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Hassan, N., Li Wen, T., Khairil Anwar, N. A. K., and Idris, A. (2021). "Ionic solution pretreatment of oil palm empty fruit bunch to produce sugars," BioResources 16(1), 1816-1824.


Ferric (III) chloride, when prepared as an ionic solution, was used to pretreat oil palm empty fruit bunch (OPEFB) before undergoing enzymatic hydrolysis to convert into value-added products. The pretreatment was assisted with microwave irradiation to improve the degradation of recalcitrant structure of lignocellulosic materials with minimum time. The effects of salt concentration, temperature, and duration of pretreatment on the chemical composition of OPEFB and total reducing sugar (TRS) yield were investigated. The results revealed that the best pretreatment occurred at pretreatment time of 10 min, 100 °C, and ferric chloride concentration of 10 w/v%. The TRS yield achieved using the pretreated OPEFB was approximately 0.485 g/g, which was three times higher than the non-pretreated OPEFB, which was only 0.154 g/g. Thus, the ionic solution pretreatment method is a promising alternative for replacing other pretreatment methods.

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Ionic Solution Pretreatment of Oil Palm Empty Fruit Bunch to Produce Sugars

Nursia Hassan, Tan Li Wen, Nur Amirah Khairina Khairil Anwar, and Ani Idris *

Ferric (III) chloride, when prepared as an ionic solution, was used to pretreat oil palm empty fruit bunch (OPEFB) before undergoing enzymatic hydrolysis to convert into value-added products. The pretreatment was assisted with microwave irradiation to improve the degradation of recalcitrant structure of lignocellulosic materials with minimum time. The effects of salt concentration, temperature, and duration of pretreatment on the chemical composition of OPEFB and total reducing sugar (TRS) yield were investigated. The results revealed that the best pretreatment occurred at pretreatment time of 10 min, 100 °C, and ferric chloride concentration of 10 w/v%. The TRS yield achieved using the pretreated OPEFB was approximately 0.485 g/g, which was three times higher than the non-pretreated OPEFB, which was only 0.154 g/g. Thus, the ionic solution pretreatment method is a promising alternative for replacing other pretreatment methods.

Keywords: Format; Lignocellulosic biomass; Ionic solution pretreatment; Ferric chloride solution; Microwave irradiation

Contact information: Institute of Bioproduct Development, School of Chemical and Energy Engineering, Faculty of Engineering, Universiti Teknologi Malaysia, Skudai, 81310 Johor, Malaysia;

* Corresponding author:


Oil palm is one of the important crops in Southeast Asian countries, as it generates increased income every year to these countries. Malaysian Palm Oil Board reported that oil palm trees planted area in Malaysia has reached 5.9 million hectares (ha) in 2019. As the oil palm plantation area increased, various biomass residues generated from oil palm industry also increased throughout the year. Oil palm empty fruit bunches (OPEFB), are the remains of the fresh fruit bunches after the fruits have been removed for oil pressing. It is the most abundant biomass waste generated at all palm oil mills. The amount of OPEFB throughout 2017 were around 15.8 to 17 million tons per year (Derman et al. 2018). Thus, huge amounts of OPEFB should be converted into value-added products, such as bioethanol, bio-oil, and sugars to reduce environment problems.

The utilization of OPEFB as a renewable carbon resource has attracted much attention to produce valuable products such as sugars. Lignocellulosic biomass is made up of complex biopolymers that consist of three major components of lignin, cellulose, and hemicellulose. However, conversion of lignocellulosic biomass, such as EFB, into sugars is still a challenge because the pretreatment methods currently available are still far from efficient and economical. Currently, there are two types of routes for EFB conversion. The first type is to de-polymerize lignocellulose directly without differentiating its components and only regard biomass as a mixture of C, H, and O elements; the degraded products are still complex and require an upgrading process; the methods used usually include gasification and thermal pyrolysis (Li and Chen 2018; Ferreira et al. 2020). The second type of conversion involves fractionation of lignocellulose biomass to lignin, cellulose, and hemicellulose (Hassan et al. 2020). Pretreatment is the most important process in the bioconversion of lignocellulose for sugars production. The purpose of pretreatment is to remove the recalcitrant lignin to ease the biomass hydrolysis process, improve the enzymatic digestibility, and increase the yield of reducing sugars produced. There are three kinds of lignocellulose pretreatment methods, which are physical (microwave, ultrasound, pyrolysis, etc.), chemical (dilute acid, mild alkali, ionic liquids, etc.) and physicochemical (steam explosion, oxidative, wet oxidation, etc.). However, different biomasses will present different standards of hemicellulose or lignin removal and thus different enzymatic digestibility, even under the same pretreatment process at the same conditions (Kang et al. 2012).

Dilute acid pretreatment is the most frequently used method to convert biomass to fermentable sugars by hydrolyzing hemicellulose using dilute acid followed by enzymatic hydrolysis. However, this method may generate toxic substances, which will inhibit fermentation. In addition, dilute acid pretreatment causes corrosion. Thus, expensive materials for construction are required, which lead to the increase in capital cost. Besides the aforementioned methods, imidazole-type ionic liquids have also been used in pretreatment processes (Cao et al. 1994; Binder and Raines 2010; Muranaka et al. 2015; Kuroda et al. 2016; Qian et al. 2016). Unlike an ionic liquid, which is expensive (Wan et al. 2018) and has melting point below the boiling point of water, an ionic solution is simply a solution containing ions where ionic compound is dissolved in water; dissociating into cations and anions.

Recently, the trend to use ionic solutions, such as ZnCl2, and FeClaqueous solutions, in the pretreatment of biomass for producing value-added product such as bioethanol has become attractive. An ionic solution has the ability to increase cellulose and hemicellulose conversion rates as well as improve hydrolysis yields (Liu et al. 2009; Hassan et al. 2020). Additionally, inorganic salt solutions are less corrosive and can achieve higher rate of enzymatic digestibility than inorganic acids and it can be recycled (Liu et al. 2009; Kamireddy et al. 2013; Hassan et al. 2020). A Lewis acid, such as FeCl3, has been proposed to be the most effective inorganic salt that can replace acid pretreatment (Liu et al. 2009; Zhu et al. 2020). FeCl3 can undergo hydrolysis and generate hydronium ions, leading to depolymerisation of hemicellulose. FeCl3 is able to degrade hemicellulose into monomeric and oligomeric sugars in liquid hydrolysate with a large amount of xylose and produce cellulose-rich solid substrate that can be converted to glucose in enzyme hydrolysis process. Moreover, pretreatment using these ionic solutions results in a significant improvement of the enzyme activity due to the metal cations forming complexes with the lignin, reducing the unproductive enzyme–lignin links (Kamireddy et al. 2013).

A perusal of published literature showed that a majority of the current pretreatment methods are energy intensive in nature (Kang et al. 2013; Liu et al. 2009). Thermal energy is crucial in accelerating the hydrolytic process, which results in substantial breakdown of the lignocellulosic structure, hydrolysis of the hemicellulose fraction and depolymerization of the lignin components. Thus, in this study a ferric salt solution and microwave heating were combined during the pretreatment, as this approach mediates in breaking of bonds among the molecules. The influence of salt concentration, temperature, and pretreatment time towards enzymatic hydrolysis of pretreated OPEFB were also investigated. The effectiveness of pretreatment process is measured in terms of total reducing sugar (TRS) yield.



A 250-mL three-necked flask was used for pretreatment of OPEFB. The solid-to-liquid ratio was set at 1:10, and 5 g of dry biomass was added in 50 mL prepared FeClsolution. The FeClsolution concentrations were varied (5, 10, and 15% w/v). The flask was placed in a microwave oven to heat at various desired temperatures (75, 90, and 100 °C) at different pretreatment times (5, 10, and 15 min). Upon the end of reaction, the flask was then cooled to below 40 °C by placing it inside the water bath. The treated OPEFB fibres were then washed with water to remove FeCl3 until pH reached 7. The pretreated solid samples were dried at 60 °C and then stored in a plastic container for later use. The mean values of results were calculated from duplicate analysis.

Composition Analysis of OPEFB

Holocellulose content was determined using the procedure used by Teramoto et al. (2009), and α-cellulose amount was determined using TAPPI T203 cm-09 (2009). The National Renewable Energy Laboratory standard method (Sluiter et al. 2008) was used to determine lignin amount inside the OPEFB sample. The mean values of results were calculated from duplicate analysis.

Enzymatic Hydrolysis

Enzymatic hydrolysis of OPEFB samples was performed to obtain the TRS yield. The hydrolysis of OPEFB samples was performed in 250-mL conical flasks containing sodium citrate buffer (pH 4.8, 0.05 M), 100 µL of sodium azide (2% w/v), and Cellic CTec2 enzymes (20 FPU/g OPEFB). Cellic CTec2 is an enzymes blend consist of cellulases, ß-glucosidases, and hemicellulose. The enzymatic hydrolysis was performed at temperature of 50 °C. The sampling was performed every 1, 4, 24, 48, 72 h of enzymatic hydrolysis and the TRS yields were analyzed at the time mentioned using 3,5-dinitrosalicylic acid (DNS) method. Then, TRS was then calculated accordingly to Eq. 1:


All hydrolysis experiments were conducted in triplicate, and errors were within ± 5%. The data are presented in average values.


Analysis of OPEFB Composition Before and After Pretreatment

Based on the data in Table 1, the pretreated OPEFB had a higher content of cellulose and lignin than non-pretreated OPEFB, where the treated OPEFB contained 65.6% of cellulose and 18.9% of lignin. The results in Table 2 also shows that there was a high loss of hemicellulose in pretreated OPEFB compared to the non-pretreated one, which was from 23.41 ± 0.59% to 3.78 ± 0.38%. The data strongly demonstrated that FeCl3 solution has the ability to solubilize hemicellulose compound in OPEFB at a higher degree. This result agrees with that of Zhang et al. (2018), who determined that FeCl3 pretreatment could easily and effectively solubilize hemicellulose in lignocellulosic sugarcane bagasse. The increase in lignin and cellulose content after FeCl3 pretreatment can be ascribed to the solubilization of hemicellulose fraction. Similar lignin and cellulose content increment phenomenon were reported by Moodley and Kana (2017) during the pretreatment of sugarcane leaves waste using microwave assisted-FeClpretreatment.

Various reaction mechanisms have been suggested for metal salts. Metal chlorides such as FeCl3 dissociate into complex ions in aqueous solvent such as water. Fe3+ can form coordinate covalent bonds with six water molecules as monodentate ligands. This metal cation behaves as a Lewis acid in an aqueous environment, which facilitate the cleavage of the glycosidic linkages (Kamireddy et al. 2013). Meanwhile, the resulting water molecule from the hydrated cation species plays a key role in forming xylose by participating as nucleophiles (Zhu et al. 2020; Moodley et al. 2020). Secondly, FeCl3 can undergo hydrolysis and generate hydronium ions, leading to depolymerisation of hemicellulose by selective hydrolysis of glycosidic linkages to form acetic and uronic acids. The released acids could also catalyse hemicellulose hydrolysis (Loow et al. 2015). Thirdly, Fe3+ cations are good electron acceptors and able to coordinate with oxygen donor atoms of carbohydrates and their derivatives without losing the protons that make up the hydroxyl groups of the ligand to form saccharide-metal cation intermediate, leading to reduced activation energy requirements for biomass hydrolysis (Loow et al. 2015; Zhu et al. 2020).

Table 1. Chemical Composition of OPEFB Before and After Pretreatment

TRS Yield Comparison of Pretreated and Non-pretreated OPEFB

Figure 1 illustrates the TRS yield of pretreated and non-pretreated OPEFB. Both curves show the same trend but at different rates.

Fig. 1. TRS yield of pretreated and non-pretreated OPEFB

The rate of enzymatic hydrolysis increased rapidly initially but gradually decreased after 20 h. The finding shows that a high amount of reducing sugars can be harvested from the pretreated OPEFB compared to the non-pretreated OPEFB. The maximum reducing sugar generated from the pretreated OPEFB was 0.485 g/g, which was three times higher than the non-pretreated OPEFB where only 0.154 g/g of sugar was produced. The data obtained remarkably proved microwave assisted ionic solution pretreatment method was effective in removing the recalcitrant structure of OPEFB, which in turn facilitated and accelerated the enzymatic hydrolysis.

Effect of FeCl3 Concentration on TRS Yield

Figure 2 illustrates the TRS yield against time at constant temperature (100 °C) and pretreatment time (10 min). The findings show that the trend of TRS yield for different FeCl3 concentrations (0%, 5% w/v, 10% w/v, and 15% w/v) were almost similar. Initially, the total reducing sugar yield increased consistently until it reached 72 h of hydrolysis. The maximum sugar yield was achieved at 10% w/v, followed by the 15% w/v and 5% w/v FeCl3 concentrations. The final amount of reducing sugars produced were 0.4924 g/g, 0.4642 g/g, and 0.2935 g/g, respectively. Meanwhile, the use of hot liquid water (0% of FeCl3) during pretreatment yielded low TRS accumulation, as only 0.1571 g/g TRS was produced.

Fig. 2. Effect of FeCl3 concentration on TRS yield; pretreatment was performed at temperature of 100 °C and for 10 min

The use of a higher amount of FeCl3 in the pretreatment resulted in higher accumulation of TRS. The TRS yield increased from and 0.2935 ± 0.0120 g/g to 0.4924 ± 0.0021 g/g when the FeCl3 concentration was increased from 5 to 10%. High concentration of Fe3+ in pretreatment solution accelerated the generation of reactive H3O+ (Ravindran et al. 2017). Thus, substantial removal of hemicellulose was achieved, which enhanced enzymatic hydrolysis by improving the enzymes access. However, low sugar yield was obtained when the FeCl3 concentration was further increased to 15% w/v with TRS yield of 0.4642 ± 0.0059 g/g. A similar observation was reported by Kang et al. (2015), where FeCl3 was used during the pretreatment to enhance the enzymatic hydrolysis of Miscanthus straw. They found that the glucose conversion yield was negatively affected when high loading of FeCl3 was used. In their study, the increase of the FeCl3 concentration beyond 2.0% caused much glucose loss, as almost 80% of glucan was hydrolyzed. It is concluded that pretreatment at 10% w/v FeCl3 is suitable to obtain higher yield of reducing sugars compared to 5% w/v and 15% w/v FeCl3.

Effect of Pretreatment Temperature and Pretreatment Time on Total Sugar Yield

Figures 3 and 4 illustrate the time course of enzymatic hydrolysis of OPEFB at different pretreatment temperatures. From these graphs, all the total reducing sugar yield trends for different pretreatment temperatures and time were almost similar. Initially, the TRS yield increased gradually until it reached 72 h of hydrolysis.

Fig. 3. Effect of temperature on TRS yields; pretreatment was performed using 10% w/v of FeCland pretreatment time of 10 min

Fig. 4. Effect of pretreatment time on the enzymatic hydrolysis of OPEFB; pretreatment was performed at FeCl3 concentration of 10% w/v and pretreatment time of 10 min

The optimum TRS yield was achieved at pretreatment temperature of 100 °C, followed by 90 °C and 75 °C, with the amount of reducing sugar produced of 0.4839 ± 0.0020 g/g, 0.4317 ± 0.0081 g/g, and 0.3189 ± 0.0079 g/g, respectively. The maximum TRS yield was achieved by pretreating at 10 min, followed by 15 min and 5 min, with the amount of reducing sugar produced at 0.4853 ± 0.0040 g/g, 0.4416 ± 0.0059 g/g, and 0.2413 ± 0.0098 g/g, respectively.

It can be observed that, at temperature of 100 °C, 0.4839 g/g of TRS was produced, which was much higher than 90 °C with 0.4317 g/g of TRS produced. However, pretreatment time for 10 min generated more TRS yield compared to pretreatment time of 15 min, where 0.4853 g/g of TRS was produced at 10 min, with only 0.4416 ± 0.0059 g/g of TRS at 15 min. This scenario could be ascribed to cellulose degradation, which was the main cause of slightly lower TRS yield. Overall, the combination of high pretreatment temperature and longer pretreatment time was not a good choice. It is suggested to slightly decrease the pretreatment time when pretreating at high temperature to obtained high yield of TRS. Finally, the best pretreatment temperature and pretreatment time were set at 100 °C for 10 min.


  1. Factors such as pretreatment time, temperature, and ferric chloride concentration influenced the total sugar yield, and ionic solution pretreatment was best performed at 10 min, 100 °C, and 10% w/v, respectively.
  2. Pretreatment at the recommended process variables achieved a TRS yield of 0.4853 g/g, which was three times higher than the non-pretreated OPEFB, for which only 0.1541 g/g was obtained.
  3. The remarkable difference in reducing sugars yield between the pretreated and non-pretreated OPEFB showed that ionic solution pretreatment can improve the enzymatic hydrolysis process.
  4. Overall, ionic solution pretreatment method is a potential and promising alternative pretreatment method.


The authors would like to thank the Malaysia Ministry of Higher Education (R.J130000.7851.5F244) for providing the funds for this research.


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Article submitted: November 9, 2020; Peer review completed: December 20, 2020; Revisions accepted: January 18, 2021; Published: January 22, 2021.

DOI: 10.15376/biores.16.1.1816-1824