Fractionation of Cellulose-Rich Products from an Empty Fruit Bunch (EFB) by Means of Steam Explosion Followed by Organosolv Treatment

In this study an empty fruit bunch (EFB) was subjected to a two-step pretreatment to defragment cellulose-rich fractions as well as lignin polymers from its cell walls. First pretreatment: acid-catalyzed steam explosion (ACSE) pretreatment of EFB was conducted under the temperature range of 180–220 °C and residence time of 5–20 min. The ACSE-treated EFB was further placed into the reactor containing 50% aq. ethanol and NaOH as a catalyst and heated at a temperature of 160 °C for 120 min for the second pretreatment: alkali-catalyzed organosolv treatment (ACO). The mass balance and properties of treated EFB were affected by the residence time. The lowest yield of a solid fraction was obtained when the residence time was kept at 15 min. Xylose drastically decreased, especially under the ACSE pretreatment. However, the crystallinity of cellulose increased by increasing the severity factor of the pretreatment and was 47.8% and 57% udner the most severe conditions. The organosolv lignin fractions also showed the presence of 14 major peaks via their pyrolysis-GC analysis. From here, it can be suggested that this kind of pretreatment can indeed be one potential option for lignocellulosic pretreatment.


Introduction
Over the recent decades, the utilization of empty fruit bunch (EFB) to produce renewable energy source has been studied extensively. This, EFB typically, is a leftover of the fresh fruit bunches after their fruit harvesting. Moreover, it is the major downstream residue from the Southeast Asian palm oil industry. This industry, recently, has produced 70 million metric tons of crude palm oil (CPO) just within one production year 2017-2018 amounting their production even up to 35% of the total production of edible vegetable oils [1]. Theoretically, palm oil plantations can produce more than 100 million metric tons of EFB since 1.42-1.88 tons of EFB are generated for every one ton of CPO produced [2]. However, unfortunately, most of these EFB is dumped and pose serious environmental problems. Therefore, it is highly desirable to utilize these biomasses for sustainable energy production.
Biochemical conversion of lignocelluloses involving chemical pretreatment followed by enzymatic hydrolysis and finally anaerobic fermentation is a well-established production line for lignocellulosic bioethanol production. In this classical approach, due to an inherited complex cell wall structure, effective fractionation of major lignocellulosic biomass components (cellulose, hemicellulose, and lignin) is crucial for effective subsequent processing. Undoubtedly, there have been several pretreatment studies involving EFB to deconstruct its native web into its constituents. In this regard, steam explosion, which uses high-pressure steam to breakdown biomass structure, has been widely adapted strategy [3]. The effects of several factors like biomass particle size, moisture, pressure, residence time, reaction temperature and catalysts on the steam explosion were consistently investigated for several decades [4]. Previously, Choi et al. used alkaline reagent (3% NaOH at 160 • C) to catalyze steam explosion pretreatment and dissolved out C5 sugar-based components as well as lignin components from biomass [5]. Their yields, after enzymatic hydrolysis, for glucose and xylose heightened up to 93% and 78% respectively. In another study, the steam explosion of rose gum tree (Eucalyptus grandis) was carried out under the temperature range of 200-210 • C and residence time of 2-5 min after impregnation with 0.087% and 0.175% (w/w) H 2 SO 4 [6]. Their results showed that 0.175% (w/w) H 2 SO 4 -impregnated chips at 210 • C for 2 min was the optimal condition for hemicellulose extraction. Today steam explosion is seen as one of the most widely employed and cost-effective process for lignocellulose pretreatment, yet the process still needs to be improved to realize industrialization [7]. In addition to this steam explosion strategy, another effective fractionation scheme for lignocelluloses is organosolv treatment, so-called organosolvation. This process, in principle, extracts lignin from lignocellulose with organic solvents, such as alcohols, acetone and organic peracids [8]. Previous studies have shown considerable effects of organosolv treatment (diethylene glycol) of rice straw and EFB as compared to soda pulping by Gonzalez et al. [9] keeping in view the heterogeneous yet complex cell wall structure of EFB, it is highly likely to investigate an appropriate pretreatment strategy for commercial production of biofuels [10]. Therefore, it has to be investigated the proper pretreatment process of EFB. In this study we adapted a two-step pretreatment strategy to fractionate EFB biomass into its sugar and non-sugar components. For this two-step pretreatments, EFB was first subjected to acid-catalyzed steam explosion (ACSE) followed by alkali-catalyzed organosolv treatment (ACO). Pretreatment efficacy was evaluated in terms of yields, crystallinity, and chemical composition of each solid fraction.

Feedstock Analysis
As raw biomass for this study, EFB (≤6% moisture) was provided by the Korea Institute of Science and Technology, in a setting of 0.5-2 mm fiber. Holocellulose, lignin, and ash content in the EFB sample were analyzed according to the National Renewable Energy Laboratory (NREL) standard procedures [11][12][13]. Carbon, hydrogen, and nitrogen contents were measured by using a CHNS-932 analyzer (LECO Corp., St. Joseph, MI, USA) and inorganic elements were analyzed by inductively coupled plasma emission spectroscopy (iCAP7400 Duo, Thermo Fisher Scientific, Waltham, MA, USA) [14]. Thermogravimetric analysis was performed under an inert atmosphere (50 mL/min N 2 flow) at a heating rate of 10 • C/min up to 800 • C with a TGA/DSC 3+ (METTLER TOLEDO, Columbus, OH, USA) [14]. All the results with the corresponding values are listed in Table 1.

ACSE-First Pretreatment
Initially, we screened out experimental conditions based on our initial trials and proceeded accordingly as described below for the pretreatment. The complete fractionation processes are delineated in Figure 1. Briefly, for ACSE treatment, EFB was first washed to remove inorganic elements using a method suggested by Moon et al. [15] Later on it was impregnated in distilled water for 120 min with slow stirring, and finally the feedstock was dried in an oven at 75 • C. It was then impregnated in dilute sulfuric acid (0.5, 1, 2 wt%) for 4 h to make it ready for steam explosion pretreatment. The effect of dilute acid concentration on impregnation was examined by the component analysis (holocellulose, lignin, and sugar) [12].
For ACSE experiments, autoclave reactors with 2 L high pressure steam were loaded first with 150 g of raw EFB biomass samples on dry basis as a control. The final temperature in the reactor was applied to reach in the interval of 180-220 • C and the residence time was 5-15 min. After the intended reaction time, a valve was opened to release the pressure immediately. Solid fractions (E-S) from the steam explosion was washed with distilled water, and the resulting aqueous fraction (E-L) was also collected. Then, acid impregnated EFB, with the same solids loading, was steam-treated under the temperature of 220 • C and 20 min of residence time, the most severe conditions that could be stably maintained with the reactor. After the reaction and cooling, solid fraction (EA-S), and aqueous fraction (EA-L) were also collected.

ACO Second Pretreatment
Organosolv treatment experiments, as a 2nd treatment for both the E-S & EA-S solid fractions obtained from 1st treatment, were performed as follows: both the solid fractions were placed, one by one, in a solvent-circulating reactor with a solvent of a 50:50% (w/w) ethanol:water mixture maintaining a (10:1 (w/w) solvent:solid ratio) along with a 2 wt% of NaOH as a catalyst. The reaction was carried out at a temperature of 160 • C and 120 min residence time. After the desired reaction time, cellulose-rich solid fractions were collected and labelled as E-S-CS and EA-S-CS and liquid fractions were labelled as E-S-OL and EA-S-OL corresponding to E-S and EA-S biomass samples, respectively. The organosolv lignin was precipitated from E-S-OL and EA-S-OL by acidification with a 2 M of hydrochloric acid maintaining pH = 2. The precipitated organosolv lignin was freeze-dried and collected as a powder and saved for further experiments.

Analysis
Yields of each solid fraction were calculated using the following equation: Yield (%) = weight of a solid fraction (g)/weight of dry feedstock (g) × 100 Holocellulose, lignin, and sugar content in each solid fraction were analyzed by the same process mentioned in Section 2.1. X-ray powder diffraction patterns (XRD) were analyzed by a Bruker D8 Advance with DAVINCI using Cu Kα radiation (λ = 1.5418 Å), operated at 40 kV and 40 mA with a scan speed of 0.5 s/min in a range of 2-50 degrees (2 thetas). Chemical compounds in liquid fraction were quantified and qualified using gas chromatography-mass spectrometry systems (5975C Series GC/MSD System, Agilent Technologies, Santa Clara, CA, USA) [14]. Organosolv lignin was analyzed by a coil-type CDS Pyroprobe 5000 (CDS Analytical Inc., Oxford, PA, USA) [16].

Effect of Residence Time and Temperature on Fractionation
The effects of residence time and temperature on the fractionation process were studied at various intervals (residence time 5, 10, 15 min and temperature 180, 200, 220 • C). Figure 2 shows the mass distribution of solid fractions obtained from EFB at various temperatures and residence time conditions.  [17]. The severity factor obtained ranged from 3.1 to 4.7. There was a scant negative correlation between the severity factor and the solid fraction yield, showing that increasing temperature and residence time results in the removal of compounds from solid fractions.
In general, both mechanical and chemical effects take place when an acetyl group derived from hemicellulose acts as an acid at high temperature during the steam explosion [18]. These effects play important roles in the hydrothermal degradation of holocellulose. Hemicellulose and lignin are isolated by the high temperature in the steam condition. Organosolv treatment, however, on the other hand, solubilize lignin and provide holocellulose by using organic solvent mixture and inorganic catalysts [7]. Therefore, holocellulose and lignin compositions of solid fractions were also analyzed to examine the fractionation efficiency of the process.
Holocellulose and lignin contents of E-S and E-S-CS were calculated for each experiment ( Table 2). Percentage of holocellulose composition decreased with increasing severity of steam explosion. A maximum of 24.1 wt% of holocellulose (220-15) was dissolved in E-L. On the contrary, the percentage of lignin composition increased with increasing severity factor. It can be anticipated that lignin melts under a high temperature and pressure, then condensed with other compounds like extractives or ash while cooling. Solid fractions which have undergone organosolvation showed an unremarkable change of holocellulose contents. In addition, a considerable amount of lignin content still remained in the solids. Maximum 7.5% of lignin (220-15) was solubilized in 50% ethanol mixture. Lignin fractionation efficiency of organosolv treatment from EFB was falling short of our expectation because of its low lignin removal. These results showed EFB presents a high level of recalcitrance to the fractionation process. Thus, dilute acid pretreatment to level down the EFB recalcitrance had considerable effects and the results are discussed in Section 3.1.2.

Effect of Dilute Acid Impregnation on Fractionation
Acid impregnation has been considered to play a vital role for steam explosion treatment [4,19]. Keeping this in view, the effects of dilute acid on the fractionation process was studied with the feedstock impregnation in dilute sulfuric acid (0.5, 1, 2 wt%) for 4 h. Steam explosion process was further operated at residence time of 20 min and temperature of 220 • C, which showed the highest severity score so that the effect of dilute acid concentration can be compared clearly. Figure 3 shows the mass distribution of solid fractions obtained from EFB at various acid concentrations.
Mass composition of EA-S did not affect by the acid concentration clearly until the concentration reaches 2.0%. Moreover, at 0.5 and 1.0% dilute acid concentrations, EA-S from ACSE also showed no remarkable changes in the mass distribution. In the range of 0%-1.0% dilute acid concentration, yields of EA-S decreased from 73.0 to 70.8 wt%. However, EA-S-CS from ACO decreased with an increase in the acid concentration. In the same range of 0%-1.0% dilute acid concentration, EA-S-CS decreased from 59.0 to 45.9 wt%. At 2.0% dilute acid concentration, EA-S and EA-S-CS decreased with an increase in the acidity.  Table 3 shows the holocellulose and lignin contents of EA-S and EA-S-CS calculated for each experimental run. The total amount of holocellulose harshly decreased with increasing dilute acid concentration. At 0.5% dilute acid concentration, 41.3 and 26.8 wt% of holocellulose still remained in EA-S and EA-S-CS, respectively. When the concentration increased to 2.0%, however, only 28.1 and 16.0 wt% of holocellulose remained in EA-S and EA-S-CS, respectively. Lignin exhibited somewhat different behavior in mass decrease. The amount of lignin did change slightly by the steam treatment. Hwoever, after ACO, 4.3%-6.7% of lignin was removed from the solid fractions. The removal efficiency of lignin between 0.5% and 2.0% acid concentration respectively. Similar studies were reported for the dilute acid treatment of rice straw [20] and corn stover [21]. However, the proper acid concentration or removal efficiencies of hemicellulose and lignin were different by feedstock properties. Since the purpose of this experiments is to lower only hemicellulose and lignin contents in EFB, so a high acid concentration was not adapted to avoid the loss of holocellulose contents.  Tables 4 and 5 show the total sugar contents of each product fractions (EA-S, EA-S-CS, EA-L, and EA-S-OL). Glucose content in the solid fractions showed no significant change while lignin content increased compared to the raw material. It means that the glucose in the EFB took limited damage by the steam explosion process and the organosolv treatment. It is possible that long reaction times increased the accumulation of degradation by-products and mass losses by volatilization [22]. As the acid concentration increased, however, the amount of glucose in the solid steadily decreased. When the acid concentration increased to 2.0%, the glucose amount decreased from 415 to 380 mg/g fraction. In contrast, concentration of glucose in EA-L dropped with 0.5 wt% acid pretreatment but increased by increasing acid concentration. Xylosyl groups were the most affected hemicellulose components during acid impregnation and the pretreatment process. The amount of xylose in the solid drastically decreased with increasing dilute acid concentration. As a result, concentration of xylose in the liquid fraction increased to twice of the control (10.1-21.9 mg/mL liquid). At 2.0% dilute acid concentration, more than 70% of the xylan in the EFB was hydrolyzed during two-step pretreatments. Arabinosyl, galactosyl, and mannosyl residues were removed from the solids by steam explosion with more than 0.5% acid concentration (Table 5).

Cellulose Crystallinity
The change of cellulose crystallinity as a result of the steam explosion and organosolv treatment was determined by analyzing the x-ray diffraction patterns ( Figure 4 and Table 6). By the XRD peak height method, crystallinity index was calculated from the ratio of the height of the 002 peaks (22.7 • ) and the height of the minimum (18.3 • ) between the 002 and the 101 peaks [23]. The crystallinity of EFB fiber decreased due to the steam explosion and organosolv treatment. The lowest crystallinity was shown in E-S-CS under the condition of 180-10 (35.9%). The crystallinity of EFB increased by increasing the severity factor. This might be caused by the removal of the amorphous structure of cellulose or components like hemicellulose.
Cellulose crystallinity after the dilute acid impregnation followed by two-step pretreatment was determined by analyzing the X-ray diffraction patterns. The crystallinity of EFB fiber also decreased by both ACSE process and ACO. There was little change of crystallinity, however, between the raw and organosolv treatment.

Chemical Properties of Organosolv Lignin
Qualitative and quantitative analysis of obtained organosolv lignins from E-S-OL were performed via pyrolysis-GC-MS analysis, and the results are listed in Table 7. Organosolv lignin obtained under steam explosion conditions of 200 • C and 5-10 min exhibited more than 14 peaks. Phenolic compounds, such as toluene (1), phenol (2), guaiacol (5), syringol (8), and isoeugenol (10), were the major compounds. As shown in Table 7, the number of chemical compounds and the overall peak size in the organosolv lignin decreased by increasing residence time. This might be explained by the fact that the lignin compounds can be condensed in harsher conditions and less decomposed by heat.

Conclusions
Two-step pretreatment, ACSE followed by ACO of EFB, was performed under a temperature range of 180-220 • C and residence time of 5-20 min after impregnation with 0.5%-2.0% dilute sulfuric acid. The mass balance and the chemical composition of pretreated EFB fractions were noticeably affected by the reaction temperature, residence time, and acid concentration during the steam explosion process. Moreover, glucose and xylose contents decreased under the acid-catalyzed pretreatment conditions. All the analyses together with the XRD peaks showed that the crystallinity of cellulose increased by increasing severity factor. In this experiment, the effect of two-step pretreatment was in no way remarkable but showed potential to advance it. Further study is needed to sort out the optimal conditions for this two-step EFB pretreatment to conclusively suggest its potential for EFB fractionation.