Enzymatic Hydrolysis and Physiochemical Characterization of Corn

Jan 6, 2016 - Faculty of Mechanical Engineering & Mechanics, Ningbo University, Ningbo, Zhejiang 315211, China. Energy Fuels , 2016, 30 (2), pp 1154â€...
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Enzymatic Hydrolysis and Physiochemical Characterization of Corn Leaf after H‑AFEX Pretreatment Chao Zhao,*,† Zhongqing Ma,† Qianjun Shao,*,†,‡ Bin Li,† Jiewang Ye,† and Hehuan Peng† †

National Engineering Research Center for Wood-Based Resource Utilization, School of Engineering, Zhejiang A&F University, Lin’an, Zhejiang 311300, China ‡ Faculty of Mechanical Engineering & Mechanics, Ningbo University, Ningbo, Zhejiang 315211, China ABSTRACT: Hydrogen peroxide presoaking prior to ammonia fiber expansion (H-AFEX) was applied to enhance fermentable sugars production from corn leaf. Effects of temperature and H2O2 loading on solid recovery, chemical constitution, and polysaccharide conversion were investigated. Physiochemical characterizations of raw material and H-AFEX-treated substrates were obtained using digital microscopy, X-ray diffraction (XRD), and Fourier transform infrared spectroscopy (FTIR). The results showed that the H-AFEX process was an effective pretreatment for improving enzymatic hydrolysis of corn leaf. The HAFEX-treated substrates under optimal conditions following enzymatic hydrolysis achieved 91.6% glucan conversion, 80.6% xylan conversion, and a total monosaccharide yield of 411.8 g per 1000 g dry biomass. Further studies showed that the H-AFEX process induced morphological modification of corn leaf by removing minerals/extractives, relocating melted lignin, and generating pores. XRD data revealed that the crystallinity index of H-AFEX-treated substrate decreased. The disruption of crystalline structure, the removal of amorphous substances, and the transition of crystalline region to amorphous region were observed. FTIR data demonstrated that H-AFEX pretreatment disrupted hydrogen bonds inter-/intra-cellulose chains, altered the supramolecular structure of cellulose, and caused destruction to the molecular structure of residual lignin. As a result, the HAFEX process significantly reduced the recalcitrance of corn leaf and facilitated biomass conversion to fermentable sugars.

1. INTRODUCTION

complexes; (5) increasing the specific surface area and porosity, and so on.2,10,12 Over the last few decades, various pretreatments including physical, chemical, thermochemical, and biological methods have been developed to enhance enzymatic hydrolysis of corn stover. In summary, the pretreatment of corn stover has three developing trends. (1) From a single method to combined pretreatment. The combined methods may be a feasible way, while a single method is difficult to treat three major constituents simultaneously. Therefore, the combined pretreatments such as alkali-assisted extrusion,13 peracetic acid treatment prior to hydrotropic process,6 combined aqueous ammonia and hydrogen peroxide,14 and microwave irradiation assisted steam explosion (SE-MI)15 are proposed. Compared with single process, the combined pretreatments improve enzymatic digestibility of corn stover in different degrees. (2) From whole corn stover to different parts/tissues. Different parts/tissues of corn stover have differences in morphological cell and tissue organization, which causing different responses to pretreatment and enzymatic hydrolysis.16−18 Investigation by Li et al. shows that the heterogeneous properties of different tissues of corn stover (for example rind, pith, leaf, and stalk) influence their different biological and physiochemical behaviors during biorefinement.17 (3) From reaction parameters optimization to reaction mechanism study. Early studies have focused on the optimization of pretreatment process while they have neglected the elucidation of pretreatment mechanism. At

The transition of the energy system from fossil fuel driven to one that includes more renewable alternatives will alleviate greenhouse gas emissions, enhance energy security, and provide sustainable development.1−3 The annual solar energy captured by lignocellulosic biomass through photosynthesis is almost 10fold than that of the total energy utilized by human beings. Thus, the production of biofuels from renewable lignocellulosic biomass will push forward an immense influence on the future energy system.2 Among them, cellulosic ethanol has taken a lead position as a promising alternative to diminishing supplies of fossil fuels. Agricultural residue is an important part of lignocellulosic biomass.4,5 Maize is the largest crop in terms of global grain production, and there is about 820 million MT byproduct of corn stover per year, which could potentially be available for cellulosic ethanol production.2 The bioconversion of lignocellulosic biomass to bioethanol usually contains four major steps: pretreatment, hydrolysis, fermentation, and purification.6−8 The lignocellulosic plant cell wall is intrinsically recalcitrant for hydrolysis owing to the complicated structure of three major constituents (cellulose, hemicellulose, and lignin) and their complex cross-linking. Therefore, a pretreatment step is critical to reduce biomass intrinsic recalcitrance to enzymatic hydrolysis and make high sugar yield possible.9−11 For an effective pretreatment, several crucial properties should be taken into consideration: (1) breaking the lignin seal with cellulose/hemicellulose; (2) partial removing/dissolving lignin and hemicellulose; (3) disrupting hydrogen bonds inter-/intra-cellulose chains and reducing its crystallinity; (4) cleaving cross-linking in lignin-carbohydrate © XXXX American Chemical Society

Received: December 2, 2015 Revised: January 4, 2016

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DOI: 10.1021/acs.energyfuels.5b02817 Energy Fuels XXXX, XXX, XXX−XXX

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Figure 1. Schematic diagram for H-AFEX pretreatment, enzymatic hydrolysis, and composition analysis of corn leaf. substances were of analytical grade and not further purified before being used as received. 2.2. H-AFEX Pretreatment. Figure 1 shows a schematic diagram interpreting H-AFEX pretreatment, enzymatic hydrolysis, and composition analysis. The pretreatment of corn leaf was performed in a 1 L high-pressure reactor, and the details have been described by Shao et al.7 The pretreatment parameters were as follows: H2O2 loading (the mass ratio of 30 wt % hydrogen peroxide solution to dry material) was 0.1, 0.4, and 0.7, temperature was 90 °C, 110 °C, and 130 °C, water loading (the mass ratio of water to dry material) was 0.7, ammonia loading (the mass ratio of ammonia to dry material) was 1.0, and the reaction time was 10 min. The solid recovery was calculated by eq 1.

present, the chemical modifications within the cell wall and the molecular structure basis causing biomass recalcitrance of some pretreatments such as ammonia fiber expansion,19 hydrothermal pretreatment,20 and liquid hot water and dilute acid4 have been investigated. The pretreatment of hydrogen peroxide presoaking prior to ammonia fiber expansion (H-AFEX) has been assessed for bamboo7 and corn stover21 in our laboratory, which leads to enhanced lignin removal and high enzymatic digestibility. The reagents (ammonia and hydrogen peroxide) used in the HAFEX process contain no carbon and release no greenhouse gases. The large-scale H-AFEX pretreatment manufacturing these chemicals will be sustainable and environmentally friendly. Although high polysaccharide conversion of HAFEX-treated bamboo and corn stover can be achieved, the mechanism of H-AFEX pretreatment still requires further research. Examining physiochemical characterization of raw material and H-AFEX-treated substrate in tandem with the results of enzymatic hydrolysis could provide insight into the fundamental mechanism of H-AFEX pretreatment. The objective of this work was to understand physiochemical characterization changes of corn leaf resulting from H-AFEX pretreatment. The influences of temperature and chemical loading on solid recovery, chemical constitution, and polysaccharide conversion were also investigated. Various physical and chemical characterizations of corn leaf including microscopic morphology, crystallinity, and chemical structure before and after H-AFEX pretreatment were measured by digital microscope system, XRD and FTIR spectroscopy.

Sr(%) = m1/m2 × 100%

(1)

Sr is solid recovery (%). m1 and m2 are the total mass of pretreated biomass and feedstock (g, dry basis), respectively. 2.3. Enzymatic Hydrolysis. Without water washing, enzymatic hydrolysis of H-AFEX-treated substrate was performed following the standard National Renewable Energy Laboratory (NREL) protocol.22 The enzymatic hydrolysis of raw material was also carried out for comparison. Hydrolysis was operated in an incubator shaker (ZHWY111B, Zhicheng Co. Ltd., Shanghai, China) at 150 rpm and 50 °C. Three kinds of enzymes including cellulase (15 FPU/(g of glucan)), βglucosidase (64 CGU /(g of glucan)), and xylanase (1000 IU/(g of glucan)) were added. The total volume of hydrolyzate was adjusted to 15 mL in 20 mL screw-cap vial using 0.05 M sodium citrate buffer (pH 4.8). 40 mg/L of tetracycline and 30 mg/L of cycloheximide were added to prevent bacterial growth. After enzymatic hydrolysis for 72 h, the supernatant was filtered by a 0.2 μm poly(ether sulfone) syringe filter and then frozen at −4 °C until high-performance liquid chromatography (HPLC) analysis. All the experiments were operated in duplicate, and the average value was reported. 2.4. Analysis Methods. The chemical analysis of untreated and HAFEX-treated substrate using two-step acid hydrolysis was conducted following the NREL analytical protocol.22 The moisture content was determined by Sartorius, model MA35, Beijing, China. Acid and enzymatic hydrolyzates were analyzed by the Agilent HPLC system (Agilent 1200 Series) equipped with a Bio-Rad Aminex HPX-87H column and refractive index detector. The chromatographic column temperature was maintained at 50 °C. The mobile phase was 0.005 M H2SO4, and the flow rate was 0.5 mL/min. The glucan and xylan conversions are calculated by eqs 2 and 3.

2. MATERIALS AND METHODS 2.1. Feedstock and Chemicals. Corn leaf, harvested in August 2013 (the growth period about 120 days), was kindly supplied by Santai farm in Mianyang City (31.23, 104.43), Sichuan Province, China. Leaves (sheath included) were peeled off stalks. Air-dried raw material was cut to a nominal size of 3 cm by a shredder. Then, the leaves were ground by a Hammer Mill (FZ102, Taisite Instrument Co. Ltd., Tianjin, China) with a 40 mesh screen sieve. The screened samples were collected in zip-lock bags and stored at −20 °C until further use. Glucose and xylose were obtained from Sigma-Aldrich Chemical Co. Ltd., Shanghai, China; 99 wt % liquid ammonia and 30 wt % hydrogen peroxide solution, from Hangzhou Longshan Chemical Co. Ltd., Zhejiang, China; 98 wt % sulfuric acid, from Kunshan Jincheng Chemical Co. Ltd., Jiangsu, China. Cellulase (Novozyme 50013) and β-glucosidase (Novozyme 50010) were bought from Novozyme Investment Co. Ltd., Beijing, China; xylanase, from Zesheng Bioengineering Technology Co. Ltd., Shandong, China. All chemical

Gc(%) = (mg × 0.9)/pg × 100%

(2)

Xc(%) = (m x × 0.88)/px × 100%

(3)

Gc is glucan conversion (%), mg is the monosaccharide of glucose released in pretreatment and enzymatic hydrolysis (g), pg is the initial polysaccharide of glucan in raw material (g), 0.9 is the conversion factor of glucose to equivalent glucan; Xc is xylan conversion (%), mx is B

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Energy & Fuels Table 1. Effects of H-AFEX Pretreatment on Solid Recovery and Composition Changes in Corn Leaf content (%) methods

H2O2 loadingb (g/g dry biomass)

temperature (°C)

solid recovery (%)

glucanc,d

xylanc

ligninc

glucan recoveryc (%)

xylan recoveryc (%)

lignin removalc (%)

untreated H-AFEXa

0.1

90 110 130 90 110 130 90 110 130

100 98.1 96.1 96.0 97.0 96.2 95.7 96.3 95.3 93.4

28.2 27.6 27.3 26.9 27.1 26.9 25.8 27.4 26.5 24.9

15.6 15.3 15.2 14.9 15.1 14.6 14.4 14.9 14.4 14.0

19.4 18.3 17.3 16.6 17.4 17.0 16.4 17.0 15.7 14.9

97.9 96.8 95.4 96.1 95.4 91.5 97.2 94.0 88.3

98.1 97.4 95.5 96.8 93.6 92.3 95.5 92.3 89.7

5.7 10.8 14.4 10.3 12.4 15.5 12.4 19.1 23.2

0.4

0.7

a

H-AFEX pretreatment conditions: 10 min residence time, 0.7 water loading, 1.0 ammonia loading. bThe H2O2 loading was the mass ratio of 30 wt % hydrogen peroxide solution to dry biomass. cData in table represent percent contents and removal rates based on dry untreated biomass. dHere the glucan was including other glucose-based monosaccharide and oligosaccharides present in corn leaf. the monosaccharide of xylose released in pretreatment and enzymatic hydrolysis (g), px is the initial polysaccharide of xylan in raw material (g), and 0.88 is the conversion factor of xylose to equivalent xylan. The total sugar yield is calculated by the total mass of fermentable sugars (g) including glucose and xylose released per 1000 g dry biomass. 2.5. Analysis on Physiochemical Characterizations of Corn Leaf. 2.5.1. Digital Microscope Analysis. The images of untreated and H-AFEX-treated corn leaf were performed by a Keyence digital microscope system (Keyence VHX-2000, Japan, 50−5000 times). The samples were oven dried at 40 °C for 24 h, and then pasted onto a slide glass through double-sided adhesive for observation. 2.5.2. XRD Analysis. X-ray diffraction of untreated and H-AFEXtreated corn leaf was conducted by a XRD-6000 diffractometer (Shimadzu, Japan). The samples were positioned on a quartz sample holder, and the radiation source was 0.154 nm. Samples were scanned operating at 40 kV and 200 mA under room temperature. The scanning diffraction angle ranged from 10° to 40° at a speed of 2°/min and with a step size of 0.02°. The biomass crystallinity index (CrI) was calculated by eq 4.

CrI = Ic/(Ic + KIa) × 100%

(4)

in which, Ic is the accumulated diffraction intensity of crystalline region in biomass, Ia is the accumulated diffraction intensity of amorphous region, and K is the correction factor, K = 10. 2.5.3. FTIR Analysis. To quantify chemical structure changes in HAFEX-treated substrate, the spectra of untreated and H-AFEX-treated corn leaf were collected by a Fourier transform infrared spectrometer (IR Prestige-21, Shimadzu, Japan). The oven drying samples were mixed with KBr pellet (the mass ratio of sample to KBr was 1:100) and prepared through pressing tablets. Sample spectra were collected at a resolution of 4 cm−1 over the range of 4000−400 cm−1, and 32 scans per sample were conducted.

3. RESULTS AND DISCUSSION 3.1. Effect of H-AFEX Pretreatment on Solid Recovery. The corn leaf used in this study contained about 28.2% glucan, 15.6% xylan, and 19.4% Klason lignin. The composition analysis of raw material corresponded to polymeric form as weight percentage of total dry weight. The maximum potential sugar yield of glucose and xylose were 313.3 and 177.3 g per 1000 g dry biomass, respectively. The solid recovery changes of corn leaf after H-AFEX pretreatment are presented in Table 1. It can be seen from Table 1 that the solid recovery descended gradually with pretreatment severity increasing. The most severity pretreatment conditions (130 °C and 0.7 H2O2 loading) gave the lowest solid recovery (93.4%), and the solid recovery ranged

Figure 2. Glucan/xylan conversion and sugar yield following enzymatic hydrolysis of untreated and H-AFEX-treated corn leaf under different H2O2 loadings: (a) 0.1 H2O2 loading, (b) 0.4 H2O2 loading, (c) 0.7 H2O2 loading. Other pretreatment conditions: 1.0 ammonia loading, 0.7 water loading, and 10 min. The enzymatic hydrolysis was run in duplicate with the mean value reported. The error bar shows the standard deviation.

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Table 2. Crystallinity Changes in Untreated and H-AFEXTreated Corn Leaf methods untreated H-AFEXa

temperature (°C)

Ic (cps)

Ia (cps)

CrI (%)

90 110 130

1.49 1.36 1.30 1.28

13.75 13.24 14.18 14.51

52.04 50.68 47.82 47.05

a

H-AFEX pretreatment conditions: 10 min, 0.7 water loading, 1.0 ammonia loading, and 0.4 H2O2 loading.

Figure 5. FTIR spectra of untreated and H-AFEX-treated corn leaf under different temperatures: (a) untreated, (b) 90 °C, (c) 110 °C, (d) 130 °C. Other pretreatment conditions: 10 min, 0.7 water loading, 1.0 ammonia loading, and 0.4 H2O2 loading.

Table 3. Assignment of Peaks to Chemical Functional Groups by FTIR wave numbers (cm−1)

Figure 3. Microscope pictures of untreated and H-AFEX-treated corn leaf: (a) untreated (200×), (b) H-AFEX-treated (200×). (H-AFEX pretreatment conditions: 130 °C, 10 min, 0.7 water loading, 1.0 ammonia loading, and 0.7 H2O2 loading).

3420

O−H stretching

2920

C−H (CH3 and CH2) stretching aromatic ring CC stretching phenolic O−H stretching amorphous cellulose absorbance C−O−C stretching crystalline cellulose absorbance ratio of amorphous to crystalline cellulose

1650 1380 1098 1050 897 1098/897a a

assignment

associated functional group rupture of hydrogen bonds of cellulose rupture of methyl/methylene group of cellulose/lignin lignin removal and changes in lignin structure changes in lignin structure amorphous region changes in biomass hemicelluloses crystalline region changes in biomass supramolecular structure changes in cellulose

The ratio of intensity at two band positions.

composition changes by H-AFEX process at 1.0 ammonia loading, 0.7 water loading, and 10 min reaction time are also presented in Table 1. In general, the gulcan and xylan recovery decreased with rising temperature and H2O2 loading. The lowest gulcan and xylan recovery, 88.3% and 89.7% respectively, were both obtained at the most severity at 130 °C and 0.7 H2O2 loading. Under other described testing conditions, the glucan and xylan recoveries were both higher than 90%, which suggested that the H-AFEX process could effectively retain polysaccharides. In addition, the lignin removal as seen from Table 1 was 5.7%−23.2% in the range of experimental conditions, and it increased with temperature and H2O2 loading rising. On the one hand, the delignification of H-AFEX-treated corn leaf was

Figure 4. X-ray diffraction spectra of untreated and H-AFEX-treated corn leaf under different temperatures: (a) untreated, (b) 90 °C, (c) 110 °C, (d) 130 °C. Other pretreatment conditions: 10 min, 0.7 water loading, 1.0 ammonia loading, and 0.4 H2O2 loading.

from 93.4% to 98.1% under experimental conditions. Solid recovery was an important factor to evaluate the effectiveness of pretreatment. For a higher solid recovery, the retention of most carbohydrates in treated substrate has become possible, which facilitates downstream of enzymatic hydrolysis.23 3.2. Effect of H-AFEX Pretreatment on Composition Changes. The effects of H2O2 loading and temperature on D

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Figure 6. Overall mass balance of untreated/H-AFEX-treated corn leaf followed by enzymatic hydrolysis (H-AFEX pretreatment conditions: 130 °C, 10 min, 0.7 water loading, 1.0 ammonia loading, and 0.4 H2O2 loading).

about doubled compared with AFEX-treated corn stover.21 Investigation by Chundawat et al. reported that AFEX process significantly destroys cell wall structure and cleaves chemical bonds adjacent in biomass rather than removing lignin and/or hemicellulose.19 However, during the H-AFEX process, lignin easily reacted with hydrogen peroxide, and low molecular weight degradation products were produced. Moreover, the copresence of ammonia and hydrogen peroxide promoted the oxidation and degradation of lignin. Most studies supported that lignin plays a negative role in enzymatic hydrolysis for biofuel production.24,25 Enzyme inhibition of lignin such as wrapping cellulose as a physical barrier hindering cellulase close to glycosidic bond and producing irreversible adsorption on cellulase has been widely recognized. Thus, the partial removal of lignin in pretreatment was beneficial for downstream enzymatic saccharification. On the other hand, the lignin removal of H-AFEX-treated corn leaf (5.7%−23.2%) was much lower than that of H-AFEX-treated corn cob (49.5%−67.4%) under the same pretreatment conditions.26 Thus, there was a significant difference in delignification from different parts of corn stover for the same pretreatment method. This difference possibly resulted from the composition and structure of lignin (either from different subunit composition and/or different interunit bonds adjacent). This result is consistent with the conclusion of Pu et al.4 and Zeng et al.18 Recently, some research focused on unveiling lignin compositional and structural changes during pretreatment, which can reveal an inhibition mechanism caused by lignin, and this has already been implemented in pretreatments such as catalytic hydrothermal pretreatment,20 acid pretreatment,4,27 ethanol organosolv pretreatment,28 and ionic liquid pretreatment.29 The composition and structure changes of lignin during H-AFEX pretreatment and its effect on enzymatic hydrolysis needed to be further studied. 3.3. Effect of H-AFEX Pretreatment on Polysaccharide Conversion. Figure 2 depicts the effects of temperature and H2O2 loading on glucan/xylan conversion and total sugar yield by H-AFEX pretreatment at 1.0 ammonia loading, 0.7 water loading, and 10 min reaction time. The glucan and xylan conversions of untreated corn leaf were 48.9% and 26.2% respectively. It can be seen from Figure 2 that the glucan/xylan conversion and total sugar yield increased synchronously with reaction temperature and/or H2O2 loading rising. The glucan conversion was higher than 80%, and that of xylan was higher

than 70% in the range of experimental conditions, which suggests that the H-AFEX process could effectively facilitate polysaccharide conversion to fermentable sugars. As shown in Figure 2a, the glucan and xylan conversions of H-AFEX-treated corn leaf at 90 °C and 0.1 H2O2 loading were 82.8% and 71.6% respectively, which indicated that the H-AFEX-treated substrate was ready to enzymatically hydrolyze even at low temperature and H2O2 loading. The maximum glucan conversion (93.4%) and xylan conversion (83.4%) were both obtained at 130 °C and 0.7 H2O2 loading (Figure 2c), while the maximum sugar yield (411.8 g/kg dry biomass) occurred at 130 °C and 0.4 H2O2 loading (Figure 2b). When the temperature was 130 °C, higher H2O2 loading (0.7) caused higher conversions of glucan and xylan in the H-AFEX-treated substrate, but resulted in a higher weight loss which caused polysaccharide reduction (Table 1). As a result, the total sugar yield at 0.4 H2O2 loading was slightly higher than that of at 0.7 H2O2 loading. Therefore, as an index of pretreatment efficiency, the total sugar yield depended on both polysaccharide conversion and solid recovery. This result is in line with the investigation by Zhang et al.23 Actually, H-AFEX pretreatment belonged to alkaline peroxide pretreatment. During the H-AFEX process, ammonia could disrupt the crystalline structure of cellulose causing its swelling and simultaneously provided an alkaline environment which was beneficial for hydrogen peroxide decomposition.19 The decomposition of hydrogen peroxide in alkaline environment readily occurs. The active radicals such as hydroxyl radical, perhydroxyl anion, and superoxide anion radical are provided. These active radicals could effectively degrade and oxidize lignin, and simultaneously depolymerize carbohydrates by cleaving diferulate linkages which cross-link polysaccharides.30,31 The cell wall was modified by H-AFEX pretreatment under the copresence of hydrogen peroxide and ammonia. On the basis of all positive changes of H-AFEX-treated substrate, the accessibility of cellulase to embedded cellulosic microfibrils significantly increased, thus improving enzymatic digestibility. 3.4. Effect of H-AFEX Pretreatment on Physiochemical Characterizations of Corn Leaf. 3.4.1. Effect of H-AFEX Pretreatment on Microscopic Morphology. Figure 3 shows micrographs of untreated and H-AFEX-treated corn leaf. As observed in Figure 3a, the surface of untreated corn leaf was contiguous and smooth. A layer of epidermal waxes arranged in parallel and leaf hairs staggered on the sample. Great changes E

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ammonia recycle percolation,32 and aqueous ammonia with hydrogen peroxide.14 The increase of crystallinity in aqueousammonia-based pretreatments is ascribed to the removal of amorphous substances in biomass. The study by Kim et al. reported that there is no basic crystalline structure change in aqueous ammonia pretreatment.33 However, H-AFEX pretreatment broke van der Waals and hydrogen bonds between adjacent cellulose chains under high temperature, changing the crystalline structure and reducing CrI. This is in agreement with the research by Kumar et al.; they state that higher pH pretreatments and thermochemical pretreatments can change the cellulose crystalline structure by disrupting hydrogen bonds of cellulose chains.34 3.4.3. Effect of H-AFEX Pretreatment on Chemical Structure. The FTIR spectra of untreated and H-AFEX-treated corn leaf under varied temperatures at 0.4 H2O2 loading, 1.0 ammonia loading, 0.7 water loading and 10 min reaction time are presented in Figure 5, and the assignment peaks to chemical functional groups according to the literatures are shown in Table 3.6,34,36 The peak near 3400 cm−1 was assigned to O−H stretching, and the H-AFEX process had broadened and decreased in intensity of this peak, suggesting that hydrogen bonds of cellulose were disrupted. The band position at 2920 cm−1 was related to C−H stretching, and the H-AFEX pretreatment produced a significant drop for each substrate (especially under high temperature), indicating that the methyl and methylene portions of cellulose/lignin were ruptured. The band around 1650 cm−1 which was attributed to the aromatic ring CC stretching in lignin decreased obviously after HAFEX pretreatment, suggesting that partial removal/dissolution of lignin. This was consistent with composition analysis in section 3.2. Also, the separation of the peak around 1650 cm−1 (Figure 5b,c) was observed, indicating that the structure of residual lignin had changed. The phenolic unit of lignin at 1380 cm−1 after H-AFEX pretreatment was weaker than that of untreated corn leaf. The study by Mou et al. reported that the phenol hydroxyl groups of lignin have a stronger inhibitory effect on enzymatic digestibility.6 Therefore, the partial elimination of phenolic lignin by H-AFEX pretreatment had a clear benefit for improving enzymatic digestibility. The peak near 1050 cm−1 was attributed to C−O−C stretching of hemicellulose, and the H-AFEX process had decreased the intensity of this peak, suggesting that partial degradation or dissolution of hemicellulose. The slight decrease of the band around 897 cm−1 (ascribed to crystalline cellulose) while a more intense of band around 1098 cm−1 (ascribed to amorphous cellulose) after the H-AFEX process both suggested that an alteration of the supramolecular structure of constitutive cellulose occurred. Compared with the untreated sample, the ratio of amorphous to crystalline region of H-AFEX-treated samples at 130 °C, which is measured by comparing intensities at bands of 1098 and 897 cm−1, increased from 0.82 to about 0.88. The increase in amorphous region was consistent with the result of XRD analysis. 3.5. Mass Balance. Figure 6 depicts the overall mass balance of corn leaf for H-AFEX pretreatment (pretreatment conditions: 1.0 ammonia loading, 0.7 water loading, 130 °C, 10 min, and with a presoaking of 0.4 H2O2 loading) and enzymatic hydrolysis (using cocktail enzymes including cellulase, βglucosidase and xylanase for 72 h). As a result, the glucan and xylan conversions of H-AFEX-treated substrate reached 91.6% and 80.6% respectively. Compared with raw material, the glucose and xylose yield increased from 153.3 to 274.8 g and

were observed in biomass morphology after H-AFEX pretreatment (Figure 3b). First, the surface of H-AFEX-treated leaf had a more rough appearance, which was possibly caused by redeposition of melted waxes. Second, a porous structure was formed on the biomass surface, which was probably because of the removal of extractives/minerals or the dissolution some contents of hemicellulose even probably cellulose. Lastly, some lignin droplets or pseudolignin appearing on the leaf surface suggested that partial lignin melted during H-AFEX pretreatment and relocated on the surface. This was supported by compositional analysis (Table 1). As a result, the disruption of biomass network, the separated and fully exposed microfibrils, and the porosity structure generated by H-AFEX pretreatment are bound to increase the accessibility between biomass and enzymes, thus facilitating biomass conversion to fermentable sugars. This is consistent with previous studies after ammoniabased pretreatments. The fully exposed microfibrils and porosity structure created by ammonization treated corn stover are observed by SEM images.32−34 Chundawat et al. visualized the highly porous structure generated by AFEX pretreatment.19 The creation of this porosity structure can improve the accessibility of biomass to enzymes, thus improving the total fermentable sugar yield by 4−5 fold. The study by Rollin et al. reported that increasing surface accessibility of cellulose is even more important than lignin removal for improving enzymatic digestibility.35 3.4.2. Effect of H-AFEX Pretreatment on Crystallinity. The XRD spectra of untreated and H-AFEX-treated corn leaf under varied temperatures at 0.4 H2O2 loading, 1.0 ammonia loading, 0.7 water loading, and 10 min reaction time are presented in Figure 4. The XRD spectrum of untreated corn leaf showed a main broad peak at 22° (2θ) and two broad humps centered at 16° (2θ) and 27° (2θ, Figure 4a). From a structural point of view, the disappearance of the 16° peak of H-AFEX-treated substrate was possibly due to the partial disruption of hydrogen bonds inter-/intra-cellulose chains and/or possible decrystallization. The shift in peaks (22° and 27°) to lower values was possibly due to an increase in spacing between stacked sheets of cellulose molecules and/or a transition of crystallographic structure. The crystallinity indexes (CrI) of untreated and H-AFEXtreated corn leaf are given in Table 2. The CrI of untreated corn leaf was 52.04%, and the CrI was slightly decreased after H-AFEX pretreatment as illustrated in Table 2. The CrI of HAFEX-treated corn leaf decreased from 50.68% to 47.05% with the temperature increase from 90 to 130 °C. Lower crystallinity of H-AFEX-treated samples clearly here seemed to be correlated with the enhancement of enzymatic hydrolysis. As seen from Table 2, when the temperature was 90 °C, the cumulative diffraction intensity of crystalline region of HAFEX-treated substrate deceased from 1.49 to 1.36 cps compared with untreated sample, and the corresponding part of the amorphous region deceased from 13.75 to 13.24 cps. Therefore, the H-AFEX pretreatment could not only destroy the crystalline structure, but also remove the amorphous substances. When the temperature was 130 °C, the cumulative diffraction intensity of crystalline region continuously fell to 1.28 cps, but the corresponding part of amorphous region rose to 14.51 cps. This result was ascribed to the transition of partial crystalline region to amorphous region. The result of XRD analysis for H-AFEX-treated substrate is inconsistent with the result of aqueous ammonia-based pretreatment, which shows that the crystallinity increased after by aqueous ammonia,33 F

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Energy & Fuels

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46.5 to 137.0 g per 1000 g dry biomass after H-AFEX pretreatment. The total monosaccharide released equaled 411.8 g per 1000 g dry biomass from H-AFEX-treated corn leaf.

4. CONCLUSION H-AFEX pretreatment was an effective pretreatment for improving enzymatic hydrolysis of corn leaf. The carbohydrates were well preserved, while lignin was partially removed. The total sugar yield depended on both polysaccharide conversion and solid recovery. The H-AFEX-treated corn leaf under optimal pretreatment conditions resulted in 91.6% glucan conversion, 80.6% xylan conversion, and a total sugar yield of 411.8 g per 1000 g dry biomass. H-AFEX pretreatment induced morphological and textural changes in lignocellulosic biomass, the fully exposed microfibrils, the porosity structure, and the relocalization of melted lignin, which were demonstrated according to microscopic analysis. The CrI of corn leaf decreased after the H-AFEX process; the disruption of crystalline structure, the removal of amorphous substances, and the transition of crystalline region to amorphous region were observed. FTIR analysis indicated that the H-AFEX process could disrupt hydrogen bonds in inter-/intra-cellulose chains, alter the supramolecular structure of cellulose, partly degrade or dissolve hemicellulose, and cause destruction of the molecular structure of residual lignin. The physiochemical characterization changes of H-AFEX-treated substrates effectively reduced lignocellulosic recalcitrance and thus facilitated biomass conversion to fermentable sugars.

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AUTHOR INFORMATION

Corresponding Authors

*Tel.: +86 571-6374-6877. E-mail: [email protected] (C.Z.). *Tel.: +86 574-8760-0872. E-mail: [email protected] (Q.S.). Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The authors are grateful for Project 31500491 supported by National Natural Science Foundation of China, preresearch Project 2013SWZ03 supported by Research Center of Biomass Resource Utilization, Zhejiang A&F University, and Project 2015FR004 supported by Research Foundation of Talented Scholars of Zhejiang A & F University.

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DOI: 10.1021/acs.energyfuels.5b02817 Energy Fuels XXXX, XXX, XXX−XXX

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DOI: 10.1021/acs.energyfuels.5b02817 Energy Fuels XXXX, XXX, XXX−XXX