlights

 A new process for sugar recovery from oil palm empty fruit bunch fibers.

 Peracetic acid/alkaline peroxide used for 98% delignification at 35 C.

 Higher cellulose/hemicellulose retention than with other reported processes.

 Sugar yield was nearly 630 mg glucose g1 original dry EFB biomass.
 2

Cellulose and hemicellulose recovery from oil palm empty fruit bunch (EFB) fibers and

production of sugars from the fibers

Suriya Palamaea, Pongsathorn Dechatiwongseb,c, Wanna Choorita,c*, Yusuf Chistid, Poonsuk

Prasertsane

a
Biotechnology Program, Agricultural Technology, Walailak University, Tasala, Nakhon Si

Thammarat 80161, Thailand

b
Department of Chemical and Process Engineering, School of Engineering and Resources,

Walailak University, Tasala, Nakhon Si Thammarat 80161, Thailand

c
Oil Palm Research Unit, Walailak University, Tasala, Nakhon Si Thammarat 80161,

Thailand

d
School of Engineering, Massey University, Private Bag 11 222, Palmerston North, New

Zealand

e
Department of Industrial Biotechnology, Faculty of Agro-Industry, Prince of Songkla

University, Songkhla 90112, Thailand

*Corresponding authors. Tel: +66-75-672355; E-mail address: cwanna35@gmail.com

 3

Abstract

A sequential two-step treatment with peracetic acid (PA) and alkaline peroxide (AP) at mild

temperatures (20–35 C) removed more than 98% of the lignin from oil palm empty fruit

bunch (EFB) fiber. For each kilogram of EFB fiber treated, 200–250 g of a solids fraction and

120–170 g of a precipitate fraction were recovered after the treatment. Subsequent enzymatic

hydrolysis (45 C, 72 h) of the recovered solids (excluding the precipitate) resulted in a

glucose yield of 629.8±0.5 g per kg of the original dry EFB biomass. Enzymatic hydrolysis of

untreated EFB yielded only 3.00.0 g glucose per kg of dry EFB. Therefore, the PA-AP

pretreatment enhanced glucose recovery from EFB by nearly 210-fold. The total treatment

time was 93 h (a 9 h PA treatment at 35 C, a 12 h treatment with AP (20 C, 4% NaOH), 72

h of enzymatic hydrolysis).

Keywords: Cellulose; hemicellulose; lignin; delignification; enzymatic hydrolysis; oil palm

empty fruit bunch

 4

1. Introduction

Oil palm (Elaeis guineensis) is an important commercial crop. The process of

extracting crude oil from fresh palm fruit bunches leaves behind a lot of lignocellulosic

byproduct, mainly the fibrous empty fruit bunches (EFB). Nearly 1.1 metric tons of EFB

needs to be disposed of per ton of oil produced (Shinoj et al., 2011; Vijaya et al., 2008).

Around 37.7 million tons of EFB is generated annually worldwide (Cui et al., 2014; Sumathi

et al., 2008). This EFB biomass has limited uses. It is mostly either mulched to produce an

organic fertilizer or burnt to generate electricity (Palamae et al., 2014). Some EFB biomass is

processed into a substrate for mushroom production.

EFB typically comprises of cellulose (24–65%), hemicellulose (21–34%) and lignin

(14–31%) (Chang, 2014). In view of its high content of cellulose and hemicellulose, EFB is a

potential source of fermentable sugars for making biofuels such as ethanol and other products

via microbial processes (Cui et al., 2014; Hamzah et al., 2011; Hassan et al., 2013; Kim and

Kim, 2013; Piarpuzán et al., 2011; Ying et al., 2014).

The bioconversion of EFB fibers to fermentable sugars requires an initial

delignification pretreatment followed by enzymatic hydrolysis of the recovered cellulose and

hemicellulose to sugars. A prior delignification treatment is essential for effective production

of sugars as lignin prevents the hydrolytic enzymes from contacting much of cellulose and

hemicellulose (Cui et al., 2014). After delignification, the cellulose and hemicellulose parts of

the fibers are generally separated for effective use. Hydrolysis of hemicellulose produces

mostly pentose sugars (e.g. xylose) whereas hydrolysis of cellulose produces mostly the 6-

carbon sugar glucose. These sugars are generally used in separate fermentation processes as

glucose fermenting microorganisms do not usually ferment pentose. Hemicellulose is also a

source of xylooligosaccharides that have various applications (Ho et al., 2014).

 5

Enzymatic processes for producing sugars from cellulosic biomass are preferable to

thermochemical processes such as acid hydrolysis (Hassan et al., 2013; Piarpuzán et al.,

2011) as the latter produce unwanted byproducts that are inhibitory to microorganisms used

in subsequent fermentation (Lenihan et al., 2010). Enzymatic hydrolysis of lignocellulosic

biomass has been discussed extensively in the literature (Cui et al., 2014; Hamzah et al.,

2011; Hassan et al., 2013; Maitan-Alfenas et al., 2015; Menon and Rao, 2012; Van Dyk and

Pletschke, 2012; Yang et al., 2011).

In an earlier work, we developed a peracetic acid (PA) pretreatment of EFB to

consistently remove nearly 53% of the total lignin and leave behind most of the hemicellulose

and cellulose (Palamae et al., 2014). In the present work, the PA-treated delignified EFB

solids are further treated using a newly developed mild alkaline peroxide (AP) pretreatment to

improve their enzymatic conversion to fermentable sugars.

2. Materials and methods

2.1. Preparation of the EFB fiber

EFB fiber used in this work had been kindly provided by the Southern Palm Co., Ltd

(1978), Surat Thani province, Thailand. On receipt, the EFB fiber had a moisture content of

22% by weight. The as received EFB fiber was placed on trays and left in an oven (model

UM-500; Memmert GmbH, Schwabach, Germany) at 105 °C to reduce the moisture content

to around 13% by weight. The dried EFB fiber was stored at room temperature (35±3 °C).

The EFB fibers were ground in a hammer mill (Kritsakonloha Inc., Chachoengsao,

Thailand) driven by a 15 kW electric motor. The rotational speed of the mill was 2,900 rpm.

The ground material left the mill through a screen of 3 mm mesh size (catalog no. 8321810-

22, Thomas Scientific, Swedesboro, NJ, USA). A 9 A suction fan rotating at 1,460 rpm was

located at the exit of the hammer mill to facilitate the flow of the ground biomass. The ground
 6

fibers were sieved (number 30 sieve, 0.5 mm aperture, ASTM E11, the International ISO 565

scale) and the material that passed through was collected. This ground material was dried to a

constant weight in an oven at 105 °C and stored at room temperature (35±3 °C), until used

(Palamae et al., 2014).

A sample of the above EFB fiber was used to determine the acetone extractible

material in accordance with the method of the National Renewable Energy Laboratory

(NREL; version 08-03-2012) (Sluiter et al., 2008). Thus a specified mass of the EFB solids

was packed in a Soxhlet extractor (Foss SoxletTM 2050, Denmark) and extracted with acetone

such that 16.0 mL of acetone was used for each g of the EFB fiber. The extract was filtered to

remove any suspended solids and a measured volume of the clarified extract was evaporated

in a fume hood in a preweighed aluminum dish. Afterwards the aluminum dish was weighed

to determine the mass of the extracted material.

2.2. Preparation of the reagents

The following chemicals were used: acetone (AR reagent, 99.5% pure), glacial acetic

acid (AR reagent, 99.5% pure), sodium hydroxide (AR reagent, 99% pure), hydrochloric

acid (AR reagent, 99.5% pure) and sulfuric acid (AR reagent, 98% pure). All these had been

purchased from RCI Labscan (Bangkok, Thailand). Hydrogen peroxide (laboratory grade,

300.0 g H2O2 L1) and nitric acid (AR reagent, 700.0 g L1) were purchased from Ajax

Finechem, Thailand. Ethanol (commercial grade reagent, 95% pure) was purchased from

Union Chemical, Thailand.

Peracetic acid (PA) was prepared by mixing 600.0 mL of glacial acetic acid with

400.0 mL of an aqueous solution of hydrogen peroxide (300.0 g H2O2 L1) and adding 15.0

mL of sulfuric acid as a catalyst. The above reagent was prepared at room temperature (35±3

°C) by mixing for 72 h (Palamae et al., 2014). Nitric acid-acetic acid solution was prepared
 7

by mixing 90.0 mL of nitric acid (700.0 g nitric acid L1) and 732.0 mL of glacial acetic acid

and making up the volume to 1 L with deionized water (Wright and Wallis, 1998).

2.3. Peracetic acid pretreatment

Delignification of the EFB fiber was carried out in a 5 L stainless steel tank held in a

35 C thermostated water bath. EFB fiber (150.0 g) was mixed with 3 L of PA solution

(equivalent to 20.0 mL of PA solution per g of EFB fiber) and stirred (150 rpm) using an

overhead stirrer (VELP Scientific Inc., Bohemia, NY, USA) for 9 h. The resulting slurry was

filtered through a nylon mesh (45 µm pore size). The recovered EFB residue was washed

with distilled water, neutralized with 6 M NaOH, and further washed with distilled water. The

solids were then dried in an oven at 80 C for 48 h. These PA-pretreated solids, referred to as

―delignified EFB fiber‖, were stored at room temperature (35±3 C) until used.

2.4. Alkaline peroxide pretreatment

Alkaline peroxide (AP) pretreatment was carried out in 500 mL Erlenmeyer flasks.

Delignified EFB fiber (2.0 g) was mixed with 34.8 mL of sodium hydroxide (either 40.0 or

80.0 g NaOH per L of water) and 5.2 mL of hydrogen peroxide (350.0 g H2O2 L1). The

flasks were incubated at either 20 C or 40 C in an incubator shaker at an agitation speed of

90 rpm for 12 h. Then, the resulting mixture was filtered through the nylon mesh. The filtrate

was retained. The collected solids were washed with distilled water, neutralized to final pH of

5.5‒6.0 with 6 M hydrochloric acid, and further washed with distilled water. The final

collected solids fraction was dried in an oven at 80 C for 48 h.

The filtrate of the above process was adjusted to pH 5.5 with glacial acetic acid. Three

volumes of 95% ethanol were added and the filtrate was allowed to stand for 48 h at room

temperature (35±3 C). The precipitate formed was recovered by centrifugation (2,991 g, 10
 8

min). The precipitate was then washed with 70% ethanol on a suction filter, placed on an

aluminum foil as a thin layer and dried at 35 C for 24 h. The recovered solids were stored in

a desiccator for further analysis.

2.5. Analyses of EFB fiber before and after treatments

2.5.1. Lignin, cellulose and hemicellulose

The acid insoluble lignin and hemicellulose in EFB fiber, the delignified EFB fiber

and the solid fraction remaining after AP treatment were determined according to the methods

of the National Renewable Energy Laboratory (NREL; version 08-03-2012) (Sluiter et al.,

2008). Cellulose content of the samples was determined as described by Wright and Wallis

(1998).

2.5.2. Surface morphology

Scanning electron microscope (Jeol JSM 5600 SEM, JEOL Ltd., Tokyo, Japan) was

used to observe the surface morphology of the EFB fiber, the delignified EFB fiber and the

solid fraction remaining after the PA-AP treatment (4% NaOH, 20 C, 12 h). For this, a finely

ground sample of the relevant solid material was mounted on the metal stub, placed in the

sample holder and metalized with a thin layer of gold.

2.5.3. FT-IR spectroscopy

A small amount (approximately 1–2 mg) of the finely ground dried EFB fiber, the

delignified EFB fiber and the solid fraction remaining after the PA-AP treatment (4% NaOH,

20 C, 12 h) was each separately homogenized with 225 mg of KBr for 1 minute (Brienzo et

al., 2009) and pressed into a disc. The FT-IR spectra of these samples were recorded with 16

scans at the resolution of 4 cm‒1 between the wavenumbers of 400 and 4000 cm‒1 with a
 9

standard DTGS detector for transmittance measurements (PerkinElmer Spectrum One FT-IR

spectrometer; Perkin Elmer, Akron, OH, USA).

2.6. Hydrolysis

2.6.1. Enzymatic hydrolysis

The enzymatic hydrolysis or saccharification of the pretreated EFB residue samples

was performed in 100 mL Erlenmeyer flasks containing 25.0 mL of sodium citrate buffer (pH

4.8, 50.0 mM), 1250 mg of the sample and 25.0102 mL of the enzyme Accellerase® 1500 or

Accellerase® XC (Genencor International Inc., Palo Alto, CA, USA). (Accellerase® 1500 is a

crude enzyme mixture with multiple activities produced by a genetically engineered strain of

Trichoderma reesei. Accellerase® 1500 has strong cellulase and -glucosidase activities.

Accellerase® XC is a crude enzyme preparation produced by a strain of Penicillium

funiculosum. Accellerase® XC has endoglucanase and xylanase activities to supplement the

action of Accellerase® 1500.) The flasks were held in a shaker incubator (45 °C, 100 rpm) for

48 h. The flasks were sampled at specified intervals and the sample was immediately

quenched by heating in a boiling water bath for 10 minutes. The quenched sample was

centrifuged (20,220 g, 10 min) and the clear supernatant was used to measure sugars, acetic

acid, furfural and 5-hydroxymethylfurfural (HMF) by high-performance liquid

chromatography.

2.6.2. High-performance liquid chromatography (HPLC)

Glucose, xylose, acetic acid, furfural and HMF concentrations were determined using

an HPLC system (model 2690, Waters, MA, USA) equipped with a refractive index detector

(model 2414, Waters, MA, USA). A metaCarb H plus column (Varian, CA, USA) was used

to measure glucose, xylose and acetic acid. The column was held at 68 °C. The mobile phase
 10

was 42.5104 M H2SO4 at a flow rate of 4.0101 mL min‒1. Furfural and HMF were

determined according to the method of the National Renewable Energy Laboratory

(NREL/TP‒510–42623) (Sluiter et al., 2008). A Shodex Sugar SH1011 column (Showa

Denko K.K., Japan) was used. The column temperature was 65 °C. The mobile phase was

5.0103 M H2SO4 and the flow rate was 1.0 mL min‒1.

2.6.3. Enzymatic assays and protein determination

Activities of cellulase and xylanase in the commercial enzyme preparations (i.e.

Accellerase® 1500 and Accellerase® XC) were measured. The cellulase activity was

determined in terms of ―filter-paper units‖ (FPU) per milliliter of the original (undiluted)

enzyme solution (Adney and Baker, 2008). One FPU was the amount of enzyme required to

release 2 mg of glucose equivalent from 50 mg of Whatman no.1 filter paper in 1 minute at

50 °C and pH 4.8 (50.0 mM citrate buffer, pH 4.8).

The xylanase activity (U mL‒1 of enzyme solution) was measured as explained for

cellulose, but using xylan as substrate. One unit (U) of activity was defined as the amount of

the enzyme (mL) required to release 1 µmol of reducing sugar (expressed as xylose

equivalents) from birch wood xylan (300 µL of 1% birch wood xylan solution) in 1 minute at

50 °C and pH 4.8 (50.0 mM citrate buffer pH 4.8).

The protein concentrations in the enzyme solutions were estimated by the Coomassie

Brilliant Blue method (Bradford, 1976) using bovine serum albumin (BSA) as the standard.

2.7. Calculations

Mass of hemicellulose in solid fraction (MH), mass of cellulose in solid fraction (MC)

and mass of lignin in solid fraction (ML) were calculated using the following equations:

(1)
 11

(2)

(3)

where H (% wt) is hemicellulose in solid fraction Ei; m (g) is the mass of the fraction Ei; C (%

wt) is cellulose in Ei; and L (% wt) is lignin in Ei.

The percentages of lignin removal (Lr), hemicellulose (Hr) removal and cellulose (Cr)

removal from EFB fiber were calculated using the following equations:

(4)

(5)

(6)

In the above equations, the solid fractions Ei are as follows: E1 is original EFB fiber; E2 is

EFB solid residue of PA treatment; E3 is EFB solid residue of AP treatment of E2; E4 is the

same as E3 but using different conditions of AP treatment of E2; E5 is same as E3 but using

different conditions of AP treatment of E2; and E6 is same as E3 but using different conditions

of AP treatment of E2.

3. Results and discussion

3.1. Chemical compositions of the EFB fiber and delignified EFB fiber

Chemical composition of the EFB fiber (E1 in Table 1) prior to any treatment was as

follows (w/w, dry basis): 36.6±0.6% hemicellulose, 28.3±1.0% cellulose and 35.1±0.8%

lignin. These values were generally comparable to ranges reported by Chang (2014). The

hemicellulose content was <3% higher than the value of 34% reported by Chang (2014) and

lignin was about 4% more than the value of 31% reported by Chang (2014). As EFB fiber is a

natural product, its composition can vary (Ishola et al., 2014; Law et al., 2007; Shinoj et al.,

2011) depending on many factors including the maturity status of the fresh fruit bunches used
 12

to recover the EFB; the geographic location where the source plant was grown; and the

season of fruit collection (Palamae et al., 2014).

 13

Table 1

Chemical compositions of EFB fiber (E1), delignified EFB fiber (E2), and the solid fractions (E3–E6) obtained after pretreatment of the

delignified EFB fiber with AP.a

Sample Pretreatment Solid fraction Composition (% w/w) Mass of components in solid fraction (g)

conditions (g)b

Hemicellulose Cellulose Lignin Hemicellulose Cellulose Lignin

E1 None 100.0 36.6±0.6 28.3±1.0 35.1±0.8 36.6±0.6 28.3±1.0 35.1±0.8

E2 PA (35 C, 9 h) 49.8±0.1 37.5±1.5 42.5±0.6 15.7±0.4 18.7±1.5 21.2±0.6 7.8±0.4

E3 AP (20 °C, 4% 25.5±0.0 11.2±0.5 81.9±0.7 2.8±0.0 2.9±0.5 20.9±0.7 0.7±0.0

NaOH, 12 h)

E4 AP (40 °C, 4% 23.4±0.0 9.4±0.1 82.5±0.5 3.3±0.1 2.2±0.1 19.3±0.5 0.8±0.1

NaOH, 12 h)

E5 AP (20 °C, 8% 21.5±0.0 8.6±0.2 84.2±1.1 2.9±0.1 1.8±0.2 18.1±1.1 0.6±0.1

NaOH, 12 h)

E6 AP (40 °C, 8% 20.3±0.1 7.0±0.1 85.0±0.8 3.4±0.0 1.4±0.1 17.3±0.8 0.7±0.0

NaOH, 12 h)
 14

a
Average values  standard deviations of triplicate experiments.
b
Per 100.0 g of lignin, cellulose and hemicellulose.

E1, original EFB fiber (including the acetone extractables which constituted 1.1±0.04% of the total mass).

E2, EFB solid residue of PA treatment.

E3, EFB solid residue of AP treatment of E2.

E4, same as E3 but using different conditions of AP treatment of E2.

E5, same as E3 but using different conditions of AP treatment of E2.

E6, same as E3 but using different conditions of AP treatment of E2.

The fractions E2–E6 did not contain any acetone extractable material.
 15

An earlier work showed that pretreatment of EFB fiber with PA (1.0 g EFB fiber +

20.0 mL PA, 9 h at 35 C) effectively removed lignin but did not remove most of the

hemicellulose originally present in the EFB fiber (Palamae et al., 2014). In the present work,

the solid residue (i.e. E2, Table 1) left after an identical treatment with PA had 37.5±1.5%

hemicellulose, 42.5±0.6% cellulose and 15.7±0.4% lignin (Table 1). Thus, 77.8% of the total

lignin in the original EFB fiber was removed by the treatment. The PA pretreatment resulted

also in a loss of nearly 49% of the original hemicellulose in the EFB and nearly 25% of the

original cellulose in EFB was lost. In some of the other pretreatment methods reported for

EFB, the losses of hemicellulose and cellulose are too high. For example, 73.1 to 93.9%

delignification of EFB was reported by Formiline pretreatment (uses 58% to 88% formic acid

with boiling for 1.5 h) (Cui et al., 2014), but loss of hemicellulose ranged from nearly 67% to

90%. Similarly, a sequential pretreatment of EFB with 4% H2SO4 at 121 C for 60 min and

then with 40% NaOH at 121 C for 15 min removed 70% of the lignin, but nearly 88% of the

original hemicellulose was lost (Kim and Kim, 2013). Any loss of hemicellulose and

cellulose is of course not wanted as it reduces the yield of the fermentable sugars from the

original EFB.

3.2. Chemical composition of the delignified EFB fiber pretreated with AP

Two solid fractions were obtained after the AP treatment of delignified EFB fiber.

These fractions were the residual solids and the precipitate recovered from the filtrate of the

treatment process. Based on the initial 100.0 g dry weight of EFB fiber, the dry weights of the

residual solids and the precipitate fractions were 20.3–25.5 g (E3–6, Table 1) and 11.7–17.4 g

(E3–6, Table 2), respectively. As the total solids recovered after the PA treatment weighed

49.8 g (E2, Table 1), the sum of the insoluble solids and the precipitate recovered after the AP

treatment represented 70 to 78% recovery of the original solids.

 16

Table 2

Chemical composition of the precipitate fractions (E3–E6) obtained after pretreating the

delignified EFB fiber (E2) with AP.a

Sample Precipitate fraction (g)b Hemicellulose Cellulose (% wt) Lignin (% wt)

(% wt)

E3 12.5±0.5 86.0±1.6 4.3±0.3 4.1±0.0

E4 11.7±0.5 81.3±0.5 6.3±0.3 4.2±0.1

E5 17.4±0.7 84.2±0.3 4.8±0.3 2.6±0.1

E6 16.1±0.7 74.9±0.6 8.4±0.2 3.6±0.0

a
Average values  standard deviations of triplicate experiments.
b
Per 100.0 g of original material on dry basis.

The solids in the precipitate were predominantly (i.e. 75 to 86%) hemicellulose (Table

2) whereas the insoluble solids were mainly (82%) cellulose (E3–6, Table 1). Therefore the

treatment effectively separated the cellulose and hemicellulose components such that the

lignin content of these components was less than 5% (Table 1, Table 2). Of the four AP

treatments tested (Table 1), the treatment at 20 C with 4% NaOH for 12 h seemed best as the

precipitate of this treatment (E3, Table 2) was nearly 86% hemicellulose with <5% of

contaminating cellulose. Overall, >98% of the lignin was removed from the EFB biomass

after the combined PA and AP treatments, while nearly 74% of the original cellulose was

retained in the residual solids (E3, Table 1).

 17

This enrichment of the precipitate with hemicellulose was a consequence of the

tendency of mildly alkaline solutions to preferentially solubilize hemicellulose and lignin, but

not cellulose (García et al., 2013; Kim and Kim, 2013; Su et al., 2015). For example,

according to Garcia et al. (2013) nearly 39.4% of the hemicellulose present in the wheat

straw could be recovered by precipitation from the liquid fraction of an alkaline treatment

(10% alkali solution, 40 °C, 90 min) process.

3.3. Morphological changes

The SEM images of the EFB fiber, the delignified EFB fiber and the AP treated (20

°C, 4% NaOH, 12 h) delignified EFB fiber are shown in Figure 1. The untreated EFB fibers

had a relatively smooth surface (Fig. 1a) with the outer layer made of lignin that protected the

fiber against rupture. Both the PA treatment (Fig. 1b) and the PA-AP treatment (Fig. 1c)

substantially altered the fiber morphology. The PA treatment damaged and removed much of

the lignin-based outer layer (Fig. 1b) (Palamae et al., 2014; Wan Azelee et al., 2014).

Removal of lignin revealed the circular silica body deposits on the surfaces of the fibers (Fig.

1b). Silica bodies were solubilized by AP treatment solution to reveal the cellulose fibers

(Fig. 1c). Thus, the pretreatment exposed the cellulose fibers and opened up spaces between

them by removing some of the interfiber material. This allowed the hydrolytic enzymes to

better contact the fibers.

 18

Fig. 1. Scanning electron micrographs of: (a) the untreated EFB fiber (E1); (b) the delignified

EFB fiber (E2); and (c) the AP treated delignified EFB fibers (E3). A silica body and the hole

left by removal of a silica body are shown in (b). Magnification: 1300.

 19

3.4. FTIR spectra of EFB fractions

The FTIR spectra of EFB fiber, the delignified EFB fiber and the AP treated (20 °C,

4% NaOH, 12 h) delignified EFB fiber are shown in Fig. 2. These spectra were generally

consistent with expectations. For example, the absorption bands at 1433 and 1513 cm−1

associated with the aromatic rings in lignin (Sun et al., 2005) occurred only in the spectrum

of the EFB fiber sample, confirming effective delignification of the PA-delignified EFB fiber

and the same fiber further treated with AP (20 °C, 4% NaOH, 12 h).

100 E1
1433
a
E2
Transmittance (%)

90
E3
b
1733 c
80
897 467
2923 1638 656
70 1428
1328 1044
a = 1513 cm1
1168
b = 1378 cm1
1106
60 c = 1258 cm1 1064
3426

4000 3000 2000 1000 0

Wavenumber (cm1)

Fig. 2. The FTIR spectra of the untreated EFB fiber (E1), the delignified EFB fiber (E2) and

the AP treated (20 °C, 4% NaOH, 12 h) delignified EFB (E3).

 20

The absorption bands associated with hemicellulose, i.e. the bands at 1044, 1258,

1328, 1378, 1428, 1733, 2923 and 3426 cm−1 (Peng et al., 2012; Su et al., 2015; Xu et al.,

2007), did not appear in all spectra. For example, no bands were seen at 1044, 1258 and 1733

cm1 in the spectra of the delignified EFB fiber and the AP treated (20 °C, 4% NaOH, 12 h)

delignified EFB fiber because these samples had only low levels of hemicellulose. The

absorptions bands at 897, 1064, 1106 and 1168 cm−1 are associated with cellulose (Liu et al.,

2006) and were present in all three spectra in keeping with expectations.

3.5. Enzymatic hydrolysis of the EFB fibers

The measured activities of the two commercial enzymes used in this study are shown

in Table 3. The total protein levels of both enzyme preparations were similar (Table 3). In

terms of volumetric activities, Accellerase 1500 had a nearly 5.7-fold higher cellulase

activity but Accellerase XC had a nearly 12.5-fold higher xylanse activity. Comparing

specific enzymatic activity (i.e. activity µg1-protein), Accellerase 1500 had 5.3-fold higher

cellulase activity but Accellerase XC had a nearly 13.5-fold higher xylanse activity. In

effect, the enzymes had complementary activities.

Table 3

Protein concentrations and enzymatic activities of Accellerase 1500 and Accellerase XC

solutions.a

Activity Accellerase 1500 Accellerase XC

Filter paper activityb (FPU mL1) 37.0±0.0 6.5±0.1

Xylanase activityc (U mL1) 222.2±0.0 2777.8±0.1

Protein (µg mL1) 3200.8±0.0 2969.7±0.0

 21

a
Average values  standard deviations of triplicate experiments.
b
Cellulase activity based on filter paper substrate.
c
Xylanase activity based on birch wood xylan substrate.

The AP treated (20 °C, 4% NaOH, 12 h) delignified EFB fiber was used as substrate

to further evaluate the enzymes individually. As shown in Fig. 3, Accellerase® 1500 was

more effective than the other enzyme in hydrolyzing the substrate to fermentable sugars.

With Accellerase® 1500, in 48 h the hydrolysis medium attained a glucose level of 328 mg

g‒1 dry substrate and a xylose level of 30 mg g‒1 dry substrate. These values were more than

2-fold greater than achieved with Accellerase XC. In view of its better performance, further

tests were done only with Accellerase® 1500.

 22

400 40
Accellerase 1500 (glucose)
dry biomass)

dry biomass)
Accellerase 1500 (xylose)
Accellerase XC (glucose)
300 Accellerase XC (xylose) 30
1

200 20

1
Glucose (mg g

Xylose (mg g
100 10

0 0
0 12 24 36 48

Time (h)

Fig. 3. Production of glucose and xylose via digestion of the AP treated (20 °C, 4% NaOH,

12 h) delignified EFB (E3). The enzymes Accellerase 1500 and Accellerase XC were used

separately to digest EFB (E3) solids. EFB solids (12.5101 g) were suspended in 25.0 mL

buffer, pH 4.8, at 45 C and an agitation speed of 100 rpm. Either Accellerase 1500 or

Accellerase XC solution (250 L) was added to the slurry (25.0 mL) of the suspended

solids.

Using all the same conditions as in Fig. 3 but an Accellerase® 1500 solution volume

of 1.5 mL in 25.0 mL of the final reaction medium and a reaction time of 72 h, the

digestibility of the various substrates (i.e. E1, the EFB fiber; E2, the PA treated (35 C, 9 h)

EFB fiber; E3, the PA-AP treated EFB fiber) was evaluated. The data are shown in Table 4.
 23

The digestibility of the untreated substrate was exceedingly low (14%; E1, Table 4) because

lignin effectively prevented the enzyme from accessing cellulose and hemicellulose.

Delignification by PA treatment enhanced digestibility of substrate to nearly 60% (E2, Table

4), increased glucose yield from the substrate by more than 100-fold relative to the untreated

substrate and also greatly increased the xylose yield. The AP treatment of the delignified

substrate (E3, Table 4) improved digestibility relative to the untreated delignified substrate

(E2) by only about 17% but increased the yield of glucose by nearly 2-fold to 630 mg g1 of

the AP-PA treated substrate. The yield of xylose was reduced (Table 4) because of the loss of

hemicellulose as a consequence of the treatment process.

Table 4

Digestibility and hydrolysis products of the samples treated with 6% (v/v)a Accellerase 1500

enzyme.b

Sample Digestibility Hydrolysis products (mg g1 dry biomass sample)

(% ) Glucose Xylose Acetic acid

E1 14.1±1.6 3.0±0.0 0.0±0.0 12.8±0.4

E2 60.1±0.9 303.8±0.4 143.0±0.2 34.4±0.1

E3 70.1±0.5 629.8±0.5 61.2±0.1 3.2±0.0

a
1.5 mL of enzyme solution per 25.0 mL of the total reaction volume.
b
All treatments were for 72 h. Average values  standard deviations of triplicate experiments.
 24

In principle, any pretreatment method that improves accessibility of the substrate to

the enzymes without causing a loss of the substrate, is expected to improve production of

fermentable sugars from a given mass of the substrate (Su et al., 2015; Zhao et al., 2009).

Although higher sugar yields than reported in Table 4 may be obtained from EFB, they

necessitate severe and expensive pretreatments. For example, an exceptionally high glucose

yield of 708 mg glucose g1 dry pretreated EFB was reported by Kim and Kim (2013), but the

two-step pretreatment required the use of 4% H2SO4 at 121 C for 60 min and then with 40%

NaOH at 121 C for 15 min. This glucose yield was only about 13% higher than our result

(Table 4), but our treatment temperature was always 35 C. Another study that claimed to

optimize the treatment conditions for obtaining a maximum sugar yield from EFB fibers

reported a maximum yield of only around 310 mg glucose g1 treated EFB (Hassan et al.,

2013), or 49% of our yield.

In addition to glucose and xylose, the enzymatic hydrolysates contained low levels of

acetic acid (Table 4) but furfural and HMF were not detected. The acetic acid may have been

a carryover from the earlier PA and AP treatments, but more likely it was formed during

enzymatic hydrolysis. Enzymatic hydrolysis of xylooligosaccharides is known to produce

acetic acid (Balat, 2011).

In summary, the proposed two-step pretreatment had the following advantages

compared to the other available treatments: (1) it used readily available and inexpensive

industrial chemicals (peracetic acid, alkaline peroxide) compared to chemicals such as

sodium bisulfite (Tan et al., 2013), ammonia (Zulkiple et al., 2016), formiline (Cui et al.,

2014), and ionic liquids (Katinonkul et al., 2012) used by others; (2) the processing

temperatures were mild (35 C) compared to other treatments that have commonly used

temperatures of 100 C (Kim and Kim, 2013; Piarpuzán et al., 2011; Shamsudin et al.,

2012; Tan et al., 2013; Zulkiple et al., 2016); and (3) the processing was carried out at
 25

standard atmospheric pressure in vessels open to atmosphere, as opposed to expensive

pressurized closed-system operations used by others (Kim and Kim, 2013; Piarpuzán et al.,

2011; Shamsudin et al., 2012; Tan et al., 2013; Zulkiple et al., 2016). Notwithstanding its

mild and inexpensive processing conditions, the proposed process effectively removed more

than 98% of the lignin from EFB. Furthermore, the conversion of the recovered solids to

glucose was high, as previously noted, in comparison with some of the other high-yielding

processes.

4. Concluding remarks

Oil palm empty fruit bunch (EFB) fiber is a major biomass residue in some regions.

EFB has little commercial value and is mostly disposed of by burning as a boiler fuel in the

palm oil mills. Direct combustion of EFB is currently legally restricted as it generates air

pollution. In principle, using the simple and mild PA-AP pretreatment developed in this

work, the EFB can be used to produce higher value fermentable sugars. While other

pretreatment methods have been proposed for sugar recovery from EFB, they are impractical

in view of the severe processing conditions (e.g. typically temperatures of >70 C)

demanded. Glucose yield from the PA-AP pretreated fibers was 629.8±0.5 g glucose kg1 dry

EFB biomass. Enzymatic hydrolysis of untreated fibers yielded only low levels of glucose

(3.0±0.0 g kg1 dry EFB biomass). If only the PA treatment was used, the glucose recovery

was 303.8±0.4 g kg1 dry EFB. The PA-AP pretreatment therefore enhanced glucose yield by

nearly 210-fold relative to yield from the untreated EFB.

Acknowledgments
 26

This research was funded by Walailak University (Code 11/2558), The Royal Golden

Jubilee (RGJ) PhD Program (PHD/0105/2554 Code 6.Q.WL/54/A.1) and Thailand Research

Fund (RTA5780002). The authors gratefully acknowledge these supports.

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