Mild Pretreatment Coupled with Ligninases Synergism Enhances Ethanol Production from Wheat Straw via Simultaneous Glucose and Xylose Fermentation

Mild Pretreatment Coupled with Ligninases Synergism Enhances Ethanol Production from Wheat Straw via Simultaneous Glucose and Xylose Fermentation | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Short Report Mild Pretreatment Coupled with Ligninases Synergism Enhances Ethanol Production from Wheat Straw via Simultaneous Glucose and Xylose Fermentation Wenxuan Zhao, Zhigang Ju This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-9171419/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 8 You are reading this latest preprint version Abstract Considering the global energy crisis, developing renewable biomass energy has become an urgent strategic need. In order to better utilize biomass resources, wheat straw was used as raw material. Findings from enzymatic hydrolysis, saccharification assays, and Scanning Electron Microscopy (SEM) characterization of pretreated wheat straw confirmed that the weak alkali-oxidation pretreatment (2% NaHCO + 2% HO) effectively eliminated lignin with simultaneous preservation of cellulose and hemicellulose fractions of wheat straw under relatively mild pretreatment conditions (50 min, 130°C, and solid-liquid ratio of 1:10). Enzymatic hydrolysis and saccharification experiments revealed that ligninases enhanced the hydrolysis efficiency of pretreated wheat straw, with laccase emerging as the most impactful component among these ligninases. Under the conditions of cellulase 20 U/g, xylanase 10 U/g, laccase 4 U/g, lignin peroxidase 6 U/g and manganese peroxidase 2 U/g, the combined sugars reached a maximum of 108.4 ± 0.6 mmol/L, which was 17.8% higher than that of the group without ligninases. Finally, ethanol was produced by fermenting both glucose and xylose in the fermentation broth, and ethanol fermentation conditions of engineered Saccharomyces cerevisiae strain WXY12 were optimized, with peptone as the nitrogen source and MgSO as the metal ion inducer, temperature at 32°C, rotational speed at 130 r/min, and inoculation amount of 1% (v/v). Under these conditions, the highest ethanol concentration reached 7.6 ± 0.2 g/L, corresponding to an ethanol yield of 20.9 g per 100 g of raw wheat straw, thereby establishing a robust theoretical basis for the efficient valorization of biomass resources. wheat straw pretreatment ligninases simultaneous saccharification and fermentation ethanol Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 1. Introduction Facing growing depletion of fossil fuels, the efficient valorization of lignocellulosic biomass has emerged as a critical research priority for sustainable biomass energy development; among these biomass resources, as one of the world’s most abundant agricultural residues, wheat straw remains largely underexploited with limited valorization [ 1 – 2 ]. Wheat straw primarily consists of cellulose, hemicellulose and lignin—a dense three-dimensional cross-linked network based on phenylpropane units that encapsulates cellulose and hemicellulose [ 3 ]. This structural barrier not only enhances plant rigidity and pathogen resistance but also inhibits the enzymatic hydrolysis of carbohydrates, the rate-limiting step for ethanol production [ 4 – 5 ]. Our previous work on wheat straw pretreatment has highlighted its potential for bioethanol production, but challenges in lignin removal still limit the large-scale application of wheat straw [ 6 ]. Thus, our study prioritizes mild pretreatment strategies that selectively remove lignin while preserving cellulose and hemicellulose, thereby improving the utilization efficiency of wheat straw and other lignocellulosic biomass resources [ 7 – 8 ]. Cellulase and xylanase are well-recognized as core enzymes for lignocellulose hydrolysis, but their efficiency is often constrained by residual lignin after pretreatment [ 9 ]. Notably, the synergistic effects between lignin-degrading enzymes (laccase, lignin peroxidase, manganese peroxidase) and polysaccharide hydrolases remain understudied in wheat straw systems. Given that ligninases can effectively hydrolyze lignin and improve the accessibility of cellulose and hemicellulose, we hypothesize that adding ligninases can further break down lignin residues, thereby boosting overall sugar yields for subsequent fermentation. In addition, most prior studies on lignocellulosic ethanol have focused solely on glucose conversion, neglecting xylose (a major product of hemicellulose hydrolysis) and leading to incomplete resource utilization [ 10 – 12 ]. In contrast, the engineered S. cerevisiae strain WXY12 selected in this study enables simultaneous glucose and xylose fermentation—overcoming this limitation and improving the economic viability of wheat straw valorization [ 13 ]. Building on the above, wheat straw was employed as the raw material for subsequent pretreatment and enzymatic hydrolysis. First, we aim to develop a safe and efficient pretreatment process by optimizing methods and conditions, which can selectively remove lignin while maintaining the cellulose and hemicellulose fractions, thus boosting the comprehensive utilization efficiency of wheat straw. Second, the synergistic effects of ligninolytic enzymes combined with cellulase and xylanase were investigated based on the sugar yield in wheat straw hydrolysate, while the optimal dosage of lignocellulase during the hydrolysis of pretreated wheat straw was determined via orthogonal experiments. Finally, on the basis of obtaining the optimal pretreatment and hydrolysis conditions, the engineered S. cerevisiae strain WXY12 was used to ferment pretreated wheat straw hydrolysate to produce ethanol, and the ethanol yield was enhanced by optimizing the fermentation conditions of pretreated wheat straw. This study aims to develop a mild and efficient pretreatment method for wheat straw, optimize the synergistic lignocellulase system for high sugar yield, and achieve efficient ethanol production via co-fermentation of glucose and xylose by S. cerevisiae WXY12. The results are expected to provide a reliable theoretical basis for facilitating the high-value utilization of lignocellulosic biomass resources. 2. Material and methods 2.1 Materials Cellulase (4×10 5 U/g) and lignin peroxidase (100 U/g) were purchased from Shanghai Yuanye Bio-Technology Co., Ltd, and xylanase (2500 U/g), manganese peroxidase (200 U/g), laccase (400 U/g) were purchased from Sigma-Aldrich (Shanghai) Trading Co., Ltd. Glucose and xylose quantification kits were purchased from Elabscience Biotechnology Co., Ltd. The engineered S. cerevisiae strain WXY12 (high-efficiency ethanol-producing) was kindly provided by Capital Normal University [ 13 ]. 2.2 Methods 2.2.1 Pretreatment of wheat straw Wheat straw was oven-dried and sieved to a particle size of 10 mesh for subsequent use. To screen for mild and efficient pretreatment methods, wheat straw was subjected to various pretreatment regimes (with distilled water as the control group): strong acid treatments (2% H 2 SO 4 , 2% HCl), weak acid treatment (2% CH 3 COOH), strong alkali treatments (2% NaOH, 2% KOH), weak alkali treatment (2% NaHCO 3 ), oxidative treatment (2% H 2 O 2 ), strong acid coupled with oxidative treatments (2% H 2 SO 4 + 2% H 2 O 2 , 2% HCl + 2% H 2 O 2 ), weak acid coupled with oxidative treatment (2% CH 3 COOH + 2% H 2 O 2 ), strong alkali coupled with oxidative treatments (2% NaOH + 2% H 2 O 2 , 2% KOH + 2% H 2 O 2 ), and weak alkali coupled with oxidative treatment (2% NaHCO 3 + 2% H 2 O 2 ) [ 14 ]. All pretreatments were conducted at a solid-to-liquid ratio of 1:10 (w/v) at 120°C for 50 min. The pretreated wheat straw was washed to neutrality, then oven-dried and weighed. The lignin, cellulose, and hemicellulose compositions of raw and pretreated wheat straw were measured according to the procedure issued by the National Renewable Energy Laboratory (NREL) [ 15 ]. Wheat straw recovery rate was calculated based on the ratio of sample weight before and after pretreatment. All treatments were performed in triplicate. To evaluate the efficiency of the pretreatment, the pretreated wheat straw was subjected to enzymatic hydrolysis using 20 U of cellulase and 20 U of xylanase per gram of cellulose and hemicellulose, respectively. For a 40 mL hydrolysis system, 1.5 g of pretreated wheat straw was combined with 2 mL of 0.05 M citrate buffer. The total volume was adjusted to 40 mL with ultrapure water, the pH was set to 4.8, and the mixture was autoclaved at 115°C for 15 min. Afterward, the corresponding dosages of the two enzymes were determined according to the cellulose and hemicellulose contents, and the enzymes were supplemented once the mixture had cooled. Hydrolysis was carried out at 50°C with shaking at 120 rpm. Glucose and xylose concentrations were quantified using the corresponding kits at 12-h intervals for a total duration of 72 h [ 16 ]. After the 72-h hydrolysis, the reaction was terminated by heating at 100°C for 10 min to denature the enzymes, ensuring no further degradation of carbohydrates during sample handling. For further optimization of the pretreatment conditions, the treatment time (40, 50, 60, 70 and 80 min), the treatment temperature (100, 110, 120, 130, and 140°C), and the solid-liquid ratio (1:8, 1:9, 1:10, 1:11, and 1:12) were evaluated, with the monosaccharide yield after enzymatic hydrolysis as the key evaluation indicator. Additionally, to further validate the effectiveness of the pretreatment methods developed in this work, the morphological and structural variations of wheat straw before and after pretreatment were characterized using SEM [ 17 ]. 2.2.2 Enzymatic hydrolysis of pretreated wheat straw by lignocellulase The individual effects of three lignin-degrading enzymes, namely laccase (Lac), lignin peroxidase (LiP), and manganese peroxidase (MnP), on the monosaccharide yield of wheat straw hydrolysate were first investigated. Each ligninolytic enzyme was supplemented at 6 U per gram of pretreated wheat straw into the hydrolysis system to evaluate its promoting effect on the hydrolysis of the substrate. Then, a five-factor, four-level orthogonal test was used to optimize the addition of five enzymes (cellulase, xylanase, Lac, LiP, MnP), so as to establish a high-efficiency complex enzymatic hydrolysis system for lignocellulosic materials. On the basis of cost savings, the maximum enzymatic hydrolysis efficiency of the pretreated wheat straw was achieved, thereby improving the monosaccharide yield (glucose and xylose). 2.2.3 Ethanol production via fermentation of pretreated wheat straw During the experiment of ethanol production via fermentation from pretreated wheat straw, the preparation of ethanol standard solutions and the gas chromatographic (GC) analysis of ethanol content were performed according to a previously reported method [ 18 ]. The retention time of ethanol in GC analysis was 2.13–2.16 min, and the regression equation was fitted based on the peak areas corresponding to different ethanol concentrations: y = 3.1571x − 0.0308, R 2 = 0.9996. The sterile enzyme solution with the optimal ratio was added into the fermentation medium containing 30 g/L pretreated wheat straw, 10 g/L yeast extract, and 1 g/L KCl. The engineered S. cerevisiae strain WXY12—a high-efficiency ethanol producer capable of co-fermenting glucose and xylose—was also inoculated into the fermentation medium. Subsequently, separate hydrolysis and fermentation (SHF, in which enzymatic hydrolysis was conducted for 3 days before fermentation) was compared with simultaneous saccharification and fermentation (SSF) for ethanol production from pretreated wheat straw [ 19 – 20 ]. The fermentation conditions were set as follows: a temperature of 30°C, rotational speed of 120 r/min, an inoculation amount of 2% (v/v), natural pH (unadjusted). The ethanol concentration in the fermentation broth was determined by GC according to the established standard curve. Afterwards, ethanol concentration was measured at 12 h intervals until it began to decrease, and the fermentation medium components and process conditions (nitrogen source, metal ion inducer, temperature, rotational speed and inoculum dosage) were then optimized to improve ethanol yields. 3. Results 3.1 Pretreatment efficacy of various methods on wheat straw To enhance the pretreatment efficacy, different pretreatment strategies were performed on wheat straw, including strong acid treatments (2% H 2 SO 4 , 2% HCl), weak acid treatment (2% CH₃COOH), strong alkali treatments (2% NaOH, 2% KOH), weak alkali treatment (2% NaHCO 3 ), single oxidative treatment (2% H 2 O 2 ), as well as acid-oxidative and alkali-oxidative systems: strong acid coupled with oxidation treatments (2% H 2 SO 4 + 2% H 2 O 2 , 2% HCl + 2% H 2 O 2 ), weak acid coupled with oxidation treatment (2% CH 3 COOH + 2% H 2 O 2 ), strong alkali coupled with oxidation treatments (2% NaOH + 2% H 2 O 2 , 2% KOH + 2% H 2 O 2 ), and weak alkali coupled with oxidation treatments (2% NaHCO 3 + 2% H 2 O 2 ), with water-treated and untreated wheat straw serving as controls. Following the pretreatment, the recovery rate of wheat straw and the contents of lignin, cellulose, and hemicellulose before and after treatment were measured. As shown in Table 1 , after pretreatment with 2% NaOH (strong alkali), wheat straw presented the highest hemicellulose retention of 26.7 ± 0.1%, with cellulose retention of 46.7 ± 0.2%, lignin residue of 5.2 ± 0.5% and wheat straw recovery rate of 79.7 ± 0.4%. In contrast, pretreatment with the combination of strong acid and oxidant (2% HCl and 2% H 2 O 2 ) resulted in the highest cellulose retention in wheat straw (61.7 ± 0.3%), along with hemicellulose retention of 13.7 ± 0.4% and a lignin residue of 4.2 ± 0.4%. Although the proportions of cellulose and hemicellulose in wheat straw treated by the weak alkali‑oxidant combined process (2% NaHCO 3 and 2% H 2 O 2 ) did not reach the maximum values, both components maintained relatively high retention rates (49.1 ± 0.2% for cellulose and 26.1 ± 0.4% for hemicellulose), with lignin residue of 4.7 ± 0.3%. Moreover, this pretreatment achieved the highest wheat straw recovery rate of 82.6 ± 0.2%, which was beneficial for improving the subsequent utilization rate of wheat straw. Therefore, the 2% NaHCO 3 + 2% H 2 O 2 pretreatment was primarily identified as the optimal strategy and employed in subsequent experiments. Table 1 Compositional analysis of wheat straw before and after various pretreatments NO. Sample Cellulose (%) Hemicellulose (%) Lignin (%) Recovery rate (%) 1 Untreated wheat straw 41.3 ± 0.3 21.1 ± 0.3 12.9 ± 0.4 100 ± 0.0 2 H 2 O 42.3 ± 0.2 23.8 ± 0.1 11.3 ± 0.1 94.6 ± 0.4 3 2% H 2 SO 4 57.3 ± 0.4 13.9 ± 0.2 6.9 ± 0.3 77.3 ± 0.5 4 2% HCl 55.6 ± 0.2 15.7 ± 0.3 7.2 ± 0.2 79.1 ± 0.5 5 2% CH 3 COOH 46.9 ± 0.5 19.8 ± 0.2 11.3 ± 0.2 86.2 ± 0.3 6 2% H 2 O 2 48.3 ± 0.1 21.8 ± 0.3 8.2 ± 0.3 84.7 ± 0.2 7 2% NaOH 46.7 ± 0.2 26.7 ± 0.1 5.2 ± 0.5 79.7 ± 0.4 8 2% KOH 45.8 ± 0.3 25.9 ± 0.2 6.4 ± 0.4 81.1 ± 0.3 9 2% NaHCO 3 44.5 ± 0.4 24.8 ± 0.2 8.9 ± 0.3 85.2 ± 0.2 10 2% H 2 SO 4 + 2% H 2 O 2 59.3 ± 0.2 16.1 ± 0.4 4.1 ± 0.2 65.7 ± 0.5 11 2% HCl + 2% H 2 O 2 61.7 ± 0.3 13.7 ± 0.4 4.2 ± 0.4 70.3 ± 0.4 12 2% CH 3 COOH + 2% H 2 O 2 55.6 ± 0.1 14.7 ± 0.2 8.9 ± 0.5 81.2 ± 0.2 13 2% NaOH + 2% H 2 O 2 52.0 ± 0.4 24.1 ± 0.1 3.7 ± 0.3 67.1 ± 0.6 14 2% KOH + 2% H 2 O 2 49.4 ± 0.3 25.6 ± 0.4 4.4 ± 0.2 73.5 ± 0.5 15 2% NaHCO 3 + 2% H 2 O 2 49.1 ± 0.2 26.1 ± 0.4 4.7 ± 0.3 82.6 ± 0.2 Note: values are expressed as mean ± standard deviation (n = 3) To further explore the effect of each pretreatment method on wheat straw, cellulase and xylanase were used to perform enzymatic saccharification on the above 15 groups. Following 72 h of hydrolysis, the concentrations of glucose and xylose in the hydrolysate were measured, and the combined sugar (glucose and xylose) content was calculated. Untreated and water-pretreated wheat straw were employed as controls. Higher glucose and xylose concentrations in the hydrolysate indicated that the pretreatment was more effective for enzymatic saccharification. As shown in Fig. 1 , water treatment exerted a slight influence on enzymatic hydrolysis and saccharification. The hydrolysates of wheat straw pretreated with 2% HCl + 2% H 2 O 2 (group 11), 2% NaOH + 2% H 2 O 2 (group 13), and 2% NaHCO 3 + 2% H 2 O 2 (group 15) showed a higher sugar concentration. Wheat straw pretreated with 2% NaOH + 2% H 2 O 2 exhibited the highest combined sugar content of 83.3 ± 1.5 mmol/L (glucose 58.2 ± 0.9 mmol/L and xylose 25.1 ± 0.7 mmol/L) after enzymatic hydrolysis and saccharification. This was followed by the 2% HCl + 2% H 2 O 2 group, which yielded 81.2 ± 1.3 mmol/L combined sugar (glucose 61.9 ± 0.4 mmol/L and xylose 19.3 ± 0.7 mmol/L). Wheat straw pretreated with 2% NaHCO 3 + 2% H 2 O 2 yielded a combined sugar content of 80.1 ± 0.9 mmol/L (glucose 52.9 ± 0.7 and xylose 27.2 ± 0.3 mmol/L) after enzymatic hydrolysis and saccharification. Comparative analysis showed that the combined sugar content of wheat straw pretreated with 2% NaOH + 2% H 2 O 2 and 2% HCl + 2% H 2 O 2 was slightly higher than that pretreated with 2% NaHCO 3 + 2% H 2 O 2 following enzymatic hydrolysis and saccharification, but the difference was not significant (p > 0.05). Moreover, NaOH is a strong alkali and HCl is a strong acid, which causes environmental pollution and instrument damage. Meanwhile, wheat straw pretreated with 2% NaHCO 3 + 2% H 2 O 2 exhibited a recovery rate of 82.6 ± 0.2%, which was significantly higher than that of the 2% NaOH + 2% H 2 O 2 (67.1 ± 0.6%) and 2% HCl + 2% H 2 O 2 (70.3 ± 0.4%) groups ( p < 0.05). Based on the above results, 2% NaHCO 3 + 2% H 2 O 2 was selected as the pretreatment method for wheat straw in subsequent experiments. Following the selection of 2% NaHCO 3 + 2% H 2 O 2 as the optimal pretreatment method, in order to obtain a better pretreatment effect, the conditions of time, temperature and solid-liquid ratio were optimized, and the effect was also verified by enzymatic hydrolysis and saccharification tests. Higher glucose and xylose concentrations in the hydrolysate indicated a better effect of wheat straw pretreatment on enzymatic saccharification. As shown in Fig. 2 a and 2 b, the maximum combined sugar content of 81.2 ± 1.2 mmol/L (glucose 55.7 ± 0.8 and xylose 25.5 ± 0.6 mmol/L) was achieved at a pretreatment time of 60 min, followed by a value of 80.1 ± 0.9 mmol/L (glucose 52.9 ± 0.7 and xylose 27.2 ± 0.3 mmol/L) at 50 min. Considering that the difference in the final combined sugar content between them was not significant (p > 0.05) and a shorter time favored energy conservation, 50 min was selected as the optimal pretreatment time for wheat straw.As shown in Fig. 2 c and 2 d, the combined sugar content reached a maximum of 92.0 ± 0.6 mmol/L (glucose 60.7 ± 0.5 mmol/L and xylose 31.3 ± 0.3 mmol/L) at 130 ℃, while lower sugar contents were observed at other temperatures. Correspondingly, 130 ℃ was determined as the optimal pretreatment temperature. As shown in Fig. 2 e and 2 f, the highest combined sugar content of 93.9 ± 1.4 mmol/L (glucose 61.2 ± 0.8 and xylose 32.7 ± 0.7 mmol/L) was obtained at a solid-liquid ratio of 1:9, followed by a value of 92.0 ± 0.9 mmol/L (glucose 60.7 ± 0.8 and xylose 31.3 ± 0.4 mmol/L) at 1:10. Although the difference in glucose and xylose contents in the hydrolysate between these two solid-liquid ratios was not significant (p > 0.05), the material utilization rate was higher at 1:10, and this ratio was chosen for wheat straw pretreatment. In summary, 2% NaHCO 3 + 2% H 2 O 2 was finally selected, and the pretreatment conditions were as follows: time of 50 min, temperature of 130 ℃, solid-liquid ratio of 1:10. The selected pretreatment method removed lignin and retained cellulose and hemicellulose components under relatively mild conditions and exhibited a better pretreatment effect. Meanwhile, the yield of monosaccharides in the hydrolysate was higher, which would lay a solid foundation for subsequent fermentation and promote the efficient comprehensive utilization of wheat straw. To verify the efficiency of lignin removal by the selected pretreatment method, SEM was employed to characterize the surface microstructure of wheat straw before and after pretreatment. Untreated and water-pretreated wheat straw were used as control groups. SEM revealed that untreated wheat straw had a uniform surface, with cellulose in a compact and ordered structure (Fig. 3 a). The structure of wheat straw after water pretreatment (Fig. 3 b) was slightly damaged, but the overall structure remained intact. In contrast, the compact structure of wheat straw pretreated with 2% NaHCO 3 + 2% H 2 O 2 was obviously destroyed (Fig. 3 c), and its surface exhibited a broken, rough, and hollow morphology. These structural modifications improved the accessibility of cellulose and hemicellulose to enzymes in the subsequent enzymatic hydrolysis, thus promoting the efficient degradation of wheat straw and enhancing its utilization efficiency. 3.2 Effect of ligninolytic enzymes on enzymatic hydrolysis and saccharification of pretreated wheat straw To further improve the hydrolysis efficiency of pretreated wheat straw, the effects of individual lignin-degrading enzymes on glucose, xylose and combined sugar (combined glucose and xylose concentrations in the hydrolysate) were first investigated, with different ligninolytic enzymes added separately to the hydrolysis system. Cellulase and xylanase were added to the hydrolysis system alone (Fig. 4 A). Cellulase, xylanase and Lac were added to the hydrolysis system (Fig. 4 B). Cellulase, xylanase and Lip were added to the hydrolysis system (Fig. 4 C). Cellulase, xylanase and Mnp were added to the hydrolysis system (Fig. 4 D). Cellulase, xylanase, Lac, Lip and Mnp were added to the hydrolysis system simultaneously (Fig. 4 E). It can be seen from Fig. 4 that adding any kind of ligninolytic enzyme to the enzymatic hydrolysis system increased the sugar content in the hydrolysate, and laccase exhibited the most significant promotion effect, with the hydrolysate combined sugar concentration increasing by 8.9% to 100.2 ± 0.6 mmol/L, and laccase had a greater impact on the xylose content, increasing by 15.7% to 36.2 ± 0.4 mmol/L. Adding Lip to the enzymatic hydrolysis system increased the hydrolysate combined sugar concentration by 4.3%, and adding Mnp to the enzymatic hydrolysis system increased the hydrolysate combined sugar concentration by 2.2%. When the three lignin-degrading enzymes were added simultaneously, the hydrolysate combined sugar concentration reached 105.0 ± 0.7 mmol/L, which was 14.1% higher than that without adding ligninases, indicating that the addition of ligninolytic enzymes could indeed promote the hydrolysis of pretreated wheat straw. A five-factor and four-level orthogonal experiment was designed to optimize the addition of five kinds of lignocellulases (Table 2 ), to reduce production costs, maximize the enzymatic hydrolysis efficiency of pretreated wheat straw and increase the monosaccharide (combined glucose and xylose) yield. It can be seen that cellulase had the largest impact on the combined sugar content in wheat straw hydrolysate (R = 22.7), followed by xylanase (R = 14.9), among ligninolytic enzymes, Lac showed the largest impact on the combined sugar content in the hydrolysate (R = 8.7), followed by Lip (R = 5.5), and Mnp (R = 0.9) had the smallest impact, which corresponded to the results of the single factor test. At the same time, the results of orthogonal experiment further confirmed that the addition of lignin-degrading enzymes to the wheat straw hydrolysis system truly improved the efficiency of enzymatic hydrolysis saccharification. According to the results of the orthogonal test, the optimal addition dosage of lignocellulases during enzymatic hydrolysis and saccharification of pretreated wheat straw was as follows: cellulase 20 U/g, xylanase 10 U/g, Lac 4 U/g, Lip 6 U/g, Mnp 8 U/g. Under this condition, the combined sugar content in the hydrolysate was 109.0 ± 0.4 mmol/L (glucose 69.8 ± 0.3 mmol/L, xylose 39.2 ± 0.5 mmol/L). However, considering that the addition amount of Mnp showed the least impact on the hydrolysis efficiency of wheat straw, thus the amount of Mnp was optimized based on the results of orthogonal experiment, tests showed that the addition of 8 U/g or 2 U/g Mnp had no significant difference on the hydrolysis efficiency ( p > 0.05). From the perspective of cost saving, the actual addition amount of Mnp in the process of enzymatic hydrolysis and saccharification was 2 U/g. It was verified that under this condition, the content of combined sugar in the hydrolysate of pretreated wheat straw was 108.4 ± 0.6 mmol/L (glucose 70.0 ± 0.5 mmol/L and xylose 38.4 ± 0.7 mmol/L), which was 17.8% higher than that before adding ligninolytic enzymes. Through optimized pretreatment methods and the addition amount of ligninolytic enzymes, a higher sugar content was obtained. Table 2 Orthogonal test analysis of wheat straw hydrolysis by lignocellulase Level Cellulase (U/g) Xylanase (U/g) Lac (U/g) LiP (U/g) MnP (U/g) Combined sugars content (mmol/L) 1 25 25 8 8 8 65.1 ± 0.7 2 25 20 6 6 6 74.3 ± 0.6 3 25 15 4 4 4 82.6 ± 0.5 4 25 10 2 2 2 91.5 ± 0.3 5 20 25 6 4 2 86.2 ± 0.4 6 20 20 8 2 4 98.2 ± 0.2 7 20 15 2 8 6 106.1 ± 0.8 8 20 10 4 6 8 109.0 ± 0.4 9 15 25 4 2 6 99.3 ± 0.6 10 15 20 2 4 8 100.3 ± 0.7 11 15 15 8 6 2 96.2 ± 0.9 12 15 10 6 8 4 103.7 ± 0.8 13 10 25 2 6 4 95.2 ± 0.6 14 10 20 4 8 2 104.7 ± 0.4 15 10 15 6 2 8 103.1 ± 0.6 16 10 10 8 4 6 101.2 ± 0.5 k1 78.4 86.5 90.2 94.9 94.4 k2 99.9 94.4 91.8 93.7 95.2 k3 99.9 97.0 98.9 92.6 94.9 k4 100.1 101.4 98.3 98.0 94.7 R 22.7 14.9 8.7 5.5 0.9 Note: All values in the "Combined sugars content" column are presented as the mean ± standard deviation (n = 3). k1–k4 represent the average value of combined sugars content at each level of the corresponding factor, R denotes the range of k values (R = max(k) – min(k)). 3.3 Ethanol production from saccharification and fermentation of pretreated wheat straw Sequential hydrolysis and fermentation (SHF) was compared with simultaneous saccharification and fermentation (SSF) for ethanol production from pretreated wheat straw. Ethanol production by SHF increased rapidly within 12–24 h, whereas that by SSF increased markedly during 36–48 h; the ethanol production rate of both processes declined gradually after 72 h (Fig. 5 a). Ethanol production by SSF reached 6.9 ± 0.2 g/L (Supplementary Materials 1 and 2), which was 9.7% higher than the 6.2 ± 0.1 g/L obtained by SHF (Supplementary Material 3).Therefore, the SSF method was selected for ethanol production. Using pretreated wheat straw as the sole carbon source, key factors including nitrogen sources and metal ions in the culture medium were further optimized. Five nitrogen sources were evaluated, including yeast powder, peptone, NH 4 HCO 3 , (NH4) 2 SO 4 , and NH 4 H 2 PO 4 .The highest ethanol yield of 7.0 ± 0.1 g/L was achieved with peptone as the nitrogen source (Fig. 5 b). KCl, NaCl, MgSO4, MnSO4 and CuSO 4 were added to the medium as the metal ion additives, and the highest ethanol yield (7.1 ± 0.2 g/L) was obtained with MgSO4 as metal ion additive (Fig. 5 c). We further optimized the fermentation conditions for this process, including temperature, rotational speed and inoculation amount. The results of fermentation temperature optimization showed that the ethanol yield was highest (7.2 ± 0.1 g/L) at 32 ℃ (Fig. 5 d). The optimization of rotational speed indicated that the ethanol yield was highest (7.5 ± 0.2 g/L) at 130 r/min (Fig. 5 e). Finally, the highest ethanol yield of 7.6 ± 0.2 g/L was achieved at an inoculation amount of 1% (Fig. 5 f). The optimized fermentation conditions were: peptone as nitrogen source, MgSO 4 as metal ion additive, temperature 32°C, rotational speed 130 r/min, and inoculation amount 1%. Under these conditions, an ethanol yield of 7.6 ± 0.2 g/L was obtained (Supplementary Material 4), which was 10.1% higher than that before optimization. Under these optimized conditions, the overall ethanol yield reached 20.9 g per 100 g of raw wheat straw through the integrated process of pretreatment, saccharification, and fermentation. All fermentation experiments were performed in triplicate (n = 3). 4. Discussion Firstly, to improve the utilization efficiency of wheat straw, we optimized the wheat straw pretreatment methods. The results showed that the combined treatment with 2% NaHCO 3 + 2% H 2 O 2 effectively removed lignin, while better retaining the cellulose and hemicellulose components of wheat straw under relatively mild conditions, thus achieving higher recovery rates of these two polysaccharides. Compared with conventional pretreatments, this superior performance can be attributed to two key factors: first, acid pretreatment tends to cause hemicellulose dissolution [ 21 ]; second, the alkaline peroxide-based pretreatment used in this study is highly effective at selectively removing lignin from wheat straw [ 22 ]. SEM observations confirmed that the morphological structure of pretreated wheat straw enhanced the enzymatic accessibility to cellulose and hemicellulose during subsequent enzymatic hydrolysis, consistent with previous findings [ 23 ], which further explains the increased monosaccharide yield in the hydrolysate of pretreated wheat straw. Meanwhile, the pretreatment method was relatively mild, which reduced water consumption during washing and alleviated environmental pollution—an advantage that conventional acid/strong alkali pretreatment methods lack [ 16 , 21 ]. Based on these results, it is further verified that the pretreatment method selected in this study exhibited superior performance under relatively mild conditions, and increased the utilization efficiency of wheat straw effectively. Secondly, in order to hydrolyze pretreated wheat straw as much as possible, the effect of ligninolytic enzymes on the sugar content in hydrolysate of pretreated wheat straw was discussed. The addition of ligninolytic enzymes promoted the hydrolysis of pretreated straw, and laccase had the most significant effect on the sugar content in the hydrolysate system, which was consistent with previous findings [ 24 ], while it was different from the previous research result that Mnp had little effect on the sugar content in wheat straw hydrolysate [ 25 ], the reason may be that the mild alkaline peroxide pretreatment used in this study preserved the specific morphological structure of wheat straw, whereas previous studies adopted harsh pretreatment methods that altered the binding sites of MnP, thus leading to different enzymatic hydrolysis outcomes. Finally, the pretreated wheat straw hydrolysate was used for ethanol fermentation. Compared with the previous concept of removing lignin and hemicellulose so as to improve the utilization rate of cellulose [ 26 ], our concept of efficient simultaneous conversion of cellulose and hemicellulose is of great significance for the efficient conversion of lignocellulosic biomass resources. Under optimal conditions, the ethanol yield reached 7.6 ± 0.2 g/L, corresponding to a conversion efficiency of 20.9 g ethanol per 100 g raw wheat straw. Compared with the result that 7.143 tons of raw wheat straw may be required to produce 1 ton of ethanol from Patel A et al, we saved 33.1% of raw materials [ 27 ]. Compared with the result that 15.5 g ethanol was harvested from 100g wheat straw in the optimal conditions by Qiu J et al [ 28 ], the yield of ethanol in our study was increased by 35.0%. The reason may be that the cellulose which can transform glucose and hemicellulose which can transform xylose were both used efficiently in the process of ethanol production of wheat straw in our study. This high conversion efficiency can be attributed to the synergistic effect of three key steps. First, the mild pretreatment retained both cellulose and hemicellulose; second, the optimized ligninolytic enzymes system enhanced the accessibility of polysaccharides; third, the SSF process promoted the simultaneous utilization of glucose and xylose. The mild pretreatment coupled with ligninases synergism not only improves ethanol yield but also reduces the production cost of bioethanol. This optimized process is expected to provide a feasible technical scheme for the industrialization of straw-derived biofuels, which is conducive to promoting the development of renewable fuel industry. It also provides a novel perspective for the high-value utilization and conversion of lignocellulosic biomass, further expanding the application potential of agricultural waste in renewable fuel production. 5. Conclusion This study developed an efficient integrated process for ethanol production from wheat straw, involving mild pretreatment, optimized enzymatic hydrolysis, and SSF. The key conclusions are as follows. Firstly, the optimal pretreatment conditions were 2% NaHCO 3 + 2% H 2 O 2 at 130°C for 50 min with a solid-to-liquid ratio of 1:10. This mild pretreatment effectively removed lignin while retaining cellulose and hemicellulose, reducing water consumption during washing and alleviating environmental pollution compared to conventional methods. Secondly, the maximum combined sugar concentration (108.4 ± 0.6 mmol/L) was achieved under the optimized enzymatic hydrolysis conditions: cellulase 20 U/g, xylanase 10 U/g, laccase 4 U/g, lignin peroxidase 6 U/g, and manganese peroxidase 2 U/g. The addition of ligninolytic enzymes significantly enhanced the accessibility of polysaccharides. Thirdly, the optimal SSF conditions were peptone as the nitrogen source, MgSO 4 as the metal ion additive, 32°C, 130 r/min, and 1% inoculation amount, yielding a maximum ethanol concentration of 7.6 ± 0.2 g/L (corresponding to 20.9 g ethanol per 100 g raw wheat straw). Overall, the synergistic effect of mild pretreatment, optimized enzyme system, and SSF enabled efficient simultaneous conversion of cellulose and hemicellulose, providing a promising and environmentally friendly approach for the high-value utilization of wheat straw and other lignocellulosic biomass. Declarations Acknowledgment The research was supported by the First-Class Undergraduate Course Construction Project of Guizhou University of Traditional Chinese Medicine (Grant No.: GZYKC-25037) Declaration of Interest Statement The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. Author contributions Wenxuan Zhao developed the idea for the study, performed the research, conducted data analysis, and prepared the manuscript. Zhigang Ju helped to validate the data and conduct feasibility analysis. All authors read and approved the final manuscript. Data Availability Statement The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation. The chromatograms related to this study have been uploaded as Supplementary Materials. 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Int J Biol Macromol 330:147895. https://doi.org/10.1016/j.ijbiomac.2025.147895 Qiu YW, Dong C, Guo JJ et al (2025) Enhancing xylose fermentation to maximize net energy gain of lime-pretreated wheat straw by delayed fed-batch simultaneous saccharification and fermentation. Biomass Bioenergy 198:107865. https://doi.org/10.1016/j.biombioe.2025.107865 Zhao WX, Ju ZG, Zheng YQ et al (2022) Screening and efficacy evaluation of a high-efficiency lignin-degrading strain from wheat straw. J Shandong Agric Univ (Nat Sci Ed) 53:386–392. https://doi.org/10.3969/j.issn.1000-2324.2022.03.008 Yu H, Xu Y, Jin X et al (2025) Insight into the enzymatic digestion limitation of wheat straw after diluted lactic acid pretreatment from the perspective of residual lignin. Int J Biol Macromol 337:149446. https://doi.org/10.1016/j.ijbiomac.2025.149446 Fan M, Ni B, Cheng Z et al (2026) Effect of molecular scale behavior of lignin during alcohols pretreatment of wheat straw on carbohydrates conversion. Renew Energy 257:124740. https://doi.org/10.1016/j.renene.2025.124740 Zhao WX, Jiao HG, Zheng YQ (2022) Effects of pretreatment and ligninase on the enzymatic saccharification of corn stover. J Jilin Agric Univ 44:548–556. https://doi.org/10.13327/j.jjlau.2020.6019 Zheng P, Xiang L, Chang J et al (2021) Nanomechanics of lignin-cellulase interactions in aqueous solutions. Biomacromolecules 22:2033–2042. https://doi.org/10.1021/acs.biomac.1c00140 Yang W, Deng Y, Wang Z et al (2025) Synergistic bio-mechanical pretreatment and fed-batch semi-SSF strategy for enhanced cellulosic ethanol production from ramie straw. Int J Biol Macromol 320:146014. https://doi.org/10.1016/j.ijbiomac.2025.146014 Yang Z, Chen JM, Zhang YH et al (2023) MoO3–catalyzed transformation of corn stalk cellulose to glycolic acid: an experimental and DFT study. Cellulose 30:3523–3537. https://doi.org/10.1007/s10570-023-05139-2 Cao L, Tang X, Zhang X et al (2014) Two-stage transcriptional reprogramming in Saccharomyces cerevisiae for optimizing ethanol production from xylose. Metab Eng 24:150–159. https://doi.org/10.1016/j.ymben.2014.05.001 Li X, Yan X, Ye M et al (2022) Enhancement of anaerobic digestion performance of corn straw via combined sodium hydroxide-cellulase pretreatment. Biochem Eng J 187:108652. https://doi.org/10.1016/j.bej.2022.108652 Sluiter A, Hames B, Ruiz R et al (2008) Determination of structural carbohydrates and lignin in biomass. Golden, National Renewable Energy Laboratory Wang X, Wang G, Yu X et al (2017) Pretreatment of corn stover by solid acid for d-lactic acid fermentation. Bioresour Technol 239:490–496. https://doi.org/10.1016/j.biortech.2017.04.089 Liu C, Liu M, Wang P et al (2020) Effect of steam-assisted alkaline pretreatment plus enzymolysis on converting corn stalk into reducing sugar. Renew Energ 159:982–990. https://doi.org/10.1016/j.renene.2020.06.084 Zhao WX, Zhao FG, Zhang ST et al (2019) Ethanol production by simultaneous saccharification and co-fermentation of pretreated corn stalk. J Basic Microbiol 59:744–753. https://doi.org/10.1002/jobm.201900117 Burman NW, Sheridan CM, Harding KG (2020) Feasibility assessment of the production of bioethanol from lignocellulosic biomass pretreated with acid mine drainage (AMD). Renew Energ 157:1148–1155. https://doi.org/10.1016/j.renene.2020.05.086 Qin Y (2022) Pretreatment strategies to enhance enzymatic hydrolysis and cellulosic ethanol production for biorefinery of corn stover. Int J Mol Sci 23:13163. https://doi.org/10.3390/ijms232113163 Monika B, Seema D, Sneh G et al (2015) Acid pretreatment of wheat straw for bioethanol production. Int J Trop Agric 33:1275–1278. https://doi.org/10.18805/ijtab1038 Toquero C, Bolado S (2014) Effect of four pretreatments on enzymatic hydrolysis and ethanol fermentation of wheat straw. Influence of inhibitors and washing. Bioresour Technol 157:68–76. https://doi.org/10.1016/j.biortech.2014.01.090 Kurniati A, Darmokoesoemo H, Puspaningsih NNT (2016) Scanning Electron Microscope analysis of rice straw degradation by a treatment with α-L-arabinofuranosidase. Procedia Chem 18:63–68. https://doi.org/10.1016/j.proche.2016.01.011 Al-Zuhair S, Ahmed K, Abdulrazak A et al (2013) Synergistic effect of pretreatment and hydrolysis enzymes on the production of fermentable sugars from date palm lignocellulosic waste. J Ind Eng Chem 19:413–415. https://doi.org/10.1016/j.jiec.2012.09.022 Kyoungseon M, Yong HK, Jiye K et al (2021) Effect of manganese peroxidase on the decomposition of cellulosic components: direct cellulolytic activity and synergistic effect with cellulase. Bioresour Technol 343:126138. https://doi.org/10.1016/j.biortech.2021.126138 Qiu J, Wang Q, Shen F et al (2017) Optimizing phosphoric acid plus hydrogen peroxide (PHP) pretreatment on wheat straw by response surface method for enzymatic saccharification. Appl Biochem Biotechnol 181:1123–1139. https://doi.org/10.1007/s12010-016-2273-7 Patel A, Patel H, Shah A (2021) Production of cellulosic ethanol from alkali-treated wheat straw using P-SSF process and bioconversion of hemicellulosic fraction into high-value products. Curr Biotechnol 10:122–132. https://doi.org/10.1201/9781003234582-11 Qiu J, Dong T, Fei S et al (2018) Bioethanol production from wheat straw by phosphoric acid plus hydrogen peroxide (PHP) pretreatment via simultaneous saccharification and fermentation (SSF) at high solid loadings. Bioresour Technol 268:355–362. https://doi.org/10.1016/j.biortech.2018.08.009 Additional Declarations No competing interests reported. Supplementary Files Graphicalabstract.png Cite Share Download PDF Status: Under Review Version 1 posted Reviews received at journal 12 May, 2026 Reviewers agreed at journal 20 Apr, 2026 Reviews received at journal 18 Apr, 2026 Reviewers agreed at journal 15 Apr, 2026 Reviewers invited by journal 13 Apr, 2026 Editor assigned by journal 28 Mar, 2026 Submission checks completed at journal 21 Mar, 2026 First submitted to journal 19 Mar, 2026 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-9171419","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Short Report","associatedPublications":[],"authors":[{"id":626630932,"identity":"8558542e-c100-4142-a405-eeeafafecf38","order_by":0,"name":"Wenxuan Zhao","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA+0lEQVRIiWNgGAWjYDACCST2gQ8GNnJs7O0HiNbC+HBGRZoxH8+ZBKK1MBtznDmcOE/CwQCvDvnZzQ8f81TcSdxw/OwxacY25vQ2CYYEhh8V23BqYZxzzNiY58wzY4MzeWnShW1suW3SjQcYe87cxqmFWSLBTDq37bCcwYEcM+mZbTy5bTIHEpgZ23BrYZNI/yad++8wj8H5N2bSvG0S6WwSCQZ4tfBIAA3PbQDaciMH5EKDBIJaJCRyio3/HDtsLHnjjSEwkBMM24CBfBCfX+RnpG98OKPmcGLf+RwDYFT+l5dvbz/44EcFbi3YwQES1Y+CUTAKRsEoQAMAYOZZUtSPNbcAAAAASUVORK5CYII=","orcid":"","institution":"Guizhou University of Traditional Chinese Medicine","correspondingAuthor":true,"prefix":"","firstName":"Wenxuan","middleName":"","lastName":"Zhao","suffix":""},{"id":626630936,"identity":"c566712b-6da7-4ac6-b1ca-f0a4728bd360","order_by":1,"name":"Zhigang Ju","email":"","orcid":"","institution":"Guizhou University of Traditional Chinese Medicine","correspondingAuthor":false,"prefix":"","firstName":"Zhigang","middleName":"","lastName":"Ju","suffix":""}],"badges":[],"createdAt":"2026-03-19 15:56:21","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-9171419/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-9171419/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":107704378,"identity":"77fbc2bf-d12b-47cf-974b-38b9d781ce30","added_by":"auto","created_at":"2026-04-24 08:45:05","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":30113,"visible":true,"origin":"","legend":"\u003cp\u003eEffects of different pretreatment methods on enzymatic saccharification of wheat straw\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-9171419/v1/a527d8b40d7882bf84dee76a.png"},{"id":107421370,"identity":"122fec18-7132-4797-9b53-91616e8583c8","added_by":"auto","created_at":"2026-04-21 10:37:03","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":117548,"visible":true,"origin":"","legend":"\u003cp\u003eOptimization of pretreatment conditions for wheat straw (a) Glucose content under different pretreatment times (b) Xylose content under different pretreatment times (c) Glucose content under different pretreatment temperatures (d) Xylose content under different pretreatment temperatures (e) Glucose content under different solid-liquid ratios (f) Xylose content under different solid-liquid ratios\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-9171419/v1/ed413d8dc9189bf5e62f2831.png"},{"id":107421372,"identity":"6c872ead-1064-43bc-959b-7123af7a7cf0","added_by":"auto","created_at":"2026-04-21 10:37:03","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":538217,"visible":true,"origin":"","legend":"\u003cp\u003eSEM images of untreated and pretreated wheat straw (a) Untreated (b) Water pretreated (c) 2% NaHCO\u003csub\u003e3\u003c/sub\u003e + 2% H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e pretreated\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-9171419/v1/e7d38c554ca11152d31572da.png"},{"id":107488426,"identity":"86fa63b4-4199-44a6-8c98-d81c803dda24","added_by":"auto","created_at":"2026-04-22 02:44:40","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":21519,"visible":true,"origin":"","legend":"\u003cp\u003eEffect of lignin-degrading enzymes on enzymatic hydrolysis and saccharification of pretreated wheat straw\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-9171419/v1/4955aff4344aae23df6228fd.png"},{"id":107421373,"identity":"f48c34be-6f57-448e-9bd9-652d6dd558ae","added_by":"auto","created_at":"2026-04-21 10:37:03","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":110045,"visible":true,"origin":"","legend":"\u003cp\u003eOptimization of ethanol fermentation conditions from pretreated wheat straw (a) Effect of SSF and SHF on ethanol yield (b) Effect of different nitrogen sources on ethanol yield (c) Effect of different metal ions on ethanol yield (d) Effect of different temperatures on ethanol yield (e) Effect of different rotation speed on ethanol yield (f) Effect of different inoculation amounts on ethanol yield\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-9171419/v1/b76fed1989f86c5a63055c54.png"},{"id":108803444,"identity":"9229409a-6a99-4c6f-947f-b6f6fc7567e7","added_by":"auto","created_at":"2026-05-08 14:54:30","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1200438,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-9171419/v1/2e856be7-5fdf-4288-a1a6-7db19d88ea21.pdf"},{"id":107456976,"identity":"b0fdf4a2-2f52-48aa-bea3-b95aee9dc321","added_by":"auto","created_at":"2026-04-21 16:06:14","extension":"png","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":235021,"visible":true,"origin":"","legend":"","description":"","filename":"Graphicalabstract.png","url":"https://assets-eu.researchsquare.com/files/rs-9171419/v1/1320b0df731ce36f8840a048.png"}],"financialInterests":"No competing interests reported.","formattedTitle":"Mild Pretreatment Coupled with Ligninases Synergism Enhances Ethanol Production from Wheat Straw via Simultaneous Glucose and Xylose Fermentation","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eFacing growing depletion of fossil fuels, the efficient valorization of lignocellulosic biomass has emerged as a critical research priority for sustainable biomass energy development; among these biomass resources, as one of the world\u0026rsquo;s most abundant agricultural residues, wheat straw remains largely underexploited with limited valorization [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eWheat straw primarily consists of cellulose, hemicellulose and lignin\u0026mdash;a dense three-dimensional cross-linked network based on phenylpropane units that encapsulates cellulose and hemicellulose [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. This structural barrier not only enhances plant rigidity and pathogen resistance but also inhibits the enzymatic hydrolysis of carbohydrates, the rate-limiting step for ethanol production [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. Our previous work on wheat straw pretreatment has highlighted its potential for bioethanol production, but challenges in lignin removal still limit the large-scale application of wheat straw [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. Thus, our study prioritizes mild pretreatment strategies that selectively remove lignin while preserving cellulose and hemicellulose, thereby improving the utilization efficiency of wheat straw and other lignocellulosic biomass resources [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eCellulase and xylanase are well-recognized as core enzymes for lignocellulose hydrolysis, but their efficiency is often constrained by residual lignin after pretreatment [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. Notably, the synergistic effects between lignin-degrading enzymes (laccase, lignin peroxidase, manganese peroxidase) and polysaccharide hydrolases remain understudied in wheat straw systems. Given that ligninases can effectively hydrolyze lignin and improve the accessibility of cellulose and hemicellulose, we hypothesize that adding ligninases can further break down lignin residues, thereby boosting overall sugar yields for subsequent fermentation.\u003c/p\u003e \u003cp\u003eIn addition, most prior studies on lignocellulosic ethanol have focused solely on glucose conversion, neglecting xylose (a major product of hemicellulose hydrolysis) and leading to incomplete resource utilization [\u003cspan additionalcitationids=\"CR11\" citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. In contrast, the engineered \u003cem\u003eS. cerevisiae\u003c/em\u003e strain WXY12 selected in this study enables simultaneous glucose and xylose fermentation\u0026mdash;overcoming this limitation and improving the economic viability of wheat straw valorization [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eBuilding on the above, wheat straw was employed as the raw material for subsequent pretreatment and enzymatic hydrolysis. First, we aim to develop a safe and efficient pretreatment process by optimizing methods and conditions, which can selectively remove lignin while maintaining the cellulose and hemicellulose fractions, thus boosting the comprehensive utilization efficiency of wheat straw. Second, the synergistic effects of ligninolytic enzymes combined with cellulase and xylanase were investigated based on the sugar yield in wheat straw hydrolysate, while the optimal dosage of lignocellulase during the hydrolysis of pretreated wheat straw was determined via orthogonal experiments. Finally, on the basis of obtaining the optimal pretreatment and hydrolysis conditions, the engineered \u003cem\u003eS. cerevisiae\u003c/em\u003e strain WXY12 was used to ferment pretreated wheat straw hydrolysate to produce ethanol, and the ethanol yield was enhanced by optimizing the fermentation conditions of pretreated wheat straw. This study aims to develop a mild and efficient pretreatment method for wheat straw, optimize the synergistic lignocellulase system for high sugar yield, and achieve efficient ethanol production via co-fermentation of glucose and xylose by \u003cem\u003eS. cerevisiae\u003c/em\u003e WXY12. The results are expected to provide a reliable theoretical basis for facilitating the high-value utilization of lignocellulosic biomass resources.\u003c/p\u003e"},{"header":"2. Material and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1 Materials\u003c/h2\u003e \u003cp\u003eCellulase (4\u0026times;10\u003csup\u003e5\u003c/sup\u003e U/g) and lignin peroxidase (100 U/g) were purchased from Shanghai Yuanye Bio-Technology Co., Ltd, and xylanase (2500 U/g), manganese peroxidase (200 U/g), laccase (400 U/g) were purchased from Sigma-Aldrich (Shanghai) Trading Co., Ltd. Glucose and xylose quantification kits were purchased from Elabscience Biotechnology Co., Ltd. The engineered \u003cem\u003eS. cerevisiae\u003c/em\u003e strain WXY12 (high-efficiency ethanol-producing) was kindly provided by Capital Normal University [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.2 Methods\u003c/h2\u003e \u003cdiv id=\"Sec5\" class=\"Section3\"\u003e \u003ch2\u003e2.2.1 Pretreatment of wheat straw\u003c/h2\u003e \u003cp\u003eWheat straw was oven-dried and sieved to a particle size of 10 mesh for subsequent use. To screen for mild and efficient pretreatment methods, wheat straw was subjected to various pretreatment regimes (with distilled water as the control group): strong acid treatments (2% H\u003csub\u003e2\u003c/sub\u003eSO\u003csub\u003e4\u003c/sub\u003e, 2% HCl), weak acid treatment (2% CH\u003csub\u003e3\u003c/sub\u003eCOOH), strong alkali treatments (2% NaOH, 2% KOH), weak alkali treatment (2% NaHCO\u003csub\u003e3\u003c/sub\u003e), oxidative treatment (2% H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e), strong acid coupled with oxidative treatments (2% H\u003csub\u003e2\u003c/sub\u003eSO\u003csub\u003e4\u003c/sub\u003e\u0026thinsp;+\u0026thinsp;2% H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e, 2% HCl\u0026thinsp;+\u0026thinsp;2% H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e), weak acid coupled with oxidative treatment (2% CH\u003csub\u003e3\u003c/sub\u003eCOOH\u0026thinsp;+\u0026thinsp;2% H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e), strong alkali coupled with oxidative treatments (2% NaOH\u0026thinsp;+\u0026thinsp;2% H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e, 2% KOH\u0026thinsp;+\u0026thinsp;2% H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e), and weak alkali coupled with oxidative treatment (2% NaHCO\u003csub\u003e3\u003c/sub\u003e\u0026thinsp;+\u0026thinsp;2% H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e) [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. All pretreatments were conducted at a solid-to-liquid ratio of 1:10 (w/v) at 120\u0026deg;C for 50 min. The pretreated wheat straw was washed to neutrality, then oven-dried and weighed. The lignin, cellulose, and hemicellulose compositions of raw and pretreated wheat straw were measured according to the procedure issued by the National Renewable Energy Laboratory (NREL) [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. Wheat straw recovery rate was calculated based on the ratio of sample weight before and after pretreatment. All treatments were performed in triplicate.\u003c/p\u003e \u003cp\u003eTo evaluate the efficiency of the pretreatment, the pretreated wheat straw was subjected to enzymatic hydrolysis using 20 U of cellulase and 20 U of xylanase per gram of cellulose and hemicellulose, respectively. For a 40 mL hydrolysis system, 1.5 g of pretreated wheat straw was combined with 2 mL of 0.05 M citrate buffer. The total volume was adjusted to 40 mL with ultrapure water, the pH was set to 4.8, and the mixture was autoclaved at 115\u0026deg;C for 15 min. Afterward, the corresponding dosages of the two enzymes were determined according to the cellulose and hemicellulose contents, and the enzymes were supplemented once the mixture had cooled. Hydrolysis was carried out at 50\u0026deg;C with shaking at 120 rpm. Glucose and xylose concentrations were quantified using the corresponding kits at 12-h intervals for a total duration of 72 h [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. After the 72-h hydrolysis, the reaction was terminated by heating at 100\u0026deg;C for 10 min to denature the enzymes, ensuring no further degradation of carbohydrates during sample handling.\u003c/p\u003e \u003cp\u003eFor further optimization of the pretreatment conditions, the treatment time (40, 50, 60, 70 and 80 min), the treatment temperature (100, 110, 120, 130, and 140\u0026deg;C), and the solid-liquid ratio (1:8, 1:9, 1:10, 1:11, and 1:12) were evaluated, with the monosaccharide yield after enzymatic hydrolysis as the key evaluation indicator. Additionally, to further validate the effectiveness of the pretreatment methods developed in this work, the morphological and structural variations of wheat straw before and after pretreatment were characterized using SEM [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section3\"\u003e \u003ch2\u003e2.2.2 Enzymatic hydrolysis of pretreated wheat straw by lignocellulase\u003c/h2\u003e \u003cp\u003eThe individual effects of three lignin-degrading enzymes, namely laccase (Lac), lignin peroxidase (LiP), and manganese peroxidase (MnP), on the monosaccharide yield of wheat straw hydrolysate were first investigated. Each ligninolytic enzyme was supplemented at 6 U per gram of pretreated wheat straw into the hydrolysis system to evaluate its promoting effect on the hydrolysis of the substrate.\u003c/p\u003e \u003cp\u003eThen, a five-factor, four-level orthogonal test was used to optimize the addition of five enzymes (cellulase, xylanase, Lac, LiP, MnP), so as to establish a high-efficiency complex enzymatic hydrolysis system for lignocellulosic materials. On the basis of cost savings, the maximum enzymatic hydrolysis efficiency of the pretreated wheat straw was achieved, thereby improving the monosaccharide yield (glucose and xylose).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section3\"\u003e \u003ch2\u003e2.2.3 Ethanol production via fermentation of pretreated wheat straw\u003c/h2\u003e \u003cp\u003eDuring the experiment of ethanol production via fermentation from pretreated wheat straw, the preparation of ethanol standard solutions and the gas chromatographic (GC) analysis of ethanol content were performed according to a previously reported method [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. The retention time of ethanol in GC analysis was 2.13\u0026ndash;2.16 min, and the regression equation was fitted based on the peak areas corresponding to different ethanol concentrations: y\u0026thinsp;=\u0026thinsp;3.1571x\u0026thinsp;\u0026minus;\u0026thinsp;0.0308, R\u003csup\u003e2\u003c/sup\u003e\u0026thinsp;=\u0026thinsp;0.9996.\u003c/p\u003e \u003cp\u003eThe sterile enzyme solution with the optimal ratio was added into the fermentation medium containing 30 g/L pretreated wheat straw, 10 g/L yeast extract, and 1 g/L KCl. The engineered S. cerevisiae strain WXY12\u0026mdash;a high-efficiency ethanol producer capable of co-fermenting glucose and xylose\u0026mdash;was also inoculated into the fermentation medium. Subsequently, separate hydrolysis and fermentation (SHF, in which enzymatic hydrolysis was conducted for 3 days before fermentation) was compared with simultaneous saccharification and fermentation (SSF) for ethanol production from pretreated wheat straw [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. The fermentation conditions were set as follows: a temperature of 30\u0026deg;C, rotational speed of 120 r/min, an inoculation amount of 2% (v/v), natural pH (unadjusted). The ethanol concentration in the fermentation broth was determined by GC according to the established standard curve. Afterwards, ethanol concentration was measured at 12 h intervals until it began to decrease, and the fermentation medium components and process conditions (nitrogen source, metal ion inducer, temperature, rotational speed and inoculum dosage) were then optimized to improve ethanol yields.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e"},{"header":"3. Results","content":"\u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003e3.1 Pretreatment efficacy of various methods on wheat straw\u003c/h2\u003e \u003cp\u003eTo enhance the pretreatment efficacy, different pretreatment strategies were performed on wheat straw, including strong acid treatments (2% H\u003csub\u003e2\u003c/sub\u003eSO\u003csub\u003e4\u003c/sub\u003e, 2% HCl), weak acid treatment (2% CH₃COOH), strong alkali treatments (2% NaOH, 2% KOH), weak alkali treatment (2% NaHCO\u003csub\u003e3\u003c/sub\u003e), single oxidative treatment (2% H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e), as well as acid-oxidative and alkali-oxidative systems: strong acid coupled with oxidation treatments (2% H\u003csub\u003e2\u003c/sub\u003eSO\u003csub\u003e4\u003c/sub\u003e\u0026thinsp;+\u0026thinsp;2% H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e, 2% HCl\u0026thinsp;+\u0026thinsp;2% H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e), weak acid coupled with oxidation treatment (2% CH\u003csub\u003e3\u003c/sub\u003eCOOH\u0026thinsp;+\u0026thinsp;2% H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e), strong alkali coupled with oxidation treatments (2% NaOH\u0026thinsp;+\u0026thinsp;2% H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e, 2% KOH\u0026thinsp;+\u0026thinsp;2% H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e), and weak alkali coupled with oxidation treatments (2% NaHCO\u003csub\u003e3\u003c/sub\u003e\u0026thinsp;+\u0026thinsp;2% H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e), with water-treated and untreated wheat straw serving as controls. Following the pretreatment, the recovery rate of wheat straw and the contents of lignin, cellulose, and hemicellulose before and after treatment were measured. As shown in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e, after pretreatment with 2% NaOH (strong alkali), wheat straw presented the highest hemicellulose retention of 26.7\u0026thinsp;\u0026plusmn;\u0026thinsp;0.1%, with cellulose retention of 46.7\u0026thinsp;\u0026plusmn;\u0026thinsp;0.2%, lignin residue of 5.2\u0026thinsp;\u0026plusmn;\u0026thinsp;0.5% and wheat straw recovery rate of 79.7\u0026thinsp;\u0026plusmn;\u0026thinsp;0.4%. In contrast, pretreatment with the combination of strong acid and oxidant (2% HCl and 2% H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e) resulted in the highest cellulose retention in wheat straw (61.7\u0026thinsp;\u0026plusmn;\u0026thinsp;0.3%), along with hemicellulose retention of 13.7\u0026thinsp;\u0026plusmn;\u0026thinsp;0.4% and a lignin residue of 4.2\u0026thinsp;\u0026plusmn;\u0026thinsp;0.4%. Although the proportions of cellulose and hemicellulose in wheat straw treated by the weak alkali‑oxidant combined process (2% NaHCO\u003csub\u003e3\u003c/sub\u003e and 2% H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e) did not reach the maximum values, both components maintained relatively high retention rates (49.1\u0026thinsp;\u0026plusmn;\u0026thinsp;0.2% for cellulose and 26.1\u0026thinsp;\u0026plusmn;\u0026thinsp;0.4% for hemicellulose), with lignin residue of 4.7\u0026thinsp;\u0026plusmn;\u0026thinsp;0.3%. Moreover, this pretreatment achieved the highest wheat straw recovery rate of 82.6\u0026thinsp;\u0026plusmn;\u0026thinsp;0.2%, which was beneficial for improving the subsequent utilization rate of wheat straw. Therefore, the 2% NaHCO\u003csub\u003e3\u003c/sub\u003e\u0026thinsp;+\u0026thinsp;2% H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e pretreatment was primarily identified as the optimal strategy and employed in subsequent experiments.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eCompositional analysis of wheat straw before and after various pretreatments\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"6\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNO.\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eSample\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eCellulose (%)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eHemicellulose\u003c/p\u003e \u003cp\u003e(%)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eLignin\u003c/p\u003e \u003cp\u003e(%)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003eRecovery rate\u003c/p\u003e \u003cp\u003e(%)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eUntreated wheat straw\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e41.3\u0026thinsp;\u0026plusmn;\u0026thinsp;0.3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e21.1\u0026thinsp;\u0026plusmn;\u0026thinsp;0.3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e \u003cp\u003e12.9\u0026thinsp;\u0026plusmn;\u0026thinsp;0.4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c6\"\u003e \u003cp\u003e100\u0026thinsp;\u0026plusmn;\u0026thinsp;0.0\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eH\u003csub\u003e2\u003c/sub\u003eO\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e42.3\u0026thinsp;\u0026plusmn;\u0026thinsp;0.2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e23.8\u0026thinsp;\u0026plusmn;\u0026thinsp;0.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e \u003cp\u003e11.3\u0026thinsp;\u0026plusmn;\u0026thinsp;0.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c6\"\u003e \u003cp\u003e94.6\u0026thinsp;\u0026plusmn;\u0026thinsp;0.4\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e2% H\u003csub\u003e2\u003c/sub\u003eSO\u003csub\u003e4\u003c/sub\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e57.3\u0026thinsp;\u0026plusmn;\u0026thinsp;0.4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e13.9\u0026thinsp;\u0026plusmn;\u0026thinsp;0.2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e \u003cp\u003e6.9\u0026thinsp;\u0026plusmn;\u0026thinsp;0.3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c6\"\u003e \u003cp\u003e77.3\u0026thinsp;\u0026plusmn;\u0026thinsp;0.5\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e2% HCl\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e55.6\u0026thinsp;\u0026plusmn;\u0026thinsp;0.2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e15.7\u0026thinsp;\u0026plusmn;\u0026thinsp;0.3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e \u003cp\u003e7.2\u0026thinsp;\u0026plusmn;\u0026thinsp;0.2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c6\"\u003e \u003cp\u003e79.1\u0026thinsp;\u0026plusmn;\u0026thinsp;0.5\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e2% CH\u003csub\u003e3\u003c/sub\u003eCOOH\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e46.9\u0026thinsp;\u0026plusmn;\u0026thinsp;0.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e19.8\u0026thinsp;\u0026plusmn;\u0026thinsp;0.2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e \u003cp\u003e11.3\u0026thinsp;\u0026plusmn;\u0026thinsp;0.2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c6\"\u003e \u003cp\u003e86.2\u0026thinsp;\u0026plusmn;\u0026thinsp;0.3\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e2% H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e48.3\u0026thinsp;\u0026plusmn;\u0026thinsp;0.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e21.8\u0026thinsp;\u0026plusmn;\u0026thinsp;0.3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e \u003cp\u003e8.2\u0026thinsp;\u0026plusmn;\u0026thinsp;0.3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c6\"\u003e \u003cp\u003e84.7\u0026thinsp;\u0026plusmn;\u0026thinsp;0.2\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e2% NaOH\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e46.7\u0026thinsp;\u0026plusmn;\u0026thinsp;0.2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e26.7\u0026thinsp;\u0026plusmn;\u0026thinsp;0.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e \u003cp\u003e5.2\u0026thinsp;\u0026plusmn;\u0026thinsp;0.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c6\"\u003e \u003cp\u003e79.7\u0026thinsp;\u0026plusmn;\u0026thinsp;0.4\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e2% KOH\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e45.8\u0026thinsp;\u0026plusmn;\u0026thinsp;0.3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e25.9\u0026thinsp;\u0026plusmn;\u0026thinsp;0.2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e \u003cp\u003e6.4\u0026thinsp;\u0026plusmn;\u0026thinsp;0.4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c6\"\u003e \u003cp\u003e81.1\u0026thinsp;\u0026plusmn;\u0026thinsp;0.3\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e2% NaHCO\u003csub\u003e3\u003c/sub\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e44.5\u0026thinsp;\u0026plusmn;\u0026thinsp;0.4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e24.8\u0026thinsp;\u0026plusmn;\u0026thinsp;0.2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e \u003cp\u003e8.9\u0026thinsp;\u0026plusmn;\u0026thinsp;0.3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c6\"\u003e \u003cp\u003e85.2\u0026thinsp;\u0026plusmn;\u0026thinsp;0.2\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e2% H\u003csub\u003e2\u003c/sub\u003eSO\u003csub\u003e4\u003c/sub\u003e\u0026thinsp;+\u0026thinsp;2% H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e59.3\u0026thinsp;\u0026plusmn;\u0026thinsp;0.2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e16.1\u0026thinsp;\u0026plusmn;\u0026thinsp;0.4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e \u003cp\u003e4.1\u0026thinsp;\u0026plusmn;\u0026thinsp;0.2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c6\"\u003e \u003cp\u003e65.7\u0026thinsp;\u0026plusmn;\u0026thinsp;0.5\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e11\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e2% HCl\u0026thinsp;+\u0026thinsp;2% H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e61.7\u0026thinsp;\u0026plusmn;\u0026thinsp;0.3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e13.7\u0026thinsp;\u0026plusmn;\u0026thinsp;0.4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e \u003cp\u003e4.2\u0026thinsp;\u0026plusmn;\u0026thinsp;0.4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c6\"\u003e \u003cp\u003e70.3\u0026thinsp;\u0026plusmn;\u0026thinsp;0.4\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e12\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e2% CH\u003csub\u003e3\u003c/sub\u003eCOOH\u0026thinsp;+\u0026thinsp;2% H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e55.6\u0026thinsp;\u0026plusmn;\u0026thinsp;0.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e14.7\u0026thinsp;\u0026plusmn;\u0026thinsp;0.2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e \u003cp\u003e8.9\u0026thinsp;\u0026plusmn;\u0026thinsp;0.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c6\"\u003e \u003cp\u003e81.2\u0026thinsp;\u0026plusmn;\u0026thinsp;0.2\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e13\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e2% NaOH\u0026thinsp;+\u0026thinsp;2% H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e52.0\u0026thinsp;\u0026plusmn;\u0026thinsp;0.4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e24.1\u0026thinsp;\u0026plusmn;\u0026thinsp;0.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e \u003cp\u003e3.7\u0026thinsp;\u0026plusmn;\u0026thinsp;0.3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c6\"\u003e \u003cp\u003e67.1\u0026thinsp;\u0026plusmn;\u0026thinsp;0.6\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e14\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e2% KOH\u0026thinsp;+\u0026thinsp;2% H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e49.4\u0026thinsp;\u0026plusmn;\u0026thinsp;0.3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e25.6\u0026thinsp;\u0026plusmn;\u0026thinsp;0.4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e \u003cp\u003e4.4\u0026thinsp;\u0026plusmn;\u0026thinsp;0.2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c6\"\u003e \u003cp\u003e73.5\u0026thinsp;\u0026plusmn;\u0026thinsp;0.5\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e15\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e2% NaHCO\u003csub\u003e3\u003c/sub\u003e+ 2% H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e49.1\u0026thinsp;\u0026plusmn;\u0026thinsp;0.2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e26.1\u0026thinsp;\u0026plusmn;\u0026thinsp;0.4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e \u003cp\u003e4.7\u0026thinsp;\u0026plusmn;\u0026thinsp;0.3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c6\"\u003e \u003cp\u003e82.6\u0026thinsp;\u0026plusmn;\u0026thinsp;0.2\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003ctfoot\u003e \u003ctr\u003e\u003ctd colspan=\"6\"\u003e\u003cem\u003eNote: values are expressed as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation (n\u0026thinsp;=\u0026thinsp;3)\u003c/em\u003e\u003c/td\u003e\u003c/tr\u003e \u003c/tfoot\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eTo further explore the effect of each pretreatment method on wheat straw, cellulase and xylanase were used to perform enzymatic saccharification on the above 15 groups. Following 72 h of hydrolysis, the concentrations of glucose and xylose in the hydrolysate were measured, and the combined sugar (glucose and xylose) content was calculated. Untreated and water-pretreated wheat straw were employed as controls. Higher glucose and xylose concentrations in the hydrolysate indicated that the pretreatment was more effective for enzymatic saccharification. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e, water treatment exerted a slight influence on enzymatic hydrolysis and saccharification. The hydrolysates of wheat straw pretreated with 2% HCl\u0026thinsp;+\u0026thinsp;2% H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e (group 11), 2% NaOH\u0026thinsp;+\u0026thinsp;2% H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e (group 13), and 2% NaHCO\u003csub\u003e3\u003c/sub\u003e\u0026thinsp;+\u0026thinsp;2% H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e (group 15) showed a higher sugar concentration. Wheat straw pretreated with 2% NaOH\u0026thinsp;+\u0026thinsp;2% H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e exhibited the highest combined sugar content of 83.3\u0026thinsp;\u0026plusmn;\u0026thinsp;1.5 mmol/L (glucose 58.2\u0026thinsp;\u0026plusmn;\u0026thinsp;0.9 mmol/L and xylose 25.1\u0026thinsp;\u0026plusmn;\u0026thinsp;0.7 mmol/L) after enzymatic hydrolysis and saccharification. This was followed by the 2% HCl\u0026thinsp;+\u0026thinsp;2% H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e group, which yielded 81.2\u0026thinsp;\u0026plusmn;\u0026thinsp;1.3 mmol/L combined sugar (glucose 61.9\u0026thinsp;\u0026plusmn;\u0026thinsp;0.4 mmol/L and xylose 19.3\u0026thinsp;\u0026plusmn;\u0026thinsp;0.7 mmol/L). Wheat straw pretreated with 2% NaHCO\u003csub\u003e3\u003c/sub\u003e\u0026thinsp;+\u0026thinsp;2% H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e yielded a combined sugar content of 80.1\u0026thinsp;\u0026plusmn;\u0026thinsp;0.9 mmol/L (glucose 52.9\u0026thinsp;\u0026plusmn;\u0026thinsp;0.7 and xylose 27.2\u0026thinsp;\u0026plusmn;\u0026thinsp;0.3 mmol/L) after enzymatic hydrolysis and saccharification. Comparative analysis showed that the combined sugar content of wheat straw pretreated with 2% NaOH\u0026thinsp;+\u0026thinsp;2% H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e and 2% HCl\u0026thinsp;+\u0026thinsp;2% H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e was slightly higher than that pretreated with 2% NaHCO\u003csub\u003e3\u003c/sub\u003e\u0026thinsp;+\u0026thinsp;2% H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e following enzymatic hydrolysis and saccharification, but the difference was not significant (p\u0026thinsp;\u0026gt;\u0026thinsp;0.05). Moreover, NaOH is a strong alkali and HCl is a strong acid, which causes environmental pollution and instrument damage. Meanwhile, wheat straw pretreated with 2% NaHCO\u003csub\u003e3\u003c/sub\u003e\u0026thinsp;+\u0026thinsp;2% H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e exhibited a recovery rate of 82.6\u0026thinsp;\u0026plusmn;\u0026thinsp;0.2%, which was significantly higher than that of the 2% NaOH\u0026thinsp;+\u0026thinsp;2% H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e (67.1\u0026thinsp;\u0026plusmn;\u0026thinsp;0.6%) and 2% HCl\u0026thinsp;+\u0026thinsp;2% H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e (70.3\u0026thinsp;\u0026plusmn;\u0026thinsp;0.4%) groups (\u003cem\u003ep\u0026thinsp;\u0026lt;\u003c/em\u003e\u0026thinsp;0.05). Based on the above results, 2% NaHCO\u003csub\u003e3\u003c/sub\u003e\u0026thinsp;+\u0026thinsp;2% H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e was selected as the pretreatment method for wheat straw in subsequent experiments.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eFollowing the selection of 2% NaHCO\u003csub\u003e3\u003c/sub\u003e\u0026thinsp;+\u0026thinsp;2% H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e as the optimal pretreatment method, in order to obtain a better pretreatment effect, the conditions of time, temperature and solid-liquid ratio were optimized, and the effect was also verified by enzymatic hydrolysis and saccharification tests. Higher glucose and xylose concentrations in the hydrolysate indicated a better effect of wheat straw pretreatment on enzymatic saccharification. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea and \u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eb, the maximum combined sugar content of 81.2\u0026thinsp;\u0026plusmn;\u0026thinsp;1.2 mmol/L (glucose 55.7\u0026thinsp;\u0026plusmn;\u0026thinsp;0.8 and xylose 25.5\u0026thinsp;\u0026plusmn;\u0026thinsp;0.6 mmol/L) was achieved at a pretreatment time of 60 min, followed by a value of 80.1\u0026thinsp;\u0026plusmn;\u0026thinsp;0.9 mmol/L (glucose 52.9\u0026thinsp;\u0026plusmn;\u0026thinsp;0.7 and xylose 27.2\u0026thinsp;\u0026plusmn;\u0026thinsp;0.3 mmol/L) at 50 min. Considering that the difference in the final combined sugar content between them was not significant (p\u0026thinsp;\u0026gt;\u0026thinsp;0.05) and a shorter time favored energy conservation, 50 min was selected as the optimal pretreatment time for wheat straw.As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ec and \u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ed, the combined sugar content reached a maximum of 92.0\u0026thinsp;\u0026plusmn;\u0026thinsp;0.6 mmol/L (glucose 60.7\u0026thinsp;\u0026plusmn;\u0026thinsp;0.5 mmol/L and xylose 31.3\u0026thinsp;\u0026plusmn;\u0026thinsp;0.3 mmol/L) at 130 ℃, while lower sugar contents were observed at other temperatures. Correspondingly, 130 ℃ was determined as the optimal pretreatment temperature. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ee and \u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ef, the highest combined sugar content of 93.9\u0026thinsp;\u0026plusmn;\u0026thinsp;1.4 mmol/L (glucose 61.2\u0026thinsp;\u0026plusmn;\u0026thinsp;0.8 and xylose 32.7\u0026thinsp;\u0026plusmn;\u0026thinsp;0.7 mmol/L) was obtained at a solid-liquid ratio of 1:9, followed by a value of 92.0\u0026thinsp;\u0026plusmn;\u0026thinsp;0.9 mmol/L (glucose 60.7\u0026thinsp;\u0026plusmn;\u0026thinsp;0.8 and xylose 31.3\u0026thinsp;\u0026plusmn;\u0026thinsp;0.4 mmol/L) at 1:10. Although the difference in glucose and xylose contents in the hydrolysate between these two solid-liquid ratios was not significant (p\u0026thinsp;\u0026gt;\u0026thinsp;0.05), the material utilization rate was higher at 1:10, and this ratio was chosen for wheat straw pretreatment. In summary, 2% NaHCO\u003csub\u003e3\u003c/sub\u003e\u0026thinsp;+\u0026thinsp;2% H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e was finally selected, and the pretreatment conditions were as follows: time of 50 min, temperature of 130 ℃, solid-liquid ratio of 1:10. The selected pretreatment method removed lignin and retained cellulose and hemicellulose components under relatively mild conditions and exhibited a better pretreatment effect. Meanwhile, the yield of monosaccharides in the hydrolysate was higher, which would lay a solid foundation for subsequent fermentation and promote the efficient comprehensive utilization of wheat straw.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eTo verify the efficiency of lignin removal by the selected pretreatment method, SEM was employed to characterize the surface microstructure of wheat straw before and after pretreatment. Untreated and water-pretreated wheat straw were used as control groups. SEM revealed that untreated wheat straw had a uniform surface, with cellulose in a compact and ordered structure (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ea). The structure of wheat straw after water pretreatment (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eb) was slightly damaged, but the overall structure remained intact. In contrast, the compact structure of wheat straw pretreated with 2% NaHCO\u003csub\u003e3\u003c/sub\u003e\u0026thinsp;+\u0026thinsp;2% H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e was obviously destroyed (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ec), and its surface exhibited a broken, rough, and hollow morphology. These structural modifications improved the accessibility of cellulose and hemicellulose to enzymes in the subsequent enzymatic hydrolysis, thus promoting the efficient degradation of wheat straw and enhancing its utilization efficiency.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003e3.2 Effect of ligninolytic enzymes on enzymatic hydrolysis and saccharification of pretreated wheat straw\u003c/h2\u003e \u003cp\u003eTo further improve the hydrolysis efficiency of pretreated wheat straw, the effects of individual lignin-degrading enzymes on glucose, xylose and combined sugar (combined glucose and xylose concentrations in the hydrolysate) were first investigated, with different ligninolytic enzymes added separately to the hydrolysis system. Cellulase and xylanase were added to the hydrolysis system alone (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA). Cellulase, xylanase and Lac were added to the hydrolysis system (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB). Cellulase, xylanase and Lip were added to the hydrolysis system (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC). Cellulase, xylanase and Mnp were added to the hydrolysis system (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eD). Cellulase, xylanase, Lac, Lip and Mnp were added to the hydrolysis system simultaneously (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eE). It can be seen from Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e that adding any kind of ligninolytic enzyme to the enzymatic hydrolysis system increased the sugar content in the hydrolysate, and laccase exhibited the most significant promotion effect, with the hydrolysate combined sugar concentration increasing by 8.9% to 100.2\u0026thinsp;\u0026plusmn;\u0026thinsp;0.6 mmol/L, and laccase had a greater impact on the xylose content, increasing by 15.7% to 36.2\u0026thinsp;\u0026plusmn;\u0026thinsp;0.4 mmol/L. Adding Lip to the enzymatic hydrolysis system increased the hydrolysate combined sugar concentration by 4.3%, and adding Mnp to the enzymatic hydrolysis system increased the hydrolysate combined sugar concentration by 2.2%. When the three lignin-degrading enzymes were added simultaneously, the hydrolysate combined sugar concentration reached 105.0\u0026thinsp;\u0026plusmn;\u0026thinsp;0.7 mmol/L, which was 14.1% higher than that without adding ligninases, indicating that the addition of ligninolytic enzymes could indeed promote the hydrolysis of pretreated wheat straw.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eA five-factor and four-level orthogonal experiment was designed to optimize the addition of five kinds of lignocellulases (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e), to reduce production costs, maximize the enzymatic hydrolysis efficiency of pretreated wheat straw and increase the monosaccharide (combined glucose and xylose) yield. It can be seen that cellulase had the largest impact on the combined sugar content in wheat straw hydrolysate (R\u0026thinsp;=\u0026thinsp;22.7), followed by xylanase (R\u0026thinsp;=\u0026thinsp;14.9), among ligninolytic enzymes, Lac showed the largest impact on the combined sugar content in the hydrolysate (R\u0026thinsp;=\u0026thinsp;8.7), followed by Lip (R\u0026thinsp;=\u0026thinsp;5.5), and Mnp (R\u0026thinsp;=\u0026thinsp;0.9) had the smallest impact, which corresponded to the results of the single factor test. At the same time, the results of orthogonal experiment further confirmed that the addition of lignin-degrading enzymes to the wheat straw hydrolysis system truly improved the efficiency of enzymatic hydrolysis saccharification. According to the results of the orthogonal test, the optimal addition dosage of lignocellulases during enzymatic hydrolysis and saccharification of pretreated wheat straw was as follows: cellulase 20 U/g, xylanase 10 U/g, Lac 4 U/g, Lip 6 U/g, Mnp 8 U/g. Under this condition, the combined sugar content in the hydrolysate was 109.0\u0026thinsp;\u0026plusmn;\u0026thinsp;0.4 mmol/L (glucose 69.8\u0026thinsp;\u0026plusmn;\u0026thinsp;0.3 mmol/L, xylose 39.2\u0026thinsp;\u0026plusmn;\u0026thinsp;0.5 mmol/L). However, considering that the addition amount of Mnp showed the least impact on the hydrolysis efficiency of wheat straw, thus the amount of Mnp was optimized based on the results of orthogonal experiment, tests showed that the addition of 8 U/g or 2 U/g Mnp had no significant difference on the hydrolysis efficiency (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026gt;\u0026thinsp;0.05). From the perspective of cost saving, the actual addition amount of Mnp in the process of enzymatic hydrolysis and saccharification was 2 U/g. It was verified that under this condition, the content of combined sugar in the hydrolysate of pretreated wheat straw was 108.4\u0026thinsp;\u0026plusmn;\u0026thinsp;0.6 mmol/L (glucose 70.0\u0026thinsp;\u0026plusmn;\u0026thinsp;0.5 mmol/L and xylose 38.4\u0026thinsp;\u0026plusmn;\u0026thinsp;0.7 mmol/L), which was 17.8% higher than that before adding ligninolytic enzymes. Through optimized pretreatment methods and the addition amount of ligninolytic enzymes, a higher sugar content was obtained.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eOrthogonal test analysis of wheat straw hydrolysis by lignocellulase\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"7\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eLevel\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eCellulase\u003c/p\u003e \u003cp\u003e(U/g)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eXylanase\u003c/p\u003e \u003cp\u003e(U/g)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eLac\u003c/p\u003e \u003cp\u003e(U/g)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eLiP\u003c/p\u003e \u003cp\u003e(U/g)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003eMnP\u003c/p\u003e \u003cp\u003e(U/g)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c7\"\u003e \u003cp\u003eCombined sugars content\u003c/p\u003e \u003cp\u003e(mmol/L)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e25\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e25\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c7\"\u003e \u003cp\u003e65.1\u0026thinsp;\u0026plusmn;\u0026thinsp;0.7\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e25\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e20\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c7\"\u003e \u003cp\u003e74.3\u0026thinsp;\u0026plusmn;\u0026thinsp;0.6\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e25\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e15\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c7\"\u003e \u003cp\u003e82.6\u0026thinsp;\u0026plusmn;\u0026thinsp;0.5\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e25\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c7\"\u003e \u003cp\u003e91.5\u0026thinsp;\u0026plusmn;\u0026thinsp;0.3\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e20\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e25\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c7\"\u003e \u003cp\u003e86.2\u0026thinsp;\u0026plusmn;\u0026thinsp;0.4\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e20\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e20\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c7\"\u003e \u003cp\u003e98.2\u0026thinsp;\u0026plusmn;\u0026thinsp;0.2\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e20\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e15\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c7\"\u003e \u003cp\u003e106.1\u0026thinsp;\u0026plusmn;\u0026thinsp;0.8\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e20\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c7\"\u003e \u003cp\u003e109.0\u0026thinsp;\u0026plusmn;\u0026thinsp;0.4\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e15\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e25\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c7\"\u003e \u003cp\u003e99.3\u0026thinsp;\u0026plusmn;\u0026thinsp;0.6\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e15\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e20\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c7\"\u003e \u003cp\u003e100.3\u0026thinsp;\u0026plusmn;\u0026thinsp;0.7\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e11\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e15\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e15\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c7\"\u003e \u003cp\u003e96.2\u0026thinsp;\u0026plusmn;\u0026thinsp;0.9\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e12\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e15\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c7\"\u003e \u003cp\u003e103.7\u0026thinsp;\u0026plusmn;\u0026thinsp;0.8\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e13\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e25\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c7\"\u003e \u003cp\u003e95.2\u0026thinsp;\u0026plusmn;\u0026thinsp;0.6\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e14\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e20\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c7\"\u003e \u003cp\u003e104.7\u0026thinsp;\u0026plusmn;\u0026thinsp;0.4\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e15\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e15\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c7\"\u003e \u003cp\u003e103.1\u0026thinsp;\u0026plusmn;\u0026thinsp;0.6\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e16\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c7\"\u003e \u003cp\u003e101.2\u0026thinsp;\u0026plusmn;\u0026thinsp;0.5\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ek1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e78.4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e86.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e90.2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e94.9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e94.4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ek2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e99.9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e94.4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e91.8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e93.7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e95.2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ek3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e99.9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e97.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e98.9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e92.6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e94.9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ek4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e100.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e101.4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e98.3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e98.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e94.7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eR\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e22.7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e14.9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e8.7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e5.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003ctfoot\u003e \u003ctr\u003e\u003ctd colspan=\"7\"\u003e\u003cem\u003eNote: All values in the \"Combined sugars content\" column are presented as the mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation (n\u0026thinsp;=\u0026thinsp;3). k1\u0026ndash;k4 represent the average value of combined sugars content at each level of the corresponding factor, R denotes the range of k values (R\u0026thinsp;=\u0026thinsp;max(k) \u0026ndash; min(k)).\u003c/em\u003e\u003c/td\u003e\u003c/tr\u003e \u003c/tfoot\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003e3.3 Ethanol production from saccharification and fermentation of pretreated wheat straw\u003c/h2\u003e \u003cp\u003eSequential hydrolysis and fermentation (SHF) was compared with simultaneous saccharification and fermentation (SSF) for ethanol production from pretreated wheat straw. Ethanol production by SHF increased rapidly within 12\u0026ndash;24 h, whereas that by SSF increased markedly during 36\u0026ndash;48 h; the ethanol production rate of both processes declined gradually after 72 h (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ea). Ethanol production by SSF reached 6.9\u0026thinsp;\u0026plusmn;\u0026thinsp;0.2 g/L (Supplementary Materials 1 and 2), which was 9.7% higher than the 6.2\u0026thinsp;\u0026plusmn;\u0026thinsp;0.1 g/L obtained by SHF (Supplementary Material 3).Therefore, the SSF method was selected for ethanol production. Using pretreated wheat straw as the sole carbon source, key factors including nitrogen sources and metal ions in the culture medium were further optimized. Five nitrogen sources were evaluated, including yeast powder, peptone, NH\u003csub\u003e4\u003c/sub\u003eHCO\u003csub\u003e3\u003c/sub\u003e, (NH4)\u003csub\u003e2\u003c/sub\u003eSO\u003csub\u003e4\u003c/sub\u003e, and NH\u003csub\u003e4\u003c/sub\u003eH\u003csub\u003e2\u003c/sub\u003ePO\u003csub\u003e4\u003c/sub\u003e.The highest ethanol yield of 7.0\u0026thinsp;\u0026plusmn;\u0026thinsp;0.1 g/L was achieved with peptone as the nitrogen source (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eb). KCl, NaCl, MgSO4, MnSO4 and CuSO\u003csub\u003e4\u003c/sub\u003e were added to the medium as the metal ion additives, and the highest ethanol yield (7.1\u0026thinsp;\u0026plusmn;\u0026thinsp;0.2 g/L) was obtained with MgSO4 as metal ion additive (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ec). We further optimized the fermentation conditions for this process, including temperature, rotational speed and inoculation amount. The results of fermentation temperature optimization showed that the ethanol yield was highest (7.2\u0026thinsp;\u0026plusmn;\u0026thinsp;0.1 g/L) at 32 ℃ (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ed). The optimization of rotational speed indicated that the ethanol yield was highest (7.5\u0026thinsp;\u0026plusmn;\u0026thinsp;0.2 g/L) at 130 r/min (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ee). Finally, the highest ethanol yield of 7.6\u0026thinsp;\u0026plusmn;\u0026thinsp;0.2 g/L was achieved at an inoculation amount of 1% (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ef). The optimized fermentation conditions were: peptone as nitrogen source, MgSO\u003csub\u003e4\u003c/sub\u003e as metal ion additive, temperature 32\u0026deg;C, rotational speed 130 r/min, and inoculation amount 1%. Under these conditions, an ethanol yield of 7.6\u0026thinsp;\u0026plusmn;\u0026thinsp;0.2 g/L was obtained (Supplementary Material 4), which was 10.1% higher than that before optimization. Under these optimized conditions, the overall ethanol yield reached 20.9 g per 100 g of raw wheat straw through the integrated process of pretreatment, saccharification, and fermentation. All fermentation experiments were performed in triplicate (n\u0026thinsp;=\u0026thinsp;3).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"4. Discussion","content":"\u003cp\u003eFirstly, to improve the utilization efficiency of wheat straw, we optimized the wheat straw pretreatment methods. The results showed that the combined treatment with 2% NaHCO\u003csub\u003e3\u003c/sub\u003e\u0026thinsp;+\u0026thinsp;2% H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e effectively removed lignin, while better retaining the cellulose and hemicellulose components of wheat straw under relatively mild conditions, thus achieving higher recovery rates of these two polysaccharides. Compared with conventional pretreatments, this superior performance can be attributed to two key factors: first, acid pretreatment tends to cause hemicellulose dissolution [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]; second, the alkaline peroxide-based pretreatment used in this study is highly effective at selectively removing lignin from wheat straw [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. SEM observations confirmed that the morphological structure of pretreated wheat straw enhanced the enzymatic accessibility to cellulose and hemicellulose during subsequent enzymatic hydrolysis, consistent with previous findings [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e], which further explains the increased monosaccharide yield in the hydrolysate of pretreated wheat straw. Meanwhile, the pretreatment method was relatively mild, which reduced water consumption during washing and alleviated environmental pollution\u0026mdash;an advantage that conventional acid/strong alkali pretreatment methods lack [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e, \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. Based on these results, it is further verified that the pretreatment method selected in this study exhibited superior performance under relatively mild conditions, and increased the utilization efficiency of wheat straw effectively.\u003c/p\u003e \u003cp\u003eSecondly, in order to hydrolyze pretreated wheat straw as much as possible, the effect of ligninolytic enzymes on the sugar content in hydrolysate of pretreated wheat straw was discussed. The addition of ligninolytic enzymes promoted the hydrolysis of pretreated straw, and laccase had the most significant effect on the sugar content in the hydrolysate system, which was consistent with previous findings [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e], while it was different from the previous research result that Mnp had little effect on the sugar content in wheat straw hydrolysate [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e], the reason may be that the mild alkaline peroxide pretreatment used in this study preserved the specific morphological structure of wheat straw, whereas previous studies adopted harsh pretreatment methods that altered the binding sites of MnP, thus leading to different enzymatic hydrolysis outcomes.\u003c/p\u003e \u003cp\u003eFinally, the pretreated wheat straw hydrolysate was used for ethanol fermentation. Compared with the previous concept of removing lignin and hemicellulose so as to improve the utilization rate of cellulose [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e], our concept of efficient simultaneous conversion of cellulose and hemicellulose is of great significance for the efficient conversion of lignocellulosic biomass resources. Under optimal conditions, the ethanol yield reached 7.6\u0026thinsp;\u0026plusmn;\u0026thinsp;0.2 g/L, corresponding to a conversion efficiency of 20.9 g ethanol per 100 g raw wheat straw. Compared with the result that 7.143 tons of raw wheat straw may be required to produce 1 ton of ethanol from Patel A et al, we saved 33.1% of raw materials [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. Compared with the result that 15.5 g ethanol was harvested from 100g wheat straw in the optimal conditions by Qiu J et al [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e], the yield of ethanol in our study was increased by 35.0%. The reason may be that the cellulose which can transform glucose and hemicellulose which can transform xylose were both used efficiently in the process of ethanol production of wheat straw in our study. This high conversion efficiency can be attributed to the synergistic effect of three key steps. First, the mild pretreatment retained both cellulose and hemicellulose; second, the optimized ligninolytic enzymes system enhanced the accessibility of polysaccharides; third, the SSF process promoted the simultaneous utilization of glucose and xylose.\u003c/p\u003e \u003cp\u003eThe mild pretreatment coupled with ligninases synergism not only improves ethanol yield but also reduces the production cost of bioethanol. This optimized process is expected to provide a feasible technical scheme for the industrialization of straw-derived biofuels, which is conducive to promoting the development of renewable fuel industry. It also provides a novel perspective for the high-value utilization and conversion of lignocellulosic biomass, further expanding the application potential of agricultural waste in renewable fuel production.\u003c/p\u003e"},{"header":"5. Conclusion","content":"\u003cp\u003eThis study developed an efficient integrated process for ethanol production from wheat straw, involving mild pretreatment, optimized enzymatic hydrolysis, and SSF. The key conclusions are as follows.\u003c/p\u003e \u003cp\u003eFirstly, the optimal pretreatment conditions were 2% NaHCO\u003csub\u003e3\u003c/sub\u003e\u0026thinsp;+\u0026thinsp;2% H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e at 130\u0026deg;C for 50 min with a solid-to-liquid ratio of 1:10. This mild pretreatment effectively removed lignin while retaining cellulose and hemicellulose, reducing water consumption during washing and alleviating environmental pollution compared to conventional methods.\u003c/p\u003e \u003cp\u003eSecondly, the maximum combined sugar concentration (108.4\u0026thinsp;\u0026plusmn;\u0026thinsp;0.6 mmol/L) was achieved under the optimized enzymatic hydrolysis conditions: cellulase 20 U/g, xylanase 10 U/g, laccase 4 U/g, lignin peroxidase 6 U/g, and manganese peroxidase 2 U/g. The addition of ligninolytic enzymes significantly enhanced the accessibility of polysaccharides.\u003c/p\u003e \u003cp\u003eThirdly, the optimal SSF conditions were peptone as the nitrogen source, MgSO\u003csub\u003e4\u003c/sub\u003e as the metal ion additive, 32\u0026deg;C, 130 r/min, and 1% inoculation amount, yielding a maximum ethanol concentration of 7.6\u0026thinsp;\u0026plusmn;\u0026thinsp;0.2 g/L (corresponding to 20.9 g ethanol per 100 g raw wheat straw).\u003c/p\u003e \u003cp\u003eOverall, the synergistic effect of mild pretreatment, optimized enzyme system, and SSF enabled efficient simultaneous conversion of cellulose and hemicellulose, providing a promising and environmentally friendly approach for the high-value utilization of wheat straw and other lignocellulosic biomass.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgment\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe research was supported by the First-Class Undergraduate Course Construction Project of Guizhou University of Traditional Chinese Medicine (Grant No.: GZYKC-25037)\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDeclaration of Interest Statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWenxuan Zhao developed the idea for the study, performed the research, conducted data analysis, and prepared the manuscript.\u003c/p\u003e\n\u003cp\u003eZhigang Ju helped to validate the data and conduct feasibility analysis.\u003c/p\u003e\n\u003cp\u003eAll authors read and approved the final manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData Availability Statement\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe raw data supporting the conclusions of this article will be made available by the authors, without undue reservation. The chromatograms related to this study have been uploaded as Supplementary Materials.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eZhang Z, Zhao X, Huang C et al (2025) Liquid hot water pretreatment technology: opening a new chapter in the green transformation and high-value utilization of biomass resources. Biomass Bioenergy 203:108278. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.biombioe.2025.108278\u003c/span\u003e\u003cspan address=\"10.1016/j.biombioe.2025.108278\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLi E, Na N, Wu N et al (2026) Dynamics of bacterial community and fermentation quality of wheat straw silage treated with lactic acid bacteria and/or sucrose. 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Bioresour Technol 268:355\u0026ndash;362. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.biortech.2018.08.009\u003c/span\u003e\u003cspan address=\"10.1016/j.biortech.2018.08.009\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"bioenergy-research","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"bere","sideBox":"Learn more about [BioEnergy Research](https://www.springer.com/journal/12155)","snPcode":"12155","submissionUrl":"https://submission.nature.com/new-submission/12155/3","title":"BioEnergy Research","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"wheat straw, pretreatment, ligninases, simultaneous saccharification and fermentation, ethanol","lastPublishedDoi":"10.21203/rs.3.rs-9171419/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-9171419/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"Considering the global energy crisis, developing renewable biomass energy has become an urgent strategic need. In order to better utilize biomass resources, wheat straw was used as raw material. Findings from enzymatic hydrolysis, saccharification assays, and Scanning Electron Microscopy (SEM) characterization of pretreated wheat straw confirmed that the weak alkali-oxidation pretreatment (2% NaHCO\u0026thinsp;+\u0026thinsp;2% HO) effectively eliminated lignin with simultaneous preservation of cellulose and hemicellulose fractions of wheat straw under relatively mild pretreatment conditions (50 min, 130\u0026deg;C, and solid-liquid ratio of 1:10). Enzymatic hydrolysis and saccharification experiments revealed that ligninases enhanced the hydrolysis efficiency of pretreated wheat straw, with laccase emerging as the most impactful component among these ligninases. Under the conditions of cellulase 20 U/g, xylanase 10 U/g, laccase 4 U/g, lignin peroxidase 6 U/g and manganese peroxidase 2 U/g, the combined sugars reached a maximum of 108.4\u0026thinsp;\u0026plusmn;\u0026thinsp;0.6 mmol/L, which was 17.8% higher than that of the group without ligninases. Finally, ethanol was produced by fermenting both glucose and xylose in the fermentation broth, and ethanol fermentation conditions of engineered Saccharomyces cerevisiae strain WXY12 were optimized, with peptone as the nitrogen source and MgSO as the metal ion inducer, temperature at 32\u0026deg;C, rotational speed at 130 r/min, and inoculation amount of 1% (v/v). Under these conditions, the highest ethanol concentration reached 7.6\u0026thinsp;\u0026plusmn;\u0026thinsp;0.2 g/L, corresponding to an ethanol yield of 20.9 g per 100 g of raw wheat straw, thereby establishing a robust theoretical basis for the efficient valorization of biomass resources.","manuscriptTitle":"Mild Pretreatment Coupled with Ligninases Synergism Enhances Ethanol Production from Wheat Straw via Simultaneous Glucose and Xylose Fermentation","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-04-21 10:36:55","doi":"10.21203/rs.3.rs-9171419/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"editorInvitedReview","content":"","date":"2026-05-12T17:49:50+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"2965467234867642692806077068053367525","date":"2026-04-21T00:20:30+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-04-18T23:47:08+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"162646849252090167828190922591058360575","date":"2026-04-15T17:52:36+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2026-04-13T17:37:59+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2026-03-28T23:33:07+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2026-03-21T12:57:11+00:00","index":"","fulltext":""},{"type":"submitted","content":"BioEnergy Research","date":"2026-03-19T15:51:20+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"bioenergy-research","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"bere","sideBox":"Learn more about [BioEnergy Research](https://www.springer.com/journal/12155)","snPcode":"12155","submissionUrl":"https://submission.nature.com/new-submission/12155/3","title":"BioEnergy Research","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"a13db6c1-4cae-4ff5-b8f6-ce3a30021e55","owner":[],"postedDate":"April 21st, 2026","published":true,"recentEditorialEvents":[{"type":"editorInvitedReview","content":"","date":"2026-05-12T17:49:50+00:00","index":14,"fulltext":""}],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2026-04-21T10:36:56+00:00","versionOfRecord":[],"versionCreatedAt":"2026-04-21 10:36:55","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-9171419","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-9171419","identity":"rs-9171419","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}