Calcium peroxide pretreatment to facilitate the delignication and enzymatic hydrolysis of wheat straw

Calcium peroxide (CaO 2 ) pretreatment was employed to remove lignin and subsequently facilitate enzymatic digestibility of wheat straw. An optimal condition was obtained at 130°C for 10 min with 0.35 g CaO 2 /g dried material of wheat straw and a 1:8 solid-liquid ratio. Under this condition, 57.8% of initial lignin, 7.2% of initial glucan, and 30.6% of initial xylan were removed from CaO 2 pretreatment, respectively, meanwhile, a glucose recovery of 90.6 % and a xylose recovery of 65.9 % were obtained from the subsequent enzymatic hydrolysis of treated wheat straw, respectively. CaO 2 pretreatment was proved to be a very effective method in delignication and improving enzymatic digestibility. Compared to raw material, the complex structure of lignocellulose was drastically disrupted with a wide emergence of scaly bulges and fully exposed microbers, which still retained in the solid.


Introduction
At present, the severe challenges posed by the scarcity of energy and resources and the increasingly serious environmental problems have aroused widespread concern among researchers in related elds. Bioethanol is regarded as one of the effective ways to achieve sustainable use of clean energy because of its renewable, low cost and abundance [1] . Wheat straw represents about 110-120% dry weight of the harvested wheat, which is a potential feedstock for bioethanol production in China and other wheatproducing countries. Wheat is one of the most widely cultivated food crops throughout the world, with about 40% of the world's populations depending on it as their staple food. China is the leading producer and consumer of wheat in the globally. In 2020, wheat yield was about 127 million tons (Mt), and the total available wheat straw was about 146 Mt in China. Wheat straw can be easily collected from the process of harvesting wheat and consists ~ 26% cellulose and ~ 26% hemicellulose, but a recalcitrant structure in which lignin is intertwined with cellulose and hemicellulose, hinders the polymer to produce monosaccharides in the step of enzymatic hydrolysis [2] . Therefore, pretreatment is a key process to remove lignin and disrupt the resistance barrier of the wheat straw to improve their enzymatic digestibility for enhancing fermentable sugar production [3,4] .
Presently, a variety of pretreatments to improve enzymatic digestibility by enhancing deligni cation, increasing porosity of the lignocellulosic biomass and disrupting its recalcitrant structure have been investigated [5] . Previous research showed that lignin is the main reason for depressing enzymes e ciency during enzymatic sacchari cation [6,7] . Alkali and oxidant agents are widely used to remove lignin from biomass, which have many favorable advantages such as less sugar loss, few inhibitors production and effective deligni cation [8,9] . Among these pretreatments, alkaline hydrogen peroxide (AHP) has attracted much attention due to its high e ciency, low secondary pollution and effective lignin removal at mild temperatures [10] . Additionally, AHP pretreatments still have a fast reaction rate of lignin removal at mild conditions [6] . It was noted that the structure of AHP treated biomass was destroyed, which enhanced the substrate accessibility to enzymes and reduced the non-productive adsorption of cellulase, which signi cantly improved the enzymatic digestibility [9,11,12] . It was reported that AHP pretreatment resulted in 91.53% lignin removal of corn stover at 30°C for 24 h with 0.5 g H 2 O 2 /g substrate (pH = 11.5), and increased the cellulose accessible pore volume and the area of exposed cellulose, which effectively enhanced the enzymatic hydrolysis e ciency [10] . However, as the principal reagent of AHP pretreatment (e.g. NaOH/H 2 O 2 ), H 2 O 2 is not only expensive but also di cult to store. Calcium peroxide (CaO 2 ) reacts with water to form calcium hydroxide and hydrogen peroxide, and the latter further produces highly oxidizing hydroxyl radical under alkaline conditions [8] . The mechanism is as follows [10,13] : 1 2 3 Based on the described mechanism, CaO 2 possesses the function of alkaline hydrogen peroxide.
Moreover, CaO 2 is more cost-effective and easier to transport and store due to its better thermal stability in comparison with hydrogen peroxide. CaO 2 pretreatment removed major part of lignin and preserved most of carbohydrates in the kenaf core powder [5] . Therefore, CaO 2 treatment is a promising method to remove lignin and improve enzymatic sacchari cation of lignocellulose.
In this work, the feasibility of enhancing enzymatic digestibility of the wheat straw by application of CaO 2 pretreatment was discussed at different conditions. The in uences of reaction time, temperature and CaO 2 loading on lignin reduction and carbohydrates degradation were determined. Treated samples were subjected to enzymatic hydrolysis with different enzymes loadings to evaluate the effects of CaO 2 pretreatment. Finally, macro-structural changes in the untreated and treated wheat straw was analyzed by scanning electron microscopy (SEM).

Wheat straw
Wheat straw was collected from rural areas of Hebei Province, China. Wheat straw was air-dried and ground to pass a 20 meshes screen in preparation for the experiment. The principal compositions of the raw wheat straw were cellulose (36.92 ± 0.98%), hemicellulose (25.11 ± 0.77%), and lignin (17.77 ± 1.26%). pretreatments were performed at varying pretreatment temperatures (90, 110, 130 and 150°C) and times (10, 30, 60, 90, 120, 150 min). After the reaction, the reactor was taken out of the heating jacket and immediately put into ice water to complete the reaction. The pretreated slurry was quantitatively transferred to a 500 mL glass beaker. The reactor was washed several times with distilled water after the transfer. Then, the mixture in the glass beaker was neutralized with acid (0.1 mol/L HCl) to pH 7.0, added distilled water to 300 mL, and ltered. The ltrate obtained was used to determine sugar and their degradation products. Wet solids were washed to remove the residual sugar. The way of washing the solids was centrifuged in two weight volumes, decanted the water and repeated six times. The washed solids were stored in a sealed plastic bag and placed in a 4°C freezer for chemical compositional analysis and subsequent enzymatic hydrolysis.

Enzymatic hydrolysis
Enzymic sacchari cation tests were performed in sodium citrate buffer (0.1 M, pH 4.8) with using of cellulase (Novozyme NS50013, 77.25 FPU/mL), β-glucosidase (Novozyme NS50010, 107.13 pNPGU/mL) and xylanase (UTC-X50, 25,000 IU/g) in a shaking incubator. Enzyme doses of cellucase 20 FPU/gsubstrate, of β-glucosidase 30 pNPGU/g-sbustrate, and of xylanase 60 IU/g-substrate were used. Weigh out CaO 2 -treated wheat straw to the equivalent of 0.2000 g on an oven dry weight basis. To each sacchari cation vial was pipetted prepared substrate, 5.0 mL (pH 4.8) sodium citrate buffer, 400 Fg tetracycline and 300 g cycloheximide. After addition of the enzymes, added distilled water to a total volume of 10 mL. Closed tightly vials were stirred thoroughly, and then returned to the shaking incubator (50°C, 150 rpm) for 48 h. After enzymatic sacchari cation test, the aliquot was withdrawn, and then diluted 10-fold. Diluted samples were boiled in a water bath for 10 min to put an end to the enzymatic digestion before being centrifuged and ltered for sugar analysis. All the sacchari cation tests were conducted in duplicate.
The recoveries of xylose and glucose were calculated as the percent conversion of gulcan and xylan in enzymatic sacchari cation process, as following equations (4) and (5), respectively.
where n 1 is the amount of glucose released in enzymatic hydrolysis, m 1 is the amount of gulcan in untreated wheat straw, n 2 is the amount of xylose released in enzymatic hydrolysis, m 2 is the amount of xylan in untreated wheat straw, 0.9 and 0.88 are the conversion factors for polymer to monomer sugars by the water of hydrolysis [14] .

Chemical analysis
Total solid and constituents (structural carbohydrates, lignin and ash) of untreated and treated wheat straw were determined referring to LAP standard procedures established by NREL [15] . Sugar was measured by HPLC (Lab Alliance Series III) equipped with refractive-index detection and a Biorad Aminex HPX-87H column. The operating procedure in detail was similar to previous report [3] . All samples were measured in duplicate, with the averages of measured values.

SEM
Surface morphological feature changes caused by CaO 2 pretreatment were captured at a magni cation of 1000 using a eld emission SEM (Nova NanoSEM 50, USA). Prior to imaging, the sample was coated by spraying the palladium-gold mixture to it conductive. Coated sample was placed in the chamber of scanning electron microscope and then observed under vacuum.

Results And Discussion
Effects of CaO 2 loading on pretreatment and enzymatic hydrolysis The CaO 2 loading is a critical factor in pretreatment as it has a great in uence on deligni cation, cellulose and hemicellulose degradation of the biomass, as well as the cost of the reagent. The effects of CaO 2 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) it is di cult to increase the active ingredient by increasing the concentration of a certain value. Yang et al. [16] observed similar results in the pretreatment of sisal waste with alkaline H 2 O 2 , which reported that the lignin removal increased signi cantly as H 2 O 2 loading increased from 0.1-0.6 g/g, and little above 0.6g/g. Increasing CaO 2 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 signi cantly affected by the CaO 2 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 CaO 2 pretreatment.
Pretreated wheat straw by CaO 2 with different loadings was subjected to enzymatic sacchari cation test.
Enzymatic hydrolysis processes were performed in 48 h with complex enzyme of cellulase (20 FPU/gsubstrate), β-glucosidase enzyme (30 pNPGU/g-sbustrate) and xylanase (60 IU/g-substrate). The recoveries of glucose and xylose for the CaO 2 loading studies were presented in Fig. 1 [16] . It could be attributed to the addition of xylanase improved the cellulose and hemicelluloses enzymatic hydrolysis effect. These results demonstrated that CaO 2 pretreatment was an e cient 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 crosslinking structure of wheat straw during CaO 2 pretreatment, which effectively improved the accessible surface area of cellulose and hemicellulose straw, and thereby enhanced enzymes digestibility [17,18] . In terms of enzymatic sacchari cation test, 0.35 g/g DM as an optimum CaO 2 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 in uenced the effect of pretreatment and energy consumption [19] . In uences of pretreatment temperature (90, 110, 130, 150°C) in relation to CaO 2 pretreatment e cacy were investigated for 120 min with the CaO 2 loading of 0.35 g/g DM (Fig. 2). As the temperature increased from 90°C to 150°C, the lignin removal rate increased signi cantly from 18.7-60.8%. In addition, the temperature rising from 90°C to 130°C has a greater impact on deligni cation 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 [20] .
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 signi cantly affect glucose recovery (91.4%). It was considered that CaO 2 pretreatment 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 [5] . 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 CaO 2 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. Both xylose and glucose recoveries for enzymatic hydrolysis gradually ascended rst 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 e ciency. For sugar recovery of enzymatic hydrolysis, the reaction time of 120 min was selected as the optimization of CaO 2 pretreatment time for the further work. It is important to note that CaO 2 pretreatment of wheat straw only resulted in the loss of a small amount of carbohydrates, and the conversion rate of cellulose and hemicellulose increased signi cantly during the enzymatic digestion process.

SEM
Scanning electron microscope (SEM) was used to study the in uence 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 micro bers, it still retained in the solid, which implied that there is no signi cant loss of cellulose. This was in agreement with the result of the degradation rate of cellulose in the pretreatment. Therefore, CaO 2 pretreatment effectively increased the accessibility of enzymes to cellulose and hemicellulose, which, in turn, enhanced enzymatic hydrolysis. Therefore, CaO 2 pretreatment was e cient in attacking the cellulose and hemicellulose bers, which signi cantly improved the enzymatic hydrolysis.

Conclusion
CaO 2 pretreatment showed effective deligni cation of wheat straw and largely retained the cellulose and hemicellulose fraction. The pretreated wheat straw achieved a higher glucose recovery and xylose recovery in enzymatic hydrolysis. Meanwhile, the surface structure of cellulose bundle was disrupted by the CaO 2 pretreatment, which raised the accessibility of cellulose and hemicellulose to enzymes, and pro tably enhanced the hydrolysis e ciency. Therefore, CaO 2 is a potential pretreatment method for improving enzymatic hydrolysis of lignocellulose.

Con ict of interest
The authors declare no competing nancial interest with the work submitted.