Investigation of Microwave Irradiation and Ethanol Pre-Treatment toward Bioproducts Fractionation from Oil Palm Empty Fruit Bunch

Abstract

Lignocellulosic biomass (LCB), such as the oil palm empty fruit bunches (OPEFB), has emerged as one of the sustainable alternative renewable bioresources in retrieving valuable bioproducts, such as lignin, cellulose, and hemicellulose. The natural recalcitrance of LCB by the disarray of lignin is overcome through the combinative application of organosolv pre-treatment followed by microwave irradiation, which helps to break down LCB into its respective components. This physicochemical treatment process was conducted to evaluate the effect of ethanol solvent, microwave power, and microwave duration against delignification and the total sugar yield. The highest delignification rate was achieved, and the optimum level of total sugars was obtained, with the smallest amount of lignin left in the OPEFB sample at 0.57% and total sugars at 87.8 mg/L, respectively. This was observed for the OPEFB samples pre-treated with 55 vol% of ethanol subjected to a reaction time of 90 min and a microwave power of 520 W. Microwave irradiation functions were used to increase the temperature of the ethanol organic solvent, which in turn helped to break the protective lignin layer of OPEFB. On the other hand, the surface morphology supported this finding, where OPEFB samples pre-treated with 55 vol% of solvent subjected to similar microwave duration and power were observed to have higher opened and deepened surface structures. Consequently, higher thermal degradation can lead to more lignin being removed in order to expose and extract the total sugars. Therefore, it can be concluded that organosolv pre-treatment in combination with microwave irradiation can serve as a novel integrated method to optimize the total sugar yield synthesized from OPEFB.

1. Introduction

According to the Global Status Report (GSR) on energy consumption, approximately 80% of energy consumption is met by non-renewable fossil fuels, such as coal, petroleum, and natural gases, as opposed to merely 18% of the energy that is obtained from renewable energy sources, such as solar, wind, hydropower, and biomass [1]. Despite this fact, there are several limitations that are associated with the dependency on fossil fuels, such as finite reserves being located in politically unstable regions of the world [2], fluctuating and uncertain fossil fuel prices, fossil fuels being non-renewable energy resources, and the emission of greenhouse gases that pollutes the environment. These drawbacks on fossil fuel use limit its applicability in meeting future energy and sustainable product requirements.

Currently, the industry, as well as society, is moving toward the concept of “reuse and recovery” of resources from the primitive conception of “take, make and dispose of” resources [3]. This is to ensure a healthy and environmentally healthy society, at the same time ensuring socio-economic prosperity. Biomass has emerged as one of the sustainable alternative renewable bioresources. As such, due to the renewable nature of biomass, a term for a parallel economy based on bio-based products has been coined by state policy decision-makers, economists, and scientists: “bio-based economy” [4].

Lignocellulosic biomass (LCB) is composed of various polymers, such as polysaccharides (comprising cellulose and hemicellulose) and phenol aldehyde lignin polymer, along with some non-polar and polar substances, in which they are all interwoven together. Hence, it can be deduced that the three main components of lignocellulosic biomass are cellulose, hemicellulose, and lignin [5]. The natural recalcitrance of LCB by the disarray of lignin can be overcome through the application of suitable pre-treatment methods, which help to break down LCB into its respective components. Consequently, the accessibility of hydrolyzing enzymes to the cellulosic and hemicellulosic components can be further improved from the pre-treatment methods of LCB, in which the sugars can be fermented to produce biofuels [6]. On the other hand, the polyphenolic lignin and other hydrolysates that are extracted from the biomass can be converted into value-added chemicals that may eventually be applied as building blocks for economically vital chemicals in the industry [7].

According to Diyanilla et al. (2020), the most popular biomass is the oil palm empty fruit bunch (OPEFB) [8]. This is due to the ease of performing pre-treatment on such biomass as a result of having a comparatively low lignin content ranging from 14.2% to 38.4%. There are various pre-treatment methods that can be employed on OPEFB [8]. These include physical, chemical, physicochemical, and biological pre-treatment methods. A widely applied pre-treatment method for the environmentally friendly and efficient fractionation of bioproducts from biomass, such as OPEFB, is biological pre-treatment, such as enzymatic pre-treatment, due to its ability to maximize the sugar yield. However, studies on different pre-treatment methods have shown that the combination of chemical pre-treatments with an increase in physical conditions, where higher temperatures and pressures are applied, can lead to a better delignification process [8]. Examples of combined pre-treatment methods include steam explosion with alkaline delignification, hot water with alkaline pre-treatment, alkaline pre-treatment with ultrasonication, electron beam irradiation with ionic liquid pre-treatment, and steam-alkali-chemical pre-treatment.

Extensive research has been conducted over recent years on the bioproducts recovery from LCB pre-treated with microwave-assisted irradiation or the organosolv pre-treatment. A study was conducted on the sugar extraction from bamboo wood subjected to microwave pre-treatment at 121 °C, where a 36.9% conversion with 16.8 g/100 g of sugar yield was obtained [9]. Furthermore, a three-component deep eutectic solvent (DES) comprising of lactic acid, glycerol, and choline chloride, subjected under microwave irradiation at 393 K for a duration of 30 min, with a solid-to-liquid ratio of 1:50, was able to achieve a delignification rate of 45 wt% for the pre-treatment of wheat straw [10]. Moreover, the use of a physical pre-treatment such as the microwave irradiation on wheat bran, corn stalks, and miscanthus stalks was able to achieve hemicellulose solubilization of approximately 30%, which in turn led to an enhanced recovery of cellulose in these low-lignin yielding plants [11].

On the other hand, organosolv pre-treatment is regarded as a promising technique for the pre-treatment of biomass through the application of organic solvents, such as ethanol, glycols, acetone, and small amounts of acids, to stimulate the pre-treatment efficiency [11]. Wei et al. (2021) compared the acid- and alkali-catalyzed ethylene glycol organosolv pre-treatment for the production of sugars from bagasse. The results showed the efficiency of the ethylene glycol-hydrochloric acid (HCl) pre-treatment in the removal of lignin (67.1%) and hemicellulose (99.3%) due to the synergistic effect of the ethylene glycol solvent with the HCl acid when compared with the single ethylene glycol pre-treatment [12]. Furthermore, the organosolv pre-treatment of mustard biomass with acetone and acid catalyst at a constant autoclave temperature of 121 °C was able to yield 80% glucose [13]. Additionally, the acid-catalyzed ethanol pre-treatment of poplar biomass was able to release up to 78% of the polysaccharides [14]. A two-stage acid hydrolysis on ethylene glycol pre-treated degraded OPEFB was also conducted on the sugar-based substrate recovery, where stage I 45 wt% of the acid coupled with high cellulose loading of 21.25 w/v% with 12 wt% of acid at 65 °C for a duration of 30 min was able to release 89.8% of optimum sugar yield [15].

Thus, it can be realized that microwave-assisted pre-treatment methods have not been widely explored and employed for the fractionation of biomass. As a result, this research project was conducted to investigate this novel and facile process intensification to address the issue that such a knowledge gap exists, especially for the re-utilization of biomass waste such as OPEFB to remove the lignin while simultaneously facilitating higher total sugar yield. As such, the main purpose of this research was to investigate the effectiveness of the synergistic relationship between microwave irradiation and organosolv pre-treatment (a physicochemical process) on OPEFB for delignification and total sugar yield, as well as to characterize the physicochemical properties of the bioproducts extracted from the pre-treatment method through various characterization studies. Consequently, the novelty of this research work lies in the focus on the effectiveness of the microwave-assisted organosolv pre-treatment method as opposed to the commonly applied singular approach of either microwave irradiation (physical process) or organosolv pre-treatment (chemical method) on OPEFB samples.

2. Experimental Material and Methods

2.1. Materials

The OPEFB samples were provided and collected from the Research and Development Centre at Universiti Putra Malaysia-Malaysian Technology Development Corporation (UPM-MTDC, Serdang, Malaysia). Chemicals such as phenol solution 89%, glucose standard solution, iodine solution (starch indicator), sodium thiosulphate (Na₂S₂O₃) 99%, potassium permanganate (KMnO₄) 99%, and potassium iodide (KI) 99% were obtained from Sigma-Aldrich, St. Louis, MO, USA.

2.2. Materials Preparation

The OPEFB was cut into smaller pieces before being dried in the oven at 80 °C overnight (for 24 h). This was done to avoid fungal or microbial growth, as well as to remove moisture from samples prior to grinding and blending. The dried pieces of biomass waste were then ground, blended, and sieved using an appropriate mesh with a size of 150 μm to ensure a uniform size of feedstock material before the pre-experiment feedstock characterization and microwave-assisted organosolv pre-treatment were conducted.

2.3. Microwave-Assisted Organosolv Pre-Treatment

The pre-treatment methods for the conversion of OPEFB biomass into the required bioproducts are regarded as the most critical step due to the complicated structure of the lignocellulosic biomass [16]. The pre-treatment method carried out in this research project is the combination of the organosolv pre-treatment and microwave-assisted irradiation. The organosolv pre-treatment of OPEFB samples using ethanol was added to the samples before being placed into a modified microwave reactor. The solutions were pre-treated using 35%, 55%, and 75% volume in aqueous solution of ethanol concentrations with various microwave powers (65, 130, 260, 390, and 520 Watts) at various reaction times (30, 60, and 90 min). The residue obtained from the pre-treatment process was separated from the solvent via filtration, and the solid residues were dried at 80 °C for 24 h to obtain a constant weight. The pre-treated solid residues were then subjected to lignin analysis via the Kappa number analysis, as well as the subsequent characterization. The schematic design of the research activity and the research methodology flow chart (inclusive of the pre-treatment stage) are displayed in Figure 1 and Figure 2, respectively.

Each set of experiments was conducted in triplicates, where a mean value was obtained for the characterization of lignin and total sugars analysis. Additionally, the standard deviation was computed for each set of data using the mean value. The calculated standard deviation did not exceed a value of 10, ensuring that the repeatability tests are reliable.

2.4. Lignin Analysis

Kappa number analysis was conducted to analyze the lignin content based on the different conditions employed during the pre-treatment process. A 250 mL solution was prepared (200 mL distilled water with 0.1 g pre-treated biomass waste + 25 mL of 0.1 N potassium permanganate (KMnO₄) + 25 mL of 4.0 N sulphuric acid (H₂SO₄)). After obtaining 10 min of reaction from the solutions by gently stirring the solutions contained inside the volumetric flask, potassium iodide (KI) was added to the solution. Blank (standard) was carried out using the same steps without the inclusion of the OPEFB sample. Titration was then carried out using 0.5 N sodium thiosulphate (Na₂S₂O₃) and 1.0 N indicator starch. The lignin content was calculated using the Equations (1)–(3):

$$p=\frac{\left(b-a\right)\times N}{0.1},$$

(1)

$$\mathrm{L}\mathrm{o}\mathrm{g} k=Log \left(\frac{p}{W}\right)+0.00093 \left(p-12.5\right),$$

(2)

$$\% \mathrm{L}\mathrm{i}\mathrm{g}\mathrm{n}\mathrm{i}\mathrm{n}=0.147\times k,$$

(3)

where a and b are the volume of Na₂S₂O₃ for OPEFB and blank titration, respectively (in mL), N is the normality of Na₂S₂O₃, p is the volume of Na₂S₂O₃ (in mL), W is the weight of the OPEFB sample used, and k is the Kappa number (volume of 0.1 N KMnO₄ consumed by 1 g of OPEFB).

2.5. Total Sugars Analysis

The total sugars analysis was carried out using the phenol–sulfuric acid method. A glucose standard solution of 2 mL was prepared in various concentrations (0, 10, 20, 30, and 60 ppm) mixed with 1 mL phenol solution (5% vol concentration) in respective test tubes. After that, 5 mL of concentrated H₂SO₄ was dropped to the surface of the solution vertically, and the mixture was left for 10 min before the measurement of the UV-Vis spectrophotometer (PG Instruments T60) using deionized water as blank at wavelength 480 nm, with this result of the measurement known as the standard curve. Furthermore, the sugar solution was prepared by mixing the OPEFB sample and distillate water at a composition ratio of 1:250 as a sample solution. A 1 mL sample solution was extracted and mixed with 1 mL distilled water in a test tube. 5 mL of H₂SO₄ and 1 mL of phenol solution (5% vol concentration) were added simultaneously to a test tube containing 1 mL sample solution mixed with distilled water prior to their UV-Vis spectrophotometer measurement.

2.6. Sample Characterizations

All the prepared samples (both pre-treated and without) were characterized using a scanning electron microscope (SEM, Hitachi, S-3400N, Tokyo, Japan) to observe their surface morphology. The samples were sputter-coated with gold prior to imaging with SEM, with a magnification power ranging from ×100 up to ×2000.

Furthermore, Fourier transform infrared spectroscopy (FTIR, Nicolet, IS10, Waltham, MA, USA) was used to characterize and determine the functional groups that were attached to the samples. The FTIR spectra were obtained using a potassium bromide (KBr) pellet/disc containing the grounded sample. The sample was then scanned, with the spectra being recorded between 400 cm⁻¹ and 4000 cm⁻¹ with a resolution of 4 cm⁻¹.

3. Results and Discussion

3.1. SEM Characterization Study

Figure 3 shows the surface morphological changes of the untreated and microwave-treated OPEFB samples in the SEM images through the observation of the external surfaces of the samples. Figure 3a shows that the OPEFB fibers are made up of many oriented microfibrils adhered to each other, with their surface morphology observed as having smooth surfaces with few flakes, as well as no opened structures. Figure 3b,c also display similar morphologies observed on the surface of OPEFB samples without being subjected to microwave power and/or organosolv pre-treatment. Contrary to expectations, a flakier surface was observed on the surface of OPEFB samples when microwaved at 130 W for 90 min, as seen in Figure 3d. Moreover, an opened and deepened structure was observed in Figure 3e when the samples were microwaved at a slightly higher power of 390 W for 90 min. Furthermore, Figure 3f demonstrates a more opened and deepened surface structure along with flakier surfaces when the samples are microwaved at an even higher power of 520 W for the same duration. Cavities were observed for OPEFB samples when subjected to microwave irradiation, revealing opened and deepened surface structures. This is due to the microwave energy that heats up the inner part of OPEFB samples, resulting in the disruption of the plant material’s cell wall as the pressure increases with higher power [17].

Microwave-irradiation heating can be explained via the thermal effect and athermal (non-thermal) effect. The lignin and cell wall components are disrupted through an increase in the temperature and pressure applied during the thermal effect, whereas relaxation and polarization of dielectric substances under the influence of electromagnetic fields generate non-thermal effect forces. The disruption of hydrogen bonds is caused by displacement resulting from the realignment of the polar molecules, where the cell wall components, such as the lignin and cellulose crystallinity breakdown, are destroyed, enhancing the hydrolysis process. Hence, it can be said that the dielectric property is the principal factor that affects the heating property of the biomass under microwave irradiation [2].

A study by Xiaokang et al. (2020) compared freeze-drying and microwave-assisted extraction on mushroom samples, where the images obtained from the SEM showed that all extractions led to the deformation of mushroom samples to some extent [17]. In the case of the microwave extraction method, some cavities were observed due to the microwaves that help heat up the inner part of the samples, which led to pressure build-up inside the cells and resulted in cell wall disruption [18]; hence, this demonstrated that the opened surface structure is indicative of the biomass cell walls being disrupted, which was in agreement with the literature.

Figure 4 and Figure 5 show the surface morphological changes of the microwave-assisted organosolv pre-treatment of OPEFB samples in the SEM images through the observation of external surfaces of the samples at the lower and higher microwave power of 65 W and 520 W, respectively. Flaky surfaces were seen on the OPEFB samples in Figure 4a when being pre-treated with lower ethanol concentration (35 vol%) at lower microwave power (65 W), whereas flakier surfaces with more opened and deepened surface structures were observed on the surface of OPEFB samples in Figure 5b when pre-treated with higher ethanol concentration (55 vol%) at higher microwave power (520 W).

A deepened surface structure indicated that the delignification process successfully took place in order for the total sugar analysis to be conducted for subsequent analysis. A more deepened surface structure indicated that a higher delignification rate was achieved for samples pre-treated with a slightly higher ethanol concentration of 55 vol% at higher microwave powers due to the higher thermal degradation attacking the lignin protective layer. Since OPEFB is a lignocellulosic biomass comprised mainly of cellulose, hemicellulose, and lignin, the functional groups in the ethanol organic solvent break through the lignin layer, allowing for the extraction of cellulose and hemicellulose. On the other hand, microwave power assists in increasing the temperature of the organic solvent to degrade the OPEFB, helping the ethanol solvent to be more active in breaking the lignin protective layer of the biomass. The inclusion of an organic solvent such as ethanol as a pre-treatment process in the microwave extraction of biomass helps shield the samples from the high-penetrating power of the microwaves. Subsequently, this can lead to a higher recovery of the sugar content from the biomass.

A study by Gonçalves et al. (2015) observed that untreated coconut shell samples showed a more highly ordered structure of the coconut fibers, whereas treated coconut shell samples showed modified disoriented sample structures [19]. Another study by De et al. (2014) used microwave irradiation in the presence of glycerol solvent for the pre-treatment of bagasse, where they found that the physicochemical properties of the biomass changed considerably before and after the pre-treatment processes, and a large amount of sugar was released during hydrolysis [20]. Binod et al. (2012) studied the effect of microwave, microwave-acid, microwave-alkali, and microwave-acid-alkali methods on enzymatic saccharification, as well as the removal of lignin from bagasse. Their results pointed out that microwave-assisted extraction methods, as opposed to microwave irradiation alone, can lead to a significant yield of fermentable sugars and lignin removal [21]. This further substantiates the efficiency of a physicochemical method such as the microwave-assisted organosolv pre-treatment in the extraction of valuable bioproducts from OPEFB. The results from the SEM images clearly demonstrated the disruption of the biomass cell wall, hence indicating the successful removal of the lignin layer in order to extract the sugar content.

3.2. FTIR Characterization Study

It is imperative to first understand the structures of the cellulose, hemicellulose, and lignin components of OPEFB before analyzing the FTIR bands. The structures of the three main components of an LCB such as OPEFB, cellulose, hemicellulose, and lignin fibers, are illustrated in Figure 6. The lignocellulosic polymers are linked to each other via specific bonds and contribute to over 70% of the total biomass [2]. Cellulose is a beta β-(1,4)-linked chain of glucose molecules. The resistance of the crystalline cellulose being subjected to degradation is contributed by the hydrogen bonds between the different layers of this particular polysaccharide.

On the other hand, hemicellulose is composed of various five- and six-carbon sugars, such as galactose, arabinose, glucose, xylose, and mannose. As for lignin, the three major phenolic compounds that constitute this particular component include p-Coumaryl alcohol, Coniferyl alcohol, and Sinapyl alcohol. Lignin is synthesized through the polymerization of these three major phenolic compounds, where their ratio within the polymer varies according to the different parts of the plants, such as the wood tissues and cell wall layers [2]. The linkage between lignin and polysaccharides is regarded as a lignin–polysaccharide complex due to the composite links that connect lignin to the polyoses side groups of arabinose, galactose, and 4-O-methylglucoronic acid, as depicted in Figure 7 [22]. Hence, the difficulty in achieving a complete separation of lignin from lignocellulosic materials resulted from the sterically structured component of lignin. Thus, a combination of methods, such as a physicochemical method, is typically employed in order for the separation of lignin from lignocellulosic biomass material to be accomplished [22]. The cellulose, hemicellulose, and lignin components form structures known as microfibrils, which are then structurally organized into macrofibrils that are responsible for mediating the structural ability in the cell wall of plants.

The FTIR spectra of untreated OPEFB samples, as well as OPEFB samples pre-treated with 55 vol% of ethanol at the lower and higher microwave power of 65 W and 520 W, respectively, for a duration of 90 min, are presented in Figure 8. The absorption peak at 2850 cm⁻¹ represented the acetyl group (CH₃C=O) of hemicellulose or methyl oxide group (OCH₃) of lignin; hence, this indicated that the ethanol solvent at both the lower and higher microwave power with a longer duration was able to achieve a considerable delignification rate, with a higher intensity of absorbance peak observed for 520 W compared with 65 W. It can also be observed that the absorbance peak at 3330 cm⁻¹ was intensified. This indicated that the ethanol solvent at the higher microwave power with a longer duration was able to achieve a significant delignification rate, thus further supporting the correspondence of the absorbance peak at 3330 cm⁻¹ to the hydrogen bond of cellulose [23].

The broadening of the band at 3200 to 3400 cm⁻¹ was associated with the O-H stretching of the cellulose hydrogen bonds [24]. Furthermore, the absorption peak at 1243 cm⁻¹ indicated the presence of lignin for the OPEFB samples pre-treated with both microwave irradiation and organosolv pre-treatment due to the presence of the aryl alkyl ether (C-O-C) bond [23]. Moreover, the absorbance peak at 1243 cm⁻¹ observed for both the fresh and pre-treated OPEFB samples was also attributed to both the C-O-C and OCH₃ groups for lignin [25]. The absorption bands at 1032 cm⁻¹ were also present in both untreated and microwave-assisted ethanol pre-treated OPEFB samples, attributed to the cellulose and hemicellulose components of the biomass material [23].

However, a prominent difference was observed at an absorbance peak of 1735 cm⁻¹ for both untreated and treated OPEFB samples, where a more intense peak was observed for the untreated samples when compared to the treated samples. This observation corresponds to the C=O stretching vibration in the ester groups of hemicellulose [26], where a higher hemicellulose composition is associated with an untreated biomass sample, with the hemicellulose content still present within the biomass material. For the OPEFB samples that undergo microwave-assisted organosolv pre-treatment with ethanol solvent, the microwave power increases the temperature of the solvent in order to allow for the functional groups in the solvent to break through the lignin layer, allowing for the extraction of cellulose and hemicellulose in OPEFB. However, some of the cellulose and hemicellulose dissolved in the solvent during the microwave irradiation process; hence, the smaller amount of hemicellulose in the treated OPEFB samples was in line with the observation of the lower intensity peak of 1735 cm⁻¹.

3.3. Lignin Analysis

Figure 9 shows the amount of lignin left for OPEFB samples subjected to microwave irradiation without organosolv pre-treatment, where a decreasing trend in the amount of lignin left in the sample was observed when the samples were subjected to higher microwave powers and longer duration. A higher microwave power of 520 W recorded a higher delignification rate with 1.15% of lignin when compared to 4.61% of lignin for a lower microwave power of 65 W, both at a longer duration of 90 min. This was attributed to the working principle of microwave, where higher penetrating powers and energies are associated with higher microwave powers, thus allowing for more disruption to take place in the OPEFB cell walls, subsequently breaking the lignin protective layer and resulting in a higher delignification rate and smaller lignin amount left in the OPEFB sample. Moreover, the longer microwave duration allows for more lignin to be broken down and removed, hence resulting in a higher lignin removal.

For the OPEFB samples pre-treated with different concentrations of ethanol solvent without being subjected to microwave irradiation, a significant decreasing trend in the amount of lignin left in the sample was observed with an increase in ethanol concentrations from 35 to 55 vol%, as seen in Figure 10. However, a slight increase in the amount of lignin left in the sample was also observed when the ethanol concentration increased from 55 to 75 vol%. The OPEFB samples pre-treated with 35 vol% ethanol recorded a higher delignification rate, with 9.87% of lignin compared with 4.90% of lignin when pre-treated with 55 vol% ethanol; however, the delignification rate slightly increased by 7.52% of lignin when pre-treated with 75 vol% ethanol. This observation can be explained by the effect of the different ethanol concentrations on the Kappa number.

The Kappa number analysis estimates the number of lignin left in the sample [5]. The amount of lignin left in the sample (%) can then be calculated using the Kappa number, as displayed in Equations (1)–(3). The amount of lignin left in the OPEFB samples decreases significantly after a 35 vol% ethanol solvent is applied. This indicated an increase in the number of degraded lignin by increasing the ethanol concentration. Therefore, this observation was important for future cellulose and hemicellulose content determination. Cellulose and hemicellulose were able to be converted into sugars through acid hydrolysis, allowing us to investigate the effect of organosolv pre-treatment on sugar yield. The study conducted by Nurfahmi et al. (2016) on the effects of organosolv pre-treatment and acid hydrolysis on OPEFB reported a decrease in the Kappa number from 38 to 25.8 when the ethanol concentration increased from 35 to 55 vol%, and the Kappa number increased slightly from 25.8 to 26.4 when the ethanol concentration increased from 55 to 75 vol% [5].

Figure 11 displays the lignin amount left for OPEFB samples subjected to microwave-assisted organosolv pre-treatment. A decreasing trend in the amount of lignin was observed when the ethanol concentration increased from 35 to 55 vol%, with a smaller amount of lignin also being observed for higher microwave powers with a longer pre-treatment period. The OPEFB samples subjected to a higher microwave power of 520 W pre-treated with 55 vol% ethanol recorded a higher delignification rate, with 0.57% of lignin compared with 2.30% of lignin for the lower microwave power of 65 W pre-treated with the same concentration of ethanol, both over a longer duration of 90 min. A similar observation was also noted, with a slight increase in the amount of lignin when the ethanol concentration increased from 55 to 75 vol%.

The trend in observation for the amount of lignin left in OPEFB samples after undergoing the microwave-assisted organosolv pre-treatment process was also attributed to the strong relationship between lignin solubility with microwave power and duration [27]. The high surface area of the OPEFB samples resulted in them being more susceptible to chemical attacks, which enhanced the delignification and degradation of lignin. The mechanism of the lignin depolymerization of organic solvents (such as ethanol) was mainly conducted through the cleavage of aryl ether linkages [28]. The delignification achieved by the ethanol solvent can be explained by the Hildebrand solubility parameter [29]. The highest delignification achieved by ethanol is attributed to its closer Hildebrand solubility parameter (a value of 26.2 MPa⁻¹) when compared to that of lignin (a general value of 22.5 MPa⁻¹) [30]. It was further supported that the smaller differences in the Hildebrand solubility between the lignin and organic solvent used would result in the largest possible solubility [31]. A higher delignification rate (lower lignin amount left in the sample) is achieved through a higher solubility of lignin in the ethanol solvent.

It was also further reported that the lignin solubility increases when the ethanol concentration reaches a maximum of 70 vol% [32]. This explains the slightly higher amount of lignin left in the OPEFB samples subjected to a 75 vol% of ethanol pre-treatment due to a slightly lower solubility of the lignin with higher ethanol concentrations, leading to a slightly lower delignification rate. From the microwave-only OPEFB pre-treatment data recorded in Figure 9, it was observed that the highest delignification rate with the lowest amount of lignin left in the sample (1.15%) was achieved with the OPEFB sample subjected to the highest microwave power of 520 W for the longer duration of 90 min. Contrary to expectations, from the organosolv-only OPEFB pre-treatment data recorded in Figure 10, it was observed that the highest delignification rate with the lowest amount of lignin left in the sample (4.90%) was achieved with the OPEFB sample subjected to the 55 vol% ethanol concentration. In comparison to the physicochemical process intensification of the microwave-assisted organosolv pre-treatment of OPEFB samples, which recorded the highest delignification rate with the lowest amount of lignin left in the sample (0.57%), it was observed that the singular approaches of pre-treatment were less effective in breaking the lignin protective layer, with the microwave-only pre-treatment being able to remove the lignin approximately four times more effectively than the organosolv-only pre-treatment. This observation was attributed to the penetrative ionizing powers of the microwave that facilitated the breakdown of the lignin protective layer. With the inclusion of a combinative approach by first pre-treating the OPEFB samples with ethanol before subjecting them to microwave irradiation, the microwave powers helped increase the temperature of the ethanol solvent, which in turn helped the solvent to be more active in attacking the OPEFB lignin layer, subjecting it to a thermal degradation process that is less severe. Hence, the microwave-assisted organosolv pre-treatment was proven to be more effective in achieving the highest delignification rate, with the lowest amount of lignin left in the OPEFB sample of 0.57%.

3.4. Total Sugars Analysis

Figure 12 presents the total sugar yield obtained for OPEFB samples pre-treated with 35, 55, and 75 vol% of ethanol concentrations while subjected to various microwave powers for different durations. It showcased the optimum (highest) yield of total sugars, which was obtained at 87.8 mg/L using 55 vol% of ethanol concentration at a higher microwave power and a longer microwave duration of 520 W and 90 min, respectively. Contrary to expectations, the OPEFB samples pre-treated with 35 vol% of ethanol and subjected to a lower microwave power of 65 W for a shorter treatment duration of 30 min yielded the lowest total sugar content of 30.1 mg/L. This observation was attributed to the correlation between microwave power and temperature [33], along with the mechanism or working principle of microwave-assisted organosolv (ethanol) pre-treatment. The optimum sugar yield was obtained for the highest microwave power of 520 W, which corresponds to approximately 180 °C (see Figure 13) [17]. A temperature higher than 180 °C could lead to the degradation of OPEFB bioactive compounds, such as hemicellulose [5]. Therefore, the maximum microwave power used in this study is within the suggested range. Furthermore, the mechanism of the microwave-assisted organosolv pre-treatment entails the microwave power increasing the temperature of the ethanol solvent, effectively breaking down the lignin protective layer. Consequently, this process leads to a lower amount of lignin left in the OPEFB sample, subsequently necessitating the extraction of the total sugars from within the OPEFB biomass sample. Thus, the optimum sugar yield was attained using the combinative approach of the microwave-assisted organosolv process intensification pre-treated with 55 vol% ethanol and subjected to the maximum microwave power with a longer duration of 520 W and 90 min, respectively.

The hydroxyl groups of solvents can influence the organosolv pre-treatment process. Ethanol solvent has one hydroxyl group, where the mechanism of the hydroxyl group involves the β-O-4 linkage cleavage of lignin; as such, this degradative pathway resulted in the formation of a dissolved organosolv lignin. This relates back to the solubility justification, where a higher delignification rate was achieved with higher solubility of lignin in a higher concentration of ethanol solvent (55 vol%). However, the solubility of lignin increases when the ethanol concentration reaches a maximum of 70 vol% [32], as previously discussed in Section 3.3. Therefore, the slightly higher amount of lignin left in the OPEFB samples can be explained by the lower solubility of lignin at higher ethanol concentrations, leading to a slightly lower delignification rate. Consequently, a comparison could be made among the samples pre-treated with the lowest concentration (35 vol%), slightly higher concentration (55 vol%), and highest concentration (75 vol%) of the ethanol solvent. Samples pre-treated with 55 vol% of ethanol yielded the highest amount of sugar extracted due to the higher delignification rate achieved, followed by those pre-treated with 75 vol% and 35 vol% of ethanol.

A further process parameter study was also conducted by further increasing the microwave powers (beyond the maximum of 520 W), duration (beyond the maximum of 90 min), and ethanol concentrations (beyond the maximum of 75 vol%) in order to study the effect of these three parameters on the amount of lignin and, consequently, the total sugar yield. With an increase in microwave power to 100% (650 W) for the longer durations of 105 and 120 min, the amount of lignin left in the 85 vol% ethanol pre-treated OPEFB samples were recorded to be 0.67% and 0.65%, respectively; as a result, the total sugar yield was recorded to be 76.7 and 77.9 mg/L, respectively. As such, it was observed that there was a higher amount of lignin left in the OPEFB samples, with an increase in the process parameters, consequently leading to a lower amount of sugar extracted. This observation was attributed to the degradation of hemicellulose above 180 °C since the microwave power of 650 W corresponds to a temperature exceeding the hemicellulose stability range, which is between 180 °C and 340 °C [5,17]. Thus, this temperature range affects the total sugars extracted, leading to a lower amount of sugars yield achieved. Moreover, the crucial physicochemical property that became the subject matter of this research work, leading to the effectiveness in extracting the total amount of sugars from OPEFB samples, is the higher solubility of organosolv lignin in the ethanol solvent used. Organosolv lignin has a tendency to aggregate in most solvents, affecting the process of lignin recovery. In a study on Alcell lignin and its solubility in ethanol-water mixtures, it was demonstrated that the solubility of lignin increased as the concentration of ethanol increased to a maximum of 70 vol% [32]. When the precipitation process of lignin was conducted by diluting solvent with water, decreasing the amount of organic solvent, the solubility of lignin decreased. This led to less lignin being recovered, with more lignin left in the sample. The decrease in lignin solubility was attributed to the increased coagulation degree of lignin in the ethanol solvent [32], with the larger particle size of lignin subsequently leading to a lower surface area reaction [32] for the precipitation process to take place, thereby resulting in a lower lignin recovery; hence, a lower amount of sugar was extracted due to the presence of lignin that was not successfully recovered.

There is a correlation between microwave power and temperature, whereby the temperature increases as microwave power increases, as depicted in Figure 13. Increasing the temperature helps promote the separation of bioactive compounds from the OPEFB, which leads to a higher reaction rate to elevate and promote the separation and cleavage of lignin bonds. However, high extraction temperatures may result in the degradation of some thermolabile bioactive compounds. Nurfahmi et al. (2016) reported that the thermal stability of hemicellulose was between 180 °C and 340 °C [5]. The maximum temperature that was used in this research project was 80% microwave power (520 W), corresponding to approximately 180 °C, as observed in Figure 13 [17]; hence, the maximum microwave power did not exceed the suggested range.

Similarly, there is a correlation between microwave duration and microwave temperatures, as depicted in Figure 14. The OPEFB sample, being pre-treated with 55 vol% ethanol subjected to 80% of microwave power (520 W), was taken as an example of the correlation demonstration. This was due to the temperatures of the samples being recorded instead of the temperature of the microwave oven after each sample was subjected to microwave irradiation, as the microwave oven used had no temperature probes for the measurement of the corresponding microwave powers, resulting in different temperatures being recorded for each sample (with different pre-treatment conditions). It was observed that a cubic equation best represented the relationship between these two parameters, with a higher correlation coefficient ($R^2$ of 0.9057) being achieved in comparison to a slightly lower $R^2$ of 0.9015 achieved for a quadratic equation. The cubic correlation indicated that, with a higher microwave duration, higher temperatures were recorded for the OPEFB samples. Naturally, as seen in Figure 13, higher microwave powers corresponded to higher microwave temperatures. Likewise, from the data recorded for all the experimental sets, the temperature of the OPEFB samples increased with a rise in the microwave powers applied. Therefore, this observation, as well as the cubic relationship plotted in Figure 14, justified the correlation between microwave duration and temperatures.

Under the working principle of microwave radiation, the occurrence of dipole rotation takes place in dielectric materials containing polar molecules that encompass an electrical dipole moment. Consequently, energy in the form of electromagnetic radiation is converted into heat energy in the materials as a result of the interactions between the dipole and the electromagnetic field [33]. In order to interlink the microwave power and treatment temperature, it is imperative to understand the phenomenon of thermal runaway in a microwave irradiation process. Thermal runaway is defined as the dramatic change in temperature resulting from small changes in the geometrical sizes of the heated material or the applied microwave power [33]. This phenomenon may lead to positive feedback in the material, where warmer areas are able to better accept more energy than the colder areas.

Theoretically, the relationship between temperature and the applied microwave field strength (or microwave power) follows a characteristic “S”-shaped curve, as reported by Brodie (2011) and represented in Figure 15 [33]. As the microwave power increases steadily, the temperature also rises steadily along the stable lower arm of the power curve. In the region of the critical power range, a small increase in the electric field strength may result in the equilibrium temperature shifting from the lower limb to the higher limb of the curve, for which the resulting change in temperature could be rather rapid and substantial. This temperature jump gives rise to the phenomenon of thermal runaway. The organic solvent, along with microwave irradiation (which provides high penetrating microwave power and, thus, higher temperature), was able to extract the lignin and hydrolyses of hemicellulose through the breakage of the lignin wall and the loosening of the crystalline structure of cellulose and hemicellulose, enabling the extraction of total sugars.

According to Kumar et al. (2020), the selection of suitable pre-treatments of the biomass material is one of the most crucial factors in extracting valuable bioproducts for various applications, in which the choice of the pre-treatment relies on the physicochemical nature or properties of the biomass. This statement can be justified by the fact that different biomasses require different methods to overcome the natural recalcitrance to ensure that a higher sugar yield can be achieved [2]. Moreover, Tsubaki and Azuma (2011) compared the conventional steam explosion method to the microwave irradiation process and reported that the removal efficiency of lignin is comparable between the two methods; however, the microwave irradiation recorded a higher sugar yield since this physical pre-treatment method specifically affects the polysaccharide fraction, as it has higher hydrophilicity, and thus, is more sensitive to hydrothermal catalysis when compared to the steam explosion [34].

An experiment conducted by Tang et al. (2017) used an organic amine catalyst for their pre-treatment method of corn stover, where the synergistic effect of n-propylamine and aqueous ethanol reported a sugar yield of 83.2% [35]. Through the research conducted by Pan et al. (2005) on the biorefining of softwoods by making use of ethanol as the solvent for organosolv pulping, it was observed that more than 90% of the sugars were retrieved by using a combination of the organosolv pre-treatment and enzymatic hydrolysis on both softwoods and hardwoods [36]. Furthermore, Ravindran et al. (2018) reported that the use of an organic amine catalyst that is capable of breaking the bond joining the lignin and hemicelluloses, proving it to be an efficient use of an organic solvent for high lignin-containing biomass. The sugar yield was achieved at 29.05 mg/g through the use of ethanol-assisted organosolv pre-treatment of biomass (as spent coffee waste) [37].

Gümüşkaya investigated the chemical content of alkali sulfite pulp at temperatures ranging between 120 °C and 200 °C and reported that the sugar content in the pulp slurries increased with a rise in the treatment temperature [38]. Additionally, the lignin content was reported to decrease with increasing heating treatment temperature. Therefore, it is a good indicator to set the heating temperature above 120 °C during the delignification process to obtain a higher sugar yield [38]. A high-temperature reaction can help promote a higher rate of reaction, thus allowing for the separation of lignin from the biomass in order to extract the sugar content. However, it was also reported that the thermal stability of hemicellulose degrades at temperatures above 340 °C [5]; hence, a temperature higher than 180 °C may decrease the total sugar yield due to hemicellulose degradation [39,40]. It was also reported that a long duration of the biomass heating process by using ethanol as a solvent seemed to dissolve some amount of hemicellulose and, at the same time, led to an increase in the solubility of lignin in pulp slurries [39].

The increase in the total sugar yield as a result of increasing the ethanol concentration was also attributed to the ether chain breaking of lignin and hemicellulose molecules. This occurrence can be justified through a higher conversion of cellulose and hemicellulose into sugars being achieved as the ethanol concentrations increase [5]. However, the total sugars seemed to decrease when 75 vol% of ethanol concentration was applied in the OPEFB pre-treatment. This was due to the effect of heavy ethanol concentration that caused some hemicellulose content to be decomposed at above 60 vol% concentration of the ethanol solvent [41].

4. Conclusions

In summary, it was concluded that OPEFB samples pre-treated with 55 vol% ethanol concentration and subjected to a higher microwave power of 520 W for a longer duration of 90 min led to the highest delignification rate, with 0.57% of lignin remaining in the OPEFB samples after the microwave-assisted organosolv pre-treatment, further corresponding to the optimum yield of total sugars obtained as 87.8 mg/L. The data on surface morphology supports this finding, revealing that OPEFB samples pre-treated with similar pre-treatment conditions have a higher degree of opened and deepened surface structure. These observations were attributed to the higher thermal degradation achieved, where the microwave power functions to an increase in the temperature of the ethanol solvent, which in turn helps facilitate the breakdown of the OPEFB protective lignin layer, leading to more lignin being removed in order to expose and extract the total sugars content.

The potential decomposition mechanism behind these observations on the lignin amount and total sugar yield is the disruption of hydrogen bonds that is caused by the displacement reaction resulting from the realignment of polar molecules, where cell wall components, such as cellulose crystallinity, are destroyed, enhancing the hydrolysis process of extracting the sugar. When LCB such as OPEFB is irradiated through microwave-assisted irradiation, the atoms or molecules within the biomass are thermally vibrated, corresponding to a rise in temperature of the organic material without rapid degradation of the biomass samples, as the presence of the ethanol organic solvent along with the working principle of microwave irradiation ensures a less severe process of thermal degradation, thereby preserving the valuable bioproducts to be extracted. At the same time, the function of the ethanol solvent is to allow for the cleavage of $\beta$-O-4 linkages and ester bonds, which are the major mechanisms of lignin cleavage.

To conclude, these results suggest that OPEFB subjected to microwave-assisted ethanol pre-treatment can help to extract the total sugars for various uses in industrial applications, such as its emergence as one of the sustainable alternative bioresources that are renewable in order to reduce the dependency on non-renewable resources, such as fossil fuels. For instance, the impact of this research project could give rise to the application of bioproducts extracted from OPEFB toward bioenergy technologies, such as biofuels (biodiesel), biopower in the generation of heat and electricity, and biochemicals such as bioplastics. However, more research on other organic solvents for OPEFB microwave-assisted pre-treatment is required and necessary to extract a higher yield of total sugars.