Biomass Conversion and Biorefinery (2018) 8:255–263

https://doi.org/10.1007/s13399-017-0299-x

ORIGINAL ARTICLE

Moderate pretreatment of oil palm empty fruit bunches for optimal

production of xylitol via enzymatic hydrolysis and fermentation
Budi Mandra Harahap 1 & Made Tri Ari Penia Kresnowati 1

Received: 24 January 2017 / Revised: 7 September 2017 / Accepted: 10 September 2017 / Published online: 24 January 2018
# Springer-Verlag GmbH Germany, part of Springer Nature 2018

Abstract
Moderate pretreatment methods were evaluated on oil palm empty fruit bunches (OPEFB) in order to obtain xylose-rich
hydrolysate for xylitol production via enzymatic hydrolysis and fermentation. Assessments on the effects of catalysts
(autohydrolysis/water, acetic acid, or ammonia) and the corresponding concentration and pretreatment time at moderate
pressure/temperature were conducted while the process performance was evaluated by enzymatic hydrolysis. Most hemicellulose
was still retained in the pretreated solid at the evaluated pretreatment conditions, hydrolysis of both the pretreated solid and spent
liquor are necessary to obtain most of xylose from OPEFB. Pretreatment using 5% ammonia gave the highest xylose yield;
however, great amount of acid would be needed for pH adjustment for the following enzymatic hydrolysis process and thereby
autohydrolysis was preferred. The optimum yield was obtained from autohydrolysis at 1.5 barg/127.9 °C for 60 min, which gave
xylose yield of 0.085 g xylose/g OPEFB (or 39.1% of hemicellulose). The obtained hydrolysate could be directly used as
substrate for fermentation.

Keywords Acetic acid . Ammonia . Autohydrolysis . Moderate condition . Oil palm empty fruit bunch . Pretreatment . Xylose

1 Introduction can be converted to xylitol [9], which is a low caloric sweetener

Oil palm empty fruit bunches (OPEFBs) are the main biomass
waste of palm oil industries. About 1.1 kg of OPEFB is co-
produced along with every kilogram of crude palm oil (CPO)
[1, 2]. Conversion of biomass to biochemicals, particularly
with high added value products via fermentation is currently
a potential alternative for overcoming the environmental
problem.
Similar to other lignocellulosic biomass, OPEFB has complex
structure which consists of cellulose (36.59–43.39%), hemicel-
lulose (14.62–24.97%), and lignin (21.69–34.37%) [3–6].
Cellulose is a homopolymer that comprises of monomeric sugar,
glucose, whereas hemicellulose is a heteropolymer that com-
prises of, in particular in OPEFB, mainly xylose (82.80%) [7].
These polymers can be degraded via enzymatic hydrolysis and
subsequently fermented to bioproducts [8]. For example, xylose
* Made Tri Ari Penia Kresnowati
kresnowati@che.itb.ac.id
1
Microbiology and Bioprocess Technology Laboratory, Chemical
Engineering Department, Institut Teknologi Bandung, Labtek X
building, Jalan Ganesha 10, Bandung 40132, Indonesia
that can be used as a sugar substitution. Xylitol can be naturally
found in fruits [10] and vegetables [11]. Xylitol fermentation has
been studied, especially in yeast Candida [12–15] and
Debaryomyces [16, 17].
The complex structure of OPEFB obstructs the access of
hydrolyzing enzymes, such as cellulase and hemicellulase, to
the polymeric carbohydrates; thus, pretreatment of biomass is
required [18]. Some alternative pretreatment methods were
reported, for example by using hot water/hydrothermal pre-
treatment or by adding other chemicals such as acids, or bases
as catalyst that was accompanied by heating [19–21].
Increasing the pretreatment condition was reported to im-
prove the efficacy, but it also induced the formation of inhib-
itory compounds that may disrupt further processes [22, 23].
Another important note is that lignocellulose pretreatment de-
creased xylose recovery; for example, the use of strong acid
may inflict further decomposition of xylose [24]. Overall, the
use of water, or the so-called autohydrolysis process, or weak
acid as the catalyst in particular at moderate pretreatment con-
dition offers an advantage.
Other lignocellulosic constituent, lignin, may also obstruct
the enzyme performance in degrading hemicellulose. General
technique for destructing the recalcitrant structure of lignin is
256
pretreatment using strong base [25, 26] or high concentration
of ammonia [27–29]. These compounds are able to dissolve
lignin derivative fragments, i.e., phenolic compounds, to liq-
uid fraction [30]. However, during base pretreatment, acids,
monomeric, and oligomeric sugars from depolymerization of
hemicellulose were also detected in the liquid fraction of the
pretreatment process [31]. Furthermore, these soluble pheno-
lic compounds can inhibit hydrolysis [32] and fermentation
[33]. In order to minimize the occurrence of further inhibition
and to minimize the dissolution of hemicellulose, dilute alka-
line such as aqueous dilute ammonia can be used.
Despite the numerous studies conducted on pretreatment of
lignocellulose, including OPEFB, the pretreatment method
focusing on xylose recovery from OPEFB has been
overlooked. It is the aim of this research to obtain the optimum
moderate OPEFB pretreatment method, with the goal for pro-
ducing xylitol from OPEFB through hydrolysis and fermen-
tation. The research covered the evaluation of different cata-
lysts that were weak acid, alkaline, and autohydrolysis, and
pretreatment conditions such as pressure and time. The opti-
mum method was then applied for preparing OPEFB hydro-
lysate and xylitol fermentation.
2 Materials and methods
2.1 Raw material
In this research, OPEFB was provided by oil palm mill PT
Incasi Raya, located in West Sumatera, Indonesia. OPEFB
was prepared by washing with tap water followed by sun
drying. Dried OPEFB was ground using disk mill and sieved
to obtain OPEFB particles with the maximal size of 60 mesh.
Lignocellulosic composition of the OPEFB particles was
identified by Chesson method [34], showing that the OPEFB
used in this research composed of 43.54 ± 2.83% cellulose,
19.14 ± 0.59% hemicellulose, and 19.83 ± 4.37% lignin.
2.2 Pretreatment
The OPEFB was pretreated using water (autohydrolysis) or
two different catalysts: acetic acid (J.T. Baker, p.a.) and am-
monia (Merck, p.a.) at various concentrations (2.5, 5, and
7.5% (w/w) of acetic acid or 1, 3, and 5% (w/w) of ammonia).
The mixture of 10 g dry OPEFB and liquid solution was
heated using autoclave and was kept at certain pressure, i.e.,
1 or 1.5 barg, for a certain period of time, i.e., 15, 30, 60, or
90 min. The increase in system temperature corresponded to
the applied pressure, giving final temperature of approximate-
ly 120.2 and 127.9 °C, respectively. Unless specifically indi-
cated, OPEFB solid to liquid ratio of 25% (w/v) was applied in
the experiments. Two fractions were formed after pretreat-
ment: pretreated OPEFB solid and spent liquor. Performance
Biomass Conv. Bioref. (2018) 8:255–263
of the pretreatment method was evaluated by the yield of
xylose from OPEFB and hemicellulose conversion which
were calculated from the obtained concentration of xylose in
the hydrolysate solution, following Eqs. (1–3) [35]:
Sugar concentration  working volume
Sugar yield ½g=g OPEFB ¼
Initial OPEFB
ð1Þ
Total glucose recovery from cellulose ½%
Glucose concentration  0:9  working volume x100%
¼
Cellulose fraction of OPEFB  initial OPEFB
ð2Þ
Total xylose recovery from hemicellulose ½%
Xylose concentration released  0:88  working volume x 100%
¼
Hemicellulose fraction of OPEFB  initial OPEFB
ð3Þ
All experiments were conducted in duplicate.
2.3 Enzymatic hydrolysis
Enzymatic hydrolysis was conducted using Cellic HTec2
(Novozyme). This enzyme was measured to have xylanase
activity of 68,491 U/mL, determined by xylanase assay using
1.0% larchwood xylan [36], as well as cellulase activity of 131
FPU/mL, determined by a filter paper assay using Whatman
cellulose filter paper [37].
The hydrolysis was performed in 300-mL erlenmeyer
flasks in a rotary shaker incubator that was set at 50 °C and
150 rpm for 48 h. The solution was adjusted to 10% (w/v)
solid to liquid ratio and pH 5 prior to hydrolysis, and 1 mL
of enzyme was used in every 100 mL hydrolysis solution.
Unless specifically indicated, the hydrolysis was conducted
to the whole pretreated solution containing the pretreated
OPEFB solid and the spent liquor.
2.4 Xylitol fermentation
2.4.1 Medium formulation
OPEFB hydrolysate was used as the carbon source for fer-
mentation. OPEFB hydrolysate for fermentation media was
prepared by pretreatment, which was conducted at optimum
condition obtained from the previous step of 2.2, followed by
hydrolysis using combination of Cellic HTec 2 (Novozyme)
and Cellic CTec 2 (Novozyme), which was conducted at
50 °C and pH 5 in a rotary shaker incubator which was pre-
set at 150 rpm for 72 h. Two by 25 g of dry OPEFB was used
for preparing hydrolysate for each fermentation experiment.
Other macronutrients and micronutrients for the fermenta-
tion were provided by presterilized inorganic salt solution
containing KH2PO4, MgSO4·7H2O, (NH4)2SO4, ZnSO4·
Biomass Conv. Bioref. (2018) 8:255–263
7H2O, CoCl·6H2O, MnSO4·4H2O, CuSO4·5H2O, CaCl2·
2H2O, Na2MoO4·2H2O, H3BO3, KI, citric acid,
Al2(SO4)3, and filtered sterilized vitamin solution following
[38, 39].
2.4.2 Fermentation
Microorganism used for the fermentation was Debaryomyces
hansenii ITBCC R85, which was obtained from the culture
collection of Microbiology and Bioprocess Technology
Laboratory, Chemical Engineering, Institut Teknologi
Bandung. D. hansenii inoculum solution was prepared in
YPX medium containing 0.5 g yeast extract, 1 g peptone,
and 1 g xylose in 100 mL water.
Fermentation for xylitol production was conducted in a
New Brunswick BioFlow 110 Bioreactor that was equipped
with temperature, pH, and agitation controller, at working vol-
ume of 1.2 L. Fermentation was controlled at 30 °C, pH 5, and
agitation of 450 rpm without air supply. Samples were taken
periodically every 24 h for 7 days.
2.5 Analytical method
Monomeric sugars (xylose and glucose) and metabolic prod-
ucts (ethanol and xylitol) in the pretreated OPEFB solution,
hydrolysate, and fermentation broth were determined using
HPLC with BioRad Aminex HPX-87X column at 60 °C using
5 mM H2SO4 as the eluent and using refractive index detector
that was set at 40 °C [39]. Data was processed using
Empower™ Chromatography Data Software (Waters Corp.,
Milford, MA).
3 Results and discussion
3.1 Comparison of pretreatment catalysts: weak acid,
weak alkaline, and autohydrolysis
The performance of pretreatment types and their correspond-
ing concentration were evaluated based on sugar concentra-
tion in OPEFB hydrolysate, as shown in Fig. 1. Despite the
applied pretreatment catalysts, the pH of the spent liquor was
observed to decrease compared to its initial condition before
pretreatment. The average change of pH after autohydrolysis,
pretreatment using acetic acid, and pretreatment using ammo-
nia were from pH 7.0 to pH 6.0, pH 3.0 to pH 2.0, and pH 11.0
to pH 10.0, respectively.
The autohydrolysis process gave the yield of xylose, which
was expressed as the amount of sugar in the hydrolysate so-
lution relative to total OPEFB used for pretreatment, of 0.02 g
xylose/g OPEFB. This number corresponded to the recovery
of 9% of the initial OPEFB hemicellulose as xylose in the
hydrolysate. In this case, depolymerization of hemicellulose
257
Fig. 1 The effects of pretreatment types and the corresponding catalyst
concentration on the yield of xylose in the hydrolysate (bar) and total
xylose recovery from hemicellulose (circle); pretreatment was conducted
at 1 barg for 15 min
and cellulose is catalyzed by hydronium ions (H3O+) and ac-
celerated by acetyl groups from hemicellulose structure with
increasing pretreatment condition.
As expected, the addition of catalyst, either the weak acid
or the weak base, improved the performance of pretreatment
process. The use of 2.5% acetic acid could increase the yield
of xylose up to 0.03 g xylose/g OPEFB, which corresponded
to the recovery of 13% of the initial OPEFB hemicellulose as
xylose in the hydrolysate. Further increase of acetic acid con-
centration did not significantly increase the obtained yield.
This might be caused by further decomposition of xylose into
furfural, which was an inhibitor of hydrolysis and fermenta-
tion. Furfural content in the spent liquor of acetic acid pre-
treatment was higher than autohydrolysis [40]. Excessive use
of acetic acid may thus reduce the xylose recovery.
The use of 1% ammonia gave the yield of xylose of 0.02 g
xylose/g OPEFB. The obtained yield of xylose was signifi-
cantly affected by the concentration of ammonia used.
Increasing the ammonia concentration to 5% increased the
yield of xylose to 0.04 g xylose/g OPEFB, which
corresponded to the recovery of 19.2% of the initial OPEFB
hemicellulose as xylose in the hydrolysate. This was the best
yield among the evaluated pretreatment configurations.
The use of ammonia in the pretreatment, however, caused
an increase in the pH of the solution, and it required a lot of
acid for pH adjustment for the subsequent enzymatic hydro-
lysis process. The excessive use of acid was neither effective
nor economical to be applied in larger scale. Autohydrolysis
process was preferred, compared to the ammonia
pretreatment.
Overall, the obtained xylose yield were in the range of
0.02–0.04 g xylose/g OPEFB, or maximally only 19.2% of
hemicelullose were recovered as xylose. The relatively low
number may be related to the moderate condition applied dur-
ing the pretreatment process, which was at 1 barg (120.2 °C)
for 15 min. For comparison, at similar pretreatment condition,
258 Biomass Conv. Bioref. (2018) 8:255–263

Mardawati et al. [7] obtained up to 27% xylose yield from

OPEFB hemicellulose, however, at much lower solid loading
ratio, of 5%. This number could still be improved, for example
by increasing the pretreatment condition, extending the hydro-
lysis time or increasing the amount of enzyme used.

3.2 Composition of pretreated OPEFB and spent

liquor

Previous studies on other lignocellulosic materials, such as

wheat straw and mischantus, showed that pretreatment caused
high reduction of hemicellulose in the pretreated solid where-
as most cellulose was still retained [29, 41]. It is of prime
importance to confirm whether after the pretreatment process
most of hemicellulose is still retained in the pretreated OPEFB
solid or dissolved in the solution, or in other words released to
the spent liquor. If most of hemicellulose is dissolved in the
solution, it might be sufficient for preparing the media for
xylitol fermentation by conducting the enzymatic hydrolysis
only to the spent liquor.
Figure 2a shows that all the spent liquor contained both
xylose and glucose, and that the measured concentration of
glucose was higher than xylose. The latter was indicated by
the yield of glucose from OPEFB that was higher than the
yield of xylose from OPEFB. On the other hand, the calculat-
ed xylose recovery from hemicellulose was higher than the Fig. 2 The effects of pretreatment types and the corresponding catalyst
calculated glucose recovery from cellulose. concentration on sugar yields (bar) and total sugar recovery (circle) in the
It was expected that due to crystalinity of cellulose and spent liquor (a) and pretreated OPEFB solid (b); pretreatment was con-
ducted at 1 barg for 15 min (white bars represent glucose yield, black bars
more amorphous structure of hemicellulose [22], hemicellu- represent xylose yield, white circles represent total glucose recovery from
lose would be more vulnerable to pretreatment. One might cellulose, black circles represent total xylose recovery from
argue that the observed higher glucose concentration in the hemicellulose)
spent liquor is originated from the degradation of hemicellu-
lose that often contains a certain amount of glucose. However, Despite all, only less than 1% of cellulose was recovered as
this is not possible to determine by most analytical methods glucose in the spent liquor hydrolysate and less than 1.5% of
and further it is assumed that glucose is only originated from hemicellulose was recovered as xylose in the spent liquor
cellulose degradation. Overall, the results indicated that dur- hydrolysate (Fig. 2a). On the other hand, up to 15% of hemi-
ing the moderate pretreatment, both hemicellulose and cellu- cellulose was recovered as xylose in the pretreated solid
lose were liberated and degraded further to xylose and OPEFB hydrolysate (Fig. 2b). These showed that after the
glucose. pretreatment process, most of hemicellulose was still retained
That the xylose recovery from hemicellulose was higher in the pretreated OPEFB solid. Therefore, hydrolysis of both
the glucose recovery from cellulose was resulted from the the pretreated solid and the spent liquor were needed to en-
lower percentage of hemicellulose than cellulose in OPEFB. hance the xylose recovery.
This result also showed that hemicellulose was more easily
degraded and transported from OPEFB to the liquor, whereas 3.3 The effects of pretreatment time and pressure
the cellulose was kept in the pretreated solid OPEFB.
In contrast to the results of overall hydrolysis in which both The effects of pretreatment time and pressure on xylose yield
the pretreated solid and the spent liquor were hydrolyzed si- are presented in Table 1. As was explained previously in Sect.
multaneously (Fig. 1), ammonia pretreatment gave the lowest 2, increase in pressure was aimed to increase the temperature.
xylose and glucose yield in the spent liquor (Fig. 2a). Indeed, Different responses were observed from autohydrolysis and
ammonia pretreatment was reported to retain higher hemicel- ammonia pretreatment of OPEFB. Neither extension of am-
lulose and cellulose in OPEFB and released lignin to the li- monia pretreatment time nor increase in ammonia pretreat-
quor instead [42]. ment pressure led to significant change in xylose yield. On
Biomass Conv. Bioref. (2018) 8:255–263 259

Table 1 Effects of pretreatment condition on the yield of xylose from

OPEFB

Type of Operation condition Yield of xylose Total xylose

pretreatment (g xylose/g recovery from
Pressure Time initial OPEFB) hemicellulose
(barg)/ (min) (%)
temperature
(°C)

5% 1/120.3 15 0.042 ± 0.006 19.186 ± 2.746

ammonia
5% 1/120.3 30 0.030 ± 0.009 14.011 ± 3.949
ammonia
5% 1.5/127.9 15 0.038 ± 0.001 17.467 ± 0.652
ammonia
Water 1/120.3 15 0.020 ± 0.000 9.043 ± 0.152
Water 1/120.3 30 0.025 ± 0.000 11.350 ± 0.120
Water 1.5/127.9 15 0.031 ± 0.001 14.329 ± 0.419

Data was obtained from duplicate experiments

the other hand, extension of the autohydrolysis pretreatment

time from 15 to 30 min was shown to increase the xylose yield
in the hydrolysate. Increase in autohydrolysis pretreatment
pressure from 1 to 1.5 barg was also shown to increase the
xylose yield in the hydrolysate. The effect of pretreatment
pressure was somehow more significant than the effect of Fig. 4 Time profile of sugar consumption (a) and product formation (b)
pretreatment time. Despite all, pretreatment with 5% ammonia for 168 h fermentation (circles represent glucose, cloves represent xylose,
still gave higher xylose yield than autohydrolysis at 1–1.5 barg triangles represent ethanol, and rectangles represent xylitol; white and
black symbols represent replicate experiments)
for 15–30 min.
Further extension of autohydrolysis pretreatment time to
destructions of the OPEFB structure due to hemicellulose deg-
60 min at 1.5 barg gave up to 40% of the OPEFB hemicellu-
radation and some extent of delignification. Consequently, the
lose conversion to xylose in the hydrolysate (Fig. 3). It is
hemicellulose could be more easily accessed by the enzyme
interesting to see that the extension of autohydrolysis pretreat-
and higher xylose yield was obtained. Meanwhile, constant
ment time to 60 min significantly improved the enzyme di-
xylose yield in the spent liquor might be the result of xylose
gestibility of the pretreated OPEFB solid whereas the xylose
decomposition, for example to furfural. The obtained results
yield in the spent liquor was observed to be relatively con-
also indicated that xylose decomposition already occurred at
stant. The extension of pretreatment time caused further
the applied operating condition.
In order to confirm this matter, additional experiments were
performed by extending the pretreatment time from 60 min at
1.5 barg to 90 min at the same pressure. No significant change

Table 2 Summary of ethanol and xylitol yield

Parameters Replication 1 Replication 2

Hydrolysate (g/g)
YXylose/initial OPEFB 0.085 0.067
YGlucose/initial OPEFB 0.213 0.186
Fermentation broth [g/g]
YXylitol/Xylose 0.398 0.407
YEthanol/(glucose + xylose) 0.223 0.261
Fig. 3 The effect of autohydrolysis time extended up to 60 min at
1.5 barg on the yield of xylose in the spent liquor (white), the pretreated YXylitol/initial OPEFB 0.020 0.025
OPEFB hydrolysate (black), and total recovery of xylose from YEthanol/initial OPEFB 0.042 0.064
hemicellulose
 260

Table 3 Comparison of sugar and xylitol yield in various biomass feedstock

No. Raw materials Pretreatment Hydrolysis Sugar concentration in hydrolysate Detoxification Fermentation Ref..

Xylose Glucose Prior concentration Xylitol yield

of hydrolysate (g xylitol/g xylose)

1 Corn cob Nonisothermal autohydrolysis Acid hydrolysis with 2% H2SO4 35.30 g/L 3.20 g/L Yes Yes 0.73 [47]
to reach 202 °C (130 °C; 15 min)
followed by posthydrolysis
with 0.5% H2SO4
(125 °C; 165 min)
2 Corn cob – Acid hydrolysis with 1% H2SO4 0.25 g/ga – Yes Yes 0.75 [48]
(121 °C; 60 min)
3 Brewery spent grain – Acid hydrolysis with 3% H2SO4 24 g/L 4.4 g/L No Yes 0.58 [44]
(130 °C; 15 min)
4 Rice straw – Acid hydrolysis with 1.5% H2SO4 17.40 g/L 4.6 g/L Yes Yes 0.53 [49]
(130 °C; 20 min)
5 Corn cob 0.1% H2SO4 High temperature steaming (HTS) 0.18 g/ga 0.026 g/gb Yes Yes 0.71 [50]
(room temperature; 24 h) (160 °C; 120 min)
6 Corn cob – Acid hydrolysis with 1% H2SO4 31.25 g/L 3.34 g/L Yes Yes 0.74 [51]
(121 °C; 40 min)
7 Corn cob – Acid hydrolysis with 1% H2SO4 21.98 g/L 3.56 g/L Yes Yes 0.37 [52]
(121 °C; 30 min)
8 Corn cob Soaking in 10% aqueous Acid hydrolysis with 1% H2SO4 33.42 g/L 1.47 g/L No Yes 0.60 [53]
ammonia (70 °C; 10 h)
9 Cashew apple baggase – Acid hydrolysis with 6% H2SO4 19.02 g/L 12.09 g/L No Yes 0.328 [54]
(121 °C; 15 min)
10 Sugarcane straw – Acid hydrolysis with 1% H2SO4 18.60 g/L 3.70 g/L Yes Yes 0.67 [55]
(121 °C; 20 min)
11 OPEFB Autohydrolysis Enzymatic hydrolysis 4.63 g/L 6.79 g/L No Addition of 0.24 [4]
(120 °C; 15 min) synthetic xylose
12 OPEFB Autohydrolysis Enzymatic hydrolysis 0.08 g/ga 0.09 g/gb No No 0.41 This study
(127.9 °C; 60 min)

a
Xylose recovery from the initial biomass used
b
Glucose recovery from the initial biomass used
Biomass Conv. Bioref. (2018) 8:255–263
Biomass Conv. Bioref. (2018) 8:255–263
was observed in total xylose recovery from hemicellulose.
About 27% decrease of xylose yield in the pretreated solid
was compensated by 30% increase of xylose yield in the spent
liquor. Extending pretreatment time longer than 60 min not
only caused further hemicellulose degradation and destruction
of OPEFB structure, but also further sugar decomposition.
This was also observed as some unknown peaks in the
HPLC analysis.
The obtained results were comparable with literatures.
Other researchers reported that autohydrolysis of oil palm fi-
ber frond at 121 °C for 60 min led to 31% hemicellulose
recovered as xylose in the hydrolysate [43]. At higher pretreat-
ment temperature and time, higher xylose yield was reported.
For example, Carvalheiro et al. [44] reported up to 61% xylose
yield from brewery’s spent grain at pretreatment condition of
190 °C for 5 min, whereas Boussarsar et al. [45] reported 78%
xylose yield from sugarcane bagasse at pretreatment condition
of 170 °C for 120 min.
3.4 Xylitol fermentation on OPEFB hydrolysate
We implemented the most optimum pretreatment method:
autohydrolysis at 1.5 barg for 60 min, followed by the enzy-
matic hydrolysis for obtaining the OPEFB hydrolysate to be
used as the substrate of fermentation for xylitol production.
The fermentation was performed to confirm whether the pre-
pared hydrolysate could be directly used to produce xylitol via
fermentation using yeast D. hansenii and to estimate the xyli-
tol that can be produced.
The prepared OPEFB hydrolysate contained 4–5 g xylose/
L and 9–9.5 g glucose/L (Fig. 4a). After 168 h fermentation,
the fermentation broth contained up to 1–1.5 g xylitol/L (Fig.
4b), which corresponded the yield of xylitol from xylose of
0.4 g xylitol/g xylose or the yield of xylitol from OPEFB of
0.020–0.025 g xylitol/g OPEFB (Table 2). In addition, ethanol
was also produced up to 3 g/L.
The yield of xylitol from xylose obtained in this experiment
was higher than previous results on xylitol fermentation using
OPEFB hydrolysate as the substrate, 0.098–0.24 g xylitol/g
xylose [4, 46]. This indicated that the applied pretreatment
method did not result in fermentation-inhibiting compounds.
Some literatures reported the use of the hydrolysate of other
lignocellulosic biomass as the substrate for fermentation pro-
ducing xylitol, shown in Table 3. In most literatures, xylose in
hydrolysate was obtained by diluted sulfuric acid hydrolysis
followed by xylitol production via fermentation. The yield of
xylitol obtained was in a range of 0.3–0.7 g/g xylose. By
different treatment method prior to fermentation, without the
use of sulfuric acid, this study was able to attain the yield of
xylitol in that range, 0.41 g/g. However, this number can still
be improved further. The overall yield of xylitol from the
lignocellulosic biomass was rarely reported. According to
[56], D. hansenii also has the ability to catabolize glucose to
261
ethanol under anaerobic condition with glucose/xylose ratio
above 30%. The higher glucose in the hydrolysate may inhibit
xylitol formation and divert to ethanol production. Under an-
aerobic condition, xylose also can be converted to ethanol
[57], increasing the overall yield of ethanol. The obtained
hydrolysate contained high glucose concentration, and conse-
quently, high ethanol concentration was produced during the
fermentation. In order to avoid high ethanol production,
higher xylanase specificity and lower cellulose activity en-
zyme or prior separation of glucose would be needed.
4 Conclusions
Compared to OPEFB pretreatment using either ammonia or
acetic acid as the catalysts, autohydrolysis was more econom-
ical and appropriate to be applied in particular for producing
xylose, the prime carbon source of xylitol fermentation, via
enzymatic hydrolysis. The obtained xylose yield would be
affected by the applied autohydrolysis conditions. However,
at the evaluated pretreatment conditions, only small part of
hemicellulose dissolved to liquor. Thus, enzymatic hydrolysis
should be performed to both the spent liquor and the
pretreated OPEFB solid, in order to recover more xylose from
OPEFB to be used for xylitol fermentation. Xylose recovery
was improved by extending autohydrolysis time and temper-
ature. The best results were obtained from autohydrolysis at
127.9 °C for 60 min, giving 39.1% xylose recovery from
hemicellulose. The obtained hydrolysate could be directly
used as substrate of fermentation for producing xylitol, giving
the maximum xylitol yield of 0.025 g/g OPEFB (0.4 g/g
xylose).
Funding information This research was funded by the Indonesian Oil
Palm Plantation Fund Management Agency grant entitled BEngineering
of Oil Palm Empty Fruit Bunches Hydrolysis for the Production of Green
Xylitol.^
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