Enhancing for Bagasse Enzymolysis via Intercrystalline Swelling of Cellulose Combined with Hydrolysis and Oxidation

To overcome the biological barriers formed by the lignin–carbohydrate complex for releasing fermentable sugars from cellulose by enzymolysis is both imperative and challenging. In this study, a strategy of intergranular swelling of cellulose combined with hydrolysis and oxidation was demonstrated. Pretreatment of the bagasse was evaluated by one bath treatment with phosphoric acid and hydrogen peroxide. The chemical composition, specific surface area (SSA), and pore size of bagasse before and after pretreatment were investigated, while the experiments on the adsorption equilibrium of cellulose to cellulase and reagent reuse were also performed. Scanning electron microscopy (SEM) and high-performance liquid chromatography (HPLC) were employed for microscopic morphology observations and glucose analysis, respectively. The results showed that pretreated bagasse was deconstructed into cellulose with a nanofibril network, most of the hemicellulose (~100%) and lignin (~98%) were removed, and the SSA and void were enlarged 11- and 5-fold, respectively. This simple, mild preprocessing method enhanced cellulose accessibility and reduced the biological barrier of the noncellulose component to improve the subsequent enzymolysis with a high glucose recovery (98.60%).


Introduction
Lignocellulose biomass is an energy form that plants utilize to store energy gained via photosynthesis [1]. It is known as a carbon-neutral, green resource, with a total annual output of 146 billion tons [2]. Fuel, materials, and chemicals in solid, liquid, and gaseous states are obtained from biomass using various technical means [3][4][5]. However, biological barriers make it extremely difficult to release fermentable sugars from cellulosic biomass. As a result, large dosages of enzymes are required for hydrolysis, which diminish their cost-efficient features for commercial application [6]. Since the energy crisis in the 1970s, biofuel and biochemical production technologies based on enzymolysis from biomass have been driven in both industry and academia [7]. Although enzymolysis offers potentially higher yields [8], higher selectivity [9], lower energy costs [10], and milder operating conditions [11] compared to chemical processes, the technology still faces significant challenges. Eliminating the biological barriers of lignocellulose biomass to enzymes under mild conditions is vital [12].
Cellulose, as a linear polymer consisting of 300-15,000 D-glucose units, aggregates into 3 to 4 nm-wide elementary fibrils due to intermolecular forces [13][14][15]. The elementary fibrils are embedded in the hemicellulose matrix and are further aggregated into 10 to 25 nm-wide primary microfibrils [16]. These primary microfibrils are likewise embedded in a lignin-carbohydrate complex (LCC) matrix and are then bonded together, weaving The chemical composition of raw materials and pretreated bagasse was determined according to the standard method of the US National Renewable Energy Laboratory (NREL) [30]. This involved a two-stage extraction of samples followed by a two-stage acid hydrolysis. Residual solids were quantified as acid-insoluble lignin content. The acid-soluble lignin content was quantified by a UV spectrophotometer (Agilent Cary 3500, Agilent, Santa Clara, CA, USA) in the analytical hydrolysate. The lignin content was the sum of acid-soluble lignin and acid-insoluble lignin content. Structure of cellulose and hemicellulose were quantified as their monomeric forms in the analytical hydrolyzate using high-performance liquid chromatography (HPLC, Agilent 1260 Infinity II, Agilent, Santa Clara, CA, USA) with an HPX-87H column (Agilent, Santa Clara, CA, USA). Cellulase (Novozyme CTec2) was purchased from Sigma-Aldrich (Shanghai, China). The cellulase activity was determined by the filter paper method according to US NREL [31] and protein content was determined using the Bradford method [32].

X-ray Diffraction
The X-ray diffraction (XRD) pattern was obtained using a MiniFlex 600 advance X-ray diffractometer (Rigaku, Tokyo, Japan) with a Cu Kα radiation source operated at 40 kV and 40 mA. The measurement of 2θ ranged from 10 • to 50 • at a scanning speed of 5 • /min and step size of 0.02 • . The crystallinity index (CrI) of pretreated cellulose was calculated by subtracting the amorphous contribution from diffraction spectra using an amorphous standard according to a previous study [33]. XRD was calculated by the following formula: where I 200 represents the maximum intensity of the lattice diffraction peak at 2θ between 22.5 • , and I am represents the intensity scattered by the amorphous component in the sample, which was evaluated as the lowest intensity at 2θ at 18 • .

Degree of Polymerization
The intrinsic viscosity degree of polymerization (DP) test [34] was used to calculate the DP of cellulose. The DP was calculated according to the following equation (with an average of three measurements per sample): where [η] G is the intrinsic viscosity (mL/g), η sp is the specific viscosity, C represents the concentration (g/100 mL), and DP is the degree of polymerization.

Specific Surface Area
Nitrogen adsorption (Micromeritics ASAP2460, Norcross, Georgia) was used to measure the specific surface area (SSA) of untreated and pretreated bagasse. The samples were degassed at 90 • C for 12 h prior to analysis to remove moisture and air from the substrate pores. The test was carried out at liquid nitrogen temperature, and the SSA of the sample was calculated using the BET model [35].

Zeta Potential
The surface charge of the pretreated cellulose was evaluated by determining the zeta potential using the zeta potential mode of the Malvern Zetasizer (ZS90X, Melvin, UK) [36]. The pretreated cellulose was uniformly dispersed in a sodium citrate buffer of pH 4.8 to form a 0.5% (w/v) suspension, and the suspension was measured and scanned with a cuvette 100 times.

Additional Measurements and Characterization
An X-ray photoelectron spectrometer (XPS) (ULVAC-PHI, Chigasaki-shi, Japan) was used to determine the surface chemical analysis of pretreated cellulose [37]. A Fouriertransform infrared (FTIR) spectrometer (TENSOR II, Brook Technology, Ettlingen, Germany) was used to obtain the FTIR spectra of the untreated bagasse, pretreated bagasse, and enzymolysis residual in the frequency range of 4000-400 cm −1 with a resolution of 4 cm −1 using the KBr tablet method [38]. Scanning electron microscopy (SEM) SU8220 (Hitachi, Tokyo, Japan) was used to analyze the surface structure of the untreated bagasse and pretreated bagasse. ImageJ software (Version 2.0, National Institutes of Health, Bethesda, MD, USA) was used to determine the diameter of the nanofibers after at least 100 measurements based on SEM images. The bagasse samples were freeze-dried using the Advantage Plus EL-85 freeze-drying system (SP Scientific, Warminster, PA, USA) and the samples were sprayed with gold to improve the conductivity of the samples before observing the samples.

Adsorption Equilibrium Experiment
Two hundred milligrams of substrate were weighed into a centrifuge tube and a series of concentrations of enzyme solutions were added (0.05 M citrate buffer, refrigerated at 4 • C before use) to form a solid loading of 2% (w/v). The mixture was shaken at 130 rpm at 4 • C for 2 h. In parallel, a blank control sample was run. After adsorption, the mixture was centrifuged at 10,000 rpm for 5 min, and the supernatant was taken. The protein concentration was determined by the Bradford method and each sample was measured in duplicate. The adsorption capacity was expressed as the difference between the concentration of added enzyme protein and that of supernatant. The adsorption data were fitted using the Langmuir equation [39]: where E b is the amount of bound cellulase (mg/g substrate), E bm represents the theoretical maximum adsorption capacity of the substrate (mg/g substrate), K a is the affinity constant (L/mg), and E f is the free enzyme in the supernatant (mg/mL).

Enzymatic Hydrolysis
Enzymolysis of pretreated bagasse was carried out in a 50 mM citrate buffer (pH 4.8) with a substrate load of 2% (w/v; dry matter, DM). Cellulase was introduced at 5, 10, and 20 filter paper unit (FPU)/g cellulose, and 0.1 g/L ampicillin trihydrate was added to avoid microbial interference during hydrolysis. After enzymolysis for 0. 5,2,4,8,16,48, and 72 h, 5 mL of solid-liquid mixture was taken out and inactivated at 100°C for 30 min, passed through a 0.22 µm filter membrane, and stored at 4 • C for further measurement of glucose yield. Enzymolysis of each sample (untreated and pretreated bagasse) was run in parallel. The glucose concentration was measured at 60 • C using an HPLC system equipped with an HPX-87H column (Agilent, Santa Clara, CA, USA). The mobile phase flow rate was at 0.6 mL/min and the detection time was 30 min. The hydrolysis efficiency of the enzyme bound to the cellulose surface was calculated by the hydrolysis rate of the unit bound enzyme in the initial stage of enzymolysis (0.5 h).

Physical and Chemical Property Characterization
To assess the efficacy of the pretreatment in the removal of noncellulose components, the chemical composition of untreated and pretreated bagasse is compared in Table 1. Table 1 showed that~100% of initial hemicellulose in the bagasse was removed during the pretreatment. As shown in the FTIR results (Figure 1), the characteristic peaks at 1737 cm −1 (C=O stretching of the acetyl and urate groups of hemicellulose or the ester bond of carboxyl groups in lignin to fragrant acid and ferulic acid) and 1247 cm −1 (the alkyl ester of the acetyl group in hemicellulose) of the hemicellulose of pretreated bagasse from untreated bagasse are decreased or completely disappeared [40]. These indicate that hemicellulose removal is complete [41], which is attributed to the fact that cellulose intercrystalline swelling fully exposes the hemicellulose and promotes the hydrolysis of the hemicellulose. Similarly, 98% of the initial lignin was removed during the pretreatment ( Table 1). As seen from the FTIR results (Figure 1), the characteristic peaks at 1515 cm −1 (C=C stretching of the aromatic skeleton), 1607 cm −1 (the aromatic skeletal stretching), and 1458 cm −1 (C-H deformation of CH 3 and CH 2 ) of lignin of pretreated bagasse from untreated bagasse almost disappeared [42]. This demonstrates that lignin was efficiently removed, attributing to the oxidative degradation of lignin by peroxyphosphoric acid (H 3 PO 5 ) formed by H 3 PO 4 and H 2 O 2 [43]. The cellulose yield possibly reached 96.03% (Table 1), which is due to both the mild reaction conditions and high selectivity of the delignification and hemicellulose removal [43]. To assess the efficacy of the pretreatment in the removal of noncellulose components, the chemical composition of untreated and pretreated bagasse is compared in Table 1. Table 1 showed that ~100% of initial hemicellulose in the bagasse was removed during the pretreatment. As shown in the FTIR results (Figure 1), the characteristic peaks at 1737 cm −1 (C=O stretching of the acetyl and urate groups of hemicellulose or the ester bond of carboxyl groups in lignin to fragrant acid and ferulic acid) and 1247 cm −1 (the alkyl ester of the acetyl group in hemicellulose) of the hemicellulose of pretreated bagasse from untreated bagasse are decreased or completely disappeared [40]. These indicate that hemicellulose removal is complete [41], which is attributed to the fact that cellulose intercrystalline swelling fully exposes the hemicellulose and promotes the hydrolysis of the hemicellulose. Similarly, 98% of the initial lignin was removed during the pretreatment ( Table  1). As seen from the FTIR results (Figure 1), the characteristic peaks at 1515 cm −1 (C=C stretching of the aromatic skeleton), 1607 cm −1 (the aromatic skeletal stretching), and 1458 cm −1 (C-H deformation of CH3 and CH2) of lignin of pretreated bagasse from untreated bagasse almost disappeared [42]. This demonstrates that lignin was efficiently removed, attributing to the oxidative degradation of lignin by peroxyphosphoric acid (H3PO5) formed by H3PO4 and H2O2 [43]. The cellulose yield possibly reached 96.03% (Table 1), which is due to both the mild reaction conditions and high selectivity of the delignification and hemicellulose removal [43]. a Yield based on the initial amount of biomass/yield based on the initial amount of cellulose in biomass.  in untreated bagasse were 41.35%, 47.19%, and 11.46%, respectively. Contributions of cellulose, hemicellulose, and lignin to these peaks have been reported [44,45], with 85% of cellulose signaling to C2 and part of it to C3, 80% of hemicellulose signaling to C2 and the rest to C3, and 50% of lignin signaling to C1 and the rest to C2. The C1 content of pretreated bagasse decreased, while C2 and C3 contents increased. These phenomena suggest that the lignin is removed and the polysaccharides are exposed on the surface of the fibers [46]. This was consistent with the results of the chemical composition and FTIR analysis.
Contributions of cellulose, hemicellulose, and lignin to these peaks have been reported [44,45], with 85% of cellulose signaling to C2 and part of it to C3, 80% of hemicellulose signaling to C2 and the rest to C3, and 50% of lignin signaling to C1 and the rest to C2. The C1 content of pretreated bagasse decreased, while C2 and C3 contents increased. These phenomena suggest that the lignin is removed and the polysaccharides are exposed on the surface of the fibers [46]. This was consistent with the results of the chemical composition and FTIR analysis. The structure of untreated bagasse is complete and compact, and the fiber bundles are arranged compactly (Figure 3a). This intact structure greatly impedes the accessibility of the cellulase to the cellulose. Bagasse was pretreated in an aqueous solution of H3PO4, and the surface morphology of the pretreated bagasse changed significantly, transforming the dense bagasse into cellulose with a nanofibrils skeleton network structure ( Figure  3b,c). The widths of most nanofibers are in the range of 10-60 nm (Figure 3d). This is attributed to the fact that the H3PO4 molecules intrude between the fibrils, breaking the hydrogen bonds between adjacent fibrils [47]. The removal of hemicellulose and lignin also increases the number of channels for H3PO4 molecules to squeeze into the cell wall, causing the distance between adjacent fibrils to widen. The structure of untreated bagasse is complete and compact, and the fiber bundles are arranged compactly (Figure 3a). This intact structure greatly impedes the accessibility of the cellulase to the cellulose. Bagasse was pretreated in an aqueous solution of H 3 PO 4 , and the surface morphology of the pretreated bagasse changed significantly, transforming the dense bagasse into cellulose with a nanofibrils skeleton network structure (Figure 3b,c). The widths of most nanofibers are in the range of 10-60 nm (Figure 3d). This is attributed to the fact that the H 3 PO 4 molecules intrude between the fibrils, breaking the hydrogen bonds between adjacent fibrils [47]. The removal of hemicellulose and lignin also increases the number of channels for H 3 PO 4 molecules to squeeze into the cell wall, causing the distance between adjacent fibrils to widen. To evaluate the effect of pretreatment on cellulose aggregation, the XRD patterns of untreated and pretreated bagasse were compared (Figure 4). The peaks [48] at 16° (101), 22° (200), and 34° (004) for cellulose I were significantly strengthened in the XRD patterns of untreated and pretreated bagasse. Similarly, the CrI value of the pretreated bagasse increased from 58.84% to 74.92% (Figure 4). There were no obvious clear peaks at 2θ = To evaluate the effect of pretreatment on cellulose aggregation, the XRD patterns of untreated and pretreated bagasse were compared (Figure 4). The peaks [48] at 16 • (101), 22 • (200), and 34 • (004) for cellulose I were significantly strengthened in the XRD patterns of untreated and pretreated bagasse. Similarly, the CrI value of the pretreated bagasse increased from 58.84% to 74.92% (Figure 4). There were no obvious clear peaks at 2θ = 12.1 • (110 for cellulose II), and 20.2 • (110 for cellulose II) in the XRD patterns of pretreated bagasse as reported in the literature [49]. These indicate that the cellulose crystal structure was unchanged, and the supramolecular structure of cellulose was not visibly broken. This implies that the swelling of H 3 PO 4 in cellulose mainly occurs in the intercrystalline spaces rather than the intracrystalline spaces.

Adaptability of Pretreated Bagasse to Cellulase
To evaluate the effect of cellulose intercrystalline swelling on bagasse enzymolysis, the adaptability of pretreated bagasse to cellulase was analyzed ( Figure 5a). As seen from the enzymolysis of pretreated bagasse, the glucose yield (78.19%) achieved at a lower enzyme dosage of 5 FPU/g was 14-fold higher than that achieved with untreated bagasse (5.25%). Further increase in the cellulase dosage to 10 FPU/g resulted in a glucose yield of 95.91% that was five-times higher than that achieved with untreated bagasse (18.07%). However, with 20 FPU/g of cellulase, a glucose yield of 98.60% was obtained: this was two-fold higher than that achieved with untreated bagasse (47.27%). This indicates that pretreated bagasse is highly amenable to cellulase.
Highly selective removal of lignin (~98%) and hemicellulose (~100%) helps to reduce the unproductive adsorption and the physical barrier of bagasse to cellulase (Table 1). These, in addition to the lower noncellulose content of the pretreated bagasse, are also associated with changes in other physicochemical properties including [50,51] pore volume (PV), SSA, degree of polymerization (DP), and CrI, directly and indirectly providing information about enhanced enzymolysis of pretreated bagasse. As seen from the PV results (Figure 5b), a new mesopore (8-23 nm) appeared in the pretreated bagasse and the PV increased to 1.60 × 10 −2 cm 3 /g from 1.43 × 10 −3 cm 3 /g (Figure 5b). Pores larger than 5.1 nm allow the enzyme to enter the substrate without being restricted by size [52]. The PV of pretreated bagasse increased 11-fold, meaning that the physical channels through which the enzyme can pass are increased. The SSA of pretreated bagasse significantly increased to 1.9068 m 2 /g from 0.3633 m 2 /g ( Table 2). The increase in the SSA of cellulose means that a larger area is available for enzyme loading [53]. The SSA of pretreated bagasse increased five-fold, meaning that the available surface area of the cellulose for enzyme loading was enhanced. The DP of cellulose dropped to 300 from an initial value of 2877 during the pretreatment ( Table 2). This is attributed to the cleavage of the β-1,4 glycosidic bonds in cellulose by the acid-catalyzed hydrolysis during the pretreatment [54]. It can therefore be inferred that the cellulose was destroyed and depolymerized, meaning that the number of nodes requiring cellulase hydrolysis was reduced.

Adaptability of Pretreated Bagasse to Cellulase
To evaluate the effect of cellulose intercrystalline swelling on bagasse enzymolysis, the adaptability of pretreated bagasse to cellulase was analyzed ( Figure 5a). As seen from the enzymolysis of pretreated bagasse, the glucose yield (78.19%) achieved at a lower enzyme dosage of 5 FPU/g was 14-fold higher than that achieved with untreated bagasse (5.25%). Further increase in the cellulase dosage to 10 FPU/g resulted in a glucose yield of 95.91% that was five-times higher than that achieved with untreated bagasse (18.07%). However, with 20 FPU/g of cellulase, a glucose yield of 98.60% was obtained: this was two-fold higher than that achieved with untreated bagasse (47.27%). This indicates that pretreated bagasse is highly amenable to cellulase.
Highly selective removal of lignin (~98%) and hemicellulose (~100%) helps to reduce the unproductive adsorption and the physical barrier of bagasse to cellulase (Table 1). These, in addition to the lower noncellulose content of the pretreated bagasse, are also associated with changes in other physicochemical properties including [50,51] pore volume (PV), SSA, degree of polymerization (DP), and CrI, directly and indirectly providing information about enhanced enzymolysis of pretreated bagasse. As seen from the PV results (Figure 5b), a new mesopore (8-23 nm) appeared in the pretreated bagasse and the PV increased to 1.60 × 10 −2 cm 3 /g from 1.43 × 10 −3 cm 3 /g (Figure 5b). Pores larger than 5.1 nm allow the enzyme to enter the substrate without being restricted by size [52]. The PV of pretreated bagasse increased 11-fold, meaning that the physical channels through which the enzyme can pass are increased. The SSA of pretreated bagasse significantly increased to 1.9068 m 2 /g from 0.3633 m 2 /g ( Table 2). The increase in the SSA of cellulose means that a larger area is available for enzyme loading [53]. The SSA of pretreated bagasse increased five-fold, meaning that the available surface area of the cellulose for enzyme loading was enhanced. The DP of cellulose dropped to 300 from an initial value of 2877 during the pretreatment  (Table 2). This is attributed to the cleavage of the β-1,4 glycosidic bonds in cellulose by the acid-catalyzed hydrolysis during the pretreatment [54]. It can therefore be inferred that the cellulose was destroyed and depolymerized, meaning that the number of nodes requiring cellulase hydrolysis was reduced.
As shown in the XPS results (Table S1), the O/C ratio of the residual from the pretreated bagasse enzymolysis at 20 FPU/g for 48 h decreased from 0.62 to 0.31. In addition, the content of C1 increased (from 31.75% to 58.79%), and the content of C2 decreased (from 53.49% to 27.17%) ( Figure S1). As seen from the FTIR results (Figure 1), the characteristic absorption peaks [56,57] at 895 cm −1 (the glycosidic bond of cellulose), 2892 cm −1 (C-H tensile vibration of methyl and methylene), 1160 cm −1 (C-O-C asymmetric stretching of cellulose), and 1066 cm −1 (C-O, C-C stretching vibration) of cellulose were weakened. The characteristic absorption peaks [58,59] at 823 cm −1 (C-H bending vibration of guaiacyl), 1273 cm −1 (C-O stretching vibration of guaiacyl), and 1637 cm −1 (C=O conjugated stretching) of lignin were significantly enhanced in the FTIR of the residue (Figure 1). This is attributed to the cellulose being converted to glucose (98.6%) by cellulase and being removed, while the lignin was retained in the enzymolysis residue.   As shown in the XRD results (Figure 5c), the CrI value decreased sharply to 35.03% from 74.92% [55], while the corresponding cellulose conversion to glucose was 55.32% during the 2 h enzymolysis. When the enzymolysis time was extended to 16 h, the percentage of cellulose to glucose increased to 80.02% and the CrI value decreased to 19.34%. The remaining crystalline cellulose was greatly enzymolyzed, the glucose yield reached to 98.6%, and the CrI value of the residue dropped to 8.51%. The cellulase therefore showed a strong preference for the digestion of crystalline cellulose over amorphous cellulose. This may be because amorphous cellulose is mixed with noncellulose components, which hinders the approach of cellulase.
As shown in the XPS results (Table S1), the O/C ratio of the residual from the pretreated bagasse enzymolysis at 20 FPU/g for 48 h decreased from 0.62 to 0.31. In addition, Polymers 2022, 14, 3587 9 of 13 the content of C1 increased (from 31.75% to 58.79%), and the content of C2 decreased (from 53.49% to 27.17%) ( Figure S1). As seen from the FTIR results (Figure 1), the characteristic absorption peaks [56,57] at 895 cm −1 (the glycosidic bond of cellulose), 2892 cm −1 (C-H tensile vibration of methyl and methylene), 1160 cm −1 (C-O-C asymmetric stretching of cellulose), and 1066 cm −1 (C-O, C-C stretching vibration) of cellulose were weakened. The characteristic absorption peaks [58,59] at 823 cm −1 (C-H bending vibration of guaiacyl), 1273 cm −1 (C-O stretching vibration of guaiacyl), and 1637 cm −1 (C=O conjugated stretching) of lignin were significantly enhanced in the FTIR of the residue (Figure 1). This is attributed to the cellulose being converted to glucose (98.6%) by cellulase and being removed, while the lignin was retained in the enzymolysis residue.

Enzymolysis Kinetic Behavior of Pretreated Bagasse
The linear correlation coefficient (R 2 ) was greater than 0.963, indicating that the Langmuir [39,60] equation fits the adsorption isotherm data well (Figure 6a). The affinity constant of pretreated bagasse was 19 L/g, which was three times that of untreated bagasse (6 L/g). This suggests that pretreated bagasse adsorption enzymes require a higher enzyme concentration at saturation than untreated bagasse. The adsorption capacity of cellulase onto the pretreated bagasse decreased to~29 mg/g from~40 mg/g (Figure 6a). The adsorption behavior of cellulase onto lignin is well-understood [61] and the hydrophobic lignin enhances the hydrophobic interaction, increasing the adsorption of enzymes onto lignin [62]. The hydrophobic interaction was weakened in pretreated bagasse due to the lower lignin content (Table 1). Cellulase was negatively charged in the buffer at pH 4.8 and demonstrated an electrostatic repulsion of cellulose, which had a negatively charged surface (−45.61 mV). Due to the weakening of the hydrophobic interaction and the enhancement of the electrostatic interaction of pretreated cellulose and cellulase, the adsorption capacity of cellulase onto cellulose decreased, although the SSA of the pretreated bagasse increased ( Table 2).

Enzymolysis Kinetic Behavior of Pretreated Bagasse
The linear correlation coefficient (R 2 ) was greater than 0.963, indicating that the Langmuir [39,60] equation fits the adsorption isotherm data well (Figure 6a). The affinity constant of pretreated bagasse was 19 L/g, which was three times that of untreated bagasse (6 L/g). This suggests that pretreated bagasse adsorption enzymes require a higher enzyme concentration at saturation than untreated bagasse. The adsorption capacity of cellulase onto the pretreated bagasse decreased to ~29 mg/g from ~40 mg/g (Figure 6a). The adsorption behavior of cellulase onto lignin is well-understood [61] and the hydrophobic lignin enhances the hydrophobic interaction, increasing the adsorption of enzymes onto lignin [62]. The hydrophobic interaction was weakened in pretreated bagasse due to the lower lignin content (Table 1). Cellulase was negatively charged in the buffer at pH 4.8 and demonstrated an electrostatic repulsion of cellulose, which had a negatively charged surface (−45.61 mV). Due to the weakening of the hydrophobic interaction and the enhancement of the electrostatic interaction of pretreated cellulose and cellulase, the adsorption capacity of cellulase onto cellulose decreased, although the SSA of the pretreated bagasse increased ( Table 2).
The unit-bound enzyme efficiency was calculated based on the enzymolysis rate at 0.5 h (Figure 6b). The unit-bound enzyme efficiency of pretreated bagasse was significantly improved (Figure 4b), although the amount of bound enzyme onto bagasse remained unchanged (Figure 6c). This means that productive adsorption increases due to increased cellulose exposure to cellulase after the removal of noncellulose components (Table 1). This implies that the adequate removal of the noncellulose components is necessary for the enzyme to diffuse into or access the cellulose. The unit-bound enzyme efficiency (0.76 g/L/h/mg bound enzyme) of bagasse at low enzyme doses was significantly higher than that of the high enzyme doses of 10 and 20 FPU/g (0.68 and 0.43 g/L/h/mg bound enzyme). This suggests that pretreated bagasse is more conducive to enzyme efficiency at lower enzyme doses. The unit-bound enzyme efficiency was calculated based on the enzymolysis rate at 0.5 h (Figure 6b). The unit-bound enzyme efficiency of pretreated bagasse was significantly improved (Figure 4b), although the amount of bound enzyme onto bagasse remained unchanged (Figure 6c). This means that productive adsorption increases due to increased cellulose exposure to cellulase after the removal of noncellulose components (Table 1). This implies that the adequate removal of the noncellulose components is necessary for the enzyme to diffuse into or access the cellulose. The unit-bound enzyme efficiency (0.76 g/L/h/mg bound enzyme) of bagasse at low enzyme doses was significantly higher than that of the high enzyme doses of 10 and 20 FPU/g (0.68 and 0.43 g/L/h/mg bound enzyme). This suggests that pretreated bagasse is more conducive to enzyme efficiency at lower enzyme doses.

Evaluation of H 3 PO 4 Recyclability
'Green' and sustainable production is widely recognized by human society [63]. If the final production quality is not disturbed, the reuse of reagents for pretreatment can significantly reduce the cost. The H 3 PO 4 mixture was reused five times for subsequent pretreatment of bagasse with the appropriate addition of H 3 PO 4 , and the resulting glucose yield of pretreated bagasse via enzymolysis was similar to that of the fresh reagent ( Figure 7a). Approximately 85% of the H 3 PO 4 is recycled directly by filtration,~10% H 3 PO 4 is recovered from ethanol washing solution via rotary evaporation, and only~5% H 3 PO 4 needs to be replenished for reuse (Figure 7b). Residual H 3 PO 4 (~5%) in pretreated bagasse almost obviated the need for acid to adjust the pH to 4.8 to meet the requirements of the enzymatic hydrolysis process. Further research will be conducted to recover acid-soluble lignin from the H 3 PO 4 mixture to further improve the reuse potential of H 3 PO 4 .

Evaluation of H3PO4 Recyclability
'Green' and sustainable production is widely recognized by human society [63]. If the final production quality is not disturbed, the reuse of reagents for pretreatment can significantly reduce the cost. The H3PO4 mixture was reused five times for subsequent pretreatment of bagasse with the appropriate addition of H3PO4, and the resulting glucose yield of pretreated bagasse via enzymolysis was similar to that of the fresh reagent ( Figure  7a). Approximately 85% of the H3PO4 is recycled directly by filtration, ~10% H3PO4 is recovered from ethanol washing solution via rotary evaporation, and only ~5% H3PO4 needs to be replenished for reuse (Figure 7b). Residual H3PO4 (~5%) in pretreated bagasse almost obviated the need for acid to adjust the pH to 4.8 to meet the requirements of the enzymatic hydrolysis process. Further research will be conducted to recover acid-soluble lignin from the H3PO4 mixture to further improve the reuse potential of H3PO4.
In addition, phosphorus, in the form of phosphate, is an important nutrient for living things [64]. The treated filtrate rich in H3PO4 has the potential to be converted to phosphorus-rich fertilizer by reacting it with calcium hydroxide or ammonia water [65]. These examples indicate biomass pretreatment can be affected with milder environmental consequences.

Conclusions
Obtaining green energy and materials from renewable biomass is an indispensable pathway for human society to deal with the energy crisis and environmental issues. To overcome the biological barriers to biomass, a strategy of intergranular swelling of cellulose combined with hydrolysis and oxidation was proposed and demonstrated, which was used for enhancing the release of fermentable sugars by enzymolysis. Due to the fact that cellulose swelling, hemicellulose hydrolysis, and lignin oxidation degradation occur simultaneously during the pretreatment, they cooperate and promote each other. Bagasse was converted into cellulose with a nanoscale size, low DP, high void fraction, and high SSA. The cellulose in pretreated bagasse was sufficiently exposed to cellulase, affording a high glucose yield (98.60%), posing a competitive pretreatment method for enzymatic hydrolysis of biomass. This nanofiber network structure of cellulose provides the possibility for the combined production of fermentable sugars and nanocellulose, which will greatly improve the efficiency of biomass refining and inspire the development of novel cellulose- In addition, phosphorus, in the form of phosphate, is an important nutrient for living things [64]. The treated filtrate rich in H 3 PO 4 has the potential to be converted to phosphorus-rich fertilizer by reacting it with calcium hydroxide or ammonia water [65]. These examples indicate biomass pretreatment can be affected with milder environmental consequences.

Conclusions
Obtaining green energy and materials from renewable biomass is an indispensable pathway for human society to deal with the energy crisis and environmental issues. To overcome the biological barriers to biomass, a strategy of intergranular swelling of cellulose combined with hydrolysis and oxidation was proposed and demonstrated, which was used for enhancing the release of fermentable sugars by enzymolysis. Due to the fact that cellulose swelling, hemicellulose hydrolysis, and lignin oxidation degradation occur simultaneously during the pretreatment, they cooperate and promote each other. Bagasse was converted into cellulose with a nanoscale size, low DP, high void fraction, and high SSA. The cellulose in pretreated bagasse was sufficiently exposed to cellulase, affording a high glucose yield (98.60%), posing a competitive pretreatment method for enzymatic hydrolysis of biomass. This nanofiber network structure of cellulose provides the possibility for the combined production of fermentable sugars and nanocellulose, which will greatly improve the efficiency of biomass refining and inspire the development of novel cellulose-based materials. In addition, this study found that cellulase preferred crystalline cellulose to amorphous cellulose, which provided new evidence for further understanding the enzymatic hydrolysis mechanism of cellulose.
Author Contributions: X.L. conceived and designed the experiments; F.B. and T.D. analyzed the data and wrote the original paper; F.B. and T.D. performed the experiments; W.C., C.C. and Z.Z. edited the manuscript. All authors have read and agreed to the published version of the manuscript.
Funding: This research and the APC were funded by Innovation-driven Development Project of Guangxi Province, grant number AA18118024-1.

Institutional Review Board Statement: Not applicable.
Informed Consent Statement: Not applicable.

Data Availability Statement:
The data presented in this study are available on request from the corresponding author.