Due to the degradation of hemicellulose, a small amount of xylose ranging from 1.95 g to 5.89 g per 100 g raw material was detected. However, more than 79.24% of xylose was in the form of oligomer, suggesting that delignification pretreatment was not sufficient to degrade hemicellulose to xylose monomer [17, 18]. Meanwhile, as the delignification became severe, the glucose yield increased gradually to 0.66 g/100 g raw material, which was much lower than the xylose yield. The crystalline structure of cellulose was hard to be degraded than the amorphous hemicellulose, which consisted with the high recovery of cellulose shown in Table 1 [19]. For inhibitors, only formic and acetic acids were detected in the pretreatment liquor, and the highest concentrations of them were 1.28 and 4.36 g/L, respectively. It was reported that 2.5 g/L formic acid and 5 g/L acetic acid presented an obvious inhibition effect on glucose fermentation [20, 21]. However, the concentration of inhibitors during delignification pretreatment was lower than their impeditive loading for ethanol fermentation.
Characterization of untreated and pretreated sugarcane bagasse
X-ray diffraction curves of native and pretreated samples were conducted to calculate the crystallinity and cellulose crystallites sizes, the results were shown in Fig. 1. There were three crystalline peaks at 15.5o, 22.2o, and 34.7o, corresponding to the diffraction peaks of 101, 002, and 040, respectively, in typical cellulose I. The CrI of original sugarcane bagasse was 40.4%. After the NaOH pretreatment at 25 oC, the CrI increased to 48.3%. This phenomenon might be caused by the release of amorphous hemicellulose and lignin, leading to the increment of cellulose content in pretreated substrates [22]. As the NaOH pretreatment temperature increased from 60 to 160 oC, the CrI increased gradually from 54.6 to 60.3%, attributing to the large removal of hemicellulose and lignin, which increased the cellulose content in pretreated solids, as confirmed by the chemical composition shown in Table 1 [23]. The removed amorphous hemicellulose and lignin led to the rupture of interaction between the three main constitutes, providing more reactive sites for enzyme attack, and enhancing the enzymatic hydrolysis. Wang et al. reported that after ultrasound assisted Ca(OH)2 pretreatment, the CrI of grass clipping increased gradually, and the corresponding reducing sugar yield increased by 3.5 times compared with that of native material [4].
Furthermore, the cellulose crystallite average size (D) was also determined based on the Scherrer equation. The cellulose crystallite size of native material was 2.42 nm (002). After NaOH pretreatment, the cellulose crystallite average size increased gradually as reaction temperature was elevated, and reached 3.29 nm after 160 oC. However, this phenomenon was not agreed with previous literatures that pretreatment could reduce the crystalline size by disrupting the cellulose crystallinity [24]. That is to say, reformation or recrystallization of crystalline cellulose occurred during the NaOH pretreatment possibly contributed to the increment of cellulose crystallite average size [25, 26]. After NaClO2 and HAc pretreatment, the CrI and cellulose crystalline size of sugarcane bagasse increased to 51.1% and 2.81 nm.
To observe the morphological changes of native and pretreated materials, SEM images were detected and shown in Supplementary Fig. S1. The original sugarcane bagasse had a regular and smooth surface without any disorganized bundles, which impeded enzyme access and attack. After the pretreatment, the fibrils became loose and rough with cracks and pores on the surface. These irregular cracks and pores were probably due to the release of lignin and hemicellulose because NaOH could swell fibers and cleave the ether linkage between lignin and hemicellulose [9]. These dissolution and perforation could increase the surface area of pretreated solids [27]. Furthermore, as the pretreatment temperature was increased, more obvious cutting points and fragments appeared on the surface of fibers. For NaClO2 and HAc pretreated substrate, a large amount of fragments and crevices could be observed, attributing to the large removal of lignin and breakages between lignin and carbohydrates. These well-separated and shorten fibers provided more surface area and roughness, leading to the increase of enzymatic accessibility [24]. These observations indicated that the rupture of fibers structure and the improvement of enzymatic digestibility contributed to the enhancement of enzymatic hydrolysis, which would be confirmed in the following section.
FTIR spectroscopy was used to identify the characteristic bands in lignin and carbohydrates to analyze the structural changes before and after pretreatment, and the results were presented in Supplementary Fig. S2. After the pretreatment, the peaks at 1510 cm-1 and 1604 cm-1, representing the stretching of aromatic lignin ring, disappeared gradually from the pretreated solids due to the delignification, especially for NaClO2 and HAc pretreated solid. The band at 833 cm-1 (indicating the vibration in syringyl lignin) diminished gradually in pretreated substrate as the pretreatment temperatures increased, meaning a dissociation of lignin polymer [2]. Furthermore, the intensity of peaks at 1740 cm-1 representing the carbonyl/acetyl in hemicellulose became weaken during NaOH pretreatment. However, the NaClO2 and HAc pretreated substrate presented a reverse appearance, which was an indication of the reservation of most hemicellulose during pretreatment. Meantime, the bands at 898 cm-1 belonged to the β-glycosidic linkages vibration of cellulose, which became strong after pretreatment. This increase was attributed to the higher content of glucan (cellulose) in pretreated solids (as shown in Table 1) [28]. This phenomenon indicated that NaOH pretreatment removed the bulk of lignin and hemicellulose and simultaneously retained most cellulose in pretreated substrates by selective breaking functional groups and chemical bonds [24].
Figure 2 illustrated the TG and differential TG (DTG) curves of native and pretreated solids to determine their thermal properties. The weight loss below 120 oC was attributed to the evaporation of moisture. All samples adequately decomposed between 200 and 400 °C. As shown by the DTG results, there were two main weight loss peaks for the original sugarcane bagasse. The first one occurred at 302 °C with DTG of -0.1 %·C− 1, ascribing to the decomposition of hemicellulose. The second one proceeded at 350 °C with DTG of -095 %·C− 1 because of the decomposition of cellulose and lignin, indicating that cellulose and lignin had a high degradation temperature than hemicellulose [29]. After NaOH pretreatment at 25 °C, only one main weight loss peak could be observed at 357 °C with DTG of 1.02 %·C− 1, corresponding to the decomposition of cellulose and lignin, which suggested that an amount of hemicellulose was degraded during NaOH pretreatment. As the reaction temperature was elevated from 60 to 160 °C with NaOH, the DTG of these pretreated samples depicted similar curves with that pretreated at 25 °C, and the highest weight loss peaks of 60, 120, 160 oC pretreated samples occurred at 348, 363, and 361 °C, respectively. The raw sugarcane bagasse decomposed 50% weight occurred at 335 °C, while the decomposition temperatures with 50% weight loss were 350, 345, 362, and 356°C for NaOH pretreated solids at 25, 60, 120, and 160 °C, correspondingly. Due to the release of partial hemicellulose and lignin during NaOH pretreatment, the pretreated substrates exhibited a higher degradation temperature, indicating the higher thermal stability of pretreated substrates [30]. Additionally, when the samples were heated to 690 °C, the native and NaOH pretreated sugarcane bagasse all reserved some residues (such as lignin and ash), and the residual weights after pretreatment were higher than the raw material. This could be concluded that the degraded/dissolved hemicellulose and lignin from the raw material indeed increase the thermal stability. For NaClO2 pretreated substrate, two weight loss peaks could be observed, attributing to the decomposition of hemicellulose (302 oC) and lignin (345 oC), respectively [31]. The degradation temperature for 50% weight loss were 344 oC, which was higher than native material but lower than NaOH pretreated at severe conditions.