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Biofuels and Biomass

Structural characterization of corn stover lignin after hydrogen peroxide presoaking prior to ammonia fiber expansion pretreatment Xianliang Qiao, Chao Zhao, Qianjun Shao, and Muhammad Hassan Energy Fuels, Just Accepted Manuscript • DOI: 10.1021/acs.energyfuels.8b00951 • Publication Date (Web): 17 Apr 2018 Downloaded from http://pubs.acs.org on April 20, 2018

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Structural Characterization of Corn Stover Lignin after Hydrogen Peroxide Presoaking Prior to Ammonia Fiber Expansion Pretreatment Xianliang Qiao†, Chao Zhao*,†,‡, Qianjun Shao§, Muhammad Hassan# †

National Engineering Research Center for Wood-based Resource Utilization, School of Engineering, Zhejiang

A&F University, Linan, Zhejiang 311300, China ‡

Zhejiang Collaborative Center of Efficient Utilization of Bamboo Resources, Zhejiang 311300, China

§

Faculty of Mechanical Engineering & Mechanics, Ningbo University, Ningbo, Zhejiang 315211, China

#

US-Pakistan Centre for Advanced Studies in Energy, National University of Science and Technology, Islamabad

44000, Pakistan

*Corresponding author Tel.: +86 571-6374-6877. E-mail: [email protected]

GRAPHIC ABSTRACT

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ABSTRACT:

The hydrogen peroxide presoaking prior to ammonia fiber expansion (H-AFEX) pretreatment is an effective and promising method for agricultural residues to decrease biomass recalcitrance and enhance enzyme accessibility. To illuminate the lignin's structural changes after H-AFEX process, ball-milled wood lignins were isolated from raw material and H-AFEX-treated corn stover. The features and structure of the obtained lignins were characterized by elemental analysis, GPC (gel permeation chromatography), FT-IR (Fourier transform infrared) spectroscopy and NMR (nuclear magnetic resonance) spectroscopy. The results demonstrated that the H-AFEX-treated lignins had a higher oxygen and nitrogen contents, while a lower carbon and hydrogen contents when compared with those of untreated one. A remarkable decrease in molecular weight of H-AFEX-treated lignin was observed. Ammonolysis, hydrolysis and oxidation reactions were major chemical modifications to lignin, and the cleavages of ferulate and p-coumarate ester bonds, alkyl ether bonds and aryl ether bonds were observed during H-AFEX process. G unit lignin was more easily degraded through demethoxylation, while the inter-unit linkages of resinol and phenyl-coumaran were relatively stable. The study on lignin structure changes during H-AFEX process could reveal the pretreatment mechanism and develop new pretreatment method, with a perspective of reducing biomass recalcitrance and improving the bioconversion of biomass to biofuels or biomaterials. Keywords: Corn stover; Pretreatment; Lignin structure; Molecular weight; HSQC

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NMR analysis 1 INTRODUCTION For human being, the use of renewable energy or materials is the inevitable choice.1,

2

Lignocellulosic biomass is the most inexpensive and abundant organic

carbon source on the earth, and is also the only sustainable source of carbon-based biofuels.3,

4

There are two conversion platforms for biomass utilization:

thermochemical conversion5, 6 and biochemical conversion.7 For the latter, biofuels such as ethanol and butanol could be converted, and various kinds of bio-based materials/chemicals could be derived from glucose or 5-hydroxymethylfurfural.8 However, there is a huge technical bottleneck when biomass utilized through biochemical route, which is known as biomass recalcitrance.9,

10

The biomass

recalcitrance of plant is formed to fight for erosion/digestion of microbes/animals in the evolution of millions of years.9, 11 Researchers have proposed various pretreatment methods for reducing biomass recalcitrance to enhance enzymatic accessibility.12 Pretreatment is considered to be one of key steps in the biochemical process of biomass utilization.13 At present, lignin is thought to be one of main reasons causing biomass recalcitrance.14,

15

Lignin consist of three phenylpropanoid monomers (G unit,

guaiacyl; S unit, syringyl; H unit, p-hydroxyphenyl) which are connected by C−C or ether bonds.16, 17 Generally speaking, lignin inhibits enzymatic hydrolysis through the following ways: (1) wraps cellulose preventing cellulase molecules to access glycosidic bond as a physical barrier,15, 18 (2) connects with carbohydrates forming 3


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lignin carbohydrate complex (LCC) as a chemical barrier,14 (3) adsorbs cellulase and reduces its enzymatic activity toward cellulose.15 Most studies reported that lignin content and polysaccharide conversion rate have a strong negative correlation.14 Therefore, lignin removal is one of the criteria to determine the effectiveness of pretreatment. On the other hand, some researchers reported that some pretreatments can achieve high enzymatic hydrolysis without a large amount of lignin removal in recent years.19 H-AFEX pretreatment, which is developed in our laboratory, also supports this conclusion.20 However, this result has not gotten a reasonable explanation. Gu et al. explained it from a perspective of different lignin locations in the cell wall: the lignin located in the secondary wall where most of polysaccharide in is easier to remove than that located in intercellular layer, and high enzymatic hydrolysis could be obtained when lignin removal from the secondary wall.21 However, this hypothesis has not been confirmed, but the lignin structural difference between secondary wall and intercellular layer has been confirmed.16 Zhao et al. believes that the study on the effects of lignin monomer composition and/or structure on enzymatic inhibitory is far more important than that of lignin content.22 Therefore, the biomass recalcitrance which caused by lignin is associated with its monomer composition, structure, the linkages with polysaccharides and the distribution in biomass.23 Among the three phenylpropanoid monomers (G, S and H unit) of lignin units, the study by Akin shows that the G unit is the main source causing enzyme inhibition.24 Zhu et al. reported that the increase of H unit in steam explosion treated 4


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lignin promotes following enzymatic hydrolysis.25 The study of dilute acid (DA) treated poplar shows that the ratio of S to G unit (S/G) is the key factor causing enzymatic inhibition.26 Pu et al. reported that it is not lignin content but the linkages with polysaccharides and the distribution in the biomass which play an important role in enzymatic inhibition.23 The study of DA-treated populus trichocarpa shows that the deconstruction of lignin is mainly breaking of β−O−4 linkage.27 Investigation by Samuel et al. supports that the β−O−4 linkages in S unit are more easily break.28 From the previous studies, the research of lignin structure changes during pretreatment are mainly concentrated in the acid pretreatments, and the correlation between lignin structure and biomass recalcitrance is not formed a unified understanding. During traditional alkali pretreatment, most of lignin has been removed, and the effects of alkali pretreatment on lignin always have been neglected. However, the novel alkali pretreatments based on ammonia such as AFEX and H-AFEX retains most of lignin.29, 30

The lignin in AFEX/H-AFEX-treated substrates did not affect subsequent efficient

enzymatic hydrolysis.31 Therefore, the influence of AFEX/H-AFEX process on the structure of lignin remains to be further explored, and this has an important significance to reveal the pretreatment mechanism and develop new pretreatment methods. In this study, we would focus on the effects of H-AFEX process on lignin structure. Ball-milled wood lignins which represents lignin original structure were isolated from raw material and pretreated corn stover, and the changes in lignin structure during H-AFEX process were studied. The features and structure of lignin 5


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were characterized by elemental analysis, molecular weight, FT-IR spectroscopy and 2D-HSQC (two-dimensional heteronuclear single-quantum coherence ) NMR spectroscopy. 2 MATERIALS AND METHODS 2.1 Materials and Chemicals Corn stover, was harvested in August 2015 from Lin’an rural area (30.23, 119.72), Zhejiang Province, China. The raw materials were naturally dried. Then the corn stover was cut, oven dryed and milled. The samples were stored at −20 oC until used. Benzene (99.5%), ethanol (99.7%), dioxane (99.5%), dichloromethane (99.5%), and pyridine (99.5%) were provided by Sino-pharm Chemical Reagent Co.,Ltd, Beijing, China. Acetic acid (99.5%), aether (99.5%) and acetic anhydride (98.5%) were provided by Lingfeng Chemical Reagent Co.,Ltd, Shanghai, China. Trichloromethane (99.5%) was provided by Xi-long Scientific Chemical Reagent Co.,Ltd, Guangdong Province, China. Hydrochloric acid (36~38%) was provided by Yonghua Chemical Technology Co.,Ltd, Jiangsu Province, China. Ammonia (99 wt.%) was provided by Longsan Chemical Co. Ltd, Zhejiang Province, China, and hydrogen peroxide solutions (30 wt.%) was provided by Tongsheng Chemical Co. Ltd, Jiangsu Province, China. 2.2 H-AFEX Pretreatment H-AFEX pretreatment was approximated to AFEX pretreatment. Before AFEX process, the sample was presoaked by hydrogen peroxide solution. The H-AFEX pretreatment was detailed by previous study.22 50 g dried materials were presoaked 6


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with 30% hydrogen peroxide solution at 0.5 H2O2 loading (the mass ratio of 30% hydrogen peroxide solution to dry biomass) at room temperature. The mixture was then put into a high-pressure reactor and undergone AFEX process. The reaction conditions of the AFEX process were: 1.0 ammonia loading, 0.7 water loading, 130~170 oC treated for 10~30 min. the ammonia/water loading was defined as the mass ratio of ammonia/water to dry biomass. 2.3 Ball-milled Wood Lignin Isolation from Corn Stover With minor modifications, the procedure of ball-milled wood lignin was shown in Figure 1 according to previous studies.32, 33 It was divided into four steps: (1) Benzene-ethanol extraction

The raw material and H-AFEX-treated corn stover were

extracted with benzene/ethanol (2:1, v/v) for 24 h. Afterwards, the samples were then extracted using 60 oC hot water for 24 h. The extractive-free samples were using 40 o

C vacuum-drying oven for 72 h. (2) Ball mill The dried sample was milled using a

planetary ball mill (QM-3SP2, Nanjing Nanda Instrument Co., Ltd., Jiangsu Province, China) at 250 rpm for 72 h, the mass ratio of steel ball to biomass was 30:1. (3) Lignin isolation

The ball-milled powder were dissolved in dioxane/water (96:4, v/v)

for 24 h. Afterwards, the samples were centrifuged, and the supernatant were collected. The residues were further extracted with dioxane/water twice. Collected all the lignin solution and then evaporated to approximately 100 mL by a rotatory evaporator (RE-52AA, Yron Chemical Instrument Co., Ltd., Shanghai, China) at 40 o

C. Then, the samples were further vacuum-dried at 40 oC to obtain crude lignin. (4)

Purification

The crude lignin was dissolved in the acetic acid/water solution (9:1, 7


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v/v). The mixture was dropped into deionized water to precipitate the lignin. The lignin was separated through centrifugation and freeze-dried. The dried-solid was dissolved in dichloromethane/ethanol solution (2:1, v/v), and then the solution was dropped into ether to precipitate the lignin. Centrifugation and freeze-drying to obtain the purified lignin. 2.4 Lignin Acetylation In order to improve the solubility of lignin samples in deuterium solvent, the lignin was acetylated before the determination of structural characterization. The procedure of lignin acetylation was as follows. 50 mg lignin was dissolved in 5 mL pyridine/acetic anhydride (2:1, v/v). Stirring the mixture for 72 h in the dark at room temperature. Then, the mixture was dropped into 50 mL aether, removing the supernatant and collecting the precipitation. Afterwards, the precipitation was dissolved in 10 mL trichloromethane. The mixture was dropped again into 50 mL aether to produce precipitation, and the precipitation was successively washed by hydrochloric acid and deionized water. The precipitation was centrifuged and freeze-dried leading to acetylated lignin. 2.5 Structural Characterization of Lignin 2.5.1 Elemental Analysis The organic elements of the purified lignins isolated from untreated and H-AFEX-treated samples were determined by a combustion method using elemental analyzer (Elementar, vario MICRO cube, Germany). The elements of carbon (C), hydrogen (H) and nitrogen (N) were directly measured, while the element of oxygen 8


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(O) was deduced from the difference with respect to the total sample. 2.5.2 GPC Analysis The acetylated lignins were dissolved in THF (tetrahydrofuran, 1.0 mg/mL) for determining the molecular weight by GPC (Waters 515, Waters, Maple Street Milford, MA, USA). The GPC system was equipped with four Waters styragel columns (HR0.5, HR2, HR4, and HR6) and an ultraviolet (UV) detector (270 nm). THF was carried as eluate liquid at flow rate of 1 mL/min, and the polystyrene was employed as polymer standards. The number average (Mn) and weight average (Mw) molecular weights of the acetylated lignins were collected and processed by Win-GPC Unity software which related to the calibration curve. 2.5.3 FT-IR Analysis The FT-IR spectra of ball-milled wood lignin isolated from untreated and H-AFEX-treated samples were determined by a Fourier transform infrared spectrometer (Nicolet iS5, Madison, WI, USA). Dried sample (1~2 mg) was mix with spectroscopic grade KBr (100~200 mg). After milling, the mixture was pressed into disk. A total of 32 scans with a 2 cm−1 resolution were signal averaged and stored, and the wave number range scanned was 4000~500 cm−1. 2.5.4 2D-HSQC NMR Analysis The acetylated ball-milled wood lignin (35 mg) was dissolved in 0.5 mL of DMSO-d6, and 2D-HSQC (13C/1H) was performed using a nuclear magnetic resonance spectrometer (AVIII 500, Bruker, Germany). The acquisition parameters of HSQC were: 200 ppm spectra width in 13C dimension while 11 ppm spectra width in 9


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H dimension, 90° pulse angle, 0.15 s acquisition time, 1.5 s relaxation delay, 1JC−H

value of 145 Hz, and acquisition of 256 complex data points and 256 scans. The contours and calculations in all spectra was performed using Bruker Topspin-NMR processing software. 3 RESULTS AND DISCUSSION 3.1 Elemental Analysis of Lignin after H-AFEX Pretreatment Table 1 shows the element analysis of ball-milled wood lignins isolated from raw material and H-AFEX-treated corn stover. The results show that the H-AFEX-treated lignins had a lower C content (55.73~58.86%) and H content (7.14~7.59%) when compared with that of untreated lignin (61.48% C content and 9.40% H content respectively). However, the O content of H-AFEX-treated lignin (L2, L3) was slightly higher than that of untreated lignin. The increase of O content might be due to the oxidation of the lignin side chains. It indicated that oxidation reaction occurred during H-AFEX process in the presence of hydrogen peroxide. Similar reaction was observed during wet explosion pretreatments of wheat straw and corn stover.34 In addition, there was also a small amount of N content (0.23%) in the untreated sample (L1). Generally speaking, there is a strong chemical bond between proteins and lignin. The detection of N content in ball-milled lignin isolated from raw material was contaminated by protein. The is consistent with previous study, which reported that the nitrogen is found in the native ball-milled lignin isolated from corn stover,35 wheat straw,36 and Alfa grass.37 After H-AFEX pretreatment, the N content increased significantly with the increase of pretreatment temperature or the extension of 10


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reaction time, and L4 (170 oC for 10 min) had the highest N content (4.73%). The increase of N content was the reaction product between ammonia and lignin. Furthermore, the amount of the reaction product rose with the increase of pretreatment temperature or the extension of reaction time. From previous studies, ammonolysis reaction between ammonia and the cell wall is the main chemical reaction in anhydrous ammonia pretreatments. N content adulteration are detected in the lignin-rich streams during AFEX pretreatment19 and extractive ammonia (EA) pretreatment.38 The determination of ammonolysis reaction products needs much more detailed work, and they would be identified in Section 3.4. 3.2 Molecular Weight Changes in Lignin after H-AFEX Pretreatment Table 2 depicts the changes in the molecular weight (Mn, Mw) and polydispersity index (PI) of the lignins isolated from untreated and H-AFEX-treated corn stover. A significant decrease of molecular weight of H-AFEX-treated lignin was observed in comparison to that of untreated one. For example, the number-average molecular weight (Mn) of L2 (130 oC for 10 min) was decreased from 4699 (L1, untreated sample) to 3824 g/mol, while the corresponding part of weight-average molecular weight (Mw) was decreased from 7751 to 4954 g/mol. With further pretreatment under harsh conditions such as longer time (30 min, L3) or higher temperature (170 oC, L4), the molecular weight was further decreased. the lowest Mn and Mw, 1285 and 2760 g/mol respectively, were both found in L4 (170 oC for 10 min). These data demonstrated that the H-AFEX process had a remarkable effect on the lignin's molecular weight. Lignin subunits after H-AFEX pretreatment were released by 11


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depolymerization. The depolymerization caused lignin having a smaller shape and size. According to previous studies, the decrease of molecular weight of pretreated lignin are observed in many pretreatment methods, such as EA pretreatment of corn stover,38 ionic liquid pretreatment of birch39 and ethanol organosolv pretreatment of switchgrass.40 However, investigation by Shao et al. shows that about 5% growth in molecular weight with respect to the AFEX-treated lignin.29 Similar phenomenon is observed in DA pretreatment.23 During these pretreatment methods, the fragmentation makes the lignin molecular weight smaller, while the condensation makes it larger. Therefore, the change in the molecular weight of lignin depends on the result of competition between fragmentation and condensation.23, 29 The polydispersity index (PI) of untreated lignin was 1.65 (L1), while that of L2 (1.30) exhibited a decrease, and L4 gave the highest PI of 2.14. Therefore, L2 exhibited relatively narrower molecular weight distribution in comparison to that of L1, which indicated that the H-AFEX pretreatment under mild conditions resulted in homogeneous lignin fragments. However, under the harsh conditions such as longer time (30 min, L3) or higher temperature (170 oC, L4), the homogeneous lignin fragments further depolymerized to smaller lignin fragments with different size and shape, and the heterogeneous lignin fragments resulted in relatively wider molecular weight distribution and higher PI. This is consistent with the features of ethanol organosolv lignin, which showing a reduce in molecular weight and growth in PI.40 However, the high PI of H-AFEX-treated lignin was just due to lignin depolymerization to fragments with different size and shape. The high PI of ethanol 12


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organosolv lignin or DA-treated lignin is the result of the competition between fragmentation and condensation.23, 40 3.3 FT-IR Analysis of Lignin after H-AFEX Pretreatment Figure 2 shows the fingerprint region of FT-IR spectra of the ball-milled wood lignins isolated from untreated and H-AFEX-treated corn stover, and Table 3 provides the corresponding bands and assignments. The bands are assigned according to the published literature.35, 40, 41 The band at 1710 cm−1 was attributed to C=O stretch in ester groups. A reduce in intensity of this peak from H-AFEX-treated lignins demonstrated that the ester bonds in lignin connecting were disrupted. The bands around 1665 cm−1 was attributed to C=O stretch in conjugated carbonyl and carboxyl. A significant growth in intensity of this peak from H-AFEX-treated lignins demonstrated that oxidation happened during H-AFEX process. The bands at 1605, 1514, and 1420 cm−1 were related to aromatic skeletal vibrations, and the bands at 1460 cm−1 was attributed to the C−H deformation combined with aromatic ring vibration. As shown in Figure 2, The similarity of the fingerprint spectra for L1 to L4 indicated that the aromatic skeleton of the lignin did not change remarkably after the H-AFEX process. Generally speaking, thermochemical pretreatment such as hydrothermal process23 or AFEX process20 couldn't destroy the aromatic ring structure of lignin. A recent study reported that the aromatic ring structure of lignin is even stable in ionic liquid pretreatment.39 Syringyl (1330 cm−1), guaiacyl (1260 cm−1) and p-hydroxyphenyl (1166 cm−1) ring C−O stretch, guaiacyl (1024 cm−1) aromatic C−H in-plane deformation, were identified in the spectra of all lignin samples, suggesting 13


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that the corn stover lignin belonged to a typical G-S-H lignin. However, the H unit was a small content. These results are consistent with previous study.35 Compared with untreated sample, the intensity of syringyl (1330 cm−1) ring C−O stretch slightly increased, whereas that of guaiacyl ring C−O stretch (1260 cm−1) and C−H stretch (1024 cm−1) dramatically decreased. This result indicated that the main effect of H-AFEX process on lignin was to break the methoxy of guaiacyl. This is agree with the results of corn stalk in alkaline peroxide pretreatment, which reported that G unit was easier to destroy than S unit under thermochemical pretreatments.20 The band around 1097~1120 cm−1 was attributed to aliphatic ethers in secondary alcohols. A significant reduce in intensity of this peak from H-AFEX-treated lignins demonstrated that the ethers bonds of lignin were broken. 3.4 2D-HSQC Analysis of Lignin after H-AFEX Pretreatment Figure 3 shows the side-chain region (13C/1H, 50~90/3.0~5.5 ppm) of the HSQC spectra of ball-milled wood lignins isolated from untreated and H-AFEX-treated corn stover, while Figure 4 shows the aromatic region (13C/1H, 90~150/6.0~8.0 ppm) information. The assignments and integration value of quantitative NMR spectra were analyzed according to previous publications.32, 38, 40 The structures of the identified lignin subunits were presented in Figure 5. As shown in Figure 3, the signals for A′γ (δC/δH 64.0/4.3 ppm) after H-AFEX pretreatment (Figure 3, L2~L4) was gradually disappeared with the increase of pretreatment temperature or the extension of reaction time when compared with that of untreated one (L1). This was the direct evidence of ester bonds cleavage during 14


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H-AFEX pretreatment. This is agree with the AFEX19 and EA42 process of corn stover. They reported that the major chemical modifications to lignin in the ammonia-based pretreatment is the cleavage of various esters in the cell-wall-linkages. In the lime pretreatment of switchgrass, Samuel et al. concluded that the cleavage of ester linkages is due to the saponification reaction.43 The absence of I′γ (δC/δH 64.9/4.8 ppm) and the presence of Iγ (δC/δH 62.2/4.1 ppm) after H-AFEX pretreatment indicated that the cleavage of alkyl ether bonds. The inter-unit linkage of Aβ−G (δC/δH 84.3/4.4 ppm) had a lower signal intensity after H-AFEX pretreatment, suggesting that the disruption of aryl ether bonds or the degradation of G units. This is consistent with the ionic liquid pretreatment of poplar, G unit lignin is more easily degraded, and a slightly elevated S/G ratio is observed.44 Investigation by Shao et al. reveals that the S/G ratio of lignin isolated from AFEX-treated switchgrass elevates from 0.81 to 0.85 when comparing with that of untreated sample.29 Most publications reported that the higher S/G ratio is beneficial for the enzymatic hydrolysis of cellulose. The high S/G ratio could be achieved by different ways, such as increasing the S unit40 or decreasing the G unit44 or both. However, the inter-unit linkages of resinol (B, δC/δH 83.7/5.1 ppm), phenyl-coumaran (C, δC/δH 67.1/3.6 ppm) had shown no big difference from L1 to L4, which indicated that the substructures of B and C were relatively stable under the described reaction conditions. Yang et al. studied the lignin structure changes in corn stalk of cooking with solid alkali and H2O2 pretreatment, and agreed with the stability of resinol (B) and phenyl-coumaran (C) structures.45 The same results are also found in ionic liquid pretreatment of poplar.44 15


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As shown in Figure 4, two signals for tricin (T6, δC/δH 99.5/6.2 ppm, and T8, δC/δH 94.9/6.6 ppm) in the aromatic region were not previously reported in corn stover lignin (Figure 4, L1). Tricin also appears in the lignin isolated from wheat straw,46 elephant grass47 and bamboo.32 Wen et al. concluded that tricin occurs in less lignified regions of plants and is prevalent in grass lignins.32 The absence of T6 and T8 signals after H-AFEX pretreatment (Figure 4, L2~L4) demonstrated that the disruption of aryl ether bonds. The presence of Jβ (δC/δH 125.6/6.8 ppm) after H-AFEX process was the evidence of oxidation reaction. In the HSQC spectra of the native corn stover lignin (L1), the significant signals for C2,6/H2,6 correlation at δC/δH 104.3/6.7 ppm was attributed to S unit, and those at δC/δH 104.7/7.3 ppm was attributed to C2,6/H2,6 correlation of S′ unit. The G unit gave different signals for C2/H2, C5/H5 and C6/H6 at δC/δH 111.6/7.0, 115.4/6.8 and 119.5/6.8 ppm as labelled G2, G5, and G6, respectively. However, the H unit was not observed. Therefore, the main structures of corn stover lignin were S and G units. Most studies gave the conclusion that the corn stover lignin is a typical G-S-H grass lignin, while pure H unit occurs in low amounts (1~2%).35 According to the FT-IR analysis of L1, there was a small amount of H unit (Figure 2). However, the amount of H unit was too small to generate a signal in the HSQC spectra. By contrast, the H unit generated significant signals at δC/δH 130.2/7.1 ppm for C2,6/H2,6 correlation after H-AFEX pretreatment (L2~L4), and the most severity reaction conditions (L4) gave the highest signal intensity of H unit. These results indicated that demethoxylation happened during H-AFEX pretreatment. Combined with the previous analysis of side-chain 16


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region of HSQC spectra, the cross linkage of structure Aβ to G unit (Aβ-G) reduced significantly (Figure 3). It indicated that the demethoxylation preferentially occurred in G unit, and after demethoxylation the G unit transformed to H unit. This result is agree with the ionic liquid pretreatment of poplar, and [C2mim][OAc] pretreatment selectively degrades the G unit of lignin, especially under higher temperatures.44 In addition, in the HSQC spectra of the native corn stover lignin (Figure 4, L1), the ferulate (FA) showed prominent signal for Cα/Hα (FAα) correlation at δC/δH 145.4/7.4 ppm, and the p-coumarate (pCA) gave different correlations for C2,6/H2,6 (pCA2,6) and Cβ/Hβ (pCAβ) at δC/δH 131.1/7.5 and 114.5/6.3 ppm, respectively. Ferulate which was associated primarily with important lignin-polysaccharide crosslinking, and p-coumarate which acylated both polysaccharides and lignin, featured heavily in native corn stover lignin (Figure4, L1). These data suggested that ferulate and p-coumarate were incorporated into lignin through ester or ether interlinkages. It was observed that the signals for FAα and pCAβ were gradually disappeared with increase of pretreatment temperature or the extension of reaction time during H-AFEX pretreatment. On the other hand, the feruloyl amide (FAM) showed prominent different signals for Cα/Hα (FAMα) and C5/H5 (FAM5) correlation at δC/δH 140.0/7.3 and 119.4/6.4 ppm respectively. The p-coumaroyl amide (pCAM) showed significant signal for Cα/Hα (pCAMα) correlation with the same position of FAMα, and signal for C3,5/H3,5 (pCAM3,5) correlation with the same position of FAM5, especially in L4. This was the chemical reaction evidence between cell wall and ammonia. The ferulate and p-coumarate were ammoniated and gave the 17


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corresponding nitrogenous compounds such as feruloyl amide and p-coumaroyl amide. Therefore, the major chemical modifications to lignin during H-AFEX pretreatment was the ammonolysis of ester-linked ferulate and p-coumarate. These results are consistent with previous studies. NMR analysis of AFEX-treated lignin revealed that the major chemical reaction to lignin is to cleave the ferulate and p-coumarate esters in the cell wall, resulting in the formation of corresponding amides (FAM and pCAM).19, 48 The FAM and pCAM are also identified in the NMR spectra of EA lignin stream, which are chemical species deriving from their ester counterparts.38 Therefore, ammonolysis reaction was the major cell wall deconstruction occurring during anhydrous ammonia-based pretreatments. In summary, ammonolysis, hydrolysis and oxidation reactions were major chemical modifications to lignin during H-AFEX pretreatment. The cleavages of ferulate and p-coumarate ester bonds, alkyl ether bonds and aryl ether bonds were observed during H-AFEX process. However, inter-unit linkages of resinol and phenyl-coumaran were relatively stable. G unit lignin was preferentially degraded through demethoxylation. A slightly increase S/G ratio was observed after H-AFEX pretreatment. According to previous studies, lignin has different degradation mechanisms in different environments. For example, the major chemical modification to lignin under acidic pretreatment conditions is fragmentation by acidolysis of β−O−4 linkages.23 Similarly, most studies including organosolv pretreatment of switchgrass40 and ionic liquid pretreatment of poplar44 reveal that the main mechanism of lignin depolymerization is the cleavage of β−O−4 linkages. However, 18


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this is not the case in alkaline pretreatment. A recent study showed that EA pretreatment preserves much of sensitive β−aryl ether units of lignin, offering the potential to co-produce biofuels or biochemicals under the conception of biorefinery.42 The chemical modification to lignin under alkaline pretreatment is the cleavage of various esters in the cell-wall-linkages.38 4 CONCLUSION The H-AFEX-treated lignins had a higher oxygen and nitrogen contents, while a lower carbon and hydrogen contents compared with those of untreated sample. A remarkable reduce in molecular weight of lignin was observed after H-AFEX process. The H-AFEX pretreatment under mild conditions resulted in homogeneous lignin fragments due to depolymerization. However, under the harsh conditions, the homogeneous lignin fragments further depolymerized to smaller lignin fragments with different size and shape, resulting in a higher polydispersity index. Tricin was identified in the corn stover lignin. The cleavages of ferulate and p-coumarate ester bonds, alkyl ether bonds and aryl ether bonds were observed during H-AFEX process. G unit lignin was preferentially degraded through demethoxylation. However, inter-unit linkages of resinol and phenyl-coumaran were relatively stable, and the aromatic skeleton of the lignin did not change remarkbably after the H-AFEX process. The comprehensive analysis of lignin structure changes upon H-AFEX pretreatment provided insights into lignin structure and biomass recalcitrance, with a perspective of improving the bioconversion of biomass to biofuels or biomaterials.

19


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AUTHOR INFORMATION Corresponding Author* Tel.: +86 571-6374-6877. E-mail: [email protected]. Notes The authors declare no competing financial interest.

ACKNOWLEDGMENTS This work was supported by National Natural Science Foundation of China (No: 31500491, 51706207), Commonweal Project of Science and Technology Agency of Zhejiang Province of China (No. 2017C32068, LGN18B06000).

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3592. (38) Da Costa Sousa, L.; Foston, M.; Bokade, V.; Azarpira, A.; Lu, F.; Ragauskas, A. J.; Ralph, J.; Dale, B.; Balan, V., Isolation and characterization of new lignin streams derived from extractive-ammonia (EA) pretreatment. Green Chem. 2016, 18, (15), 4205−4215. (39) Wen, J.; Sun, S.; Xue, B.; Sun, R., Quantitative structures and thermal properties of birch lignins after ionic liquid pretreatment. J. Agr. Food Chem. 2013, 61, (3), 635−645. (40) Hu, G.; Cateto, C.; Pu, Y.; Samuel, R.; Ragauskas, A. J., Structural characterization of switchgrass lignin after ethanol organosolv pretreatment. Energ. Fuel. 2012, 26, (1), 740−745. (41) Wang, S.; Ru, B.; Lin, H.; Sun, W.; Luo, Z., Pyrolysis behaviors of four lignin polymers isolated from the same pine wood. Bioresource Technol. 2015, 182, 120−127. (42) Da Costa Sousa, L.; Jin, M.; Chundawat, S. P. S.; Bokade, V.; Tang, X.; Azarpira, A.; Lu, F.; Avci, U.; Humpula, J.; Uppugundla, N.; Gunawan, C.; Pattathil, S.; Cheh, A. M.; Kothari, N.; Kumar, R.; Ralph, J.; Hahn, M. G.; Wyman, C. E.; Singh, S.; Simmons, B. A.; Dale, B. E.; Balan, V., Next-generation ammonia pretreatment enhances cellulosic biofuel production. Energ. Environ. Sci. 2016, 9, (4), 1215−1223. (43) Samuel, R.; Foston, M.; Jiang, N.; Allison, L.; Ragauskas, A. J., Structural changes in switchgrass lignin and hemicelluloses during pretreatments by NMR analysis. Polym. Degrad. Stabil. 2011, 96, (11), 2002−2009. (44) Wen, J.; Yuan, T.; Sun, S.; Xu, F.; Sun, R., Understanding the chemical transformations of lignin during ionic liquid pretreatment. Green Chem. 2014, 16, (1), 181−190. (45) Yang, Q.; Shi, J.; Lin, L.; Peng, L.; Zhuang, J., Characterization of changes of lignin structure in the processes of cooking with solid alkali and different active oxygen. Bioresource Technol. 2012, 123, 49−54. (46) Del, R. J.; Rencoret, J.; Prinsen, P.; Martinez, A. T.; Ralph, J.; Gutierrez, A., Structural characterization of wheat straw lignin as revealed by analytical pyrolysis, 2D-NMR, and reductive cleavage methods. J Agr. Food Chem. 2012, 60, (23), 5922−5935. (47) Del, R. J.; Prinsen, P.; Rencoret, J.; Nieto, L.; Jimenez-Barbero, J.; Ralph, J.; Martinez, A. T.; 24


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Gutierrez, A., Structural characterization of the lignin in the cortex and pith of elephant grass (Pennisetum purpureum) stems. J Agr. Food Chem. 2012, 60, (14), 3619−3634. (48) Chundawat, S. P. S.; Vismeh, R.; Sharma, L. N.; Humpula, J. F.; Da Costa Sousa, L.; Chambliss, C. K.; Jones, A. D.; Balan, V.; Dale, B. E., Multifaceted characterization of cell wall decomposition products formed during ammonia fiber expansion (AFEX) and dilute acid based pretreatments. Bioresource Technol. 2010, 101, (21), 8429−8438.

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Table 1. Element analysis of the ball-milled wood lignins Samples

Elemental composition (%)

C/H

C900 formula

0.23

6.54

C900H1650O317N2.9

33.95

3.19

7.81

C900H1383O411N44.2

7.32

31.70

4.05

7.78

C900H1388O376N54.9

7.59

28.82

4.73

7.76

C900H1392O331N62.0

C

H

O

N

L1a

61.48

9.40

28.90

L2b

55.73

7.14

L3b

56.93

L4b

58.86

a

Ball-milled wood lignin isolated from raw material (L1).

b

Ball-milled wood lignins isolated from H-AFEX pretreatment substrates. H-AFEX pretreatment conditions: 0.5

H2O2 loading, 1.0 ammonia loading, 0.7 water loading. 130 oC for 10 min (L2), 130 oC for 30 min (L3), 170 oC for 10 min (L4).

26


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Table 2. Number-average (Mn) and weight-average (Mw) molecular weight and polydispersity index (PI) of the ball-milled wood lignins Samples

Mn (g/mol)

Mw (g/mol)

PIc

L1a

4699

7751

1.65

L2b

3824

4954

1.30

L3b

1799

3185

1.77

L4b

1285

2760

2.14

a

Ball-milled wood lignin isolated from raw material (L1).

b

Ball-milled wood lignins isolated from H-AFEX pretreatment substrates. H-AFEX pretreatment conditions: 0.5

H2O2 loading, 1.0 ammonia loading, 0.7 water loading. 130 oC for 10 min (L2), 130 oC for 30 min (L3), 170 oC for 10 min (L4). c

PI- polydispersity index, PI = Mw/Mn

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Table 3. Assignment of infrared absorption bands of lignin Wave number (cm−1)

Assignment

1710

C=O stretch in ester groups

1665

C=O stretch in conjugated carbonyl and carboxyl

1605,1514,1420

Skeleton vibration of aromatic rings

1460

C−H deformations asymmetric in CH3 and CH2

1370

Phenolic O−H stretching

1330

C−O stretch in syringyl ring

1260

C−O stretch in guaiacyl ring

1166

C−O stretch in p-hydroxyphenyl

1120,1097

Aliphatic ethers in secondary alcohols

1024

C−H stretch in guaiacyl ring

28


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Figure captions: Figure 1. The flowchart of ball-milled wood lignin isolated from corn stover. Figure 2. The FT-IR spectra of ball-milled wood lignin isolated from untreated (L1) and H-AFEX-treated corn stover (L2~L4). H-AFEX pretreatment conditions: 0.5 H2O2 loading, 1.0 ammonia loading, 0.7 water loading. 130 oC for 10 min (L2), 130 o

C for 30 min (L3), 170 oC for 10 min (L4).

Figure 3. The 2D-HSQC spectra of ball-milled wood lignin isolated from untreated (L1) and H-AFEX-treated corn stover (L2~L4) (side-chain region). H-AFEX pretreatment conditions: 0.5 H2O2 loading, 1.0 ammonia loading, 0.7 water loading. 130 oC for 10 min (L2), 130 oC for 30 min (L3), 170 oC for 10 min (L4). Figure 4. The 2D-HSQC spectra of ball-milled wood lignin isolated from untreated (L1) and H-AFEX-treated corn stover (L2~L4) (aromatic region). H-AFEX pretreatment conditions: 0.5 H2O2 loading, 1.0 ammonia loading, 0.7 water loading. 130 oC for 10 min (L2), 130 oC for 30 min (L3), 170 oC for 10 min (L4). Figure 5. The main structures in the lignin preparations. (A) β−O−4, aryl−alkyl ether; (A′) β−O−4, aryl−alkyl ether with acylated γ−OH with p-coumaric acid; (B) resinol structure formed by β−β/α−O−γ linkages; (C) p-coumaran structure formed by β−5/α−O−4 linkages; (G) guaiacyl unit; (S) syringyl unit; (S′) oxidized syringyl unit linked with a carbonyl or carboxyl group at Cα; (H) p-hydroxyphenyl unit; (T) tricin; (J) cinnamyl aldehyde end-groups; (I) p-hydroxycinnamyl alcohol end-groups; (I′) p-hydroxycinnamyl alcohol end-groups acylated at the γ−OH; (FA) ferulate; (pCA) p-coumarate; (FAM) feruloyl amide; (pCAM) p-coumaroyl amide. 29


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Dried corn stover Extracted with benzene/ethanol solution (2:1, v/v) for 24 h Extracted with hot water 60 o C for 24 h Vacuum-dried with P2 O 5 40 o C for 72 h

Extractive-free samples Ball milled, weight ratio of steel ball to biomass 30:1, 250 rpm for 72 h Vacuum-dried with P2 O 5 40 o C for 72 h

Ball milled powder Extracted with dioxane/water solution (96:4, v/v) for 24 h Repeat 2 times

Centrifuged at 10,000 rpm for 10 min

Cellulose-rich residue

Lignin in solution Rotatory evaporated and concentrated Vacuum-dried with P2 O 5 40 o C for 72 h

Crude lignin Purified with acetic acid/water solution (9:1, v/v) Purified with dichloromethane/ethanol solution (2:1, v/v)

Purified lignin

Figure 1.

30


1024

1120 1097

1166

1260

1330

1370

1460 1420

1514

1605

1665

L4

Absorbance

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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1710

Page 31 of 34

L3

L2

L1

1800

1700

1600

1500

1400

1300

1200

1100

1000

Wave number (cm−1)

Figure 2.

31


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Figure 3

32


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Figure 4

33


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OMe O

R MeO

γ

MeO

OH

γ O O β α OH

β

O α

OH

OMe

O

R

B

MeO

C

2

6 MeO

OMe

G

OH

O

O

R

OMe

O

OMe

2

6

O

O

O

OH

OMe

OMe

γ

A′

2

5

HO

α

O

OH 6

O

OMe

O

A

O

6

2

OMe O

R

S

R

S′

O

H

HO

MeO

O

H

RO

O

γ

γ

OMe 8

O

HO

β

O

6

OH

OH

OMe

OH

OMe

I

O

I′

J

H2N

O

O

H2N

O

O

O

β

α

α

6 OMe

α

2

6

FA

O

O

O

T

O

OMe

O

5 O

pCA

O

OMe

FAM

5

3 O

pCAM

Figure 5.

34


Structural characterization of corn stover lignin ... - ACS Publications

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