Enhancing enzymatic hydrolysis of corn stover by twin-screw extrusion pretreatment

Enhancing enzymatic hydrolysis of corn stover by twin-screw extrusion pretreatment

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Industrial Crops & Products xxx (xxxx) xxxx

Contents lists available at ScienceDirect

Industrial Crops & Products journal homepage: www.elsevier.com/locate/indcrop

Enhancing enzymatic hydrolysis of corn stover by twin-screw extrusion pretreatment Zichao Wanga, Xiaojia Hea, Liming Yana, Jinpeng Wanga, Xiaolong Hub, Qi Sunc,*, Huiru Zhanga,* a

College of Biological Engineering, Henan University of Technology, Zhengzhou, 450001, China College of Food and Biological Engineering, Zhengzhou University of Light Industry, Zhengzhou, 450001, China c College of Life Sciences, Chongqing Normal University, Chongqing, 401331, China b

A R T I C LE I N FO

A B S T R A C T

Keywords: Corn stover Twin-screw extrusion Enzymatic hydrolysis Monosaccharide

Efficient and cost-effective hydrolysis of cellulose and hemicellulose into monosaccharide is the crucial and challengeable step for corn stover utilization. In this study, twin-screw extrusion pretreatment changed the particle size distribution and spatial structure of corn stover samples, but had basically no effects on their chemical structure. Cellulose content of CS-DW and CS-DW-GNa increased, but hemicellulose content and crystalline index decreased with extrusion pretreatment. Glucose yield of CS-DW increased from 25 g/L of CS with 48 h hydrolysis time to 34 g/L by 36 h of hydrolysis time, and glucose yield of CS-DW-GNa increased to 45 g/L with 24 h hydrolysis time. Meanwhile, xylose yield of CS-DW and CS-DW-GNa increased from 19 g/L of CS (48 h) to 27 g/L (36 h) and 40 g/L (24 h), respectively. These results provide good guidance for improving the conversation of lignocelluloses to monosaccharide for further utilization.

1. Introduction

Dale, 2004), and most of which is left unused, discarded optionally or burnt directly, thus wasting resources, polluting environment and causing direct damage to the soil ecosystem (Li et al., 2018; Zhao et al., 2018). Obviously, development efficient utilization methods of corn stover would open the door towards addressing the energy and environmental crisis. Due to a three-dimensional and highly crystalline complex structure formed by cellulose, lignin and hemicelluloses in corn stover, efficient and cost-effective hydrolysis of cellulose and hemicellulose into monosaccharide via enzymatic strategy or other methods is the crucial and challengeable step for corn stover utilization (Hu et al., 2019; Karimi Alavijeh and Karimi, 2019; Xu et al., 2017). Therefore, the pretreatment process is usually applied to break down the structural barrier of corn stover and increase its hydrolysis rate. In fact, there are some pretreatment methods have been reported for hydrolysis of lignocelluloses, including acid, alkaline, supercritical carbon dioxide, thermal, thermo-chemical, biological approaches, ionic liquid, ammonia based methods and so on (Rabemanolontsoa and Saka, 2016; Sindhu et al., 2016). However, production of toxic compounds in pretreated process, long processing time and severe corrosion to production equipment usually limit the application of the above mentioned pretreatment methods for corn stover. On the other hand, extrusion pretreatment is not only a continuous process compared with batch systems, but also characterized by the non-production of fermentation

With the depletion of fossil-based energy reserves, contamination of environment and sustainable development of economy, many researchers have transferred their interests to use clean and renewable resources for bio-fuels and chemicals production (Sindhu et al., 2016; Wang et al., 2018a,b). Due to its abundant, clean, renewable, cost-effective and environmental friendly features, lignocelluloses is considered to be a promising alternative, which can derive many high value-added compounds, such as cell protein, oil, viscose rayon, xylonic acid, cellulose nitrate, cellulose acetate, cellulose nanocrystals, nanofibrillated cellulose, short chain fatty acids and bio-ethanol (Crutchik et al., 2018; Hernandez et al., 2018; Liu et al., 2018a; Wang et al., 2018a,b). As a common type of lignocelluloses, corn stover has attracted considerable attention due to its high availability and large quantity. According to statistics, the maize yield in the world was 1038.28 million tons which was much higher than that of rice and wheat in 2014. Meanwhile, based on the residue to crop ratio of 1.6, the yield of corn stover in the world can reach 1661.25 million tons (Liu et al., 2018c). In the USA, more than 90 million acres of land were used to grow corn (Karimi Alavijeh and Karimi, 2019), and approximately 216 million tons of corn stover is produced every year in China (Yang et al., 2019). Nevertheless, less than ten percent of corn stover is processed for industrial production, animal feed and bedding (Kim and ⁎

Corresponding authors. E-mail addresses: [email protected] (Q. Sun), [email protected] (H. Zhang).

https://doi.org/10.1016/j.indcrop.2019.111960 Received 4 May 2019; Received in revised form 31 October 2019; Accepted 7 November 2019 0926-6690/ © 2019 Elsevier B.V. All rights reserved.

Please cite this article as: Zichao Wang, et al., Industrial Crops & Products, https://doi.org/10.1016/j.indcrop.2019.111960

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2.4. Particle size distribution

inhibitors, large processing scale, short residence time and moderate temperature features (Cha et al., 2015; Kuster Moro et al., 2017). Furthermore and most importantly, extrusion is becoming an easily adaptable pretreatment method in the utilization of lignocelluloses. Due to the high yield of corn stover worldwide (1661.25 million tonnes in 2014) and improper utilization (Karimi Alavijeh and Karimi, 2019; Liu et al., 2018c), there is an urgent need for efficient utilization of corn stover. At the same time, based on the production of CO2 from NaHCO3 for increasing barrel pressure (Li et al., 2012a,b), and high boiling point and specific heat property of glycerol for increasing barrel temperature (Yan et al., 2012) during extrusion process, a twin-screw extrusion with addition of glycerol and NaHCO3 for corn stover pretreatment was studied in this work. In addition, corn stover samples, before and after pretreated with twin-screw extrusion, were characterized by Mastersizer, automatic fiber analyzer, Scanning electron microscopy (SEM), Fourier transform-infrared spectroscopy analysis (FT-IR), X-ray diffraction (XRD) and Differential scanning calorimeter (DSC). Furthermore, the enzymatic hydrolysis of corn stover samples to glucose and xylose by cellulase and hemicellulase was analyzed in this research.

The particle size distribution of corn stover samples in this experiment were measured using a Mastersizer 3000 (Malvern Instruments Ltd., United Kingdom). Meanwhile, the heterogeneity of corn stover samples was calculated by particle size span according to Silva et al. (Silva et al., 2012). Particle size span = (D90 – D10) / D50, where D10, D50 and D90 are the diameter of corn stover particles within the overall particle size distribution whereby 10 %, 50 %, and 90 % of particles smaller in size, respectively. 2.5. Scanning electron microscopy (SEM) observation The morphology of corn stover samples were captured by a Quant 200 scanning electron microscope (FEI, Netherlands). Corn stover sample was placed on to a specimen holder and sputter coated with gold layer (10 min, 2 mbar) before observation. Subsequently, each sample was transferred to the SEM at an accelerating voltage of 3.0 kV and 2000-fold magnifications. 2.6. Fourier transform-infrared spectroscopy (FT-IR) analysis

2. Materials and methods

In this work, a Fourier transform-infrared spectroscopy (Nexus 470, Nicolet, USA) was used to detect the changes of corn stover samples before and after treatment. Briefly, 1 mg of corn stover sample was thoroughly ground with 400 mg KBr and laminated to pellets. The scanning wave was 400–4000 cm-1 and the results were analyzed with OMNIC-8.2 software.

2.1. Materials Corn stover was collected in the suburb of Zhengzhou in 2018, and the leaves, cobs and roots were wiped out after collection. Then, the stalks were cut into small pieces and dried in an oven at 80 °C to constant weight, and the dried corn stover (termed CS) was shattered by a muller (RT-34, Hongquan Pharmaceutical Machinery Ltd., Hong Kong, China) and filtered through a 80 mesh screen. Glycerol, NaHCO3, KBr, H2SO4, CH3COOH, CH3COONa, C10H14N2Na2O8 (EDTA), Na2B4O7 10 H2O, C4H10O2, C12H25SO4Na, Na2HPO4, C19H42BrN, acetone, diatomite, decahydronaphthalene were analytical grade and bought from Sinopharm Chemical Reagent Co., Ltd. (China). Glucose and xylose were purchased from Sigma (USA), hemicellulase (Cellic HTec, 10,000 U/g) and cellulase (Cellic CTec2, 10,000 U/g) were purchased from Novozymes (USA).

2.7. X-ray diffraction (XRD) analysis X-ray diffractometer (D8advance, Bruker, Germany) was used to analyze the crystallinity of corn stover samples in this experiment. The source of radiation was Cu-Kα, the scanning angle range was 10°-60° (2θ), scanning voltage was 30 kV, scanning current was 30 mA, scanning rate was 2°/min and the step size was 0.02°. The crystalline index (%) of corn stover sample was calculated according the methods reported by other researchers (Shirkavand et al., 2017; Segal et al., 1959). CrI = [(I002 - Iam) / I002] ×100, where I002 and Iam are the maximum intensity and minimum intensity at 22.8° and 18.4°, respectively.

2.2. Extrusion pretreatment of corn stover

2.8. Differential scanning calorimetry (DSC) analysis

Part of shattered corn stover was used to produce extruded corn stover samples by a twin-screw extruder (Process 11, Thermo Fisher Scientific, Waltham, MA, USA). The moisture content of one group corn stover sample was adjusted to 25 % (w/w) with distilled water (termed CS-DW), another group of corn stover sample was added with 23 % (w/ w) distilled water, 2 % (w/w) glycerol and 1 g/L NaHCO3 (termed CSDW-GNa). The screw speed was 100 rpm, mass barrel temperature was 120 °C and the feeding rate was 2 kg/h. The extruded corn stover samples were dried in an oven at 80 °C to constant weight, ground by the muller and filtered through 80 mesh screen. CS, CS-DW and CS-DWGNa were packed and kept in a desiccator for further analysis.

Corn stover sample was placed in an aluminum crucible. The sample crucible was hermetically sealed and the total mass was recorded with Q2000 (TA Instruments, USA). Nitrogen was used as pure gas and the flow rate was 40 mL/min, the scanning rate was 10 °C/ min, and the scanning temperature was within the range of 0−200 °C. 2.9. Enzymatic hydrolysis analysis The enzymatic hydrolysis experiment of corn stover samples by hemicellulase and cellulase, respectively, in this work was performed in a 250 mL Erlenmeyer flask containing 50 mL of sodium acetate buffer (pH = 4.8), at the same time, 0.02 % (w/v) sodium azide was added to inhibit microbial growth. The enzyme activities of cellulase and hemicellulase were detected according the internationally accepted filter paper enzyme activity assay method NREL/TP-510-42628 of the National Renewable Energy Laboratory (NREL) (Ji et al., 2017; Yu et al., 2019). After which, one gram of corn stover sample was placed into the flask and enzyme loading was 15 FPU/g of dry matter. Then, the flasks were immediately incubated at 50 °C and 150 rpm for 72 h in a shaking incubator (I2400, New Brunswick Scientific Inc., Edison, NJ, USA). At an interval time of 12 h, 5 mL hydrolysate was withdrawn for glucose or xylose yield determination. At the same time, the reaction system without taking samples during enzymatic hydrolysis process

2.3. Chemical composition determination of corn stover samples Contents of cellulose, hemicellulose and lignin in different corn stover samples were determined by a Fibertec ™ 2010 automatic fiber analyzer (FOSS, Denmark) according the Van-Soest method (Le et al., 2017). Crude protein content in corn stover samples was determined according to AOAC 920.87 method (AOAC 2000), crude fat content in corn stover samples was determined according to AACC 30-25 method (AACC, 2004), and crude ash content in corn stover samples was determined according to AOAC 923.03 method (AOAC, 2000). 2

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twin-screw extruder pretreatment. On the other hand, Table 1 also showed that the solid content of original corn stover sample (CS) was 91.03 %, but after different twin-screw extrusion pretreatments, solid contents in CS-DW and CS-DW-GNa samples were 85.72 % and 79.47 %, respectively. Moreover, the pretreatment yields of CS-DW and CSDW-GNa after different twin-screw extrusions were 92.39 % and 87.53 %, representing that the removal percentages of component in CS-DW and CS-DW-GNa were 7.61 % and 12.47 % during extrusion process. The removal of component from CS-DW and CS-DW-GNa after pretreated by twin-screw extrusion might due to the removal of lignin and extractives during extrusion process reported by other researchers (Li et al., 2012a,b; Liu et al., 2018a).

was used for analyzing degradation rates of corn stover compositions. In the end of enzymatic hydrolysis, corn stover samples were filtered, washed with distilled water for five times and dried in an oven at 80 °C to constant weight. The content of cellulose, hemicellulose and lignin in different corn stover samples after hydrolyzed by cellulase and hemicellulase were also determined by the Fibertec ™ 2010 automatic fiber analyzer. 2.10. Glucose and xylose yield determination The hydrolysate was centrifuged at 12,000×g and 4 °C for 10 min, and the glucose or xylose yield in the supernatant was determined by high performance liquid chromatography (HPLC) (Agilent 1200 series, Santa Clara, CA, USA) with a Aminex HPX-87H column (300 mm × 7.8 mm; Bio-Rad Laboratories Inc, Hercules, CA, USA) and a refractive index detector. The HPLC conditions were as follows: mobile phase was 5 mmol/L H2SO4, flow rate was 0.6 mL/min, temperature was 35 °C, injection volume was 20 μL, and the sample solution was filtered through a 0.22 μm membrane before injection.

3.2. Chemical composition determination of corn stover samples As can be seen from Table 2, after pretreated by extrusion with different moistures, cellulose contents of the pretreated corn stover samples were 33.98 % (CS-DW) and 35.03 % (CS-DW-GNa), which were 1.23 % and 2.28 % higher than the original corn stover sample (CS) of 32.75 %, but the cellulose yields of CS-DW and CS-DW-GNa were 95.86 % and 93.62 %. In addition, hemicellulose contents of corn stover samples decreased by 0.88 % (CS-DW) and 2.23 % (CS-DW-GNa) from the original 31.08 % (CS) to 30.20 % (CS-DW) and 28.85 % (CS-DWGNa), hemicellulose yields of CS-DW and CS-DW-GNa were 89.77 % and 81.25 %. Although lignin contents of corn stover samples were basically invariant and maintained at around 10.0 %, lignin yields of CSeDW and CS-DW-GNa were 90.74 % and 87.01 %. The increase of cellulose contents in the pretreated corn stover samples might relate to the removal of hemicellulose, crude fat, crude protein, crude ash and other extractives during extrusion pretreatment (Table 2), but the results showed that there was almost no removal of the lignin. Except for the different content of cellulose, hemicellulose and lignin in different parts of corn stover, such as stalks, leaves and cobs (Karimi Alavijeh and Karimi, 2019), different pretreatment methods might affect the content of water soluble substances and the linkages between polysaccharide units in corn stover, thus leading to the change of cellulose, hemicellulose and lignin content (Chen et al., 2007; Liu et al., 2018b). For instance, cellulose content of corn stover sample could be decreased by breaking β-1,4 glycosidic bonds with ball milling process (Liu et al., 2018c; Yu et al., 2019), ball milling combined with phosphoric acid pretreatment could lead to depolymerization of polysaccharides through the destruction of cellulose and hemicellulose long chains (Yu et al., 2019). Meanwhile, Xu et al. (Xu et al., 2017) have reported that the ash content of corn stover decreased after the ammonia and ultrasound pretreatment strategies.

2.11. Statistical analysis Each analytical result was expressed as mean ± SD after three replicate measurements. Data were analyzed by using the Origin Pro 8.2 software. 3. Results and discussion 3.1. Particle size distribution and pretreatment yield of corn stover samples Before and after twin-screw extruder pretreatment, particle size distribution curves of corn stover samples were shown in Fig. 1. Compared with CS, particle size distribution curves of CS-DW and CS-DWGNa shifted left obviously, suggesting the particle sizes of CS-DW and CS-DW-GNa were decreased by extrusion pretreatment, and this was in according with the results reported by Ji et al. (Ji et al., 2017) when rice straw samples of BM0 to BM60 were studied by mechanical fragmentation process during the starting stage from 0 min to 60 min. Meanwhile, median particle sizes of CS, CS-DW and CS-DW-GNa were all more than 50 μm (Table 1), indicating the extrusion pretreatment destroyed corn stover samples at tissue scale (Ji et al., 2017). Moreover, particle size span of CS, CS-DW and CS-DW-GNa did not change much (Table 1), this was similar to the work of Yu et al. (Yu et al., 2019), but the small particle size span of CS, CS-DW and CS-DW-GNa demonstrated the homogeneity of corn stover samples before and after the

3.3. Morphology observation The SEM images of corn stover samples before and after pretreated by the twin-screw extruder at 2000-fold magnification were shown in Fig. 2. The surface of CS was smooth and presented sheet-like morphology which suggested that the tissues and fibrous structures in corn stover were remained intact. When pretreated by extrusion with 25 % (w/w) distilled water as humidizer, surface of CS-DW became rough with numerous cracks. Meanwhile, when 23 % (w/w) distilled water, 2 % (w/w) glycerol and 1 g/L NaHCO3 were used as humidizer for extrusion pretreatment, CS-DW-GNa changed to rhabdoid with large amounts of voids and exhibited a rough and stripe-like surface. These observations demonstrated that extrusion pretreatment effectively broke the recalcitrant structure of corn stover samples and will facilitate the subsequent enzymatic hydrolysis (Liu et al., 2018a; Yu et al., 2019). Furthermore, many researchers have obtained similar results as reported in this work, Liu et al. (Liu et al., 2018d) found that the surface became rough with large amounts of voids and a thin layer of flaky structure was observed on the surface after the corn straw was pretreated with alkaline solution of ionic liquids. Yu et al. (Yu et al., 2019)

Fig. 1. Particle size distribution curves of different corn stover samples. 3

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Table 1 Particle size and yield of different corn stover samples. Sample

D10 (μm)

D50 (μm)

D90 (μm)

Span

Solid content (%)

Pretreatment yield (%)

CS CS-DW CS-DW-GNa

53.15 ± 2.18 76.37 ± 1.45 86.98 ± 5.58

122.45 ± 5.85 145.09 ± 8.94 154.51 ± 6.88

199.88 ± 3.24 238.29 ± 15.49 262.49 ± 8.65

1.20 ± 0.37 1.12 ± 0.69 1.14 ± 0.19

91.03 ± 0.17 85.72 ± 0.31 79.47 ± 0.26

∕ 92.39 ± 0.25 87.53 ± 0.13

Table 2 Chemical composition of different corn stover samples (%, on dry matter). Sample

Cellulose

Hemicellulose

Lignin

Crude fat

Crude protein

Crude ash

CS CS-DW CS-DW-GNa

32.75 ± 0.32 33.98 ± 0.14 35.03 ± 0.75

31.08 ± 0.57 30.20 ± 0.28 28.85 ± 0.36

10.07 ± 0.91 9.89 ± 0.43 10.01 ± 0.62

2.16 ± 0.37 2.09 ± 0.52 1.95 ± 0.26

5.36 ± 0.95 5.19 ± 0.71 5.06 ± 0.59

4.08 ± 0.58 3.89 ± 0.78 3.76 ± 0.24

CS, CS-DW and CS-DW-GNa decreased along with the extrusion pretreatment (Fig. 5), this is in consistent with the results of crystalline index, further indicating that the crystal structure of corn stover samples were destroyed during extrusion pretreatment induced by the removal of hemicelluloses content. Similar result was obtained to this work when corn straw was treated with a pure bacteria system of Bacillus Subtilis by Xu et al. (Xu et al., 2018), but Xu et al. (Xu et al., 2017) gained the opposite result of crystalline index change to this work when corn cob, corn stover and sorghum stalk were pretreated by dilute aqueous ammonia combined with ultrasonic.

have revealed that the corn stover exhibited larger cracks and a layered structure after pretreated with pulverization and phosphoric acid. 3.4. Change of structure analysis by FT-IR Chemical change of corn stover samples was analyzed by FT-IR based on the position and shape of absorption peaks within the infrared spectra (Fig. 3). The absorption band at around 3008 cm-1 might due to = CeH stretching in cellulose, and the peaks of CS, CS-DW and CSDW-GNa at around 3008 cm-1 increased, indicating the increase of cellulose content in corn stover samples, which is consistent with the increase of cellulose contents from 32.75 % in CS to 33.98 % in CS-DW and 35.03 % in CS-DW-GNa (Table 2). Absorption bands at around 2924 cm-1 and 2853 cm-1 might refer to CeH stretching in cellulose, absorption band at around 2000 cm-1 might attribute to ester carbonyl. Absorption band at around 1376 cm-1 might relate to CeH vibration in hemicellulose, a slight decrease in the absorption peak at around 1376 cm-1 was observed, which demonstrates a slight decrease of hemicellulose content in corn stover samples, and this is in accordance with the results of removing hemicellulose during twin-screw extrusion pretreatment in Table 2. Absorption band at around 1322 cm-1 might belong to CeO vibration in lignin, and absorption bands at around 1164 cm-1 and 1048 cm-1 might correspond to CeOC stretching and COee stretching in cellulose. Absorption bands at around 874 cm-1 and 719 cm-1 might relate to CeO stretches and CHe stretches in cellulose, the increase of absorption peaks at around 719 cm-1 might also due to the removal of hemicellulose, crude fat, crude protein, crude ash and other extractives during extrusion pretreatment (Table 2), thus the increase of cellulose content in corn stover samples (Guo et al., 2018; Hernandez et al., 2018; Huang et al., 2019; Quiles-Carrillo et al., 2018; Yu et al., 2019). However, a characteristic peak at the wavenumber of 2600 cm-1 was detected in CS-DW and CS-DW-GNa, we speculated that which is the OeH stretching and this will be studied in our future work.

3.6. Enzymatic hydrolysis Efficient and cost-effective hydrolysis of cellulose and hemicellulose to monosaccharide by enzymes or other methods is the crucial and challengeable step for corn stover utilization (Huang et al., 2019; Maurya et al., 2015; Wang et al., 2018a). In this work, glucose yield from CS was 25 g/L with an enzymatic hydrolysis time of 48 h (Fig. 6A), and this yield reached 28 g/L at the end of 72 h hydrolysis time, but the conversion rate of cellulose to glucose was only 35 %. After pretreatment, glucose yields from CS-DW and CS-DW-GNa were 34 g/L and 45 g/L with the enzymatic hydrolysis time of 36 h and 24 h, respectively. At the end of 72 h, glucose yields from CS-DW and CS-DW-GNa reached 38 g/L and 50 g/L, representing an increase of 10 g/L and 22 g/ L compared to CS, respectively. Removal of hemicellulose during extrusion process decreased the crystalline index of corn stover samples (Fig. 4), addition with the removal of crude fat, crude protein, crude ash and other extractives (Table 2), more cellulose was exposed and degraded by cellulase, thus leading to the conversion rates of cellulose to glucose reached 47 % and 63 % at the end of 72 h, respectively. At the same time, Fig. 6B showed xylose yields from CS, CS-DW and CS-DW-GNa followed a similar trend as glucose, xylose yields were 19 g/L (CS), 27 g/L (CS-DW) and 40 g/L (CS-DW-GNa) at the hydrolysis time of 48 h, 36 h and 24 h, and these yields reached 20 g/L, 30 g/L and 43 g/L at the end of 72 h hydrolysis time, respectively. Removal of hemicellulose during extrusion retreatment (Table 2) reduced the hemicellulose content and the loosely connected hemicellulose in corn stover samples, and then made the yield of xylose was lower than that of glucose. At the hydrolysis time of 72 h, the conversion rates of hemicellulose to xylose in CS, CS-DW and CS-DW-GNa were just 29 %, 38 % and 47 %, respectively. However, conversion rates of hemicellulose to xylose in pretreated corn stover samples (CS-DW and CSDW-GNa) were all higher than that of original (CS), these results indicated that change of particle size distribution and spatial structure modification might also contribute to higher enzymatic hydrolysis rate (S. Ribeiro et al., 2015). Wang et al. (Wang et al., 2018a) reported that the glucose and xylose yields from corn stover with high-solid pretreatment by enzymatic hydrolysis reached 0.45 g/g and 0.18 g/g, respectively. In the work conducted by Yu et al. (Yu et al., 2019), glucose

3.5. Crystalline index and DSC analysis In general, amorphous lignocellulose is easy attacked by various enzymes and crystalline index is an important factor of enzymatic hydrolysis efficiency (Xu et al., 2018). As shown in Fig. 4 of the XRD results, calculated crystalline indexes of CS, CS-DW and CS-DW-GNa were 38.73 %, 33.05 % and 24.88 %, respectively. Meanwhile, a decrease trend of diffraction peaks of CS, CS-DW and CS-DW-GNa at around 22.8° in Fig. 4 of XRD results was observed, also suggesting the decreased crystalline indexes of corn stover samples. The possible reason for the decreased crystalline indexes of corn stover samples might relate to the destruction of some hydrogen bonds in cellulose and the break of the dense structure of hemicellulose during extrusion process reported by other researchers (Geng and Henderson, 2012; Wada et al., 2010). At the same time, the glass transition temperature of 4

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Fig. 3. FT-IR results of different corn stover samples.

Fig. 4. XRD results of different corn stover samples.

Fig. 2. SEM images of different corn stover samples. CS (A), CS-DW (B) and CSDW-GNa (C).

Fig. 5. DSC results of different corn stover samples.

yield in this work.

yield from corn stover with pulverization and phosphoric acid pretreatment by enzymatic hydrolysis was 41.41 mg/g. Therefore, glucose and xylose yields in this work could be competitive with other pretreatments of corn stover. However, Ji et al. (Ji et al., 2017) obtained 287.07 mg/g glucose yield from rice straw with mechanical fragmentation pretreatment, and this yield was much higher than the glucose

4. Conclusions Compared with the traditional chemical and biological strategies for lignocelluloses pretreatment, the extrusion method has advantages of 5

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Fig. 6. Glucose yield (A) and xylose yield (B) from different corn stover samples.

continuous process, non-production of fermentation inhibitors, large processing scale, short time and moderate temperature. Our results revealed that the twin-screw extrusion pretreatment not only can change the particle size distribution and spatial structure of corn stover samples, but decrease their crystalline index. Meanwhile, glucose and xylose yields of corn stover samples with pretreatment increased from 25 g/L and 19 g/L with hydrolysis time of 48 h to 45 g/L and 40 g/L with 24 h hydrolysis time, respectively.

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Declaration of Competing Interest This original research paper was not previously submitted to Industrial crops and products and we wish to be considered for publication in your distinguished journal. No conflict of interest exits in the submission of this manuscript, and manuscript is approved by all authors for publication. I would like to declare on behalf of my co-authors that the work described was original research that has not been published previously, and not under consideration for publication elsewhere, in whole or in part. All the authors listed have approved the manuscript that is enclosed. Acknowledgements This work is supported by the Science and Technology Research Program of Chongqing Municipal Education Commission (KJQN201800540), and the Natural Science Foundation of Henan Provincial Education Department (19A180015). References Cha, Y.L., Yang, J., Park, Y., An, G.H., Ahn, J.W., Moon, Y.H., Yoon, Y.M., Yu, G.D., Choi, I.H., 2015. Continuous alkaline pretreatment of Miscanthus sacchariflorus using a bench-scale single screw reactor. Bioresour. Technol. 181, 338–344. Chen, S.F., Mowery, R.A., Scarlata, C.J., Chambliss, C.K., 2007. Compositional analysis of water-soluble materials in corn stover. J. Agric. Food Chem. 55 (15), 5912–5918. Crutchik, D., Frison, N., Eusebi, A.L., Fatone, F., 2018. Biorefinery of cellulosic primary sludge towards targeted short chain fatty acids, phosphorus and methane recovery. Water Res. 136, 112–119. Geng, X., Henderson, W.A., 2012. Pretreatment of corn stover by combining ionic liquid dissolution with alkali extraction. Biotechnol. Bioeng. 109 (1), 84–91. Guo, Y., Cong, S., Zhao, J., Dong, Y., Li, T., Zhu, B., Song, S., Wen, C., 2018. The combination between cations and sulfated polysaccharide from abalone gonad (Haliotis discus hannai Ino). Carbohydr. Polym. 188, 54–59. Hernandez, C.C., Ferreira, F.F., Rosa, D.S., 2018. X-ray powder diffraction and other analyses of cellulose nanocrystals obtained from corn straw by chemical treatments. Carbohydr. Polym. 193, 39–44. Hu, J., Li, D., Lee, D.J., Zhang, Q., 2019. Gasification and catalytic reforming of corn straw in closed-loop reactor. Bioresour. Technol. 282, 530–533. Huang, W., Wachemo, A.C., Yuan, H., Li, X., 2019. Modification of corn stover for improving biodegradability and anaerobic digestion performance by Ceriporiopsis subvermispora. Bioresour. Technol. 283, 76–85. Ji, G., Han, L., Gao, C., Xiao, W., Zhang, Y., Cao, Y., 2017. Quantitative approaches for illustrating correlations among the mechanical fragmentation scales, crystallinity and enzymatic hydrolysis glucose yield of rice straw. Bioresour. Technol. 241, 262–268. Karimi Alavijeh, M., Karimi, K., 2019. Biobutanol production from corn stover in the US. Ind. Crop. Prod. 129, 641–653.

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