Evaluation of the Addition of Polyethylene Glycol in the Enzymatic Hydrolysis of Rice Husk

Abstract

This study evaluated the effect of polyethylene glycol (PEG 1500 and 4000) addition on the enzymatic hydrolysis (EH) of ground rice husk (≤250 μm). To reduce the amount of enzyme adsorbed on silicon dioxide and lignin and to evaluate the enzymatic hydrolysis, PEG 1500 and 4000 g/mol were added at three concentrations (0.3, 0.4 and 0.5 g PEG/g SiO₂). When PEG 1500 was added at 0.5 g/g SiO₂, the conversion of cellulose to cellobiose was not significantly increased (p ≥ 0.05); the conversion to glucose was 41.76%, and the conversion of hemicellulose to xylose was 93.45%, all with respect to the control assay. Addition of PEG 4000 at 0.5 g/g SiO₂ showed an increase of 14.78% in the hydrolysis of cellulose to cellobiose, 56.59% in that of cellulose to glucose, and 93.24% in that of hemicellulose to xylose. The addition of PEG shows that at a higher molecular weight and higher concentration, there are significant differences in the percentage of conversion of cellulose and hemicellulose into fermentable sugars, achieving efficiencies of ≈75%.

1. Introduction

Annual world rice production is 523.8 million tons, which generates around 104.76 million tons of husks [1], representing 20% of the grain’s weight. This bio-product is rich in cellulose [2] and hemicellulose [3]. The main component of the lignocellulosic biomass is cellulose, which is composed of linear chains of cellobiose (D-glucopyranosyl-β-1, 4-D-glucopyranose), containing approximately 10,000 glycoside units [4]. Hemicellulose is a heteropolysaccharide which can be hydrolyzed enzymatically to its monomers (xylose, glucose, galactose, mannose, arabinose and gluconic acid) and subsequently converted into food, chemical and fuel products [5].

One advantage of the EH of cellulose and hemicellulose in comparison with acid hydrolysis is its specificity, as the former does not produce undesirable compounds, which may limit the use of cellobiose, glucose and xylose. Recent advances in genetic engineering have also obtained enzymatic strains which use low reaction times and can work in slightly acid pH medium, thereby avoiding the need for expensive pretreatments [6]. However, there are several factors that affect enzyme activity and inhibit the hydrolytic process. β-glucosidase in aqueous solution has the ability to hydrolyze cellulose and hemicellulose, but its performance is limited by the presence of silicon dioxide, which inhibits the hydrolysis of cellulose by blocking the access of cellulase to cellulose [7].

Silicon dioxide (SiO₂) is the main component of rice husk ashes, representing around 18% w/w [8]. It is an inert compound and requires high concentrations and high temperatures to react with strong bases such as sodium hydroxide. It is not very reactive with mineral acids such as HCl [9]. Lignin is another component found in rice husk in a concentration of around 20%. Lignin also acts as an irreversible blocker of access by cellulase to cellulose when it occurs in concentrations of over 40% [10]. According to [11], the adsorption of the cellulase enzymes (Ctec2) by amorphous silica is a negative endothermic reaction in EH of cellulose, as cellulase is immobilized and the amount of free enzyme to carry out the hydrolytic process is reduced.

High concentrations of SiO₂ and lignin in lignocellulosic biomass lead to a decreased concentration of free enzyme during the hydrolytic process, which causes the cost of production to increase. An alternative solution is to include active additives, such as PEG [12], which bind to SiO₂ and lignin to form stable biopolymers [13] and prevent the enzyme from binding to the inhibitors present in these residues [14]. Active additives have physicochemical properties that make them ideal compounds for the adsorption of the inhibitors found in lignocellulosic residues. They generate polymerization [15], substitution [12], oxidation [10] and cross reactions [16]. In addition, they are reusable, economical and non-toxic [17]. The specificity of PEG according to their molar mass has been observed. PEG 1000 can bind to metal ions, but does not always improve enzymatic hydrolysis [18]. PEG 1500 can mitigate the inhibition caused by mineral salts and metal ions, such as Fe (II) [19], while PEG 4000, with its longer chain, is more effective at binding to lignin due to its hydrophobic interactions and hydrogen bonds [20]. The main purpose of this study was to evaluate the effects of the addition of polyethylene glycol with molecular masses of 1500 and 4000 g/mol (PEG 1500 and PEG 4000) in rice husk hydrolysis.

2. Materials and Methods

2.1. Materials

The ground rice husk (≤250 μm) used in this research was obtained from a local rice-processing industry (Machala, Ecuador). It contained 18.4% ash, made up of 88% silicon oxide. The enzyme Celluclast 1.5 L (188 FPU/mL) was purchased from Sigma-Aldrich (Darmstadt, Germany). The following chemicals were reagent grade or higher purity and were purchased from Sigma-Aldrich (Germany): polyethylene glycol with molecular weight 1500 (34 EO), 4000 (91 EO) (PEG 1500 and PEG 4000), microcrystallized cellulose, Bradford reagent, bovine serum albumin (BSA), glucose and xylose. Cellobiose was purchased from LGC Labor GmbH Augsburg Dr. Ehrenstorfer GmbH (Augsburg, Germany).

2.2. Methods

2.2.1. Analysis of Lignin, Cellulose, Hemicellulose

The content of cellulose, hemicellulose and lignin was determined using the method proposed by the National Renewable Energy Laboratory (NREL, version 08-03-2012) [21]. Cellulose was determined according to [22]. Ash content was assessed by a thermogravimetry method, and the SiO₂ content of the rice husk ashes by X-ray fluorescence spectrometry (XRF) using an S4 Pioneer Bruker-AXS spectrometer (Bruker, Billerica, MA, USA).

2.2.2. Elemental Analysis

The elemental analysis of carbon (C), hydrogen (H), nitrogen (N) and sulfur (S) in the rice husk (≤250 μm) was carried out using a Flash EA1112 elemental analyzer (Thermo Finnigan, Somerset, NJ, USA). Prior to the CHNS analysis, thermogravimetric curves were obtained at heating rates of 10, 20, 30 and 50° K/min within a temperature range of 323–1173° K [23].

2.2.3. Crystallinity of Rice Husk

The relative crystallinity of the raw and milled shell (≤250 μm) was analyzed by light diffraction. The analysis was performed on a Bruker-AXS S4 Pioneer X-ray fluorescence spectrometer (XRF). The diffraction intensity of the Cu Kα radiation (wavelength 0.154 nm, under 40 kV and 40 mA conditions) was measured with a scan range of 5° to 50° (2θ) [24].

Relative crystallinity (RC) was calculated using the following equation [25]:

$$\mathrm{R}\mathrm{C}(\%)={\displaystyle \frac{A_c}{A_c-A_a}}\times 100$$

(1)

where A_c is the crystalline area and A_a is the amorphous area on the X-ray diffractograms.

2.2.4. Enzyme Adsorption Tests

The adsorption test of silicon dioxide and lignin from RH (≤250 μm) by β-glucosidase (Celluclast 1.5 L) was carried out in a 2.0 mL Eppendorf tube containing 1 mL of 50 mM sodium citrate buffer solution (pH 4.8) with 5 mg of residue (composed of 0.9 mg/g of silicon dioxide and 0.7 mg/g of lignin) and 2 mg/mL of enzyme, was homogenized for a contact time of 1 to 6 min at 50 °C, and the concentration of free enzyme was measured every minute. This assay was used as a control.

To reduce the inhibitory effect of silicon dioxide and lignin on the enzymatic hydrolysis of RH, three different concentrations of PEG 1500 (0.3, 0.4, and 0.5 g/g SiO₂) and five concentrations of PEG 4000 (0.1, 0.2, 0.3, 0.4, and 0.5 g/g SiO₂) were tested.

For the PEG, SiO₂, and lignin adsorption tests, 1 mL of 50 mM sodium citrate buffer solution (pH 4.8) was heated with 5 mg of residue, 30 FPU of enzyme, and either PEG 1500 or PEG 4000, depending on the experiment. The mixture was then heated to 50 °C and stirred constantly at 120 rpm for 6 min.. A sample was taken every minute and immediately centrifuged at 10,000 rpm for five minutes using an Eppendorf mini centrifuge (ELMI-Sky Line) to quantify the free enzyme.

The protein content in the supernatant was determined according to the Bradford method (0.1 mL supernatant was taken and mixed with 1 mL Bradford reagent). The samples were kept at room temperature for ten minutes and read at 595 nm using an ultraviolet spectrophotometer [26]. The calibration curve was obtained by measuring progressive concentrations of bovine serum albumin as standard. The amount of enzyme adsorbed on the silicon oxide was calculated by subtracting the enzyme concentration in the supernatant from that of the control sample (cellulose crystallized without SiO₂). The following equation was used to calculate the amount of immobilized enzyme [27].

$$\mathrm{q}\mathrm{i}={\displaystyle \frac{\mathrm{V}({\mathrm{C}}_0-{\mathrm{C}}_{\mathrm{s}})}{\mathrm{w}}}$$

(2)

where V is the volume of the aqueous phase (mL), C₀ and C_s are the concentrations of cellulase added initially and cellulase in the supernatant obtained after immobilization (mg of β-glucosidases/mL of solution), and w is the weight of dry silicon oxide (g).

2.2.5. Hydrolysis of Cellulose and Hemicellulose

A pretreatment was carried out to reduce the crystallinity of the cellulose present in the rice husk (≤250 μm). Biomass pretreatments favor adsorption by reducing particle size and increasing the contact area. Subsequently, the RH was sterilized at 121 °C for 15 min. The enzymatic hydrolysis of the residue was carried out in vials of 120 mL. Several assays were carried out with a rice husk concentration of 5% (w/w) at three different concentrations of PEG 1500 and PEG 4000 (0.3, 0.4 and 0.5 g PEG/g SiO₂) in a 50 mM sodium citrate buffer (pH 4.8) in a final working volume of 100 mL. The vials were stirred for five minutes to allow the PEG to adsorb on the silicon oxide and lignin. The Celluclast 1.5 L enzyme (β-glucosidase) was then added at a concentration of 30 FPU/g residue [28] and placed on an orbital shaker at 50 °C, 200 rpm for 120 h [29]. Samples were taken every 24 h, centrifuged for five minutes at 10,000 rpm using an ELMI-Sky Line mini centrifuge, and filtered on a 0.22 μm syringe filter (Millipore, Bedford, MA, USA). The production of sugars (cellobiose, glucose and xylose) was quantified by high performance liquid chromatography (HPLC) equipped with a refractive index detector (Agilent Hewlett-Packard Model 1100, Santa Clara, CA, USA).

The percentage conversion of polysaccharides (cellulose and hemicellulose) into different sugars (cellobiose, glucose and xylose) was calculated according to the following equation [30].

$$\%\mathrm{c}\mathrm{o}\mathrm{n}\mathrm{v}\mathrm{e}\mathrm{r}\mathrm{s}\mathrm{i}\mathrm{o}\mathrm{n}={\displaystyle \frac{\left(\frac{\mathrm{m}\mathrm{g} \mathrm{s}\mathrm{u}\mathrm{g}\mathrm{a}\mathrm{r}}{\mathrm{m}\mathrm{L}}\right)\times 1.4 \mathrm{m}\mathrm{L}\times \mathrm{H}}{\left(\frac{\mathrm{m}\mathrm{g} \mathrm{p}\mathrm{o}\mathrm{l}\mathrm{y}\mathrm{s}\mathrm{s}\mathrm{a}\mathrm{c}\mathrm{h}\mathrm{a}\mathrm{r}\mathrm{i}\mathrm{d}\mathrm{e}\mathrm{s}}{\mathrm{g} \mathrm{b}\mathrm{i}\mathrm{o}\mathrm{m}\mathrm{a}\mathrm{s}\mathrm{s}}\right)\times 0.014 \mathrm{g} \mathrm{b}\mathrm{i}\mathrm{o}\mathrm{m}\mathrm{a}\mathrm{s}\mathrm{s}}}\times 100$$

(3)

where H = specific hydrolysis factor for each polysaccharide/sugar combination mg/mL of sugar from HPLC analysis, and “mg polysaccharides/g biomass” is obtained from EH.

2.2.6. Scanning Electron Microscopy (SEM)

The surface morphology and semiquantitative chemical microanalysis of PEG (1500 and 4000) adsorbed to rice husk after 120 h of enzymatic hydrolysis were determined by scanning electron microscopy (SEM) using the JSM-6400 scanning microscope (JEOL, Tokyo, Japan). The morphology and composition of the PEG precipitate was determined using primary electron beams of 20 keV and an increase of 2000× [31].

2.3. Statistical Analysis

Analysis of Variance (ANOVA)

In order to observe whether there was a significant difference (p ≤ 0.05) between the means of the experiments studied, a one-way analysis of variance was performed, and to establish whether the experiments differed from each other, Tukey’s HSD test was used.

3. Results

3.1. Relative Crystallinity of Rice Husk

X-ray diffractograms performed on raw and milled rice husk (≤250 μm) show peaks at 2θ = 21.2° and 10.7° and at 2θ = 21.9° and 10.7°, respectively, which are typical diffraction angles for this type of biomass [32]. The raw rice husk showed a higher relative crystallinity than the milled rice husk (≤250 μm). Other research has shown that glucose release increases between 24 and 36% for sizes less than 250 μm, leading to lower crystallinity values. According to [33], cellulose hydrolysis increases when a mechanical fractionation is applied, reducing the crystallinity of the cellulose present in lignocellulosic biomass to approximately 50%.

3.2. Chemical Characterization of Rice Ash

In order to determine if there are compounds capable of inhibiting EH, it is essential to know the composition of the rice husk. Ash content represented 18.4% of the rice husk. The ashes in the rice husk were analyzed using X-ray diffraction (XRF) and showed a chemical composition of 88% silicon dioxide and 12% CaO, MgO, Na₂O and K₂O.

3.3. Effect of the Addition of PEG on the Interaction of Celluclast 1.5 L with Silicon Dioxide and Lignin

The main effect of the addition of PEG is the hydrophobic interaction and hydrogen bonds that form in the presence of lignin.

The first experiment examined the effect of PEG 1500 on the adsorption of Celluclast 1.5 L (2 mg/mL) on silicon dioxide (0.9 mg/mL) and lignin (0.7 mg/mL) present in RH (≤250 μm). The percentage of free enzyme in the medium after the addition of concentrations of 0.3, 0.4 and 0.5 g PEG/g SiO₂ was 65.0 ± 0.40%, 66.01 ± 0.06% and 66.14 ± 0.19%, respectively (Figure 1).

Figure 1 shows that with 0.3 g of PEG 1500/g SiO₂, 65.0 ± 0.40% free enzyme is obtained, while with 0.2 g of PEG 4000/g SiO₂ (Figure 2) yields the same amount of free enzyme (65.09 ± 1.9%), and with 0.3 g of PEG 4000/g SiO₂, around 94% is achieved. When the concentration of PEG 4000 is increased (0.4 and 0.5 g PEG/g SiO₂), there is no significant difference in the increase in free enzyme.

Figure 2 shows the results obtained from treatment with PEG 4000 using the same concentration of rice husk (≤250 µm). The percentage of free enzyme in the medium after the addition of concentrations of 0.1, 0.2, 0.3, 0.4 and 0.5 g PEG/g SiO₂ was 41.76 ± 0.86%, 65.09 ± 1.9%, 95.55 ± 2.08%, 95.39 ± 0.90% and 95.46 ± 1.58%, respectively.

Rice husks contain around 30% inhibitors (lignin and SiO₂), which saturate PEG at concentrations of 0.1 g·g⁻¹ of inhibitors, leaving 40% of the enzyme free.

3.4. Enzymatic Hydrolysis of Rice Husk

3.4.1. Partial Hydrolysis of Cellulose

Complete cellulose hydrolysis is one of the main obstacles of biomass technology; when this homopolysaccharide is part of complex matrices, much of it is hydrolyzed to cellodextrins (cellobiose).

Several assays were carried out to study the effect of the three concentrations (0.3, 0.4 and 0.5 g/g SiO₂) of PEG 1500 and 4000 on the partial hydrolysis of cellulose. In the experiment using PEG 1500 at a concentration of 0.5 g/g SiO₂, 210.54 ± 1.92 mg/L, cellobiose was produced. No significant differences were found in the hydrolysis of rice husk for the three concentrations of the PEG 1500 studied (Figure 3b). Significant differences in the hydrolysis of cellulose to cellobiose were observed, by contrast, at the three concentrations of PEG 4000 studied (0.3, 0.4 and 0.5 g/g SiO₂). In the assay where PEG 4000 (Figure 3a) was added with a concentration of 0.5 g/g SiO₂, a cellobiose concentration of 371.28 ± 1.97 mg/L (Figure 3) was achieved, the highest of all the assays.

3.4.2. Total Hydrolysis of Cellulose

The effect of adding PEG 1500 (a) and 4000 (b) on the EH of cellulose is shown in Figure 4. In the experiments where PEG 1500 was added at a concentration of 0.5 g/g SiO₂, a glucose concentration of 1048.98 ± 11.1 mg/L was achieved. The addition of PEG 4000 at 0.5 g/g SiO₂ showed a higher glucose concentration of 1421.50 ± 8.28 mg/L.

Glucose production increases significantly after 72 h of enzymatic hydrolysis compared to the control (19% conversion), indicating that at that time, all the enzyme becomes unproductive due to its binding with SiO₂ and lignin.

3.4.3. Hydrolysis of Hemicellulose

Figure 5 presents the results of the addition of PEG 1500 (a) and 4000 (b) at three different concentrations (0.3, 0.4 and 0.5 g/g SiO₂) on the hydrolysis of hemicellulose. An increase was observed in xylose production for all the PEG concentrations tested, owing to the action of these surface-active additives, which improve enzymatic activity and prevent β-glucosidase (Celluclast 1.5 L) from binding with silicon dioxide or lignin. In the experiments with PEG 1500 at 0.5 g/g SiO₂, an increase of 80.82 ± 2.73 mg/L xylose was observed with respect to the control. Following the addition of PEG 4000 (0.5 g/g SiO₂), an increase of 79.87 ± 1.91 mg/L was achieved with respect to the control. There was no significant difference in xylose production with respect to the two PEGs used.

3.5. Effect of PEGs (1500 and 4000) on the Conversion of Cellulose and Hemicellulose

The study measured the effect of adding PEG (1500 and 4000) at three different concentrations (0.3, 0.4, and 0.5 g/g SiO₂) on the efficiency of fermentable sugar production during enzymatic hydrolysis of RH. Figure 6 shows the conversion percentages of cellulose to cellobiose and glucose, and of hemicellulose to xylose, using 30 FPU of β-glucosidase as the hydrolyzing agent. In the control test (without additives), a partial conversion of cellulose to cellobiose of 6.86 ± 0.6%, cellulose to glucose of 14.63 ± 0.6%, and hemicellulose to xylose of 75.08 ± 0.75% was observed. The experiments with PEG 1500 at the highest concentration (0.5 g/g SiO₂) obtained concentrations of 8.38 ± 0.28 for cellobiose, 41.76 ± 0.15 for glucose, and 93.45 ± 0.12 for xylose. Experiments with PEG 4000 at the highest concentration (0.5 g/g SiO₂) obtained higher conversion rates: 14.78 ± 0.19 for cellobiose, 56.59 ± 0.27 for glucose, and 93.24 ± 0.31 for xylose, indicating that the higher the molecular weight and concentration of PEG, the higher the percentage of saccharification of this residue.

Analysis of Variance in Sugar Production

To evaluate the effect of PEG type (1500 and 4000) and its concentration (0.3, 0.4, and 0.5 g/g SiO₂) on the absorption of inhibitors present in rice hulls and on increasing the percentage of conversion of cellulose and hemicellulose into fermentable sugars (cellobiose, glucose, and xylose), an analysis of variance (ANOVA) was performed on the data obtained from the six experiments.

The ANOVA performed on the percentages of conversion of cellulose and hemicellulose to fermentable sugars in the six experiments indicated a significant difference (p ≤ 0.05), with the highest mean being achieved where PEG 4000 (0.5 g/g SiO₂) was added. The results of the analysis of variance are shown in the following table (

Table 1. ANOVA of the percentage conversion of cellulose and hemicellulose.

Data	Mean	Variance	N
Control	31.29	1.20423	3
PEG 1500 (0.3 g/g SiO2)	48.38	0.39083	3
PEG 1500 (0.4 g/g SiO2)	52.24	0.52923	3
PEG 1500 (0.5 g/g SiO2)	56.73	0.1351	3
PEG 4000 (0.3 g/g SiO2)	63.9	1.0924	3
PEG 4000 (0.4 g/g SiO2)	69.05	1.71903	3
PEG 4000 (0.5 g/g SiO2)	74.79	0.35583	3
F = 7473.04684
p = 0

).

In the Supplementary Material, the results of the Tukey test (95% confidence) are found, which indicate that all means differ from each other, since no means overlap vertically.

3.6. Qualitative and Quantitative Chemical Microanalysis of PEG 1500 and 4000

In the Supplementary Material, the results of the elemental micrograph performed on the precipitate of rice husk treated with PEG 1500 at a concentration of 0.5 g/g SiO₂, obtained after 120 h of EH, show an adsorption of 69.01 ± 2.85% silicon (Si).

The micrograph also showed the presence of 26.10 ± 2.97% oxygen (O), 2.54 ± 0.60% chlorine (Cl) and 2.54 ± 0.60% potassium (K). The micrograph of rice husk treated with PEG 4000 indicated the presence of silicon (Si) in a concentration of 58.20 ± 3.77%, as well as 13.64 ± 4.16% oxygen (O) and 28.15 ± 3.77% carbon (C). The presence of carbon in the precipitate of rice husk treated with PEG 4000 rather than PEG 1500 is due to the ability of this additive to adsorb higher molecular weight compounds, such as lignin.

4. Discussion

In rice husk subjected to a grinding pretreatment (≤250 μm), solubility increased by 100% and relative crystallinity decreased by 27%, with respect to the raw material. Research carried out with wheat straw has observed that by reducing particle size (<400 μm), the crystallinity of lignocellulosic biomass decreases and the percentage of cellulose and hemicellulose conversion increases [34].

The adsorption of β-glucosidase on SiO₂, lignin and other mineral salts present in lignocellulosic waste limits the EH of holocellulose [35]. The present study evaluated the effect of PEG 1500 and PEG 4000 on the interaction of the enzyme with silicon dioxide and lignin in the enzymatic hydrolysis of RH (≤250 μm). Rice husk ashes contain silicon dioxide (88%), which is the main inhibitor of enzymatic hydrolysis because it is adsorbed by the cellulase enzyme, rendering a large amount of the enzyme unproductive and decreasing hydrolysis of cellulose and hemicellulose. Research [36] reports that the application of silicon dioxide nanoparticles provides a greater area of immobilization of β-galactosidase without significant losses of catalytic activity, as opposed to using conventional macro-scale silicon dioxide matrices. Other studies have demonstrated that substances such as silicon dioxide that are present in lignocellulosic biomass ashes can immobilize enzymes and reduce hydrolysis [37].

Studies of rice husk hydrolysis also show that the higher the molecular weight of PEG, the greater the capacity to adsorb substances that bind to cellulase, thus reducing its enzymatic activity. The use of PEG 4000 in a concentration of 0.3, 0.4 and 0.5 g/g SiO₂ reduced cellulase adsorption in lignin, leaving approximately 95.95% of free enzyme for the three concentrations of additives studied. Similar studies with other types of lignocellulosic biomass confirm that interactions between PEG 4000 and lignin are strong and leave the enzyme free [38]. Studies of wheat straw hydrolysis indicate that at concentrations of 0.1 g PEG·g⁻¹ of substrate, glucose conversion rates improve significantly [39]. The superior performance of PEG 4000 in comparison to PEG 1500 is due to its higher molecular mass, which allows it to form a greater number of hydrogen bonds that adsorb on lignin easily. Similar results have also been reported by authors working with pure cellulose rather than real residue [40]. Research into the enzymatic hydrolysis of bamboo waste pretreated with acids confirms that the addition of PEG 4000 and Tween 80 improves glucose production, because this additive intervenes in the interaction between lignin and cellulase through hydrogen bonds/Van der Waals forces and hydrophobic action [41].

E H of rice husk using Celluclast 1.5 L (30 FPU/g residue) took place in two stages: first, cellulose was converted to cellobiose, and conversion of cellobiose to glucose was detected after 48 h.

There was a significant difference in the glucose concentration for the three concentrations (0.3, 0.4 and 0.5 g/g SiO₂) of both PEG 1500 and PEG 4000. The difference in the glucose concentration obtained from the addition of PEG 1500 and 4000 at a concentration of 0.5 g/g SiO₂ was 372.52 ± 2.82 mg/L, with greater efficiency observed in relation to PEG 4000, owing to its ability to adsorb on both silicon dioxide and lignin, which are both inhibitors of the hydrolysis process studied here.

Experiments with PEG 4000 at the highest concentration studied (0.5 g/g SiO₂) obtained a conversion of 14.78 ± 0.19% cellulose to cellobiose, 56.59 ± 0.27% cellulose to glucose, and 93.24 ± 0.31% hemicellulose to xylose. Other studies carried out using a pure substrate (Avicel PH 101) and adding PEG 4000 (0.05 g/g cellulose) with a Celluclast 1.5 L enzyme reported cellulose conversions of 78.9% cellulose hydrolysis to cellobiose and glucose [42].

Similar studies report higher glucose yields when using PEG 4000 as the active additive due to higher β-glucosidase activity [43]. Research by [44] on lignocellulosic waste claims that PEG 4000 reduces the unproductive binding of the enzyme with silicon dioxide and lignin. The pre-incubation of corn stover residues with polyethylene glycol (PEG 4000) reduces the ability of lignin to adsorb enzyme and increases saccharification by 17.28% [4] (see

Table 2. Results reported by some authors using different types of PEG and their effect on the enzymatic hydrolysis of lignocellulosic residues.

Substrate	Tipe PEG	PEG Concentration	Implementation Phase	Conversion	Reference
Rice Husk	4000	5 mg/mL	Co-addition in saccharification	21.46%	[45]
Sugarcane bagasse	4000	no report	Pre-treatment	52%	[46]
Rice straw	4000	0.05–0.2 g/g substrate	Saccharification	0.288 mg/mL	[47]
Corn stover	6000	0.05 g/g substrate	Saccharification	50.14%	[48]
Lignocellulosic waste	6000	5 mg/mL	Pre-treatment	62.3%	[49]
Eucalyptus kraft pulp	400	75 mg/g substrate	Saccharification	65 g/L	[50]

).

In the EH of the rice husk, the hemicellulose was 93% hydrolyzed to xylose. From the beginning of the hydrolysis process, there was already a low concentration of xylose owing to the sterilization process used. Results from studies on hydrolysis of corn cob show a fraction of hemicellulose hydrolyzed after convective heating [51].

The scanning electron micrographs (SEM) performed on the precipitate (PEG 1500 + inhibitor) after 120 h of enzymatic hydrolysis of the rice husk (≤250 μm) show that the additive only adsorbed on silicon dioxide and 2.54% potassium. Similar studies using PEG 1500 show that the additive has high affinity, adsorbs most of the silicon oxide present [52], and forms strong crystals [53].

The micrograph of the rice husk treated with PEG 4000 had the ability to adsorb silicon dioxide and lignin, which favored EH. Studies on the enzymatic hydrolysis of pure cellulose plus lignin treated with PEG 4000 (5 mg/mL) and an enzymatic mixture indicate that binding of enzymes with lignin is disabled because the inhibitor remains attached to the PEG [47].

The ANOVA analysis indicated that there was a significant difference in the conversion percentages of cellulose and hemicellulose into fermentable sugars among the six experiments studied, with the highest average being achieved in the experiment where PEG 4000 (0.5 g/g SiO₂) was added, obtaining a conversion of 74.79%. The Tukey test (HSD) indicates that all means differ from each other.

Acid hydrolysis investigations applying sulfuric acid (1.5%) and detoxification with activated carbon at concentrations around 4% achieved conversion percentages of the cellulose/hemicellulose fraction of 68% [54]. Applying eutectic solvents to delignify the rice husk and increase the holocellulose fraction, achieved hydrolysis efficiencies of around 67.91 ± 3.28 g of sugars/100 g of carbohydrates [55].

5. Conclusions

The addition of PEG with a higher molar mass and concentration demonstrated the additive’s ability to bind to enzymatic hydrolysis inhibitors present in rice husks, such as silicon dioxide and lignin, thus freeing the enzyme (β-glucosidase), which in turn favored the partial bioconversion of cellulose to cellobiose and then the total hydrolysis of cellobiose to glucose. The same occurred with the hydrolysis of hemicellulose to xylose.

The elemental micrographs of the PEG 1500 and PEG 4000 precipitates after 120 h of enzymatic hydrolysis confirmed that PEG 1500 has the ability to adsorb on silicon dioxide, leaving lignin free to adsorb approximately 40% of the enzyme. This also shows that silicon dioxide is not the only inhibitor present in rice husk.

PEG 4000 has the ability to chemically bind to the main inhibitors present in lignocellulosic waste, which promises multiple technological and economic benefits for the biotechnological use of these resources.