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The 2nd International Conference on Food and Agricultural Sciences 2023 IOP Publishing
IOP Conf. Series: Earth and Environmental Science 1377 (2024) 012005 doi:10.1088/1755-1315/1377/1/012005

Production of ethanol from oil palm trunk by simultaneous

saccharification and fermentation process at bench scale

A E Tjahjono*, D P Meidiawati, D Paramitasari, Suparman, B Rustiaty, Musa, Y

S Pramana, Abdurachman and B Triwiyono
Research Center for Agroindustry, National Research and Innovation Agency (BRIN),
Cibinong, Bogor, West Java, Indonesia

*Corresponding author: [email protected]

Abstract.A largenumber of oil palm trees are replanted every year. Considering the extent
of replanting and OPT's high starch and sugar content, it was determined to be a potential
raw material for bioethanol production. This study conducted ethanol production by
simultaneous saccharification and fermentation of starch-rich powder extracted from oil
palm trunks. Fermentation was conducted on a 100 L bench scale to evaluate the feasibility
and further development of the process before the process is implemented at a larger scale.
The preparation of the starch-rich flour involves shredding, drying, and screening of the
trunks until the starch-rich powder passes through an 80-mesh sieve. Furthermore, starch
was liquefied and partially saccharified. Subsequent saccharification was performed
simultaneously with fermentation. The initial total sugar content was adjusted to 15 % w/v
and starch was enzymatically hydrolyzed using commercial α-amylase (Liquozyme) and
glucoamylase (Novozyme) produced by NOVO. Fermentation was performed using
Saccharomyces cerevisiaeHakken No. 1. Since the starch-rich flour contains useful
nutrients and growth factors for microbes, the fermentation medium only required the
addition of a nitrogen source (urea 1 g/L). The ethanol content after fermentation was
approximately 8.7 % v/v with a fermentation ratio of 88.8 %. These results were obtained
at a fermentation time of 60 h. One OPT yields 80 kg of starch-rich powder, equivalent to
33 liters of bioethanol.

1. Introduction
Currently, oil palm plantations in Indonesia cover more than 16 million hectares and an enormous number of
trees (over 25 years old) have been felled for replanting[1]. Oil palm trunks (OPT) generated from replanting
activities are usually left on the site, mostly without any effort to utilize it. If replanting is carried out on plants
that are 25 years old, then every year there will be 600 thousand hectares of plantation should be replanted. OPT
contains high amounts of starch.Several publications and our preliminary studies show that OPT between 25 and
30 years old contains approximately 15- 31 % starch [2, 3,4].
The anatomy of the oil palm trunk consists of vascular bundles and parenchyma. Starch is stored in
parenchyma cells, whereas vascular bundles contain a high portion of lignin [3]. The location of starch in the oil
palm trunk is the basis for its separation from the fiber. In addition to containing a considerable amount of starch,
OPT also contains various nutrients. Therefore, separating starch from other OPT components by the dry method
allows some of these nutrients to be included in the starch-rich compound and will provide sufficient nutrients
for the bioconversion of starch into various useful products[5,6].

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The 2nd International Conference on Food and Agricultural Sciences 2023 IOP Publishing
IOP Conf. Series: Earth and Environmental Science 1377 (2024) 012005 doi:10.1088/1755-1315/1377/1/012005

Considering the large area of oil palm plantations that must be replanted every year, while on the other hand,
Indonesia requires a large amount of bioethanol for gasoline substitution, OPT starch can be a candidate as a raw
material for bioethanol. Our preliminary study showed thatstarch and other sugars, mostly glucose,are
fermentable [7]. In this study, hydrolysis and fermentation of OPTstarch were conducted at a bench scale of 100
Liter to evaluate the feasibility and further development of the process. The bioconversion of starch to glucose
and finally to ethanolwas conducted by simultaneous saccharification and fermentation (SSF). SSF process
combines saccharification and fermentation steps in one vessel [2, 8]. Hydrolysis ofstarch was conducted
enzymatically, and fermentation was conducted using the yeast Saccharomyces cerevisiae.

2. Materialsand methods
2.1. Preparation of OPT’s starch-rich flour
The OPTs with their bark removed were crushed using a cutting mill. The crushed OPT was then dried
and screened using a 1 mm sieve hole. The parenchyma is the fraction that passes through a sieve. The
parenchyma fraction was then screened for a second time using an 80-mesh sieve. The flour that
passed through the filter was starch-rich flour. The OPT’s starch-rich flour contained≥ 70 % Total
Sugar.
Two commercial-grade starch-degrading enzymes, Liquozyme Supra and Dextrozyme GA, were
obtained from Novozyme. Yeast Saccharomyces cerevisiaeHakken No. 1 is a collection from the
Starch Laboratory – National Agency for Research and Innovation, Republic of Indonesia.

2.2. Preparation of inocula

Fermentation was carried out on a 100 L scale. The inoculum prepared was a 4% fermentation volume.
Saccharomyces cerevisiae Hakken No. 1 was inoculated into 40 ml molasses medium (urea, 2 g/L;
NPK, 0.2 g/L; molasses, total sugar 7 %). The mixture was incubated at 32 oC for 18 hours, transferred
into 4 L of fresh molasses medium, and incubated for another 18 hours [9].

2.3. Preparation of SSF

The SSF medium was prepared as follows. The starch-rich flour with a final total sugar content of 15%
w/v, was mixed with other medium component, namely urea (1 g/L). The mixtures were liquefied
using Liquozyme Supra 0.05%v/v starch at 90 oC for 60 min. The suspensions were then saccharified
using Dextrozyme GA (0.2%v/v starch) at 60 oC for 2 hours. After the temperature was lowered to 32o
C, inocula were added to the fermentation broth. Fermentation was performed for 72 hours [9].

2.4. Analysis
Yeast cell concentration was determined by directly counting individual cells under a light microscope
at a magnification of x400 using a hemocytometer. The total sugar content (TS) and reducing sugar
content (RS) were analyzed using the Modified Somogyi method [8]. Ethanol was measured using a
Shimadzu HPLC equipped with a refractive index detector and Shimpack SCR 101H column. Samples
from the fermentation broths were first diluted and centrifuged (1300 rpm for 15 min), and the
supernatant was injected into the HPLC [9]. The analytical values are shown as the means of duplicate
analysis.

3. Results and discussions

Analysis of the sugar content in the OPT showed that the reducing sugar content, mostly glucose and
sucrose, fructose, and xylose were present in much smaller amounts. Similar results were obtained by
Kosugi et al.[6], but Tomimura showed that glucose and xylose were the main components [10]. This
result is possibly caused by differences in sample preparation. The total sugar content was between
15% and 39%. The sugar content of the OPT is shown in Table 1The highest total sugar content was
observed at the top level and inside part (core) of the trunk. The total sugar content was gradually
increase as the trunk height gets higher (Figure 1). Similar results were obatained by several researcher
[10, 11, 12]. Starch and sugars are stored inside the parenchyma cells as food reserve [12].

2
The 2nd International Conference on Food and Agricultural Sciences 2023 IOP Publishing
IOP Conf. Series: Earth and Environmental Science 1377 (2024) 012005 doi:10.1088/1755-1315/1377/1/012005

Table1. Sugar content of OPT The palm trees were cut down at the age of 26 years.
Upper part of the Middle part of the Lower part of the
Parameter
trunk (%) trunk (%) trunk (%)
Starch 34.5 25.14 17.24
Reducing Sugar 9.28 5.21 1.01
Glucose 7.81 4.56 0.78
Sucrose 0.38 0.20 0.08
Fructose 0.21 0.14 0.10
Xylose 0.17 0.13 0.09

As shown in Table 1, all the sugars found in the trunk, except xylose, are sugars known to be
fermented by Saccharomyces cerevisiae [7].

Figure. 1. Total sugar content in OPT

Ethanol fermentation was carried out at a 100 L scale by setting the initial total sugar content to
approximately 15%, with the calculation that if the fermentation process goes well, it will be able to
obtain approximately 9% v/v ethanol. The starch-rich flour used as a fermentation substrate was
extracted from the entire trunk. The only nutrient added was the N source, urea, at 1 g/l. The
fermentation process was preceded by liquefaction using the Liquozyme from NOVO at a dose of
0.05% of the total sugar content. This enzymatic reaction was performed at 90oC for 30 min, and the
temperature was then reduced to 60oC for the saccharification process.
Saccharification was carried out by adding Dextrozyme at a dose of 0.2% of the total sugar content
for 2 hours. After 2 hoursof the saccharification process, the temperature was lowered to 32°C, and the
yeast Saccharomyces cerevisiae Hakken no. 1 was added. The fermentation process was carried out
for 72 hours. When the fermentation process began, the reducing sugar content was only around 40%
of the total sugar content. The remainder was saccharified during a fermentation process. The
fermentation performance is shown in Figure 2.

3
The 2nd International Conference on Food and Agricultural Sciences 2023 IOP Publishing
IOP Conf. Series: Earth and Environmental Science 1377 (2024) 012005 doi:10.1088/1755-1315/1377/1/012005

Ethanol
Total Sugar
Reducing Sugar

Figure 2. Time course of the fermentation of Saccharomyces cerevisiaeHakken no. 1. Initial

Total Sugar was 15.2 %.

Fermentation lasted for 60 h, producing ethanol at a concentration of 8.7% with the fermentation
ratio 88.8%, as shown in Figure. 2. At the end of fermentation, almost all reducing sugar was
consumed by the yeast (residual reducing sugar was 0.22%), and the residual total sugar was 1.71 %.
The fermentation substrate only required an additional nitrogen source, which was urea. This shows
thatthe nutrientsin starch-rich flour were sufficient. Starch extraction from 1 (one) OPT produced 80
kg of starch-rich flour with a total sugar content of 72%. Then, based on the fermentation
performance, 80 kg of starch-rich flour will produce 33 liters of bioethanol.

4. Conclusion
This research shows that using OPT as raw material for bioethanol production has great potential.
Apart from not requiring a lot of nutrients, the lignocellulose portion of OPT will be useful as a heat
source and electricity for the process by applying a combined heat and power system. Further research
that needs to be done so that the use of OPT as raw material for bioethanol production is more
competitive is to improve the extraction system for starch-rich flour from OPT and enhance the
productivity of the fermentation process, namely by shortening the fermentation time and developing a
fermentation system that makes it possible to obtain a higher concentration of ethanol such as the use
of High Gravity Fermentation method.

References
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Leading Estate Crops Commodity 2020 – 2023
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R, Sudesh K, Ibrahim WA, Saito M and Mori Y 2012 Bio. Tech.125 37-42
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chemical properties of oil palm trunk starch Starch/Starke 51 293-301
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[5] Saelee N and Sriroth K 2014 Adv. biosci. biotechnol5 957-965

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The 2nd International Conference on Food and Agricultural Sciences 2023 IOP Publishing
IOP Conf. Series: Earth and Environmental Science 1377 (2024) 012005 doi:10.1088/1755-1315/1377/1/012005

[6] Kosugi A, Tanaka R, Magara K, Murata Y, Arai T, Sulaiman O, Hashim R, Abdul Hamid ZA,
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