Characterization of Empty Fruit Bunch Treated with Ionic Liquid Prior to Enzymatic Delignification

The technological utility of enzymes for delignification can be increased by using ionic liquid to open more accessible surface area for biomass transformation into bio-based products. The present paper demonstrates application of ionic liquid (IL) [emim][DEP] 1-ethyl-3 methyllimidazolium-diethyl phospate for empty fruit bunch (EFB) pretreatment process followed by enzymatic delignification by using Laccase. It was found that [emim][DEP] increased the performance of the enzyme laccase and henced higher cellulose rich materials, whereas also reduced the lignin content in the EFB. The lowest lignin content obtained from IL-laccase treated EFB was approximately 17.92%, lower than the lignin content in the untreated EFB. Both treated and untreated EFB were characterized in chemical and physical properties by using scanning electron microscope (SEM), fourier transform infrared (FTIR), and thermogravimetric analysis (TGA/DTG) to observe the changes resulted from the pretreatment.


Introduction
Empty fruit bunch (EFB) is one of solid waste produced from palm oil industry.Oil palm tree is one of the most well-known and extensively cultivated plant families.Based on the preliminary experiment, EFB contains holocellulose about 49.06% and lignin about 25.33% (See Table 1).
EFB shows high content of holocellulose, which makes this biomass as a promising feedstock for biomaterial and biofuel products.However, the complexity in the biomass structure created recalcitrance to any chemicals or enzymatic processes.Lignocellulose is mainly composed of the rigid semi-crystalline polysaccharide cellulose, the amorphous multicomponent polysaccharide hemicellulose, and the crosslinked aromatic polymer lignin, as a primary constituents of plant cell walls, in which cellulose fibers are embedded into entangled matrix consisting of lignin and hemicellulose, forming a tight and compact structure.These inherent properties of lignocellulosic materials make them resistant to the chemicals and enzymes.
Thus, pretreatment process needs to be taken at first, the aim of this pretreatment is to change these complex properties of lignocellulosic materials by breaking the lignin chain and the crystalline structure of cellulose simultaneously increasing the porosity of the cellulose rigid structure.Briefly, the mechanism of pretreatment can affect the physical properties of biomass such as a degree of polymerisation, a crystallinity structure and surface area of the solid substrate.
Ionic liquids (ILs), a potentially attractive 'green' recyclable alternative to environmentally harmful organic solvents, have been increasingly exploited as solvents and/or (co)solvent and/or reagent in a wide range of applications including pretreatment of lignocellulosic biomass (Sun et al., 2011;Mora-Pale et al., 2011;Moniruzzaman and Ono, 2012).Moreover, this IL provides many desirable properties such as low toxicity, low corrosiveness, low melting point (<-20 o C) and low viscosity (10 pa at 80 o C) (Moniruzzaman and Ono, 2012).
Many experiments have been conducted using ILs to dissolve wood or lignocellulosic biomass at different conditions and with the combination of other processes, such as microwave radiation (Ha et al., 2011), alkali pretreatment (Lan et al., 2011) or followed by enzymatic delignification (Moniruzzaman dan Ono, 2012).ILs pretreatment are able to reduce the degree of polymerization of cellulose rich-material and leading to enhanc the enzymatic delignification efficiency of wood biomass (Moniruzzaman and Ono, 2012;Moniruzzaman et al., 2013).
The extensively explored of numerous studies focused on the dissolution of natural polymer especially cellulose in ILs as solvents, encourages this work to study more about the ability of IL to separate the cellulose and lignin in the EFB and to enhance the enzymatic delignification process with ultimate goal to obtain cellulose rich fibers with minimum structural alteration.EFB samples (treated and untreated) were characterized using SEM, FTIR and TGA analysis to see the effect of pretreatment to the physical structures of the sample and the chemical characterizations were analyzed using standard method as described in the methodology.

Dissolution of EFB in Ionic Liquid and Enzymatic Delignification
Ionic liquid was put into a 50 mL roundbottom flask with magnetic stirrer at 80°C for 1 h.EFB was added into ionic liquid (IL) with a ratio 10:1 (IL : Sample w/w).After cooling process, water:acetone mixture (1:1 v/v) was added into biomass-IL mixture and stirred for 20 min to separate the solid materials from dissolved IL and lignin.Then, the treated sample was washed with distilled water and put in the oven drying for overnight.For enzymatic delignification process, the treated sample (after IL treatment at 80°C for 1 hr) was placed into conical flask and added with sodium acetate (100 mM, pH 4.5) (ca. 5 wt.% biomass), Laccase (≥10µ/mg), and 1-hydroxybenzotriazole (HBT) (1.5 wt.% of biomass) was added as a mediator and stirred at 50 o C.After 24 h, 0.1 M sodium hydroxide (NaOH) was added into the mixture and stirred for 1 h to extract lignin from the IL-enzyme treated EFB.The mixture was then filtered and washed with distilled water until pH 7 under mild vacuum prior to oven drying.

Characterization of Untreated and Treated Materials
Concisely, the holocellulose (α-cellulose and hemicellulose) determination was performed by using acidified sodium chlorite solution at 70 o C at 1 h.In addition, α-cellulose was treated with 17.5% sodium hydroxide and 10% acetic acid.The difference values between holocellulose and α-cellulose gave hemicellulose value of the samples.The lignin content of the sample was analyzed according to ASTM D 1106-96 (Klason Lignin).The surfaces of samples were photographed by Scanning Electron Microscope (SEM) (TM 3030, Hitachi Ltd., Tokyo, Japan).Thermogravimetric analysis (TGA-Q50, TA instrument, USA) was performed to compare the degradation characteristics and thermal stability of the treated and untreated samples, by heating 5 mg of sample in a platinum pan at a rate 10 °C/min in a nitrogen atmosphere.Fourier Transform Infrared Spectroscopy (Thermo Nicolet IS10, USA) was conducted to determine any chemical changes occuring in the biomass sample during the pretreatment process.

Chemical and Physical Characterization of EFB
The chemical composition study of untreated and treated EFB is summarized in Anion from IL will disrupt the free hydroxyl group in the lignocellulosic material, whilst the cation will interrupt the hydroxyl oxygen atom, thus disrupt its three dimensional network.Consequently, hemicellulose and lignin is dissolved (Mäki-Arvela et al., 2010).Furthermore, as shown in Table 1, compared with untreated EFB the cellulose content of treated EFB with IL prior to enzymatic delignification is higher because of the removal of hemicellulose, lignin, and soluble extractives during the treatment process.

The Morphology of EFB
Fig. 1 depicts SEM images of untreated and treated EFB at which taken at 200 µm with magnification 500.The untreated EFB showed intact morphology, compact, ordered and rigid fibril structure because of the lignin coating on cellulose fibers (Fig. 1), while IL-laccase treated EFB showed loose, disordered, and curly structure.This swelling may be a result of breaking interintramolecular hydrogen bonding caused by phosphate anion from [emim][DEP] during the pretreatment, which led to open more accessible area for enzyme laccase to degrade lignin.

Surface Composition of EFB analyzed by FTIR
The FTIR spectra of treated and untreated EFB are shown in Fig. 2. As observed in the Figure , the dominant peaks at 3346 cm -1 (O-H stretch) and 2892 cm -1 (C-H stretch) represent the alipathic moieties and the prominent peaks at 1709 -1735 cm -1 attribute to polysaccharides (Labbe et al., 2005).There were subtle difference spectra between the untreated and treated EFB, particularly in the finger print region at 1508 -1588 cm -1 and 1248 cm -1 (Faix, 1991;Fengel & Ludwig, 1991;Isroi et al., 2012), which characterized the C=O aromatic skeletal for lignin's IR spectra in the untreated EFB, the bands were found disappeared in the treated EFB with ionic liquid prior to laccase treatment.
The dissipation of an IR absorbance of lignin in the treated EFB indicated the removal of some lignin during the treatment.This is consistent with the chemical composition study (see Table 1), in which the lignin percentage was obtained lower for samples with ionic liquid followed by laccase treatment compared to untreated EFB.

Thermal Stability of EFB
Thermogravimetric measures the change of biomass weight at a specific heating rate, because the physical and chemical reactions of sample when heated, showed the characteristics of the materials.EFB thermal decomposition curves for untreated and treated with ionic liquid are shown in Fig. 3.The DTG data of untreated EFB show one dominant peak with a peak value (Tmax) of 300 o C, whilst EFB treated with ionic liquid followed by laccase treatment showed temperature peak at Tmax 330 o C, which was higher than that the untreated EFB.
The value of Tmax represents the maximum decomposition rate occurs at certain temperature, in order to gauge the impact of thermal ability after ionic liquid pretreatment of biomass samples.This results implied that the thermal stability for treated EFB was more improved than that the original biomass, because of the transformation of cellulose I structure into cellulose II after the pretreatment which was reported had better thermal stability (Zhang et al., 2014).

Conclusion
In

Table 1 .
Chemical composition of untreated and treated sample Laccase treatment with sodium acetate buffer and HBT, at 50 o C for 24 h a The data represent the average of three experiments with standard deviation b Ionic liquid pretreatment condition IL : Biomass ratio 10:1 (w/w), 80 o C for 1 h c