**4. Discussion**

Organosolv pretreatment uses organic solvents to remove lignin and hemicellulose, leaving a high purity glucan-rich fraction after solid-liquid separation. The fine tuning of organosolv pretreatment can consider a manifold of parameters including the solvent type and its concentration, catalyst addition, catalyst type and its concentration, temperature, retention time, S/L ratio, biomass particle size, and pretreatment vessel design. Substantial research on organosolv pretreatment has been carried out using a wide range of organosolv systems, applied to a wide range of lignocellulosic materials. The emphasis has been on the optimization of the pretreatment system in order to recover a high-purity and highly digestible glucan fraction, while the effect of pretreatment parameters on the recovery and purity of lignin has been comparatively less investigated [28]. Moreover, research studies using acid-catalyzed ethanol organosolv for the pretreatment of OPEFB are scarce in the scientific literature.

In this work, we demonstrated that parameters including catalyst type and its concentration, temperature, retention time, and S/L ratio had an effect on the recovery and purity of lignin following ethanol organosolv pretreatment of OPEFB. Through a series of optimization steps, where factor interaction is hypothesized to take place, OPFEB was decomposed into glucan-, lignin-, and hemicellulosic compounds-rich fractions. A delignification of ca. 90% was obtained at the following conditions: 0.07% H2SO<sup>4</sup> (g/g substrate), 50% ethanol, 210 ◦C, 90 min, and S/L ratio of 1:10. The purity and recovery of glucan was 74% and 80%, respectively, and the corresponding values for lignin were 71% and 65%, respectively.

Lignin is the most abundant aromatic polymer in nature. Lignin is composed by coniferyl alcohol and sinapyl alcohol, with a small amount of *p*-coumaryl alcohol. Currently, most of the lignin (in low-purity) is produced by Kraft pulping process which reaches 50 million tons/year as low-value residuals [40]. Low-quality lignin limits its range of applications. Lignin produced through organosolv pretreatment of lignocellulose materials can be of high-purity, widening the range of potential applications. High-purity lignin can be converted into various polymers such as surfactants, plasticizers, superabsorbent gels and hydrogels, coating, and stabilizing agents [41–43]. Lignin also has the potential to be utilized as raw material for nanofiber with antioxidant capacity, resins, and vanillin synthesis [16–18]. The compositional analysis of the glucan-rich fractions revealed that loss of lignin takes place during the precipitation and centrifugation steps. This can be related to the intrinsic limitations of the recovery methods used or to the chemical characteristics of the resulting lignin. It has been reported that the dissolved lignin is polydispersed and, therefore, the used solvent for precipitation might be specific for certain lignin fractions. Reduced efficiency of water as anti-solvent has been reported for lower-molecular weight lignins with high contents of methoxyl and phenolic hydroxyl groups [44]. Therefore, establishing relationships among organosolv pretreatment parameters, lignin molecular weight and surface characteristics, and lignin recovery yields, need to be considered in future studies. Furthermore, lignin surface and compositional characteristics will influence the end-application and, consequently, influence the economic contribution of lignin to lignocellulosic biorefineries.

Among the organic solvents employed for organosolv pretreatment of lignocellulosic materials, ethanol is the most studied solvent due to its low price, good solubility of lignin, lower toxicity compared to other alcohol-based solvents, its miscibility with water, and ease of recovery [45]. Furthermore, the use of ethanol as solvent opens the possibility for integration of 1st and 2nd generation ethanol plants. Such strategy has been proposed as a result of techno-economic bottlenecks that still need to be overcome for commercialization of lignocellulosic biorefineries [46]. Accordingly, the glucan-rich fraction could be directed to yeast fermentation into ethanol, while the hemicellulosic compounds-rich fraction could be added to the yeast fermentation left-overs, following distillation, for e.g., biogas production through anaerobic digestion. Another strategy would be to take advantage of the capacity of filamentous fungi to consume pentose sugars so additional ethanol and fungal biomass for feed applications could be produced [47]. The ethanol as an end-product or as a pretreatment solvent could be recirculated through the system through already installed distillation columns and

evaporators. Such integration strategy contributes to a needed holistic approach for cost-effective organosolv pretreatment processes, where optimization of pretreatment parameters leading to efficient material fractionation, need to be coupled with efficient fraction separation and solvent recycling methods [48]. By leading to lower energy consumption, the use of distillation is preferable to the use of evaporation for solvent recycling in systems containing water/low-boiling point organic solvent such as ethanol [48]. Reasonably, the extent of dilution of the black liquor will influence the energy consumption during distillation; therefore, studies on optimization of the amount of water added to induce lignin precipitation are also of relevance.

In this study, the crystallinity of glucan-rich fraction was 24.48% lower than that of the raw OPEFB. A decrease in crystallinity was also observed for pretreated Loblolly pine using 65% (v/v) ethanol with 1.1% (w/w) H2SO<sup>4</sup> as the catalyst at 170 ◦C for 1 h [49]. Following OPEFB delignification and decreased crystallinity, 94.06 ± 4.71% glucan digestibility was achieved with an enzyme loading of 30 FPU/g within 18 h. Previous studies showed that high digestibility yield (>75%) of glucan-rich fraction from organosolv pretreatment of lignocellulosic material was obtained with enzyme loading higher than 10 FPU/g for 42–78 h of hydrolysis [50–53]. In addition to its use for ethanol production, glucan-rich fraction has also been proposed for production of acetone-butanol-ethanol (ABE) solvent, bio succinic acid, and fat-rich biomass [28].

A catalyst is usually added during organosolv pretreatment. The main effect in the addition of a catalyst is an increase in the rate and extent at which hydrolysis of hemicellulose and the cleavage of lignin-lignin bonds (α- and β-aryl ether linkages) occur [54]. Two types of acid catalysts i.e., acetic acid and sulfuric acid were employed in this study. The lignin recovery and purity of pretreatment using sulfuric acid as catalyst were significantly higher than those achieved when acetic acid was added at the same amount. To achieve similar lignin purity and recovery, about 270 times more acetic acid was needed. Even though acetic acid is more environmentally friendly, the high amount of acetic acid added can become problematic in the further utilization of lignocellulose fractions, that is, through microbial conversion where acetic acid can act as an inhibitor [55]. The higher lignin purity and its recovery after pretreatment with sulfuric acid as the catalyst is more likely due to the higher reactivity and efficiency of sulfuric acid on breaking the lignin-carbohydrate and lignin-lignin bonds than acetic acid [21]. The result from this study is in agreement with previous research by de la Torre et al., (2013) [56] who studied lignin recovery yields with different catalysts including acetic acid and sulfuric acid. Wheat straw was pretreated with 50% ethanol as a solvent and 0.001 N of each catalyst was tested for 30 min pulping. The organosolv pretreatment using acetic acid as catalyst led to 51% lignin recovery, whereas pretreatment using sulfuric acid led to 61% lignin recovery. Therefore, the results of this study support the need for alternative and effective catalysts. The catalyst should have a comparatively lower environmental footprint than that of sulfuric acid, to be used in organosolv pretreatment systems, a research gap previously identified [28].

The observations made on lignin purity and its recovery while varying the temperature and retention time agree with those reported by other studies. In this study, when the temperature was increased from 180 ◦C to 210 ◦C, lignin purity and recovery were increased by ca. 62% and 103%, respectively. This result is in agreement with an existing strong relationship between lignin solubility and temperature [57]. High delignification (>85%) was achieved during organosolv pretreatment of Silver birch wood chips and Norway spruce using ethanol as solvent and 1% sulfuric acid as catalyst at 200 ◦C [51,58]. The purity of the lignin from organosolv pretreatment of Silver birch wood chips was higher than that obtained in this work though, which was 96% [58]. A study by Goh et al., (2011) [59] reported 52% of lignin recovery from organosolv pretreatment of OPEFB using ethanol 65% (v/v) as solvent and 1.63% of sulfuric acid as the catalyst at a temperature of 190 ◦C. Generally, to obtain a better delignification rate, longer retention times are required. Results from this study showed that the use of retention time of 30 min was only able to recover 20% of lignin with a purity of less than 50%. When the pretreatment time was prolonged to 60 min, 45% of lignin was able to be recovered with 66% purity. A retention time of 60 min was reported to be necessary in order to obtain high delignification

(≥80%) on organosolv pretreatment of mixed sawmill and beechwood [60,61]. A study by Alio et al., (2019) [60] showed that 57% of lignin was recovered from organosolv pretreatment sawdust mixture of softwood species using sulfuric acid as a catalyst for 60 min. Furthermore, a retention time >60 min was needed in order to recover >30% of lignin during sulfuric acid-catalyzed organosolv pretreatment of OPEFB [59].

The S/L ratio is another key parameter for the commercialization of organosolv pretreatment. Higher S/L ratio is more favorable due to higher concentrations of glucan, hemicellulosic compounds, and lignin per unit volume. The higher concentration of substrate per volume leads concomitantly to the reduction of the amount of catalyst and solvent used per gram of substrate. However, the S/L ratio plays a role in organosolv pretreatment performance by affecting the contact between biomass and solvent. An S/L ratio higher than 1:10 was found to be detrimental to lignin purity and its recovery. Among research works applying organosolv systems for the pretreatment of lignocellulosic materials, such S/L ratio predominates [38]. The use in this study of an S/L ratio of 1:10 represented a concentration of 0.07% H2SO<sup>4</sup> (gram acid per gram of substrate), which is almost 10× lower than that normally used for organosolv pretreatment [38], while attaining similar delignification yields together with high lignin recovery and purity. For instance, a study carried out by Goh et al., (2011) [59] showed that in order to recover 52% of the lignin from organosolv pretreatment of OPEFB using ethanol 65% as solvent at 190 ◦C for 75 min, 1.63% sulfuric acid was needed as a catalyst. Such a lower amount of acid used can have impacts on corrosion potential, economic feasibility, environmental footprint, and on the application of edible fungal biomass as feed ingredients. Altogether, the results of this work show the potential of acid-catalyzed ethanol organosolv pretreatment for fractionation of OPEFB and achieved a set of conditions leading to high lignin recovery and purity and digestible glucan. Moreover, the research work demonstrates the possibility to use much lower concentrations of acid with potential economic and environmental impacts. However, the system still can benefit from more environmentally-friendly catalysts and further system optimization that allow higher solid loading to be used, lower water usage during lignin precipitation, and clear relationships between lignin characteristics and end-application. Combining these optimization and characterization strategies with integration of OPEFB in 1st generation ethanol plants can create a beneficial biorefinery system for a such readily available lignocellulosic material in Indonesia, and in other countries such as Malaysia, the second largest worldwide producer of palm oil.
