Integrated chemical and multi-scale structural analyses for the processes of acid pretreatment and enzymatic hydrolysis of corn stover

Carbohydrate Polymers 141 (2016) 1–9

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Integrated chemical and multi-scale structural analyses for the processes of acid pretreatment and enzymatic hydrolysis of corn stover Longjian Chen, Junbao Li, Minsheng Lu, Xiaomiao Guo, Haiyan Zhang, Lujia Han ∗ College of Engineering, China Agricultural University (East Campus), 17 Qing-Hua-Dong-Lu, Hai-Dian District, Beijing 100083, PR China

a r t i c l e

i n f o

Article history: Received 8 October 2015 Received in revised form 17 December 2015 Accepted 30 December 2015 Available online 2 January 2016 Keywords: Acid pretreatment Chemical analysis Corn stover Enzymatic hydrolysis Structural analysis

a b s t r a c t Corn stover was pretreated with acid under moderate conditions (1.5%, w/w, 121 ◦ C, 60 min), and kinetic enzymolysis experiments were performed on the pretreated substrate using a mixture of Celluclast 1.5 L (20 FPU/g dry substrate) and Novozyme 188 (40 CBU/g dry substrate). Integrated chemical and multiscale structural methods were then used to characterize both processes. Chemical analysis showed that acid pretreatment removed considerable hemicellulose (from 19.7% in native substrate to 9.28% in acidpretreated substrate) and achieved a reasonably high conversion efficiency (58.63% of glucose yield) in the subsequent enzymatic hydrolysis. Multi-scale structural analysis indicated that acid pretreatment caused structural changes via cleaving acetyl linkages, solubilizing hemicellulose, relocating cell wall surfaces and enlarging substrate porosity (pore volume increased from 0.0067 cm3 /g in native substrate to 0.019 cm3 /g in acid-pretreated substrate), thereby improving the polysaccharide digestibility. © 2016 Elsevier Ltd. All rights reserved.

1. Introduction Lignocellulose biomass is mainly composed of cellulose, hemicellulose, and lignin. Although carbohydrate-enriched lignocellulose biomass is likely to become the primary feedstock of biofuels obtained by biochemical conversion, the structural and compositional features of lignocellulose biomass create a protective barrier and prevent enzymatic depolymerization of structural polysaccharides (Sanderson, 2011). To improve the enzyme hydrolysis conversion efficiency, some pretreatment methods such as comminution (Zhu, Wang, Pan, & Gleisner, 2009), hydrothermal (Wan, Zhou, & Li, 2011), and thermomechanical pretreatments (Pierre, Maache-Rezzoug, Sannier, Rezzoug, & Maugard, 2011; Pierre, Sannier, et al., 2011), have been explored. The dilute acid pretreatment is one of the most efficient pretreatments for lignocellulose biomass (Kim, Seo, Kim, & Han, 2015). Pretreatment with dilute acid offers the following attractive features: ease of use, a higher reaction rate and a readily produced purified product. Biochemical conversion typically consists of two key steps:

∗ Corresponding author. E-mail addresses: [email protected] (L. Chen), [email protected] (J. Li), [email protected] (M. Lu), xmguo [email protected] (X. Guo), [email protected] (H. Zhang), [email protected], [email protected] (L. Han). http://dx.doi.org/10.1016/j.carbpol.2015.12.079 0144-8617/© 2016 Elsevier Ltd. All rights reserved.

pretreatment and enzymatic hydrolysis (Chundawat, Beckham, Himmel, & Dale, 2011). Accordingly, the combination of dilute acid pretreatment and enzymatic hydrolysis has been widely applied for the biochemical conversion of lignocellulosic biomass (Gao et al., 2014; Kapoor et al., 2015; Tan & Lee, 2015). A number of previous studies have investigated the chemical and structural changes occurring in lignocellulose during dilute acid pretreatment. For example, Kumar, Mago, Balan, and Wyman (2009) pretreated corn stover with dilute acid and then measured the chemical composition and identified structural changes in the pretreated substrate using X-ray Diffraction (XRD), Scanning Electron Microscopy (SEM), Fourier Transform Infrared Spectroscopy (FTIR), and X-ray Photoelectron Spectroscopy (XPS). Similarly, Li et al. (2010) dilute acid pretreated switchgrass and characterized the material using XRD, SEM, FTIR, Raman spectroscopy and chemical methods. As enzymolysis is the key step after pretreatment in the biochemical conversion of lignocellulose, it is also very important to chemically and structurally characterize an enzymatically hydrolyzed substrate. Some studies have attempted to explore such changes in lignocellulose during both acid pretreatment and enzymatic hydrolysis. For example, Hsu, Guo, Chen, and Hwang (2010) not only performed a chemical analysis on sugar and an inhibitor during the acid pretreatment and enzymatic hydrolysis of rice straw but also characterized their structural features using FTIR, XRD, and Specific Surface Area/Pore Volume (SSA/PV). It should be noted that rice straw is a challenging substrate for cellulosic

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conversion because it contains a high ash content, which can interfere with the dilute acid pretreatment and affect enzyme activity (Yu & Chen, 2010). A similar study was carried out by Chandel et al. (2014) in which several imaging techniques (SEM, Transmitted Light Microscopy (TLM), FTIR, Fourier Transform Near-Infrared (FT-NIR), Raman spectroscopy, and Nuclear Magnetic Resonance (NMR)) were used to investigate the structural changes induced during the acid pretreatment and enzymatic digestion of sugarcane bagasse. Corn stover, which has different chemical and structural characteristics than rice straw, is a likely feedstock for cellulosic conversion in many geographic areas, whereas bagasse is generally only used in parts of the world where sugarcane is grown. In the present study, dilute acid pretreatment and enzymatic hydrolysis of corn stover were carried out, and the material was characterized using chemical and structural analyses. To our knowledge, this is the first work in the literature combining numerous chemical and structural techniques for a global comprehensive approach to reveal chemical and structural changes at the molecular level during the acid pretreatment and enzymatic digestion of corn stover.

samples were collected at different hydrolysis times (0.5 h, 1 h, 2 h, 4 h, 8 h, 16 h, 24 h, 48 h, and 72 h). 2.4. Cellulase adsorption Cellulase absorption on native and acid-pretreated substrates were performed in 10 mL centrifuge tubes containing 5 mL 0.05 M citrate buffer (pH 4.8) with several enzyme loadings (4.78–34.76 mg/g substrate, 2%, w/v, dry basis). The mixture of substrate and enzyme was incubated for 2 h in a shaking water bath at 100 rpm and 4 ◦ C to avoid hydrolysis. Substrate blanks without enzyme was also run in parallel. After incubation, all the samples were centrifuged at 4 ◦ C for 10 min in a refrigerated centrifuge at 4000 × g. The supernatant was decanted to measure the protein concentration by the Bradford method (Bradford, 1976). Bound enzyme was calculated by subtracting the free cellulase concentration from the initial enzyme concentration loaded to each reactor. Bound enzyme concentration was correlated with free enzyme concentration using the following Langmuir equilibrium isotherm: Eb =

2. Materials and methods 2.1. Feedstock and chemicals Corn stover was collected in 2013 from the Shangzhuang agronomy farm of the China Agricultural University, located in Beijing, China. The corn stover was air dried and milled to coarse particle size (∼1–2 cm). Then it was dried in a forced-air oven at 45 ◦ C for 48 h and milled to a size less than 1 mm in a RT-34 hammer mill (Rong Tsong Precision Technology Co., Taiwan). The milled material was sieved by a JH-300A sieve shaker fitted with a 40-mesh screen (Jiahe Machinery Co., Henan province, China). The samples were stored in a sealed plastic bag at room temperature before use. All of the chemicals were purchased from Sigma–Aldrich (St. Louis, MO, USA), Beijing Chemical Works (Haidian district, Beijing, China), and J&K Scientific Ltd. (Chaoyang district, Beijing, China). 2.2. Assay methods involving enzymes Celluclast 1.5 L (cellulase) and Novozyme 188 (ˇ-glucosidase) were used for the enzymatic hydrolysis of corn stover. The filter paper unit (FPU) activity of cellulase was determined according to the National Renewable Energy Laboratory (NREL) Analytical Procedure (Adney & Baker, 2008). The cellobiase unit (CBU) activity of ˇ-glucosidase was determined using the methodology published by Kim et al. (2013). The enzyme protein content was determined by the Bradford method (Bradford, 1976). The protein content of Celluclast 1.5 L and Novozyme 188 were 35.3 mg protein/mL and 27.4 mg protein/mL, respectively. 2.3. Pretreatment and enzymatic hydrolysis Corn stover at a solid-to-liquid ratio of 1:10 was mixed with 1.5% (w/w) dilute sulfuric acid and autoclaved at 121 ◦ C for 60 min to generate the acid-pretreated sample. The water-insoluble solid was washed thoroughly with distilled water to establish a neutral pH. Enzymatic hydrolysis of the acid-pretreated solid sample was performed in a 50 mL Erlenmeyer flask containing 2 g (dry matter) of the solid sample and 38 mL of citrate buffer (50 mM, pH 4.8). The sample in citrate buffer was supplemented with cellulase loadings (Celluclast 1.5 L: 20 FPU/g dry substrate equal to 8.41 mg protein/g dry substrate; Novozyme 188: 40 CBU/g dry substrate equal to 2.36 mg protein/g dry substrate). Enzymatic hydrolysis was allowed to proceed at 50 ◦ C at 150 rpm in an incubator shaker (model SHA-B(A), Kexi instrument, Jiangsu province, China), and

Emax Ka Ef 1 + Ka Ef

(1)

where Eb is the bound enzyme (mg/g substrate), Ef is the concentration of free enzyme in solution (mg/mL), Emax is the maximum adsorption capacity (mg/g substrate), and Ka is the equilibrium constant (mL/mg). The Langmuir adsorption constants (Emax and Ka ) on native and acid-pretreated substrates were obtained by nonlinear regression of their adsorption data. Distribution coefficient (Kr in mL/g), another constant from Langmuir adsorption isotherm, could be used to estimate the relative affinity of enzyme for substrates (Zhang & Lynd, 2004). The distribution coefficient can be calculated by Kr = Emax × Ka . 2.5. Chemical and structural analysis 2.5.1. Chemical analysis The carbohydrate and lignin contents of the solid samples (native and acid-pretreated substrates) were determined using the analytical procedures in NREL/TP-510-42618 (Sluiter et al., 2011). The constituent concentrations in the dilute acid and enzymatic hydrolysates were measured by high-performance liquid chromatography (HPLC) (Hitachi L-7200 with a refractive index detector L-2490, Hitachi Ltd., Tokyo, Japan). The concentrations of glucose, xylose, arabinose, and cellobiose were determined by an HPLC system equipped with a BP-800 Pb2+ column. The mobile phase was ultrapure water. The HPLC column was operated at 80 ◦ C with a mobile phase flow rate of 0.6 mL/min. The injection volume was 20 ␮L, and the elution time per injection was 40 min. The concentrations of furfural, HMF and acetic acid were determined by an HPLC system equipped with a BP-800 H+ column. The mobile phase was 5 mM sulfuric acid in ultrapure water. The HPLC column was operated at 55 ◦ C with a mobile phase flow rate of 0.6 mL/min. The injection volume was 20 ␮L, and the elution time per injection was 50 min. All of the samples were filtered through a 0.22-␮m filter prior to analysis. The amount of total reducing sugars was estimated using a spectrophotometer (UV 2550, Shimadzu, Kyoto, Japan) following the dinitrosalicylic acid (DNS) method of Miller (Miller, 1959). 2.5.2. Structural analysis The SEM analysis was performed using a Hitachi S-3400 scanning electron microscope (Hitachi, Tokyo, Japan). The specimens were prepared for SEM inspection by adhering each sample to carbon glue followed by plating with Pt. Representative images were acquired with an accelerating voltage of 15–20 kV.

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The crystalline nature was analyzed using an X-ray diffractometer (Bruker D8 advance, Bruker AXS Inc., WI, USA) with Cu K␣ radiation (40 mA, 40 kV). The data were collected at a step size of 0.2◦ over the 2 range of 5◦ –40◦ . The crystallinity index (CrI) was determined from the XRD data and calculated by CrI = (I002 − Iam )/I002 × 100%, where I002 is the intensity of the crystalline region and Iam is the intensity of the amorphous region. The FTIR analysis was performed to detect the changes in functional groups. The samples were pelletized by mixing 300 mg of spectroscopic grade KBr with 3 mg of sample in an agate mortar. Each sample was then subjected to 10 ton of pressure for 3 min in a hydraulic press (Atlas 25T; Specac, Swedesboro, NJ, USA). Spectra were collected in the range of 4000–400 cm−1 , with 32 scans and a resolution of 4 cm−1 per sample using a Perkin Elmer Spectrum 400 Fourier transform infrared spectrometer (Perkin Elmer, MA, USA). The surface properties were examined using XPS (Quantera SXM, ULVAC-PHI, Inc., Japan) with a monochromatic Al K␣ X-ray source. The analyzed sample area was approximately 300 ␮m × 300 ␮m with an analysis depth of less than 10 nm. Survey spectra were recorded with a pass energy of 55 eV. Calibration of the binding energy scale was achieved by setting the C1s peak of saturated hydrocarbons to 284.8 eV. The elemental composition (C1s; O1s) was determined from the ratio of the relative peak areas corrected by the sensitivity factors of the corresponding elements. High-resolution C1s spectra were also recorded, and deconvolution of the high-resolution C1s spectra was performed using a Gauss–Lorentzian curve-fitting program (XPSPEAK v4.1). AFM images were obtained in ScanAsyst mode using a MultiMode 8 AFM equipped with a Nanoscope V converter (Bruker Corp., Santa Barbara, USA). The ScanAsyst mode automatically optimizes the imaging parameters, including the set point, feedback gains, and scan rate. Images were acquired in tapping mode (in air) using an etched silicon probe. All images were collected from 5 unique fibers for each treatment, with representative images shown. To eliminate external vibrational noise, the microscope was placed on an active vibration-damping table. All of the 2 ␮m × 2 ␮m amplitude images were recorded and processed using Bruker Nanoscope Analysis software (v1.4) to obtain the surface roughness factor (root mean square roughness, Rq). A JW-BK112 porosity analyzer (JWGB Sci. & Tech. Co., Beijing, China) was used to measure the SSA and PV distribution of the water-pretreated and acid-pretreated corn stover samples. The samples were degassed at 50 ◦ C for 12 h and then cooled in the presence of nitrogen gas, allowing the nitrogen gas to condense on the surfaces and within the pores. SSA was calculated using the Brunauer–Emmett–Teller (BET) model, which relates the gas pressure to the total volume of gas adsorbed. The PV distribution with respect to the pore size was estimated using the Barrett–Joyner–Halenda (BJH) model.

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Table 1 Sugar yields and by-product concentrations in the acid-pretreated hydrolysate. Sugar yield

Acid-pretreated hydrolysate

Glucose (Mean ± SD, %) Xylose (Mean ± SD, %) Arabinose (Mean ± SD, %) Total reducing sugar (Mean ± SD, g/kg DM) Acetic acid (Mean ± SD, mg/mL) Furfural (Mean ± SD, mg/mL) Hydroxymethyl furfural (Mean ± SD, mg/mL)

21.44 64.04 71.78 328.68 2.16 0.10 0.84

± ± ± ± ± ± ±

0.36 4.25 0.80 29.42 0.11 0.02 0.11

substrate. A previous study reported similar changes in carbohydrate and lignin contents before and after dilute acid pretreatment (Zhu, Sathitsuksanoh, et al., 2009). 3.2. Sugar and by-products in hydrolysates The sugar yields and by-product concentrations in the acidpretreated hydrolysates are shown in Table 1. Xylans are the predominant hemicelluloses in lignocellulose, and when some xylan hemicellulose links are attacked by H+ protons (supplied by acids), the backbones tend to be deconstructed to monosaccharides (mainly xylose and arabinose) (Hu, Lin, Liu, & Liu, 2010). Some by-products, including acetic acid, furfural, and hydroxymethyl furfural (HMF), were also detected during the acid pretreatment (Table 1). These by-products have an inhibiting effect on the efficiency of cellulose saccharification and sugar fermentation. This study removed by-products by separating hydrolysate-solid and then washing the insoluble solid to establish a neutral pH. There are many other strategies to reduce the inhibiting influences of byproducts, such as dilution, vaporization, chemical additives, and microbial treatment techniques based on the fermenting microbe (Jonsson, Alriksson, & Nilvebrant, 2013). The current study applied a moderate pretreatment condition and thereby produced very low concentrations of by-products (0.10 mg/mL furfural, 0.84 mg/mL HMF), which has limited inhibition effect on the enzymolysis and fermentation process even without by-products removal (Larsson et al., 1999). The high sugar yields and low by-product concentrations suggest that moderate acid pretreatment can achieve efficient corn stover xylan hydrolysis. As shown in Fig. 1, the glucose and total reducing sugar yields in the enzymatic hydrolysates were 58.63% and 326.02 g/kg DM, respectively, after 72 h. This high efficiency of enzymatic hydrolysis is likely attributable to the effective removal of hemicellulose.

3. Results and discussion 3.1. Carbohydrate and lignin contents of solid samples The cellulose, hemicellulose, and lignin contents of the solid samples were 32.34 ± 0.26% (mean ± SD% DM), 19.70 ± 0.11%, and 17.52 ± 1.20% for the native substrate and 47.63 ± 1.69%, 9.28 ± 0.43%, and 26.94 ± 0.83% for the acid-pretreated substrate, respectively. These values of native corn stover are within the ranges reported in another study (Jin, Zhang, Yan, Qu, & Huang, 2011). After dilute acid hydrolysis, the hemicellulose content significantly decreased to 9.28%, also confirming the efficiency of moderate acid pretreatment in removing hemicellulose from lignocellulose. The decrease in the hemicellulose content in turn increased the amount of cellulose and lignin in the acid-pretreated

Fig. 1. Glucose yield and total reducing sugar yield in the enzymatic hydrolysate with respect to time.

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Fig. 2. SEM analysis of substrates. (a) Native substrate; (b) acid-pretreated substrate; and (c) 72-h enzymatically hydrolyzed acid-pretreated substrate.

3.3. SEM analysis Fig. 2a illustrates the compact and regular surface structure of the native substrate displayed, with fibers arranged in bundles, which impeded enzyme accessibility to the cellulose. However, acid pretreatment resulted in damage to the cellular structure, as evident by the separation of fibers from the pith and the loosening of the fibrous network (Fig. 2b). A possible reason for this result is the partial removal of hemicellulose during the dilute acid pretreatment. Enzymatic hydrolysis of the acid-pretreated substrate completely destroyed the cellulose, as demonstrated by the unstructured and heterogeneous nature of the samples (Fig. 2c), which also coincides with the high cellulose conversion rate for the enzymatic hydrolysis of the acid-pretreated substrate. 3.4. XRD analysis The crystallinity index (CrI) values of the native, acid-pretreated, and enzymatic-hydrolyzed substrates are shown in Table 2. Compared with the CrI (41.48%) of the native substrate, that (54.52%) of the acid-pretreated material was considerably higher. The CrI value calculated from X-ray diffraction reflects the total sample crystallinity and is thus sensitive to other components such as hemicellulose, lignin and disordered cellulose domains, which together contribute to the amorphous phase signal of the sample. As one of the amorphous phases, the hemicellulose component was hydrolyzed by acid pretreatment, and the crystalline phase was thus indirectly increased. Increases in CrI after acid pretreatment have also been reported in previous studies (Hsu et al., 2010; Kumar et al., 2009). The extent of CrI increase depends on the substrates as well as the acid pretreatment conditions. In the study of Hsu et al. (2010), the CrI of native rice straw, which had a high CrI value (57%), ranged from 58% to 65% after acid pretreatment and

increased slightly as the operating temperature increased. Chandel et al. (2014) also observed an increased in CrI (58.82%) of acidpretreated sugarcane bagasse compared to the native substrate (48.8%). In contrast to previous studies in which only the CrI of the enzymatically hydrolyzed endpoint was measured (Bernardinelli, Lima, Rezende, Polikarpov, & deAzevedo, 2015; Chandel et al., 2014), the current study monitored CrI values at different stages of hydrolysis (2 h, 16 h, and 72 h). It was observed that the CrI values were considerably stable, ranging from 50.18% to 51.45%, during the enzymatic hydrolysis, indicating that the commercial enzyme mixture used does not preferentially digest crystalline or amorphous cellulose. This is an intriguing finding that is contrary to the general viewpoint that amorphous cellulose is less resistant to enzyme depolymerization than crystalline cellulose during cellulose-toglucose conversion. This finding was also observed by Bernardinelli et al. (2015), in a study in which the native cellulose CrI in sugarcane bagasse was evaluated by quantitative 13 C MultiCP solid-state NMR, with no significant variation during enzymatic hydrolysis. In comparison to the endpoint CrI method of Bernardinelli et al. (2015), the multipoint CrI method of the current study provides more information on the mechanism of hydrolysis of the biphasic (crystalline or amorphous) cellulose in corn stover. 3.5. FTIR analysis Cellulose-related bands in FTIR spectra were observed at approximately 898 cm−1 , 1053 cm−1 , 1164 cm−1 , 2918 cm−1 , and 3400 cm−1 (Fig. 3). The band at 898 cm−1 is characteristic of the glycosidic bond ˇ-(1 → 4) in cellulose, and the bands at 1053 cm−1 and 1164 cm−1 can be attributed to C O stretching and C O C asymmetrical stretching, respectively. Compared with the native substrate, the acid-pretreated sample showed a slightly enhanced

L. Chen et al. / Carbohydrate Polymers 141 (2016) 1–9 Table 2 XRD analysis of substrates. Sample

Crystallinity index (CrI) (Mean ± SD, %)

Native substrate Acid-pretreated substrate 2-h enzymatically hydrolyzed substrate 16-h enzymatically hydrolyzed substrate 72-h enzymatically hydrolyzed substrate

41.48 54.52 50.18 51.42 51.45

± ± ± ± ±

0.10 1.33 0.88 3.73 1.23

intensity at 1164 cm−1 . This result is also consistent with the increase in cellulose content due to the removal of hemicellulose after pretreatment with dilute acid. The peak at 2918 cm−1 is due to the asymmetrical stretching of CH2 and CH bonds, and that at approximately 3400 cm−1 is associated with the O H stretching vibration of ␣-cellulose. As the absorption peak in the native substrate was similar to that in the pretreated substrate, it appears that most of the crystalline cellulose was not disrupted by the acid pretreatment. This finding was similar to that of Bernardinelli et al. (2015), who observed no significant variations in cellulose CrI inside sugarcane bagasse after pretreatment with dilute sulfuric acid (1%, v/v, 120 ◦ C, 40 min). Nonetheless, the intensity of the 3400 cm−1 peak significantly decreased after enzymatic hydrolysis. These changes provide strong evidence for the hydrolysis of both crystalline and amorphous cellulose during the enzymatic hydrolysis, which corresponds to the XRD analysis findings. Hemicellulose-related bands in the spectra were observed at approximately 1245 cm−1 and 1733 cm−1 . The band at approximately 1245 cm−1 was due to the stretching of C O bonds, which is characteristic of hemicellulose and lignin. A band at approximately 1733 cm−1 is characteristic of the C O stretching of hemicellulose. The acid-pretreated and enzymatically hydrolyzed substrates showed lower absorbed intensity at 1245 cm−1 and 1733 cm−1 compared with the native substrate, presumably because acid pretreatment is known to remove a large portion of hemicellulose. Lignin-related bands in the spectra were observed at approximately 1458 cm−1 , 1515 cm−1 , 1604 cm−1 , and 1720 cm−1 . The band at approximately 1458 cm−1 is reportedly due to CH2 and CH3 deformations in lignin (Guo, Chen, Chen, Men, & Hwang, 2008). The band at approximately 1515 cm−1 is due to the C C stretching of the lignin aromatic ring. The band at approximately 1604 cm−1 is attributable to lignin aromatic skeletal vibration and C O stretching (Kumar et al., 2009). The fact that these three bands were observed in all of the substrates suggests that lignin was not disrupted by the acid pretreatment and enzymatic hydrolysis, a result that is also consistent with the chemical analysis. The peak at 1720 cm−1 was detected in the acid-pretreated and enzymatically

Fig. 3. FTIR spectroscopy analysis of the substrates.

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hydrolyzed substrates and some researchers have discussed this absorption band in acid-pretreated substrates (Hsu et al., 2010; Kamireddy et al., 2013). The 1720 cm−1 peak is usually attributed to the acetyl group in hemicellulose structures and the phenyl ester linkage between hemicellulose and lignin (Kumar et al., 2009). As the chemical analysis in our study showed that acid pretreatment removed most of the hemicellulose, we suggest that the absorption peak at 1720 cm−1 arose from the uncleaved phenyl ester linkages between lignin and a small amount of hemicellulose. This finding also supports the assertion that dilute acid pretreatment can remove most of the hemicellulose in biomass by cleaving acetyl linkages. Similar phenomena under moderate acid pretreatment were also reported for forage sorghum (Corredor et al., 2009) and sunn hemp (Kamireddy et al., 2013). 3.6. XPS analysis The oxygen-to-carbon (O/C) ratio of native substrate was found to be 0.27 (Fig. 4), which is comparable with the reported value of 0.33 (Kumar et al., 2009). The known theoretical O/C ratios of such biomass components as cellulose, hemicellulose, lignin, and extractives are 0.83, 0.81, 0.33, and 0.03–0.11, respectively (Laine, Stenius, Carlsson, & Strom, 1994), and the biomass surface essentially consists of waxes (Rjiba, Nardin, Drean, & Frydrych, 2007) and other lipophilic extractives (Inari et al., 2011). This may explain the low O/C ratio of native substrate, even though its main components are cellulose, hemicellulose, and lignin. After acid pretreatment, the O/C ratio increased to 0.38, similar to the results presented in the study of Kumar et al. (2009). On the one hand, this may be due to the increase in cellulose content after acid pretreatment, on the other hand, acid pretreatment destroys the tight network structure of cellulose–hemicellulose–lignin and therefore improves the cellulose surface accessibility. The surface migration of cellulose may induce an increase of the O/C ratio. The slight decrease in the O/C ratio from 0.38 to 0.34 after enzymatic hydrolysis could be a consequence of enzyme-mediated cellulose hydrolysis. Fig. 4 also lists the fractions of the component-fitted carbon regions from high-resolution C1s spectra of all the samples. Only three carbon peaks (C1, C2, and C3) were obtained upon deconvolution of the higher resolution C1s spectra. The three carbon peaks belong to the following carbon bond classes: (i) C1 corresponds to carbon atoms bonded to other carbon or hydrogen atoms (C C, C H); (ii) C2 corresponds to carbon atoms bonded to a single non-carbonyl oxygen atom (C O); and (iii) C3 corresponds to carbon atoms bonded to a carbonyl or two non-carbonyls (C O or O C O). Chundawat, Venkatesh, and Dale (2007) have reported the contributions of biomass components to these peaks, with cellulose contributes close to 85% of its signal to the C2 peak and the remainder to C3, hemicellulose close to 80% of its signal to the C2 peak and the remainder to C3, lignin 50% of its signal to C1 and the remainder to C2, and extractives most of their signals to C1. The concentrations of C1, C2, and C3 in native corn stover were 65.29%, 20.99%, and 13.72%, respectively, and acid pretreatment resulted in a decrease in the C1 content and an increase in both the C2 and C3 contents. The increase in the cellulose content and accessibility after acid pretreatment may be responsible for the increase in both the C2 and C3 contents, which indirectly decreased the C1 content. Compared with the acid-pretreated substrate, the enzymatically hydrolyzed substrate had a higher C1 content and lower C2 and C3 contents. The chemical analysis of the enzymatic hydrolysates showed that the cellulose hydrolysis was high, up to 58.63%, and the large degree of cellulose hydrolysis also enhanced the relative lignin content. This decrease in cellulose and increase in lignin may have contributed to the increase in the C1 content and the decrease in the C2 and C3 contents.

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Fig. 4. XPS analysis of substrates. (a) Wide-scan spectra of native substrate; (b) high-resolution C1s spectra of native substrate; (c) wide-scan spectra of acid-pretreated substrate; (d) high-resolution C1s spectra of acid-pretreated substrate; (e) wide-scan spectra of enzymatically hydrolyzed substrate; (f) high-resolution C1s spectra of enzymatically hydrolyzed substrate.

3.7. AFM analysis Fig. 5 shows the amplitude images of the native, acid-pretreated, and enzymatically hydrolyzed substrates. Among these substrates, native substrate has the smallest height (28.7 nm) and Rq (3.74 nm) values (Fig. 5a), suggesting an intact and uniform surface structure.

A smooth structure was also observed in SEM images (Fig. 2a). The height and Rq values significantly increased to 206.3 nm and 26.6 nm after acid pretreatment (Fig. 5b); this may be due to hemicellulose hydrolysis by the acid, which heavily damages the intact cellulose-hemicellulose-lignin network. It is also interesting to note that some hemispherical formations which may be lignin (Chandel

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Fig. 5. AFM analysis of substrates. (a) Native substrate; (b) acid-pretreated substrate; (c) enzymatically hydrolyzed substrate.

et al., 2013; Kristensen, Thygesen, Felby, Jorgensen, & Elder, 2008), appeared on the surface. Selig et al. (2007) explained their formation using lignin surface mobilization during high-temperature pretreatment. When the pretreatment temperature was within or above the range of the transition temperatures for lignin in biomass, more fluidized lignin may be forced to coalesce in an aqueous environment; upon cooling, the lignin deposits as droplets onto the biomass surface. The glass transition temperature of lignin was measured to be approximately 100 ◦ C by Hatakeyama, Izuta, Hirose, and Hatakeyama (2002), and the temperature of the pretreatment in the present study was 121 ◦ C, which is near the glass transition temperature and may cause the formation of lignin droplets. However, it should be noted that the degree of lignin rearrangement in this study was not very pronounced compared to that under a much higher pretreatment temperature (Selig et al., 2007). The moderate rearrangement of lignin on the biomass surface may be helpful for the subsequent enzymatic hydrolysis. On the one hand, the relocation of lignin moieties helps to increased cellulose exposure and possibly enhances enzymatic hydrolysis (Chandel et al., 2013). On the other hand, a limited amount of lignin on the substrate surface may not induce the severe enzyme inhibition that often occurs with the non-productive adsorption of cellulase enzymes onto lignin (Kim, Kreke, Ko, & Ladisch, 2015; Ximenes, Kim, Mosier, Dien, & Ladisch, 2011). Compared with the acid-pretreated substrate, the enzymatically hydrolyzed substrate had lower height and Rq

values (78.9 nm and 11.3 nm, respectively) (Fig. 5c). Additionally, the surface was filled with many lignin droplets. Similar results were reported by Hansen, Kristensen, Felby, and Jorgensen (2011), where any structure resembling microfibrils vanished and the surface appeared smoother after enzymatic hydrolysis. As the samples were enzymatically hydrolyzed, lignin droplets were released from the hydrolyzed cell walls and accumulated on the residual lignin or other droplets from the layers below due to their hydrophobic nature, thus increasing the concentration of lignin on the surface, as also revealed by the XPS measurements (Fig. 4). 3.8. SSA and PV analysis The SSA and PV values were 2.06 ± 0.38 m2 /g and 0.0067 ± 0.0011 cm3 /g for the native substrate and 4.33 ± 0.19 m2 /g and 0.019 ± 0.00035 cm3 /g for the acid-pretreated substrate, respectively, comparable to those reported in previous studies (Hsu et al., 2010; Yu, Chen, Men, & Hwang, 2009). The surface area of the substrate can be divided into an interior surface area reflected by the biomass porosity and an exterior surface area largely determined by the particle size. A previous study reported that over 90% of substrate enzymatic digestibility is contributed by accessible pores (Huang, Su, Qi, & He, 2010). For the acid-pretreated substrate, the pore volume accessible to cellulase with a 5.1 nm diameter (Grethlein, 1985) was 0.015 cm3 /g, which was much

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hydrolysis. First, the enzymatic hydrolysis of corn stover following moderate acid pretreatment can further disrupt the substrate structure and achieve a reasonably high cellulose conversion efficiency (chemical analysis, SEM, FTIR, XPS, AFM). Second, the commercial enzyme mixture used in this study does not exhibit a strong tendency toward digesting either crystalline or amorphous cellulose (FTIR, XRD). Thus the enzymatic hydrolysis of corn stover proceeded via simultaneous hydrolysis of crystalline and amorphous cellulose. 4. Conclusions

Fig. 6. Equilibrium adsorption of cellulase to native and acid-pretreated substrates.

larger than the value of 0.0034 cm3 /g for the native substrate. This high cellulose accessibility to cellulase after acid pretreatment may be due to significant alterations in chemical compositions and physical structures. Based on the chemical analysis, the hemicellulose content decreased significantly from 19.70% to 9.28% after pretreatment with dilute acid. It has been proposed that hemicellulose, which is distributed in the interfibrillar space through fiber pores, acts as a physical barrier that limits cellulose accessibility (Meng & Ragauskas, 2014). Pretreatment with dilute acid hydrolyzes the hemicellulose and thus increases cellulose accessibility. Some studies further suggest that this significant improvement in cellulose accessibility is due to the increase in substrate swelling and porosity caused by the acid hydrolysis of hemicellulose (Chen, Tu, & Sheen, 2011; Pu, Hu, Huang, Davison, & Ragauskas, 2013). Indeed, similar phenomena were also observed in the SEM (Fig. 2) and SSA/PV analyses.

This study investigated the dilute acid pretreatment and enzymatic hydrolysis of corn stover using integrated chemical and multi-scale structural methods (chemical analysis, SEM, XRD, FTIR, XPS, AFM, SSA/PV) to characterize both processes. The results revealed that moderate acid pretreatment can remove hemicellulose, redistribute lignin and enhance substrate porosity, significantly improving the enzymatic digestibility of corn stover. Characterization of the corn stover after enzymatic hydrolysis suggested that the currently available commercial enzyme mixture can achieve a reasonably high cellulose conversion efficiency (58.63% of glucose yield). Moreover, the commercial enzyme mixture used here does not have a strong preference for digesting either crystalline or amorphous cellulose. Acknowledgments This study was supported by the National Natural Science Foundation of China (Project No. 31571569), the Beijing Nova Program (Project No. Z131105000413056), the Beijing Youth Talent Plan Program in University (Project No. YETP0317), the Beijing Excellent Talents Cultivation Program (Project No. 2013D009007000001), and the National High-level Personnel of Special Support Program for Outstanding Young Talents (Project No. Zutingzi 2015-48).

3.9. Analysis of integrated chemical and structural characteristics Based on the integrated chemical and structural analyses, dilute acid pretreatment of corn stover has the following effects: (1) removes hemicellulose by cleaving acetyl linkages (chemical analysis, FTIR, XPS); (2) rearranges the distribution of lignin via surface mobilization (AFM, XPS); and (3) enhances substrate porosity via lignocellulose swelling and loosening (SEM, SSA/PV). These alterations will improve cellulose accessibility and thus reduce the resistance of lignocellulose to subsequent enzymatic hydrolysis. For example, the effect of acetyl groups on cellulose accessibility has been investigated, with the results indicating that acetyl groups may restrict cellulose accessibility by inhibiting productive binding by increasing the diameter of the cellulose chain or by changing its hydrophobicity (Kumar & Wyman, 2009). DeMartini et al. (2011) suggested that the pattern of lignin rearrangement could dramatically open up the structure of the cell wall matrix and improve the accessibility of the majority of cellulose microfibrils, which likely explains another critical mechanism for the enhanced digestibility of acid-pretreated substrates. Cellulase adsorption data (Fig. 6) in current study also provided corroborative evidences for the effect of acid pretreatment on cellulose accessibility to cellulase. The maximum adsorption capacity (Emax ) and distribution coefficient (Kr ) of acid-pretreated substrate were approximately three times higher than those of native substrate. The increased times of Emax and Kr were also consistent with those of SSA and PV after acid pretreatment. All these results further confirmed that cellulose accessibility to cellulase was increased after acid pretreatment. Integrated chemical and structural analysis techniques were used to characterize the material after subsequent enzymatic

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