Optimization of OPEFB lignocellulose transformation process through ionic liquid [TEA][HSO4] based pretreatment

Research on the transformation of Oil Palm Empty Fruit Bunches (OPEFB) through pretreatment process using ionic liquid triethylammonium hydrogen sulphate (IL [TEA][HSO4]) was completed. The stages of the transformation process carried out were the synthesis of IL with the one-spot method, optimization of IL composition and pretreatment temperature, and IL recovery. The success of the IL synthesis stage was analyzed by FTIR, H-NMR and TGA. Based on the results obtained, it showed that IL [TEA][HSO4] was successfully synthesized. This was indicated by the presence of IR absorption at 1/λ = 2814.97 cm−1, 1401.07 cm−1, 1233.30 cm−1 and 847.92 cm−1 which were functional groups for NH, CH3, CN and SO2, respectively. These results were supported by H-NMR data at δ (ppm) = 1.217–1.236 (N–CH2–CH3), 3.005–3.023 (–H), 3.427–3.445 (N–H+) and 3.867 (N+H3). The TGA results showed that the melting point and decomposition temperature of the IL were 49 °C and 274.3 °C, respectively. Based on pretreatment optimization, it showed that the best IL composition for cellulose production was 85 wt%. Meanwhile, temperature optimization showed that the best temperature was 120 °C. In these two optimum conditions, the cellulose content was obtained at 45.84 wt%. Testing of IL [TEA][HSO4] recovery performance for reuse has shown promising results. During the pretreatment process, IL [TEA][HSO4] recovery effectively increased the cellulose content of OPEFB to 29.13 wt% and decreased the lignin content to 32.57%. The success of the recovery process is indicated by the increasing density properties of IL [TEA][HSO4]. This increase occurs when using a temperature of 80–100 °C. The overall conditions obtained from this work suggest that IL [TEA][HSO4] was effective during the transformation process of OPEFB into cellulose. This shows the potential of IL [TEA][HSO4] in the future in the renewable energy sector.

www.nature.com/scientificreports/ the mechanism of forming a dense surface structure, so that the catalyst and cellulose enzymes do not work optimally. An illustration that explains this situation can be seen in Fig. 1.
In overcoming the pretreatment problem of OPEFB, many pretreatment methods have reported their performance. Pretreatment is an important step in opening or stretching the lignocellulose structure. Pretreatment can be done either physically, chemically or a combination of the both. Physical methods such as uncatalyzed steam-explosion 7,8 , liquid hot water (LHW) 9,10 , mechanical communication 11 , and high energy radiation 12 . Meanwhile, Chemical methods such as catalyzed steam-explosion 13 , acid pretreatment 14,15 , alkaline pretreatment 16,17 , ammonia fiber/freeze explosion (AFEX) 18,19 , organosolv 20,21 , and pH-controlled liquid hot water 10 . To improve pretreatment results, a combination of these two methods is often carried out such as the combination of the hydrothermal method with sulfuric acid. Although this method can improve the purity of cellulose and enzyme performance during the bioethanol production process, however these methods have also an impact on the high use of hazardous chemical solvents during the pretreatment process which can cause new environmental problems 22 . Another impact that has also been reported is how pretreatment such as physical pretreatment can affect the properties of cellulose 23 . Based on these problems, OPEFB pretreatment was mostly directed at the Ionic Liquid (IL) based pretreatment system. This system is reported to be an environmentally friendly biomass pretreatment system with a low amount of solvent.
IL pretreatment is a new system for the development of the OPEFB pretreatment method. This method is more environmentally friendly, effectively destroys lignin and hemicellulose bonds without damaging the glucose structure, effectively reduces cellulose crystallinity, low volatility and vapor pressure, and can be recycled 24,25 . In addition, IL has special properties such as wider fluid temperatures, high thermal stability, and negligible vapor pressure 26 . Certainly, these essential properties are needed in the lignocellulose biomass transformation. IL has been reported for biomass pretreatment among others 1-buthyl-3-methylpyridinium chloride, [   www.nature.com/scientificreports/ humidity tolerance, and in dissolving cellulose requires a low moisture content, this is in contrast to the high moisture content of the biomass.
As an alternative to the deficiency of IL, it is important to choose the type of anion. In this regard, the synthesis and application of triethylammonium hydrogen sulphate [TEA] [HSO 4 ] as IL for the pretreatment process resulted in lower production costs 30 . In addition, the length of the alkyl chain in ammonium plays an important role in the effectiveness of biomass pretreatment. Besides price, another important consideration of the broad application of IL [TEA] [HSO 4 ] to pretreatment processes is recovery and recycling 24 . Based on this, we specifically reported IL [TEA] [HSO 4 ] activity for the transformation of OPEFB. The IL synthesis stage was carried out using the one-pot method by combining triethylammonium and sulfuric acid. In the pretreatment stage, we optimized the performance of IL [TEA] [HSO 4 ] based on concentration and temperature. The recycling process is carried out in several stages, including filtering, mixing solvents, washing and separating. The purpose of our recycling process is to optimize the use of IL [TEA] [HSO 4 ].
Although our concern in this work is the transformation of OPEFB for bioethanol production, however, in the development of other renewable technologies, the pretreatment results obtained can be expanded in application such as for the manufacture of activated carbon and carbon nanotubes. Two areas of study have been reported by 35 . In addition, the results of OPEFB pretreatment can be used as ingredients to decompose food waste previously reported by 36 .
In general, the use of various types of IL in the bioethanol production stage shows good performance. The use of IL is effective in increasing the enzymatic hydrolysis of cellulose to glucose. Imidazolium-based IL is reported to increase porosity and specific surface area accessible to enzymes during hydrolysis and fermentation. The use of this type of IL resulted in a glucose content of 97.7% 37 . Another IL type that has been reported is pyrrolidonium based IL. Its use causes the enzymatic hydrolysis process to effectively produce a glucose content of 91.81% 38 . The application of amino acid-based IL was also reported to be effective in producing a glucose content of 87.7% 39 . Based on these results it is known that the application of IL in bioethanol production is influenced by the use of the IL composition and temperature during the pretreatment process.

Experimental methods
Apparatus and materials. Starting   Preparation of OPEFB pulp. The pretreatment process in this work begins with the preparation of pulp from the OPEFB. OPEFB raw materials obtained from PTPN-VII (Lampung-Indonesia) were milled and separated using a 30 mesh sieve. The separated pulp was dried in the oven for 24 h until the moisture content was below 10 wt%. Furthermore, the lignocellulose content, moisture and ash content of the pulp were analyzed using standard procedures from the National Renewable Energy Laboratory (NREL).  4 ] and H 2 O were weighed as much as 8.0 g and 2.0 g, respectively. Both are put into the Schott bottle slowly and then homogenized. After that, the OPEFB pulp was weighed as much as 2.0 g and put into the Schott bottle. This composition was homogenized with a magnetic stirrer for 17 h at 80 °C using hot medium silicone oil. Furthermore, the filtrate is separated using a vacuum pump with the addition of methanol, while the resulting residue is prepared for analysis of the amount of lignocellulose after transformation. The same treatment was also used for the variation in the composition of 85% and 91%. Lignin analysis by UV-Vis. 0.45 μm filter paper is weighed, and used to filter the sample through a vacuum filter system. The filter paper and the residue stuck on it are dried and weighed. Then the residue is ignited at 575 °C for 3 h using a furnace, and followed by weighing the weight of the ash obtained. As for the filtered filtrate, 0.03 mL was taken and put into a test tube containing 2.70 mL 4% H 2 SO 4 . Furthermore, the absorption was measured using a UV-Vis spectrophotometer at a wavelength of 205 nm. The dissolved and insoluble lignin levels are calculated using the equation:

OPEFB biomass pretreatment.
Note: (2)   4 ] synthesis process based on the one-spot method. Another chemical shift data that confirms the success of the synthesis process occurs at δ = 1.106 (CH 2 -N). Figure 3B shows 24 . So that every composition we propose always keeps the water percentage at 20%.   4 ], the tissue inside the cell wall will be disturbed thereby reducing the recalcitrant nature of the OPEFB biomass. Cellulose which regenerates after pretreatment tends to be more amorphous in its macro structure making it easier for enzyme hydrolysis. Figure 4 shows the optimization results of the IL [TEA][HSO 4 ] composition for OPEFB transformation. We have observed the success of the OPEFB transformation through an increase in cellulose content. When comparing the data on lignocellulose content before pretreatment ( The optimum pretreatment process occurred at the use of 85% composition (Fig. 4B), where in this composition there was an increase in cellulose content to 45.84% and a decrease in lignin and hemicellulose content to 32.80% and 5.87%, respectively. The same trend also occurred for the composition of 80% and 91%. However, the two compositions showed that the pretreatment rate of OPEFB to increase the cellulose content tended to be www.nature.com/scientificreports/ slower. At the 80% composition (Fig. 4A) the cellulose content only increased to 32.80%, while at 91% composition (Fig. 4C) the cellulose content only increased to 40.13%. The decrease in the amount of lignin and hemicellulose from this work tends to be lower than the work reported by 30,42 . This problem is very likely to occur due to the type of lignoselullosic biomass used 43 . We used OPEFB with very different lignocellulosic content, which suggests that the IL performance results could also be different. In addition, we suspect that the prepared OPEFB pulp (size = ± 30 mesh) contributed to this result. The particle size, moisture content and type of biomass used can affect the pretreatment process 44 . Furthermore, we pay serious attention to the use of the composition 91%. In this composition, the mass of IL [TEA] [HSO 4 ] is greater than that of 80% and 85%. The mass used in the composition of 91% is 20 g which  ]. In general, the acidic properties are reported to be related to the solubility of cellulose 45 .
Another approach used to explain the solubility of cellulose during IL-based pretreatment is the type, geometry and size of anions, and the number of cations added. Cations are reported to interfere with oxygen atoms from glycosidic and hydroxyl through disperse forces and hydrogen bonding at the axial position of the cellulose fibers 46 . Figure 5 is the result of temperature optimization during OPEFB pretreatment using IL [TEA] [HSO 4 ]. Temperature optimization is another important step during the pretreatment process. The stubborn nature of lignin during the pretreatment process which can be transformed into other forms of lignin will greatly inhibit the performance of IL [TEA] [HSO 4 ]. It is necessary to release lignin so that the fermentation process is optimal. So this problem makes pretreatment conditions such as temperature as one of the variables that have an impact on lignin solubility. Several literatures report the use of variable pretreatment temperatures, as reported by 40 . This basis is what we use to study the effect of temperature (50, 80, 100, 120 and 150 °C) on IL performance in the OPEFB transformation.
On temperature optimization, our focus is on the total cellulose content. The general trend resulting from temperature optimization is that when the total amount of cellulose increases, there will be a decrease in the total amount of lignin and hemicellulose, and vice versa. This tendency was shown at the pretreatment temperature of 120 °C. We then report these results as the optimum temperature for pretreatment of OPEFB using IL [TEA] [HSO 4 ]. These results corroborate the work of the IL [TEA] [HSO 4 ] application for biomass pretreatment, as reported by 42,47 . At this temperature, the total percentage for cellulose increase was 62.58%. As for the release of lignin and hemicellulose were 48.65% and 31.32%, respectively.
On the other side of Fig. 5, we observe how an increase in temperature causes a decrease in the total cellulose content and an increase in the total lignin content. This tendency occurs at a pretreatment temperature of 150 °C. In addition to the tendency for the deterioration of cellulose structure when the temperature increases, specifically for lignin it can be explained that there are non-lignin components that combine with lignin during pretreatment using IL [TEA] [HSO 4 ]. If we look at the data in Fig. 5, the non-lignin components are most likely cellulose and hemicellulose. Where at a temperature of 150 °C there was a significant decrease in both. This problem has also been previously reported by 40 . Another factor that causes the hydrolysis of cellulose and hemicellulose is none other than the acidic nature of IL [TEA] [HSO 4 ]. Both are hydrolyzed and then undergo a dehydration reaction to 5-HMF and furfural, and contribute to the condensation reaction.

IL [TEA][HSO 4 ] recovery.
Although several previous studies reported the ability of IL [TEA] [HSO 4 ] to be recovered and recycled, the different types of biomass make this process still interesting to study. Figure 6 shows how the IL [TEA][HSO 4 ] recovery process we did. The recovery process goes through the same steps as reported by 30 with the basic principle being a gradual washing by a solvent through the use of controlled temperatures. The temperatures we report in the recovery process are 80 °C and 100 °C. This temperature was not higher than the IL [TEA] [HSO 4 ] recovery temperature reported by 40 4 ] recovery was not optimal in increasing the total cellulose content and decreasing the total lignin content. We suspect that this is due to changes in the composition and properties of IL during OPEFB pretreatment. So that this is our priority in the future. However, when compared with data on lignocellulose content of OPEFB before pretreatment (Table 1) there was a change in the percentage of cellulose content from 27.40 to 29.13%. Likewise, the percentage of lignin content changed from 34.61 to 32.57%. A comparison scenario of the OPEFB pretreatment process using IL and conventional methods is shown in Table 2. In addition to effectively releasing lignin and hemicellulose content, the application of ILs provides advantages, including low energy, short time and processing.

Conclusions
The ability of IL [TEA] [HSO 4 ] to transform OPEFB through pretreatment process was investigated in this work. This transformation aims to increase the cellulose content by reducing the lignin and hemicellulose content. The positive impact of our work is to facilitate the enzymatic hydrolysis of cellulose to reducing sugars such as glucose. In addition, this work is an attempt to increase the usefulness of IL [TEA] [HSO 4 ] in the pretreatment process. The optimization process shows that the selection of the IL composition and temperature are important factors in obtaining high cellulose content. The increase in cellulose content of OPEFB seen in the use of the IL [TEA] [HSO 4 ] composition was 85% with a pretreatment temperature of 120 °C. During the recovery process, the temperature must be kept constant. Changes in temperature can cause the decomposition of IL. These results form our basis in developing the pretreatment process of OPEFB in the future.