Effects of CaO2 loading on pretreatment and enzymatic hydrolysis
The CaO2 loading is a critical factor in pretreatment as it has a great influence on delignification, cellulose and hemicellulose degradation of the biomass, as well as the cost of the reagent. The effects of CaO2 loadings on lignin removal and carbohydrate degradation of wheat straw were measured in the range of 0.15, 0.25, 0.35, 0.45, 0.55 g/g DM at 130°C for 120 min (Fig. 1). The delignification rate increased significantly from 26.2–57.8%, as CaO2 loading elevated from 0.15 to 0.35 g/g DM; nevertheless, with no significant decreasing trend on delignification with above 0.35 g/g DM. Due to the low solubility of CaO2, it is difficult to increase the active ingredient by increasing the concentration of a certain value. Yang et al.  observed similar results in the pretreatment of sisal waste with alkaline H2O2, which reported that the lignin removal increased significantly as H2O2 loading increased from 0.1–0.6 g/g, and little above 0.6g/g. Increasing CaO2 loading form 0.15 to 0.55 g/g DM, the degradation rates of glucan and xylan for pretreatment increased from 4.8% and 20.9–7.6% and 36.7%, respectively. The result indicated that lignin removal was significantly affected by the CaO2 loading. The degradation of hemicellulose and some part of cellulose were also observed. However, both glucan and xylan were retained in wheat straw in large proportion even after CaO2 pretreatment.
Pretreated wheat straw by CaO2 with different loadings was subjected to enzymatic saccharification test. Enzymatic hydrolysis processes were performed in 48 h with complex enzyme of cellulase (20 FPU/g-substrate), β-glucosidase enzyme (30 pNPGU/g-sbustrate) and xylanase (60 IU/g-substrate). The recoveries of glucose and xylose for the CaO2 loading studies were presented in Fig. 1. Glucose recovery increased from 52.1 to 93.2% with CaO2 loading increasing from 0.15 to 0.55 g/g DM. CaO2 loading showed a significant impact on enhancing glucose recovery. The results showed that increasing CaO2 loading from 0.15 to 0.35 g/g DM glucose recovery increased by 73.9%. However, only 2.9% glucose recovery improvement was achieved by CaO2 loading from 0.35 to 0.55 g/g DM. It was noted that xylose recovery increased with elevated CaO2 loading within 0.35 g/g DM, and then decreased with continued CaO2 loading. In totally, as CaO2 loading increased from 0.35 to 0.55 g/g DM did not evidently improve both the lignin removal and cellulose digestibility significantly; instead, decreased the hemicellulose digestibility. The maximum recovery of xylose was 65.9%, which was obtained at 0.35 g/g DM. This trend may be due to higher loss of hemicellulose from CaO2 pretreatment. The maximum recovery of xylose in this work was higher than that for alkaline H2O2 pretreated sisal waste (pH = 11.5, 6h, at room temperature, 0.6 g H2O2/g), though slightly higher delignification rate . It could be attributed to the addition of xylanase improved the cellulose and hemicelluloses enzymatic hydrolysis effect. These results demonstrated that CaO2 pretreatment was an efficient pretreatment wheat straw in enhancing the subsequent enzymatic digestibility. Lignin removal was positively correlated with enzymatic hydrolysis of cellulose. This was probably attributed to the removal of lignin and the destruction of the complex cross-linking structure of wheat straw during CaO2 pretreatment, which effectively improved the accessible surface area of cellulose and hemicellulose straw, and thereby enhanced enzymes digestibility [17, 18]. In terms of enzymatic saccharification test, 0.35 g/g DM as an optimum CaO2 loading was used for subsequent experiments.
Effect of temperature on pretreatmemt and enzymatic hydrolysis
Temperature is a vital factor in the chemical reaction, as well as in the pretreatment biomass process, which directly influenced the effect of pretreatment and energy consumption . Influences of pretreatment temperature (90, 110, 130, 150°C) in relation to CaO2 pretreatment efficacy were investigated for 120 min with the CaO2 loading of 0.35 g/g DM (Fig. 2). As the temperature increased from 90°C to 150°C, the lignin removal rate increased significantly from 18.7–60.8%. In addition, the temperature rising from 90°C to 130°C has a greater impact on delignification than the temperature rising from 130 to 150°C. As showed in Fig. 2, increasing temperature could accelerate the loss of hemicellulose and cellulose. It can be observed that the removal rate of the gulcan and xylan was quite different depending on the temperature. The maximum removal of gulcan and xylan reached 8.7 and 48.1%, respectively, which suggested that hemicellulose could be degraded more easily than cellulose at the same pretreatment condition, due to their different structures .
The recoveries of glucose and xylose for enzymatic digestibility were plotted along pretreatment temperature in Fig. 2. An increase of the pretreatment temperature favored the enzymatic hydrolysis of cellulose, which was similar to that of lignin removal. As it was observed for glucose recovery increased from 40.9–91.4%, with the temperature rising from 90 to 130°C. As temperature increased to 150°C, it didn’t significantly affect glucose recovery (91.4%). It was considered that CaO2pretreatment was effective in breaking apart lignin and hemicellulose while keeping cellulose intact at high temperature, thereby enhanced in its surface area, by which it becomes more accessible to enzymatic hydrolytic treatment . Xylose recovery initially increased with temperature, while it gradually declined with further increasing temperature. The maximum recovery of xylose was 65.9%, which was obtained at 130°C. It was attributed that a partial hemicellulose degradation during CaO2 pretreatment at 150°C resulted in the decrease of xylose recovery of enzymatic hydrolysis. In terms of enzymatic hydrolysis, 130°C was taken for the appropriate reaction time.
Effect of time on pretreatment and enzymatic hydrolysis
Reaction time affects the utilization of the reagent and the effect of pretreatment. The effects of reaction time varying from 10 to 150 min were investigated at 130°C with CaO2 loading 0.35 g/g. The results of delignification and enzymatic hydrolysis were listed in Fig. 3. Lignin removal significantly enhanced from 29.3 to 57.3%, as the reaction time rising from 10 to 90 min. However, further enhance time at 120 and 150 min, no significant increase of delignification was exhibited. The removal of gulcan and xylan slowly increased with the reaction time increasing. However, it was observed that reaction time had little effect on the degradation of cellulose and hemicellulose at 130°C with CaO2 loading 0.35 g/g. As the reaction time increasing from 10 to 120 min, the removal rates of glucan and xylan enhanced from 5.5 to7.3%, and from 26.4 to 31.4%, respectively.
Both xylose and glucose recoveries for enzymatic hydrolysis gradually ascended first and then declined with increasing reaction time, as it was seen in Fig. 3. The maximum recovery of glucose and xylose of 90.6% and 65.9%, respectively, was available at the time of 120 min. Further increasing the reaction time to 150 min, both the recovery of glucose and xylose decreased for enzymatic hydrolysis. This was caused by the loss of cellulose and hemicellulose fraction during the pretreatment process. In general, the reaction time is not a variable leading to major changes in enzymatic hydrolysis. Hence, increased reaction time did not favorably enhance enzymatic hydrolysis efficiency. For sugar recovery of enzymatic hydrolysis, the reaction time of 120 min was selected as the optimization of CaO2 pretreatment time for the further work. It is important to note that CaO2 pretreatment of wheat straw only resulted in the loss of a small amount of carbohydrates, and the conversion rate of cellulose and hemicellulose increased significantly during the enzymatic digestion process.
Scanning electron microscope (SEM) was used to study the influence of calcium oxide pretreatment on the structure and surface morphology of the wheat straw. Figure 4 showed that the untreated wheat straw had a smooth, continuous, and dense structure, while the treated wheat straw presented a loose sheet structure due to the removal of more than half of the lignin. Although the complex structure of lignocellulose was drastically disrupted with a wide emergence of scaly bulges and fully exposed microfibers, it still retained in the solid, which implied that there is no significant loss of cellulose. This was in agreement with the result of the degradation rate of cellulose in the pretreatment. Therefore, CaO2 pretreatment effectively increased the accessibility of enzymes to cellulose and hemicellulose, which, in turn, enhanced enzymatic hydrolysis. Therefore, CaO2 pretreatment was efficient in attacking the cellulose and hemicellulose fibers, which significantly improved the enzymatic hydrolysis.