Structural transformations of Hybrid Pennisetum lignin: effect of

Research Article pubs.acs.org/journal/ascecg

Cite This: ACS Sustainable Chem. Eng. XXXX, XXX, XXX−XXX

Structural Transformations of Hybrid Pennisetum Lignin: Effect of Microwave-Assisted Hydrothermal Pretreatment Dan Sun,† Bing Wang,† Han-Min Wang,† Ming-Fei Li,† Quentin Shi,‡ Lu Zheng,‡ Shuang-Fei Wang,§ Shi-Jie Liu,|| and Run-Cang Sun*,†

ACS Sustainable Chem. Eng. Downloaded from pubs.acs.org by UNIV OF WINNIPEG on 11/10/18. For personal use only.

†

Beijing Key Laboratory of Lignocellulosic Chemistry, Beijing Forestry University, No. 35 Tsinghua East Road, Haidian District, Beijing 100083, China ‡ Shanghai Dssun New Material Co., Ltd., Shanghai 200233, China § College of Light Industry and Food Engineering, Guangxi University, No. 100 Daxue East Road, Nanning 530000, China || School of Light Industry and Engineering, South China University of Technology, No. 381 Wushan Road, Tianhe District, Guangzhou 510641, China S Supporting Information *

ABSTRACT: To understand the structural features and transformations of hybrid Pennisetum (HP) lignin, an integrated treatment on the basis of microwave-assisted hydrothermal pretreatment (MHTP) (at 180−200 °C for 10−20 min) followed by 2% NaOH aqueous post-treatment (at 90 °C for 2.0 h) was proposed in this study. The isolated alkali lignins (ALs) were investigated by GPC and FT-IR and comparatively studied with residual lignins (DELs, prepared by the combination of MHTP and following double ballmilling and enzymatic hydrolysis) by HPAEC and advanced NMR techniques. Results demonstrated that the yields of ALs (35.2%−82.3%) after MHTP increased significantly, and the content of the associated polysaccharides (0.1%−1.8%) was much lower than those of the DELs (2.6%−11.1%). NMR characterization revealed that the two classes of lignin fractions were SGH-type, and the content of various linkages (e.g., β-O-4, BE, and α-O-4/β-O-4) and the ratios of S/G and pCE/FA varied regularly with increasing MHTP intensity. Meanwhile, the depolymerization and repolymerization reactions of lignin were found to be simultaneous during the MHTP process, in which the depolymerization was the dominate reaction. The efforts focused on the HP lignin in this study will maximize the utilization of energy crop resources for biorefinery industries. KEYWORDS: Hybrid Pennisetum, Microwave-assisted extraction, Lignin, Structural transformation, NMR

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β-1, and β-β).6 Lignin connects carbohydrates together by different chemical bonds (e.g., benzyl ether, phenyl glycoside, and benzyl ester), forming a lignin−carbohydrate complex (LCC) with a three-dimensional network structure. To some extent, the unique structure of lignin provides compactness and rigidity to cell walls; however, the connections limit the valueadded utilization of carbohydrates (cellulose and hemicelluloses) to produce liquid fuels and biomaterials. Therefore, the utilization of lignin mainly relies on its structural characteristics, and thorough investigation of the structural features of lignin is crucial in current and future biorefineries. Generally, it is extremely significant to obtain a representative lignin sample with high yield and purity through an economic and ecofriendly technique prior to analyzing its structural traits. Hitherto, a host of biorefinery technologies,

INTRODUCTION With the continuous development of industrialization and growing concern over the excessive emissions of greenhouse gases, the demands for energy and fuels become increasingly urgent, which stimulate researchers to explore renewable, sustainable, and comparably cleaner alternatives to improve the current energy crisis.1,2 Due to abundance and sustainability, lignocellulosic biomass from urban wastes, energy crops, forestry, and agricultural residues has been considered to be an ideal alternative resource for producing various valuable products, including chemicals, biomaterials, and energy.3,4 Hence, full utilization of the main component streams (lignin, cellulose, and hemicelluloses) of lignocellulosic biomass has become a feasible strategy to solve the forthcoming exhaustion of conventional sources and serious environment problems.5 Lignin, as the most abundant aromatic polymer, is primarily composed of three units, syringyl (S), guaiacyl (G), and phydroxyphenyl (H) units, which are linked by ether bonds (e.g., α-O-4 and β-O-4) and carbon−carbon bonds (e.g., β-5, © XXXX American Chemical Society

Received: September 14, 2018 Revised: October 29, 2018 Published: November 1, 2018 A

DOI: 10.1021/acssuschemeng.8b04695 ACS Sustainable Chem. Eng. XXXX, XXX, XXX−XXX

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ACS Sustainable Chemistry & Engineering

Figure 1. Scheme for preparation of double enzymatic lignin (DEL), DEL190(20), DEL200(20), and alkali lignins (ALs) from hybrid Pennisetum.

during the MHTP process, which is beneficial for the subsequent delignification by alkali treatment. As one of the energy crops, hybrid Pennisetum (HP) possesses good properties such as high biomass productivity and strong reproductive capacity and has been widely recognized as a promising feedstock to produce biofuels.18 However, there is still a shortage of in-depth studies with respect to the structure of the HP lignin. It is indispensable to expand the knowledge on the structure of HP lignin to realize its maximum utilization. In this study, HP was hydrothermally treated with microwaves under different conditions. The HP and six microwave-assisted hydrothermally pretreated residues were subjected to 2% NaOH aqueous post-treatment to extract alkali lignin fractions (ALs, ALControl, and AL180(10)‑200(200)). Simultaneously, the HP and two microwave-assisted hydrothermally pretreated residues were also treated with double ball-milling and enzymatic hydrolysis to successfully prepare residual lignin fractions (DELs). The structural features of ALs were comparatively studied as compared to those of DELs. More importantly, the structural differentiations between ALControl and AL180(10)‑200(200) were also elaborately investigated by an array of analytical methods, including gel permeation chromatography (GPC), high-performance anion exchange chromatography (HPAEC), Fourier transform infrared (FTIR), high-resolution two-dimensional heteronuclear singlequantum coherence (2D-HSQC), and 31P NMR spectroscopies. The efforts focused on the structural characteristics and

including mechanical ball-milling, steam explosion, hydrothermal treatment, dilute acid treatment, and mild alkali treatment have been applied to isolate lignin fraction from biomass. Previous research showed that lignin can be easily fractionated from grass stem by alkali treatment.7 The alkali treatment is characterized by the selective removal of lignin without degradation of carbohydrates. Meanwhile, the process augments the porosity of cellulose substrates and the accessibility between cellulose substrate and enzyme.8,9 However, to isolate alkali lignin (AL) with high yield and purity, it is necessary to identify an appropriate pretreatment technique for effectively degrading hemicelluloses prior to alkali treatment.10 Among various pretreatment techniques, hydrothermal pretreatment has been generally deemed as an ecofriendly and cost-efficient biorefinery technique due to its water-only medium and chemical-free features, avoiding the occurrence of corrosion phenomena and the formation of neutralization sludges.11,12 Recently, microwave-assisted hydrothermal pretreatment (MHTP) has drawn great attention due to its excellent features of quick, energy-efficient, and homogeneous heating of biomass.13,14 In contrast to the conventional heating mode, the internal heating property of microwave radiation surmounts the shortcoming of temperature gradient and hence increases the potential for industrialization.15,16 Also, a previous study also showed that microwave radiation can change the supermolecular structure of lignocellulosic material and then improve its reactivity.17 Hence, most hemicelluloses in biomass are rapidly degraded B


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Table 1. Yields of ALs and Carbohydrate Contents of All Lignin Fractions (ALs and DELs) under Various Treatment Conditions Carbohydrate contentf (%) Samples

e

Yield (%)

Ara

Gal

Glu

Xyl

Man

GalA

Total

DELa DEL190(20)b DEL200(20)b ALControlc AL180(10)d AL190(10)d AL200(10)d AL180(20)d AL190(20)d AL200(20)d

__ __ __ 35.2 39.7 49.0 44.5 57.1 82.3 66.3

1.3 ND ND 0.3 0.1 0.1 0.1 0.1 0.0 0.0

0.5 0.1 0.1 0.2 ND ND ND ND ND ND

1.4 1.6 1.1 0.3 0.1 0.2 0.1 0.3 0.1 0.1

7.4 1.4 0.8 1.0 0.8 0.5 0.4 1.0 0.2 ND

0.4 0.5 0.6 ND ND ND ND ND ND ND

0.1 ND ND ND ND ND ND ND ND ND

11.1 3.6 2.6 1.8 1.0 0.8 0.6 1.4 0.3 0.1

a

Represents the lignin prepared from hybrid Pennisetum by a double ball-milling and enzymatic hydrolysis. bRepresents the lignins prepared from hybrid Pennisetum by the combination of MHTP under various conditions given and subsequent double ball-milling and enzymatic hydrolysis. c Represents the lignin isolated from hybrid Pennisetum by a 2% aqueous NaOH treatment process without MHTP. dRepresents the lignin isolated from hybrid Pennisetum by an integrated process based on MHTP under various conditions given and subsequent 2% NaOH post-treatment. e Represents the yield of alkali lignin [(weight of lignin obtained by precipitation during the 2% NaOH treatment)/(content of Klason lignin in the raw material or the microwave-assisted hydrothermally pretreated residue)] × 100%. fAra, arabinose; Gal, galactose; Glu, glucose; Xyl, xylose; Man, mannose; GluA, glucuronic acid; ND, not detected. and precipitation in seven volumes of acidic water (pH 2.0) from the supernatant, purification with extensive acidic water, and freezedrying. According to the MHTP conditions and the media of lignin extraction, the obtained seven ALs were named ALControl, AL180(10), AL190(10), AL200(10), AL180(20), AL190(20), and AL200(20). Lignin Acetylation. Generally, the lignin polymers should be first acetylated prior to the GPC detection due to their relatively poor solubility in tetrahydrofuran (THF). The acetylation of ALs in this study was performed in light of previous literature.20 At length, 20 mg of lignin samples was dissolved in an acetic anhydride/pyridine system (v/v, 1:1, 2 mL). Next, the mixture samples were acetylated in a shaking incubator at 20 °C for 24 h in darkness. The mixture samples after acetylation were condensed to 1 mL and further dropped dropwise into 7 mL of anhydrous ether to obtain acetylated lignin samples by centrifuging. The obtained acetylated lignin samples were placed in a fume hood for 24 h to dissipate anhydrous ether. Analysis Procedures. The polysaccharides associated with the DELs and ALs were determined by dilute sulfuric acid hydrolysis of lignin preparations and analyzed by HPAEC according to the literatures.21,22 The weight-average (Mw) and number-average (Mn) molecular weights of the ALs were guaged by GPC (Agilent 1200, USA). After acetylation, ALs were gauged by GPC with an ultraviolet detector at 240 nm on a PL-gel 10 μm mixed-B 7.5 mm ID column, calibrated with PL polystyrene standards. The FT-IR spectra of ALs were performed as reported previously.23 The various hydroxyl (OH) groups in ALs and DELs were determined by 31P NMR spectra, and the concrete operation process was conducted according to a previous literature.24 2D-HSQC spectra of ALs and DELs were recorded on a Bruker AVIII 400 MHz spectrometer at 25 °C, and the quantitative analyses of the substructures and main linkages in ALs and DELs relied on some formulas as summarized in previous literatures.25,26

transformations of HP lignin in this study will expand the opportunities for further value-added utilizations of lignin.

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MATERIALS AND METHODS

Materials. Hybrid Pennisetum was supplied by the National Experiment Station for Precision Agriculture, Xiaotangshan, Beijing, China. Before treatment, the HP stems were milled, extracted with toluene/ethanol (2:1, v/v), and then oven-dried at 60 °C for 12 h. The main chemical components of the extractive free powder was 38.7% cellulose, 29.6% hemicelluloses, and 20.9% lignin (2.0% acidsoluble lignin and 18.9% Klason lignin), which was determined based on our previous report.19 Commercial cellulase (Cellic CTec2, 100 FPU/mL) was purchased from Novozymes (Beijing, China). Preparation of DELs. The preparation procedure of DELs is illustrated in Figure 1a. Specifically, the unpretreated HP stem powder and two pretreated residues were suffered from a ball-milling process for 5 h at a milling frequency of 450 rpm in a planetary ball mill (Fritsch GMBH, Idar-Oberstein, Germany), respectively, and then the milled samples (5 g) and cellulase (100 FPU/mL, 2 mL) were orderly added in sodium acetate buffer (pH 4.8, 100 mL). Next, the mixture samples were incubated at 50 °C for 48 h in a shaking incubator (ZWYR-2102C, Shanghai, China) with a rotational speed of 150 rpm. After sufficient enzymatic hydrolysis of the mixture samples, the residual lignin samples were obtained by centrifuging, washing thoroughly with sodium acetate buffer and deionized water, and then freeze-drying. The dried lignin-rich samples were subsequently suffered from a ball-milling process for 2 h and enzymatic hydrolysis once again. Ultimately, the DEL, DEL190(20), and DEL200(20) samples were collected and named as DELs. Isolation of Alkali Lignin Fractions (ALs). The MHTP processes were implemented using a microwave pressure vessel (Milestone MicroSYNTH, Italy), as shown in Figure 1b. In detail, the HP stem powder (10 g) and deionized water (100 mL) were mixed in a microwave reactor and then heated at 180, 190, and 200 °C for 10 and 20 min each. After MHTP, the residues were collected by filtrating the mixtures through a Buchner funnel. Subsequently, the HP stem powders before and after MHTP (5 g) were treated with the 2% NaOH aqueous (100 mL) at 100 °C for 2 h each. The obtained liquid fractions were neutralized within a pH range of 5.5−6.0 with 6 M HCl and then condensed to one-third of the original solution. After that, with uniform agitating, each condensed liquid was slowly dribbled into three volumes of 95% ethanol followed by the flocculent precipitate (hemicelluloses) appearing. After collecting the hemicelluloses, the alkali-soluble lignins were obtained by concentration

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RESULTS AND DISCUSSION Yields and Associated Polysaccharides of Lignins. The associated carbohydrates of DELs and ALs as well as the yields of ALs are shown in Table 1. The yields of ALControl and AL180(10)‑200(20) were acquired based on the Klason lignins in the raw material and the residues after MHTP, respectively. As shown in Table 1, only 35.2% of the alkali lignin (ALControl) from the unpretreated HP was extracted, while the yield of AL180(10)‑200(10) extracted from the integrated process on the basis of MHTP and 2% NaOH post-treatment significantly increased from 39.7% to 82.3%. The results showed that the C


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Table 2. Weight-Average Molecular Weights (Mw) (g mol−1) and Number-Average Molecular Weights (Mn) (g mol−1), and Polydispersity (Mw/Mn) of Alkali Lignin Fractions (ALs) Released during the Integrated Process Based on MHTP under Various Conditions Given and Following Alkali Post-Treatment

alliance of MHTP and following 2% NaOH post-treatment was effective in deconstructing the tight plant cell wall, contributing to the extraction of lignin with high yields in HP. Moreover, the optimum operating condition of MHTP was found to be 20 min and 190 °C, in which a highest yield of AL190(20) (82.3%) was obtained. In this case, it could be concluded that most of the lignin−carbohydrate complex linkages were fractured as MHTP performed at 190 °C for 20 min. It has been reported that the fracture of chemical bonds between lignin and hemicelluloses leads to the migration and relocation of lignin, which gives a more concentrated distribution within the plant cell wall.27 Therefore, the lignin polymers were easier to extract after MHTP. However, the yield of AL200(10) and AL200(20) decreased dramatically when the MHTP temperature continually increased to 200 °C, which may result from the degradation of lignin at the high temperature given. The reduced lignin yield may also be related to the formation of “pseudolignin”, which was caused by the recondensation reactions between carbohydrates and lignin degradation products under the harsh MHTP conditions.28 Furthermore, all the obtained ALs possessed a high purity, which could be verified by the associated polysaccharides. Distinctly, the content of associated polysaccharides in ALControl (1.8%) was apparently lower than that in DEL (11.1%) as shown in Table 1. The reason for this is that DEL contained a large percentage of unhydrolyzed xylose from hemicelluloses and a small percentage of glucose from cellulose, while these sugars were relatively free in ALControl, suggesting that the alkali-extracted lignin fraction had a higher purity than residual lignin. Also, the cleavage of lignin−carbohydrate complex linkages during MHTP not only resulted in the degradation of hemicelluloses but also in increased contact area between the microwaveassisted hydrothermally pretreated substrates and cellulases. Therefore, the contents of associated polysaccharides in DEL decreased sharply (from 11.1% to 2.6%) after MHTP, which could also be reflected by the weakened xylose signals in the 2D-HSQC spectra of DEL190(20) and DEL200(20). Analogously, the reduced trend emerged in the ALs (from 1.8% to 0.1%). Based on these considerable data, it can be inferred that MHTP is a promising method among the many pretreatment techniques. The combination of MHTP with alkali posttreatment can extract lignin polymers from HP with a high yield and purity. Molecular Weights Determination. The weight-average molecular weights (Mw) and number-average molecular weights (Mn), as well as polydispersity (Mw/Mn) were measured to estimate the effect of the integrated treatment on the molecular structure of ALs. As shown in Table 2, the Mw (2910−4220 g mol−1) of AL180(10)‑200(20) was lower than that (4980 g mol−1) of ALControl, which was mainly ascribed to depolymerization of the lignin macromolecule resulting from the cleavage of β-O-4 ether bonds of lignin, as can be confirmed by the NMR afterward.20 As the MHTP severity increased, the Mw of AL180(10)‑200(20) decreased as expected. However, the Mw of AL190(10) and AL190(20) fractions showed a slightly increased trend, implying that the depolymerization and repolymerization reactions of lignin occurred simultaneously. But importantly, the depolymerization was the dominate reaction during the MHTP process. With regards to polydispersity index (Mw/Mn, PI), the seven alkali lignin polymers presented the comparatively confined molecular distributions (1.31−1.49), suggesting that the integrated

Samples

Mw

Mn

Mw/Mn

ALControl AL180(10) AL190(10) AL200(10) AL180(20) AL190(20) AL200(20)

4980 4170 4220 3090 3760 3770 2910

3340 3120 2930 2360 2670 2560 2150

1.49 1.34 1.44 1.31 1.41 1.47 1.35

process facilitated the formation of homogeneous alkali lignin fragments. FT-IR Spectra. The structural characteristics of lignin polymers can be qualitatively determined by FT-IR spectra. Several absorption regions of FT-IR spectra in ALs were identified based on previous literatures.20,29,30 As shown in Figure 2, a broad and strong absorption band located at 3410−

Figure 2. FT-IR spectra of alkali lignins (ALs) isolated from hybrid Pennisetum.

3430 cm−1 is attributable to the stretching vibration of aliphatic OH groups. The absorbances presenting at 2844 and 2940 cm−1 belong to the vibration sketch of C−H in the methylene and methyl groups, respectively. The intense bands at 1595, 1508−1515, and 1422 cm−1 are characteristic bands of the lignin aromatic skeleton vibrations. Obviously, the intensities of the above three bands weakened with the enhancement of MHTP intensity, whereas they did not disappear even if the sample was treated at a high temperature (200 °C). This suggested that the primary aromatic structure of ALs was not broken during the integrated treatment. As compared with the ALControl, the intensities of the absorbances at 1460 cm−1 (C−H deformation combined with aromatic ring vibration) and 1328 cm−1 (syringyl and condensed guaiacyl units) in the AL180(10)‑200(20) decreased, which was caused by the demethoxylation reactions of lignin during MHTP. A weak band at 1162 cm−1 in ALs is the characteristic band of the D


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Figure 3. 2D-HSQC NMR spectra of double enzymatic lignin (DEL), DEL190(20), and DEL200(20) prepared from hybrid Pennisetum.

signals of α-ethoxylated β-aryl-ether (β-O-4, A′), oxidized syringyl units (S′), and acylated p-hydroxycinnamyl alcohol end groups (I′γ) appearing in the DEL spectra were not found in the ALControl spectra. The prominent signals of pCE, FA, and H in HP lignin (ALs and DELs) were typical of gramineous plant lignin.34 What is noteworthy was that the chemical shift of p-coumarate correlations (pCE8) signal in DEL spectra was δ113.8/6.25 (Table 3), while it shifted to δ115.2/6.29 in ALControl spectra. This suggested that the acylated pCE was cleaved under the alkaline condition to produce free pCE.35 Moreover, no carbohydrates signals were found in ALs spectra, suggesting that the combination of MHTP and following alkali delignification can effectively isolate lignin with a high purity, which was consistent with the tiny quantities of carbohydrates in Table 1. With MHTP, the intensities of some signals in ALControl and DEL spectra changed to some degree. For example, the signal of spirodienones existed in ALControl, whereas it disappeared in AL180(10)‑200(20). The result implied that the β-1 linkage was particularly sensitive to temperature, which made it easy to be degraded during the MHTP process. In this study, the representative signals, such as phydroxyphenyl (H), ferulates (FA), and p-coumarates (pCE) in ALs and DELs exhibited some regularities with the intensification of MHTP condition, which could be revealed by the relative abundances of the basic compositions (e.g., pCE/FA and S/G/H ratios) and those of main linkages (e.g., β-O-4, BE, and α-O-4/β-O-4). It is clear from Table 4 that the relative content of β-O-4 linkages in AL180(10)‑200(20) (27.2− 8.9/100Ar) was lower than that in ALControl (36.8/100Ar). Parallelly, the relative content of β-O-4 linkage in DEL decreased from 51.9 to 5.5/100Ar with MHTP. These results suggested that the β-O-4 linkage in lignin was sensitive to the high temperature, which made lignin macromolecule susceptible to depolymerization during the MHTP process. Regarding to the β-β linkage in ALs, its relative content continued to decline with the increasing pretreatment intensity. In contrary, the relative content of β-5 linkages in

antisymmetric C−O stretching of ester groups of lignin, which may be caused by the saponification of acetate groups or hydroxycinnamates (ferulic acid and p-coumaric) during the alkali treatment.20 In addition, the two bands at 1122 and 1029 cm−1 weakened with the aggravation of MHTP conditions, which further manifested that the G- and S-type lignin units were all subjected to the demethoxylation reactions during the MHTP process, especially at the high temperatures. To verify these results and obtain more explicit structural information on HP lignin fractions, the qualitative and quantitative NMR technique was utilized in the following study. 2D-HSQC NMR Spectra. In recent years, due to the substantial improvement in resolution (over 1D methods), 2DHSQC NMR has become a more advanced and popular technique for the structural characterization of natural polymers such as hemicelluloses and lignin. In this study, 2D-HSQC NMR was utilized to characterize the structural features and transformations of DELs and ALs. The DELs and ALs spectra are depicted, respectively, in Figures 3 and 4. The assignments of a large number of signals are elaborately classified in Table 3, and the identified substructures of all the lignins are sketched in Figure 5 on the basis of the earlier researches.30−33 As shown in Figure 3, the main linkages and substructures in the side-chain region of DEL spectra were β-aryl-ether (β-O-4, A and A′), phenylcoumaran (β-5, C), methoxy groups (OCH3), benzyl ether (BE), acylated and nonacylated phydroxycinnamyl alcohol end groups (I′γ and Iγ). Aside from the basic units of syringyl (S and S′), guaiacyl (G), and phydroxyphenyl (H) lignin units, the signals of p-coumarates (pCE) and ferulates (FA) in the aromatic region of DEL spectra were also distinguished. As for the ALControl, it was found that emerging signals in the spectra of ALControl and DEL exhibited great differences. Typically, in addition to the aforementioned linkages and substrates, resinol (β−β, B), spirodienone (β-1, D), and α, β-diaryl ethers (α-O-4/β-O-4, E) were also found in the spectra of ALControl (Figure 4). The E


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Figure 4. 2D-HSQC NMR spectra of alkali lignins (ALs) isolated from hybrid Pennisetum.

the variation of the average molecular weight of lignins (Table 2). Analogously, the missing signal of β-5 linkage in DEL200(20) was mostly resulted from the depolymerization of lignin. As a common LCC linkage, the signals of BE structure disappeared in DEL200(20) and AL200(20), suggesting that the BE structure was more liable to be degraded under the harsh MHTP conditions. Furthermore, the rations of pCE/FA, S/G, and S/ G/H are also the imperative parameters for tracking the structural changes of ALs and DELs besides the cleavage of ether bonds and degradation/condensation of carbon−carbon

ALControl after MHTP increased from 1.7 to 3.2/100Ar. These results implied that the cleavage of β-O-4 and β-β linkages accompanied by the repolymerization of β-5 linkages during MHTP. However, when the MHTP intensity was further increased, the relative content of β-5 linkages in AL190(20)‑200(20) fractions decreased instead, implying that the β-5 linkages were subjected to the depolymerization under the harsh MHTP conditions. Therefore, it could be inferred that the depolymerization and condensation occurred concurrently during the integrated process, which was in accordance with F


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Table 3. Assignments (ppm) of 13C−1H Cross Signals in 2D-HSQC NMR Spectra of Lignin Fractions (DEL and ALControl) from Hybrid Pennisetum Labels

DEL

ALControl

Assignments

Cβ Bβ Dβ OMe Aγ Iγ Cγ X5 A′γ I′γ Bγ Aα X2 X3 X4 Eα Dα BE Aβ(G) Bα Aβ(S) Cα S2,6 S′2,6 G2 FA2 G5 PCE8 FA8 G6 H2,6 PCE2,6 PA7+PCE7

53.1/3.44 ND ND 55.6/3.70 59.7/3.59 61.2/4.08 62.9/3.89 63.0/3.18 63.0/4.32 64.0/4.79 ND 71.8/4.86 72.8/3.00 73.9/3.25 75.4/3.50 ND ND 82.9/4.96 83.3/4.38 ND 85.8/4.10 87.3/5.45 103.5/6.70 105.1/7.15 110.9/6.98 110.9/7.36 115.5/6.78 113.8/6.25 115.1/6.40 118.9/6.78 127.7/7.18 129.9/7.45 144.5/7.44

52.9/3.48 53.6/3.04 59.8/2.78 55.6/3.70 59.7/3.60 61.2/4.08 62.2/3.71 ND ND ND 71.0/3.79−4.16 71.6/4.84 ND ND ND 78.7/5.57 80.9/5.07 82.0/4.90 84.3/4.37 84.4/4.66 85.8/4.10 87.0/5.52 103.8/6.70 ND 110.8/6.97 110.9/7.28 115.5/6.78 115.2/6.29 116.4/6.38 118.9/6.78 127.7/7.18 129.7/7.51 143.7/7.47

Cβ-Hβ in phenylcoumaran substructures (C) Cβ-Hβ in β−β (resinol) substructures (B) Cβ-Hβ in spirodienones (D) C-H in methoxyls (−OCH3) Cγ-Hγ in β-O-4 substructures (A) Cγ-Hγ in cinnamyl alcohol end groups (I) Cγ-Hγ in phenylcoumaran substructures (C) C5-H5 in β-D-xylopyranoside Cγ-Hγ- in γ-acylated β-O-4 substructures (A) Cγ-Hγ in acylated cinnamyl alcohol end groups (I′) Cγ-Hγ in β-β resinol substructures (B) Cα-Hα in β-O-4 linked to a S unit (A) C2-H2 in β-D-xylopyranoside C3−H3 in β-D-xylopyranoside C4−H4 in β-D-xylopyranoside Cα-Hα in α,β-diaryl ethers (E) Cα-Hα in spirodienones substructures (D) Cα-Hα in benzyl ether (BE) Cβ-Hβ in β-O-4 linked to G/H unit (A) Cα-Hα in β−β resinol substructures (B) Cβ-Hβ in β-O-4 linked to a S unit (A) Cα-Hα in phenylcoumaran substructures (C) C2,6-H2,6 in syringyl units (S) C2,6-H2,6 in oxidized (Cα=O) syringyl units (S′) C2-H2 in guaiacyl units (G) C2-H2 in ferulate (p-FA) C5-H5 in guaiacyl units (G) C8-H8 in p-coumarate (pCE) C8-H8 in ferulate (p-FA) C6-H6 in guaiacyl units (G) C2,6-H2,6 in H units (H) C2,6-H2,6 in p-coumarate (pCE) C7-H7 in ferulate (FA)+C7-H7 in p-coumarate (pCE)

linkages.36 In detail, the increased S/G ratios in DELs (from 1.2 to 2.4) and ALs (from 1.6 to 6.2) suggested that the degradation reaction of S-type lignin was less likely to occur as compared to that of G-type lignin. In fact, the preferentially degraded lignin was mainly the G-type with noncondensed structure and low molecular weight.37 At the same time, the content of H changes regularly with the intensification of MHTP conditions, which can be reflected by the ratio of S/G/ H. It can be deduced from the ratios of S/G and S/G/H that the demethoxylation of G and S units (especially for G unit) conduced to the increase of H-type lignin content. Additionally, the appreciable increase in the pCE/FA ratio clearly demonstrated that a large number of etherified FA units were removed with the enhancement of MHTP intensity. 31 P Spectra. The distribution and amounts of multifarious OH groups in lignin were determined by the quantitative 13P NMR technique. In this study, the 13P NMR spectra of DELs and ALs were sketched in Figures S1 and S2 (Supporting Information), respectively, and the content of the diverse OH groups was calculated in Table 5 based on previous publications.25,38,39 Table 5 showed that both the alkaliextracted lignins (ALs) and residual lignins (DELs) from HP had the following order of OH contents: aliphatic OH > phenolic OH > carboxylic OH. It has been reported that the majority of OH groups derived from the aliphatic side chain of

lignin. Therefore, an obviously increasing trend of the content of aliphatic OH groups in DELs (3.15−7.51 mmol/g) resulted from the disruption of ester or ether linkages in the side-chain of DEL during the MHTP process.40 The content of S- and condensed G-type phenolic OH in DEL increased significantly, which was mostly attributable to the fracture of β-O-4 bonds with MHTP.41 By comparison, the content of noncondensed G-type phenolic OH in DEL decreased, which was likely ascribed to the degradation of G-type lignin fragments during the MHTP process. The result could also be attested by the increasing S/G ratio of DEL190(20) and DEL200(20). In view of the above analysis, it could be concluded that both condensation and depolymerization reactions occurred during the integrated process. As a consequence of MHTP, the cleavage of pCE contributed to a higher content of phydroxyphenyl OH in DEL190(20) and DEL200(20) than that in DEL. It was found that the content of aliphatic OH in ALControl and DEL after MHTP presented an opposite trend, namely, the content of aliphatic OH in ALs decreased (from 5.38 to 1.21 mmol/g) as increment of MHTP severity. This may result from the oxidation and modification of aliphatic OH groups during the integrated process, especially under the MHTP conditions with high temperatures.42 Due to the cleavage of ether bonds, the contents of S-type and condensed G-type phenolic OH in AL180(10)‑200(20) were higher as compared to G


Research Article


Figure 5. Main substructures, different side-chain linkages, and aromatic units identified by 2D-HSQC NMR of double enzymatic lignins (DEL), DEL190(20), DEL200(20), and alkali lignins (ALs) from hybrid Pennisetum: (A) β-O-4 aryl ether linkages with a free −OH at the γ-carbon, (A′) β-O-4 aryl ether linkages with acetylated −OH at γ-carbon, (B) resinols, (C) phenylcoumarans, (D) spirodienones, (E) α, β-diaryl ethers, (BE) benzyl ether, (I) p-hydroxycinnamyl alcohol end groups, (I′) p-hydroxycinnamyl alcohol end groups acylated at the γ-OH, (H) p-hydroxyphenyl units, (G) guaiacyl units, (S) syringyl units, (S′) oxidized syringyl units with a Cα ketone, (pCE), p-coumarate, and (FA), ferulate.

Table 4. Quantification of Lignin Fractions (DELs and ALs) by 2D-HSQC NMR Samples

BE

β-O-4

β-β

β-1

β-5

α-O-4/β-O-4

pCE/FAa

S/Gb

S/G/Hc

DEL DEL190(20) DEL200(20) ALControl AL180(10) AL190(10) AL200(10) AL180(20) AL190(20) AL200(20)

11.8 3.5 ND 9.0 8.7 8.4 6.2 4.1 2.8 ND

51.9 14.5 5.5 36.8 27.2 20.6 21.5 17.1 11.6 8.9

NDd ND ND 3.4 2.5 1.0 0.5 2.7 0.3 0.1

ND ND ND 1.5 ND ND ND ND ND ND

1.3 0.8 ND 1.7 3.2 2.7 3.2 2.6 1.0 0.2

ND ND ND 7.1 4.9 2.3 2.8 2.3 ND ND

3.8 4.9 / 0.8 0.9 2.4 3.1 2.3 2.9 /

1.2 1.9 2.4 1.6 2.0 4.1 6.5 1.6 1.7 6.2

50/42/7 48/25/25 51/21/32 60/38/2 63/32/5 74/18/8 78/12/10 56/35/9 51/30/19 62/10/28

a

Represents the pCE/FA ratio obtained by the equation pCE/FA = 0.5I(pCE2,6)/I (FA2). bRepresents the S/G ratio obtained by the equation S/G = 0.5I(S2,6)/I (G2). cRepresents the S/G/H ratio obtained by the equation S/G/H = {0.5I(S2,6)/[0.5I(S2,6) + I(G2) + I(H2,6)]}/{I(G2)/[0.5I (S2,6) + I(G2) + I(H2,6)]}/{0.5I(H2,6)/[0.5I(S2,6) + I(G2) + I(H2,6)]} dRepresents not detected.

those of ALControl. In addition, the content of H-type phenolic OH (originated from pCE and H units) in ALs increased from 0.30 to 0.67 mmol/g, mostly attributed to the demethoxylation of esterified G units during the MHTP process. This was consistent with the results obtained by 2D-HSQC NMR aforementioned. Overall, the proposed biorefinery process on the basis of MHTP and following alkali post-treatment could release alkali lignins (ALs) from HP with higher yield and purity. Under the optimal MHTP condition (190 °C, 20 min), the yield of

AL190(20) arrived at the peak (82.3%), which was 2.3 times more than that of ALControl. NMR analyses showed that both the alkali-extracted lignins (ALs) and residual lignins (DELs) were SGH-type with a strong predominance of S-type lignins and different quantities of ferulates and p-coumarates. The βO-4 aryl-ether linkages were the primary linkages in these lignin units, followed by benzyl ethers and carbon−carbon bonds, which varied regularly with the aggravation of MHTP. As compared to DELs, the ALs had relative free carbohydrates and fewer β-O-4 linkages. Particularly, some linkages (α-O-4/ H


Research Article

ACS Sustainable Chemistry & Engineering Table 5. Quantification of Lignin Fractions (DELs and ALs) by

31

P NMR (mmol/g)

Samples

Aliphtic OH

Syringyl OH

Condensed Guaiacyl OH

Noncondensed Guaiacyl OH

p-hydroxyphenyl OH

Carboxylic Group

DEL DEL190(20) DEL200(20) ALControl AL180(10) AL190(10) AL200(10) AL180(20) AL190(20) AL200(20)

3.15 7.02 7.51 5.38 3.18 2.93 2.84 2.12 1.65 1.21

0.37 0.65 0.63 0.44 0.80 0.93 1.13 0.80 0.95 1.03

0.07 0.14 0.12 0.09 0.14 0.19 0.24 0.16 0.22 0.26

0.38 0.36 0.28 0.75 0.69 0.71 0.95 0.57 0.71 0.75

0.94 0.49 0.34 0.30 0.42 0.47 0.67 0.44 0.59 0.65

0.03 0.09 0.07 1.14 0.96 0.92 0.94 0.75 0.68 0.64

β-O-4, β-β, and β-1) presented in ALControl were not found in DEL. During the integrated process, the fracture of linkages such as BE and β-O-4 and the reduction of molecular weights accompanied by the depolymerization of lignin macromolecule. Additionally, the content of aliphatic OH in DELs (3.15−7.51 mmol/g) and ALs (1.21−5.38 mmol/g) presented an opposite trend with MHTP. In short, the investigation on the structures and compositions of the lignins isolated from HP under different MHTP conditions will expand the opportunities for producing lignin-based materials and chemicals for biorefinery industries.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acssuschemeng.8b04695.

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(Figure S1: 31P NMR spectra of double enzymatic lignin (DEL), DEL190(20), and DEL200(20) prepared from hybrid Pennisetum. Figure S2: 31P NMR spectra of alkali lignins (ALs) isolated from hybrid Pennisetum. PDF)

AUTHOR INFORMATION

Corresponding Author

*Tel: +86-10-62336903. Fax: +86-10-62336903. E-mail: [email protected]. ORCID

Ming-Fei Li: 0000-0003-3123-0565 Shi-Jie Liu: 0000-0003-3286-8958 Run-Cang Sun: 0000-0003-2721-6357 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We are grateful for the financial support of this study from the National Key R&D Program of China (2017YFB0307903), National Natural Science Foundation of China (31430092), and Fundamental Research Funds for the Central Universities (2015ZCQ-CL-02).

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Structural transformations of Hybrid Pennisetum lignin: effect of

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