Comparison of the Structural Characteristics of Cellulolytic Enzyme

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Comparison of the structural characteristics of cellulolytic enzyme lignin preparations isolated from wheat straw stem and leaf Bo Jiang, Tingyue Cao, Feng Gu, Wenjuan Wu, and Yongcan Jin ACS Sustainable Chem. Eng., Just Accepted Manuscript • DOI: 10.1021/ acssuschemeng.6b01710 • Publication Date (Web): 02 Nov 2016 Downloaded from http://pubs.acs.org on November 8, 2016

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ACS Sustainable Chemistry & Engineering

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Comparison of the structural characteristics of cellulolytic enzyme lignin preparations

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isolated from wheat straw stem and leaf

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Bo Jiang,† Tingyue Cao,† Feng Gu,‡ Wenjuan Wu,† Yongcan Jin*,†

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†

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Nanjing Forestry University, Nanjing 210037, China

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‡

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Yancheng 224051, China

Jiangsu Co-Innovation Center of Efficient Processing and Utilization of Forest Resources,

School of Chemistry and Chemical Engineering, Yancheng Institute of Technology,

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Mailing address: [email protected]; [email protected];

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[email protected]; [email protected]; [email protected]

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* Corresponding author

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Dr. Yongcan Jin

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Laboratory of Wood Chemistry

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Department of Paper Science and Technology

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Nanjing Forestry University

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159 Longpan Rd., Nanjing 210037, China

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E-mail address: [email protected]

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Tel.: +86(25)8542 8163; Fax: +86(25)8542 8689

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ACS Paragon Plus Environment


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ABSTRACT Lignin structure has been considered as an important factor that significantly

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influences the biorefinery processes. In this work, the effect of ball milling on the structural

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components and extractable lignin in enzymatic residues was evaluated, and the structural

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characteristics of the cellulolytic enzyme lignin preparations isolated from wheat straw

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stem (SCEL) and leaf (LCEL) were comparatively investigated by a combination of

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nitrobenzene oxidation (NBO), ozonation, infrared spectroscopy and 1H–13C heteronuclear

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single quantum coherence nuclear magnetic resonance (2D HSQC NMR). The results

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showed that 4 h ball-milled samples were good enough for structural analysis with high

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lignin yield. Both of CELs are typical p-hydroxyphenyl-guaiacyl-syringyl lignin which

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associated with p-coumarates and ferulates. However, the structure of lignin in wheat straw

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stem is rather different from that in leaf. Compared to stem lignin, leaf lignin has lower

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products yield of NBO and ozonation, lower erythro/threo ratio and higher condensation

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degree. The analysis of 2D HSQC NMR indicated that the S/G ratio of SCEL was 0.8,

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which is about twice as much as that of LCEL. The flavone tricin is incorporated into both

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stem and leaf lignins. The content of tricin in LCEL is higher than that in SCEL.

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KEYWORDS: Wheat straw, Stem, Leaf, Cellulolytic enzyme lignin (CEL), Structural

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characteristics

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INTRODUCTION Lignin is one of the most abundant aromatic biopolymers and a major component of

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plant cell walls. It is mainly composed of the monolignols p-coumaryl, coniferyl, and

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sinapyl alcohols which give rise to the p-hydroxyphenyl (H), guaiacyl (G) and syringyl (S)

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lignin units.1 The chemical utilization of lignin is supposed to be an important part of the

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lignocellulosic biorefinery, but the complex morphological structure restricts its wide

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application. Lignin is even associated with carbohydrates (in particular with hemicelluloses)

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via covalent bonds to form a tight compact structure such as lignin-carbohydrate complex

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(LCC).2 In herbaceous plants, hydroxycinnamic acids (p-coumaric and ferulic acids) are

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attached to lignin and hemicelluloses via ester and ether bonds as bridges between them

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forming carbohydrate-ether-hydroxycinnamate-ester-lignin complexes,3, 4 which result in

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the structure of non-wood lignin being much more complex than wood lignin. Therefore, it

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is nearly impossible to separate lignin from lignocellulose solely and keep their native state.

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Alternatively, cellulolytic enzyme lignin (CEL) has commonly been used for the

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structural analysis of cell wall lignin, which utilizes cellulolytic enzyme hydrolysis prior to

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dioxane/water extraction of ball-milled wood meal to remove carbohydrates and achieve

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lignin with high yield and purity.5, 6 For decades, a lot of work was devoted to

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understanding the structural features of lignin from plant cell wall, for example, the

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monomeric content of lignin polymer and some other structural features were analyzed with

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different chemical degradation methods such as alkaline nitrobenzene oxidation,7

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ozonation,8 thioacidolysis,9 and derivatization followed by reductive cleavage that uses

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acetyl bromide for derivatization and zinc for reductive cleavage.10 These wet chemical

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methods can be very precise for specific functional groups and structural moieties. 3



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However, each chemical method gives limited information (mainly uncondensed lignin

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units) and is not able to provide a general picture of the entire lignin structure.

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In recent years, the analytical methods of nuclear magnetic resonance (NMR) for

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lignin characterization have been significantly improved. NMR has the advantages of the

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analysis of the whole lignin structure and direct detection of lignin moieties, including the

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presence of aryl ether, condensed and uncondensed aromatic and aliphatic carbons.11, 12

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Additionally, two-dimensional heteronuclear single quantum coherence (2D HSQC) NMR

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has been developed to quantify the lignin structures and LCC linkages. The

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semi-quantitative 2D NMR could be an ideal experiment for the estimation of specific

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lignin structures, and provides information on the interunit linkages.13, 14 For example, del

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Río et al.15 investigated milled wood lignin from wheat straw and found it is a G-S-H type

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lignin associated with p-coumarates and ferulates. Rencoret et al.16 investigated cellulolytic

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lignin in brewer’s spent grain and found the lignin presents a predominance of G units and

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the main substructures present are β–O–4’ followed by β–5’, β−β’ and 5–5’ linkages. The

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flavone tricin was present in these lignins, as also occurred in other grasses.17, 18

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Wheat straw has been considered as one of the most important feedstocks for the

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production of chemicals, materials and fuels via biorefinery technology. Lignin structure is

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one of the key factors that influence the processes of biorefinery such as pretreatment and

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enzymatic hydrolysis. Researches indicated that the structures of herbaceous lignin in leaf

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are rather different from that in stem. For instance, Markovic et al.19 studied the structure of

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acid detergent lignin (ADL) in alfalfa leaf and stem by the attenuated total reflectance

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Fourier transform infrared (FTIR), and the results indicated the spectra of ADL from leaf

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and stem are similar in frequency of absorption bands, but different in their intensities. Min 4


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et al.20 investigated the structure of lignin in corn stover and pointed out that stem lignin

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had higher contents of p-coumaric acid, ferulic acid and β–O–4’ linkages but with lower

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contents of β–5’, β–β’ linkages and lower ratio of p-hydroxyphenyl/guaiacyl (H/G). The

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alkaline nitrobenzene oxidation (NBO) data showed stem lignin had higher products yield

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and syringaldehyde/vanillin (S/V) ratio than leaf lignin. These findings suggest that

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structural differences between stem and leaf in herbaceous lignin may result in different

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biorefinery processes. As Jin et al.21 reported that, the enzymatic sugar recovery of sodium

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carbonate-pretreated wheat leaf was higher than that of wheat stem, and the different

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structure of lignin in stem and leaf might be one of the important influence factors.

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In this paper, the CEL protocol was used to isolate lignin preparations from wheat

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straw stem and leaf. The CEL preparations were characterized by destructive (alkaline

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nitrobenzene oxidation and ozonation) and nondestructive methods (2D HSQC NMR and

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FTIR spectroscopy) for understanding the difference of the structural characteristics

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between lignin in wheat straw stem and leaf.

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MATERIALS AND METHODS

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Materials. Wheat straw (Triticum aestiuium) was collected from Yancheng, Jiangsu,

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China in May, 2011. The materials were classified into stem and leaf (sheath included) by

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hands, and then were ground using a Wiley mill. The particles passed through 20 mesh

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(0.85 mm) sieve were collected. The straw meals were extracted with ethanol/benzene (1:2,

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v/v) for 48 h to obtain extractive-free samples. No specific step was carried out to remove

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protein. The extracted samples were air dried and subsequently vacuum dried.

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Cellulase from Trichoderma reesei (NS 50013, 84 FPU/mL), β-glucosidase from

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Aspergillus niger (NS 50010, 350 CBU/mL) and xylanase (NS 50014, 850 FXU/mL) were 5



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generously provided by Novozymes (Novo Nordisk A/S, Demark). The chemicals used in

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this study were purchased from Wako Pure Chemical Industries, Ltd. (Osaka, Japan) and/or

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Tokyo Chemical Industry Co., Ltd. (Tokyo, Japan).

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Isolation of CEL. The procedure for the isolation of CEL from wheat straw stem and

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leaf is illustrated in Figure 1. The vacuum dried straw (2 g in each bowl) was milled in a

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planetary ball mill (QM-3SP2, Nanjing Nanda Instrument Plant, China) at a fixed

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frequency of 600 rpm. Two 100 mL zirconium dioxide bowls with 16 zirconium dioxide

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balls (1 cm diameter) in each bowl were used in the milling. The milling time was 2–6 h to

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obtain milled straw samples with different milling degrees. An interval of 5 min was set

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between every 15 min of milling to prevent overheating. After ball milling, the straw

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powder (MS and ML for stem and leaf, respectively) was carefully collected and dried

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under vacuum.

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The ball-milled sample (5 g) was suspended in 100 mL acetate buffer at pH 4.8, and an

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enzyme cocktail mixed by NS 50013, NS 50014 and NS 50010 with a ratio of 1 FPU : 1.2

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FXU : 1 CBU was added in a 250 mL Erlenmeyer flask and then incubated in a shaker

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(DZH-2102, Jinghong, Shanghai, China) at 180 rpm and 50 °C. The charge of mixed

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enzyme based on cellulase activity was 60 FPU/g-cellulose. After 72 h of enzymatic

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hydrolysis, the mixture was centrifuged to remove the supernatant. The residue was washed

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by centrifugation for 3 times using sodium acetate buffer and deionized water, respectively.

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The washed enzymatic residues of ball-milled stem and leaf (EMS and EML) were

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freeze-dried and then extracted twice (2 × 24 h) with 50 mL of 96% aqueous dioxane (v/v)

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under nitrogen atmosphere. The supernatants were combined and the solvent was removed

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by vacuum evaporation. The dried crude lignin samples were purified by 90% (w/w) acetic 6


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acid.22 The obtained CEL preparations from stem and leaf were named SCEL and LCEL,

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respectively. No further purification was performed for the preservation of the structural

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features of the lignin preparations.

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Extractable lignin measurement. Extractable lignin23 was used to evaluate the

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solubility of lignin in enzymatically hydrolyzed residues. Twenty milligram of sample was

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suspended in 10 mL 96% (v/v) aqueous dioxane. The mixture was magnetically stirred for

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48 h at room temperature. The extract was separated by centrifugation and 5 mL of the

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supernatant was reduced with 1 mg of NaBH4 in 1 mL of 0.05 M NaOH for 24 h and then

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was neutralized with 4 mL of glacial acetic acid. The same process was duplicated on 96%

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(v/v) aqueous dioxane without sample suspension for the preparation of reference. The UV

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absorbance at 280 nm was measured to calculate the amount of lignin using 13 L/(g·cm)

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from sweetgum23 as the gram absorptivity.

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Analytical methods. Lignin and sugar content of the samples were analyzed using the

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NREL protocol.24 The Klason lignin (KL) content was taken as the ash free residue after

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acid hydrolysis. The hydrolysate was collected for the determination of the acid-soluble

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lignin (ASL) and the structural sugars. The ASL was measured by absorbance at 205 nm in

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a UV-vis spectrometer (TU-1810, Beijing Puxi, China) and 110 L/(g·cm) as absorptivity

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value was used which is an average of several reported values. The monomeric sugars were

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quantitatively measured with a high performance liquid chromatography (HPLC, Agilent

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1200 Series, Santa Clara, CA) equipped with the refractive index detector (RID). The

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HPLC analysis was carried out using a Bio Rad Aminex HPX-87H 20n exclusion column

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(300 × 7.8 mm, Bio-Rad Laboratories, Hercules, CA) with a Cation-H Refill Cartridge

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guard column (30 × 4.6 mm, Bio-Rad Laboratories, Hercules, CA). The ash content was 7



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determined by combustion at 575 °C. Alkaline nitrobenzene oxidation and ozonation were carried out according to the

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procedure reported by Chen25 and Akiyama et al.,8 respectively. 2D NMR spectra of the CELs were recorded at 25 °C on an AVANCE III 600 MHz

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instrument (Bruker, Switzerland) equipped with a cryogenically cooled 5 mm TCI

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z-gradient triple-resonance probe. The lignin preparations (50 mg) were dissolved in 0.5

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mL of deuterated dimethyl sulfoxide (DMSO-d6) according to the method previously

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described.26, 27 The central solvent peak was used as the internal reference (δC/δH 39.5/2.50).

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The HSQC experiments used Bruker’s “hsqcetgpsp.2” adiabatic pulse program with

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spectral widths from 0 to 16 ppm (9615 Hz) and from 0 to 165 ppm (24900 Hz) for the 1H-

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and 13C-dimensions. The number of collected complex points was 2048 for the

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1

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increments were recorded in the 13C-dimension. The 1JCH used was 145 Hz. Processing

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used typical matched Gaussian apodization in the 1H-dimension and squared cosine-bell

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apodization in the 13C-dimension. Prior to Fourier transformation, the data matrices were

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zero-filled to 1024 points in the 13C-dimension.

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H-dimension with a recycle delay of 1.5 s. The number of transients was 64, and 256 time

FTIR spectra of SCEL and LCEL were recorded using a VERTEX 80V FTIR

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spectrometer (Bruker, Germany). Around 2 mg of lignin samples was mixed with 400 mg

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KBr, then determined after grinding and tabletting. The scan resolution was 4 cm–1 and the

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scan area was 4,000–400 cm–1.

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RESULTS AND DISCUSSION

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Effect of ball milling time on structural components and extractable lignin in enzymatic residues. The chemical composition of wheat straw stem was rather different 8


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from that of leaf as shown in Table 1. After enzymatic hydrolysis, the glucan and xylan

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content was very low in the hydrolyzed residues of both wheat straw stem and leaf.

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However, the target products, SCEL and LCEL, still contained a certain amount of xylan

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after 1,4-dioxane/water extraction. The removal of xylan was less than that of glucan, it

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indicated that the lignin and hemicelluloses are present in cell walls not only as a simple

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mixture but through chemical linkages.28 Ball milling leads to the structural modification of

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total lignin, such as the increase of carbonyl content, the decrease of molar mass and

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cleavage of aryl ether bonds. Lu and Ralph29 pointed out that ball milling destroys the

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side-chain structure of lignin by the cleavage of β–O–4’ bonds and the increase of

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α-carbonyl group in a certain degree. Ikeda et al.30 investigated the effect of ball milling on

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lignin structure and found the drops of etherified β–O–4’ linkages and the increases of

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phenolic β–O–4’ linkages occurred during the ball-milling process. However, the effect of

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ball milling on total lignin was different from isolated lignin preparations,31 in particular,

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the amount of β–O–4’ in the total lignin decreases progressively with ball milling, but it is

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rather constant in MWL and CEL.6, 32 Furthermore, Capanema et al.32 compared MWL and

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CEL preparations from three kinds of hardwood and pointed out that the yield of the lignin

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preparations increases linearly with the milling time in the interval of 2.5–12.5 h and the

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yields of CEL preparations are about twice as high as those of the corresponding MWLs. In

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contrast, the S/G ratio does not change in the total lignin, but fluctuate in MWL and CEL

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depending on the yield. Ball milling in this work helps improve substrate enzymatic

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digestibility because more saccharides, especially hemicelluloses, were released.

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Fujimoto et al.23 and Hu et al.6 described that if the extractable lignin yields from milled woods are the same, the structural changes of lignin caused by the ball milling are 9



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similar regardless of the difference in milling conditions and apparatus. In this study,

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extractable lignin was introduced as a general criterion to evaluate the milling degree and

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the potential yield of isolated lignin. Figure 2 shows the extractable lignin yield of

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enzymatically hydrolyzed stem and leaf. Extractable lignin yield of stem was significantly

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improved when the ball milling time improved from 2 h to 4 h. The increase of extractable

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lignin yield leveled off when the ball milling time was over 4 h. This result indicated that 4

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h ball milling is good enough for isolating lignin by 96% 1,4-dioxane/water extraction and

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the yield of extractable lignin in stem, on the basis of lignin in raw material, could reach

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68.3%. On the basis of lignin in the enzymatic residue, more than 85% of the lignin in 4 h

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ball-milled stem could be extracted after enzymatic hydrolysis. The extractable lignin of

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leaf was much lower than that of stem. This was potentially caused by the structural

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differences between leaf lignin and stem lignin, or by the more non-lignin components in

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leaf.33

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Fujimoto et al.23 studied the quantitative evaluation of milling effects on lignin

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structure during the isolation process of milled wood lignin (MWL) by ozonation. The

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results indicated that the proportion of β–O–4’ linkages showed declining trend as the

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extension of ball milling time. The ozonation products yield and erythro to threo ratio (E/T)

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decreased with the increase of extractable lignin yield. The structure of erythro form β–O–4’

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was broken preferentially in the process of ball milling, but the degree would not exceed

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25%.6 The degree for 4 h ball-milled wheat straw stem and leaf in this work was only 3.2%

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and 4.7%, respectively. Therefore, CEL preparations obtained through 4 h ball milling time

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in this work were representative for investigation of lignin interunit linkages.

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Structural characteristics of lignin during CEL isolation. Alkaline nitrobenzene 10


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oxidation and ozonation were performed to investigate the effects of ball milling and

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enzymatic hydrolysis on the structural characteristics of lignin in wheat straw stem and leaf.

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The results are given in Table 2. The difference of NBO products yield between stem (2.23

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mmol/g-lignin) and leaf (1.60 mmol/g-lignin) suggested the condensation degree of lignin

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in leaf was higher than that in stem. Compared to lignin in raw materials and enzymatic

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residues, the isolated CELs had higher NBO products yield. It implied that wheat straw

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CEL featured with a lower condensation degree than original lignin. Due to the high

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proportion of condensed guaiacyl units, only about 30% of them are converted to vanillin.

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On the contrary, the conversion of syringyl units to syringaldehyde may be as high as 90%

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due to the low proportion of condensation.25, 34 After 4 h ball milling, the decrease of NBO

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products yield was 1.3% for stem, while it was 6.3% for leaf. The effects of ball milling

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time in this work (2–6 h) on aromatic structure of lignin were not significant since no

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obvious changes of S/V/H ratio were observed. However, while CEL well represents the

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total biomass lignin in softwood6 and hardwood32, this is apparently not the case for

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non-wood lignins, specifically for the wheat straw ones in this work, as indicated S/V/H

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ratio data of CELs vs total lignin (Table 2). This is likely due to more heterogeneous

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structure of non-wood lignins.

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The ozonation products yield and E/T ratio of lignin decreased with the ball milling

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hours. For example, in 4 h ball-milled stem, the decrease of ozonation yield and E/T ratio

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were 7.1% and 3.2%, respectively. However, the ozonation products yield of LCEL (0.46

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mmol/g-lignin) was higher than that of raw material (0.33 mmol/g-lignin). Compared with

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stem, leaf had lower ozonation yield. It might be caused by all these yields were based on

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the sum of Klason lignin and acid-soluble lignin, while the Klason procedure cannot 11



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discriminate between true lignin and lignin-like materials in leaf.35 Salamanca et al.33 also

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pointed out that the Klason lignin may include both lignin and other non-hydrolyzable

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products. In untreated leaf, the Klason lignin residue originated from components highly

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resistant to degradation by H2SO4, and the Klason lignin residue greatly overestimated the

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real lignin content of leaf. Some lignin-like materials in leaf contribute to the amount of

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Klason lignin residue,36, 2 and these materials were easily removed during lignin isolation

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process,36 as a result, the extracted leaf lignin showed both higher NBO and ozonation

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products yield than lignin in original leaf and its enzymatic hydrolysis residue. The E/T

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ratio of leaf lignin was lower than that of stem lignin, it is in good agreement with the result

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of NBO analysis which showed leaf lignin had low S units content. The ratio of syringyl to

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guaiacyl stereo-chemically governed the proportion of erythro and threo forms of β–O–4’

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structures during lignin formation.8

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1

H–13C HSQC NMR analysis. 1H–13C NMR is a powerful tool to probe structures of

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lignin and its derivatives. The signals relate to the structural units and various linkages

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between units of lignin in 2D NMR spectra can be assigned according to the published

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literatures.12, 15-17, 37 The NMR spectra of CEL preparations from wheat straw stem and leaf

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are illustrated in Figure 3, and the detailed assignments of main peaks of SCEL and LCEL

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in NMR spectra (δC/δH 150–50/8.0–2.5) are listed in Table S1. Figure 4 depicts the major

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lignin substructures shown in Figure 3. A semi-quantitative analysis based on HSQC

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signals was performed using Bruker’s Topspin 2.1 processing software and the integral

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method was according to the method described by del Río et al.15

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As shown in Figure 3, signals from p-hydroxyphenyl (H), guaiacyl (G), and syringyl (S) units were observed clearly in the isolated SCEL and LCEL. The prominent signals 12


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corresponding to p-coumarate (PCA) and ferulate (FA) structures were observed which

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were typically identified in gramineous plant lignin.12, 16, 17, 27, 37 The NMR spectra indicated

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that β–O–4’ was the main interunit linkages of lignin, followed by phenylcoumarans and

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other minor linkages, such as resinols, spirodienones, dibenzodioxocins and α, β-diaryl

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ethers. The signals of the β–5’ structure in wheat straw is much more intensive than that of

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β–β’ structure. It indicates that the condensation degree of G units is higher than that of S

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units, which could be used to explain the reason of S/V increment in isolated lignin

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samples.38 The intensive signals derived from tricin (T) were detected which acted as

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antioxidants, antimicrobial and antiviral agents in vascular plants.18 Tricin is considered to

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be fully compatible with lignification reactions and is an authentic lignin monomer.17 Tricin

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linked to lignin units via 4’–O–β–ether bonds had been reported.17, 39

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Polysaccharide signals (for X), mainly originated from hemicellulose, were found in

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the spectra, including xylan correlations in the range δC/δH 65–80/2.5–4.5, which partially

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overlapped with some lignin signals. As shown in Figure 3, the polysaccharide cross-peak

285

signals of X2 (δC/δH 72.9/3.14), X3 (δC/δH 74.1/3.32), X4 (δC/δH 75.6/3.63), X5 (δC/δH

286

63.2/3.26 and 3.95) were assigned to β-D-xylopyranoside.40 These polysaccharide signals

287

evidenced that lignin was mainly linked with xylan via covalent bonds which caused

288

difficulties in effective separation of components in a technical scale. LCC is primarily

289

composed of γ-ester, benzyl ether and phenyl glycoside and is one of the main factors

290

causing recalcitrance of biorefining.41 In the process of lignin purification by 90% (w/w)

291

acetic acid, LCC substructures especially benzyl ether and phenyl glycoside structures were

292

most removed which reduced the signals overlap of lignin and carbohydrate in the 2D

293

NMR spectra of lignins. 13



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

294

The relative amounts of the main lignin interunit linkages and the molar abundances of

295

the different lignin units (H, G and S), p-coumarates, ferulates, and the molar S/G ratios of

296

the lignins in wheat straw, estimated from volume integration of contours in the HSQC

297

spectra, are given in Table 3. The percentage of these structural units was calculated by

298

referring these structural unit signals to the total number of aromatic rings (H+G+S). The

299

data indicated that the main substructures present are β–O–4’ alkyl aryl ether both in SCEL

300

(64%) and LCEL (56%), while other linkages referring as the condensed structures (β–β’

301

resinols, β–5’ phenylcoumarans and β–1’ spirodienones) were present in minor amounts. In

302

particular, the contents of β–β’, β–1’ interunit linkages were similar, but the content of β–5’

303

substructures in LCEL was higher than that in SCEL, that was caused by the higher

304

condensation degree of leaf lignin, which may correlate with S/G ratio of stem and leaf

305

lignins. Furthermore, tricin and its derivatives were believed to protect plants from

306

pathogens,42 the high amounts of tricin in wheat straw are remarkable (12-17%) which may

307

induce the isolation and purification of tricin for potential application such as food and

308

medicine fields even though the physiological function of tricin in plants remains poorly

309

understood.

310

Comparatively, in aromatic/unsaturated region of the HSQC spectra, the signal of G

311

units is obviously more intensive than H and S units both in SCEL and LCEL. The result of

312

LCEL from semi-quantitative 2D NMR analysis is consistent with the results of NBO.

313

However, the data of SCEL from NMR are not consistent with the NBO results because the

314

condensation degree of G units is higher than that of S units. Additionally, the content of G

315

units in LCEL (71%) was higher than that in SCEL (54%) which had more S units content,

316

it induced a significant difference on S/G ratio between SCEL (0.8) and LCEL (0.4). The 14


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317

NBO results showed the leaf lignin exhibits a higher degree of condensation (Table 2). The

318

more branched and condensed G units such as 5–5’, 4–O–5’ units not only acted as a

319

surface barrier, but restricted the swelling of lignocellulose and reduced the accessible

320

surface area available to the enzyme.43 However, the linear lignin contained more S units

321

that could adsorb on the cellulose surface more tightly which blocked the accessibility of

322

cellulose dramatically.44 Comprehensively, the effects of lignin on lignocellulosic

323

saccharification may depend on S/G ratio as reports pointed out that lignin with high S/G

324

ratio is negative on biomass enzymatic digestibility in Miscanthus.45, 46 Therefore, the lower

325

S/G ratio in leaf lignin may explain why the enzymatic sugar recovery of sodium

326

carbonate-pretreated21 and green liquor-pretreated47 wheat leaf was higher than that of

327

wheat stem. Comparing the ratio of S/G/H in 2D HSQC NMR spectra with the ratio of

328

S/V/H in nitrobenzene oxidation, apparent differences were observed and that was because

329

the p-coumarates and ferulates in wheat straw contributed to the H and V units under

330

alkaline nitrobenzene oxidation condition, respectively.48 Alkaline nitrobenzene oxidation

331

can only detect the non-condensed guaiacyl, syringyl and p-hydroxyphenyl units, and it

332

measures the S/V of releasable syringaldehyde (minor syringic acid) and vanillin (minor

333

vanillic acid) monomers from oxidative cleavage of the side chain.20 NMR does profile the

334

“entire” lignin (the isolated lignin) in principle including the condensed and the

335

non-condensed parts of lignin. Although some contours of aromatic parts of composing

336

units (G and H units) were overlapped with the contours of aromatic parts of p-coumarate

337

and ferulate esters, the value of S/G ratio was still calculated by using the reported

338

assignments of the aromatic contour of each composing unit.26 Even though including the

339

ferulates in G units and p-coumarates in H units when correlating 2D NMR data with the 15



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

340

NBO ones, the discrepancies were still considerably obvious. Santos et al.49 pointed out that

341

wood lignin has a good linear relation between S/G and S/V ratio, and the S/G ratio of

342

lignin can be predicted by multiplying the S/V value by a constant (0.806). However, an

343

earlier reported value of 0.59 may be more reasonable.50 In this work, the value of constant

344

was 0.62 and 0.50 for SCEL and LCEL, respectively, which was reasonable in

345

consideration of structural differences between non-wood and wood lignin. The different

346

forms of ferulic acid and lignin-like materials36 in stem and leaf lignins may cause the

347

different content of V and S units or the difference was simply derived from the enrichment

348

of certain types of subunits in the free phenolic groups.

349

FTIR spectroscopy. The FTIR spectra of LCEL and SCEL in wheat straw are shown

350

in Figure S1. The assignments of main absorption bands51-54 are listed in Table S2. The

351

spectra showed some common features but also vibrations that were specific to each lignin.

352

The stretching vibration of S-unit at 1330 cm–1 in SCEL was apparently stronger than that

353

in LCEL which means stem lignin has more S units content than that in leaf lignin, which is

354

consistent with the data of NBO products and the analysis of 2D NMR. In contrast, the

355

bending vibration of C–H and C–O at 1085 cm–1 in LCEL is showed clearly but is almost

356

undetectable in SCEL. These different stretching vibration positions between SCEL and

357

LCEL may be attributed to the difference of steric hindrance, electron cloud density and

358

field effect among functional groups. For example, Li et al.55 pointed out that S unit is

359

characterized with more methoxy groups on benzene ring which generates steric hindrance

360

effect. The oxygen atoms in phenolic hydroxyl and methoxy groups have shown strong

361

electronegativity. The unshared p electron pairs can form p-π conjugated system with the π

362

electron cloud of benzene ring. 16


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363 364


CONCLUSIONS The structure of lignin in wheat straw stem is rather different from that in leaf. Wet

365

chemistry results showed that leaf lignin has lower products yield of nitrobenzene oxidation

366

and ozonation, lower erythro/threo ratio and higher condensation degree than stem lignin.

367

The 2D HSQC NMR spectra and ozonation results showed that the stem lignin contains

368

more β–O–4’ linkages which are mainly composed by erythro form. Besides, leaf lignin has

369

less syringyl (S) content and more guaiacyl (G) content while the similar hydroxyphenyl (H)

370

content, which means the lower ratio of S/G in leaf lignin. The flavone tricin is

371

incorporated into both stem and leaf lignins. The content of tricin in leaf lignin is higher

372

than that in stem lignin. The analysis of FTIR spectra also showed that the functional

373

groups are structurally different between wheat straw stem and leaf lignins. The difference

374

of structural characteristics between stem and leaf lignins in herbaceous plants may be the

375

main influence factors on saccharification of lignocellulosic biomass which results in

376

different biorefinery processes.

377

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378 379

FUNDING SOURCES This work was supported by the National Key Technology Research and Development

380

Program of China (grant number 2015BAD15B09), the National Natural Science

381

Foundation of China (grant numbers 31370571, 31400514), China Postdoctoral Science

382

Foundation (Grant No. 2016M591853), and the Priority Academic Program Development

383

of Jiangsu Higher Education Institutions, China.

384

18


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385

Supporting Information. The FTIR spectra of LCEL and SCEL; Assignment of the

386

1

387

the assignment of absorption peaks in CELs are supplied as Supporting Information.

H−13C correlation peaks in the 2D HSQC spectra of SCEL and LCEL; The positions and

388

19



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389

REFERENCES

390

(1) Boerjan, W.; Ralph, J.; Baucher, M. Lignin biosynthesis. Annu. Rev. Plant Biol. 2003,

391 392 393 394 395 396

54, 519–546. (2) Buranov, A.U.; Mazza, G. Lignin in straw of herbaceous crops. Ind. Crop. Prod. 2008, 28, 237–259. (3) Baucher, M.; Monties, B.; Montagu, M.V.; Boerjan, W. Biosynthesis and genetic engineering of lignin. Crit. Rev. Plant Sci. 1998, 17, 125–197. (4) Sun, R.; Tomkinson, J. Comparative study of lignins isolated by alkali and

397

ultrasound-assisted alkali extractions from wheat straw. Ultrason. Sonochem. 2002, 9,

398

85–93.

399

(5) Chang, H.M.; Cowling, E.B.; Brown. W. Comparative studies on celluloytic enzyme

400

lignin and milled wood lignin of sweetgum and spruce. Holzforschung 1975, 29, 153–

401

159.

402

(6) Hu, Z.J.; Yeh, T.F.; Chang, H.M.; Matsumoto, Y.; Kadla, J.F. Elucidation of the

403

structure of cellulolytic enzyme lignin. Holzforschung 2006, 60, 389–397.

404 405

(7) Dzhumanova, Z.K.; Dalimova, G.N. Nitrobenzene oxidation of lignins from several plants of the family Gramineae. Chem. Nat. Compd. 2011, 47, 419–421.

406

(8) Akiyama, T.; Sugimoto, T.; Matsumoto, Y.; Meshitsuka, G. Erythro/threo ratio of beta–

407

O–4 structures as an important structural characteristic of lignin. I: Improvement of

408

ozonation method for the quantitative analysis of lignin side-chain structure. J. Wood

409

Sci. 2002, 48, 210–215.

410

(9) van de Pas, D.J.; Nanayakkara, B.; Suckling, I.D.; Torr, K.M. Comparison of

411

hydrogenolysis with thioacidolysis for lignin structural analysis. Holzforschung 2014,

412

68, 151–155.

413

(10) Partovi, T.; Abdol, H.M.; Seyyed, A. M. Investigation of structure of milled wood and

414

dioxane lignins of Populus nigra and Cupressus sempervirens using the DFRC method.

415

Chem. Pap. 2012, 66, 800–804.

416

(11) Capanema, E.A.; Balakshin M.Y.; Kadla J.F. A comprehensive approach for

417

quantitative lignin characterization by NMR spectroscopy. J. Agric. Food Chem. 2004,

418

52, 1850–1860. 20


Page 20 of 33

Page 21 of 33

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

419


(12) Wen, J.L.; Sun, S.L.; Xue, B.L.; Sun, R.C. Recent advances in characterization of

420

lignin polymer by solution-state nuclear magnetic resonance (NMR) methodology.

421

Materials 2013, 6, 359–391.

422

(13) Balakshin, M.Y.; Capanema, E.A.; Gracz, H.; Chang, H.M.; Jameel, H. Quantification

423

of lignin–carbohydrate linkages with high-resolution NMR spectroscopy. Planta 2011,

424

233, 1097–1110.

425 426 427

(14) Crestini, C.; Melone, F.; Sette M.; Saladino, R. Milled wood lignin: A linear oligomer. Biomacromolecules 2011, 12, 3928–3935. (15) del Río, J.C.; Rencoret, J.; Prinsen, P.; Martínez, Á.T.; Ralph, J.; Gutiérrez, A.

428

Structural characterization of wheat straw lignin as revealed by analytical pyrolysis,

429

2D-NMR, and reductive cleavage methods. J. Agric. Food Chem. 2012a, 60, 5922–

430

5935.

431

(16) Rencoret, J.; Prinsen, P.; Gutieŕrez, A.; Martıńez, A.T.; del Río, J.C. Isolation and

432

structural characterization of the milled wood lignin, dioxane lignin, and cellulolytic

433

lignin preparations from brewer’s spent grain. J. Agric. Food Chem. 2015, 63,

434

603−613.

435

(17) Wu, L.; Lu, F.C.; Regner, M.; Zhu, Y.M.; Rencoret, J.; Ralph, S.A.; Zakai, U.I.;

436

Morreel, K.; Boerjan, W.; Ralph, J. Tricin, a flavonoid monomer in monocot

437

lignification. Plant Physiol. 2015, 167, 1284–1295.

438 439

(18) Zhou, J.M.; Ibrahim, R.K. Tricin—a potential multifunctional nutraceutical. Phytochem. Rev. 2010, 9, 413–424.

440

(19) Markovic, J.P.; Štrbanovic, R.T.; Terzic, D.V.; Djokic, D.J.; Simic, A.S.; Vrvic, M.M.;

441

Živkovic, S.P. Changes in lignin structure with maturation of alfalfa leaf and stem in

442

relation to ruminants nutrition. Afr. J. Agric. Res. 2012, 7, 257–264.

443

(20) Min, D.Y.; Jameel, H.; Chang, H.M.; Lucia, L.; Wang, Z.G.; Jin, Y.C. The structural

444

changes of lignin and lignin-carbohydrate complexes in corn stover induced by mild

445

sodium hydroxide treatment. RSC Adv. 2014, 4, 10845–10850.

446

(21) Jin, Y.C.; Huang, T.; Geng, W.H.; Yang, L.F. Comparison of sodium carbonate

447

pretreatment for enzymatic hydrolysis of wheat straw stem and leaf to produce

21



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

448 449 450

fermentable sugars. Bioresour. Technol. 2013, 137, 294–301. (22) Björkman, A. Studies on finely divided wood. Part I. Extraction of lignin with neutral solvents. Svensk Papperstidn. 1956, 59, 477–485.

451

(23) Fujimoto, A.; Matsumoto, Y.; Chang, H.M.; Meshitsuka, G. Quantitative evaluation of

452

milling effects on lignin structure during the isolation process of milled wood lignin. J.

453

Wood Sci. 2005, 51, 89–91.

454

(24) Sluiter, A.; Hames, B.; Ruiz, R.; Scarlata, C.; Sluiter, J.; Templeton, D.; Crocker, D.

455

Determination of structural carbohydrates and lignin in biomass. Laboratory analytical

456

procedure, NREL, Report No. TP–510–42618, 2008.

457 458 459 460

(25) Chen, C.L. Nitrobenzene and cupric oxide oxidations. In Methods in Lignin Chemistry; Lin, S., Dence, C., Eds.; Springer-Verlag: Berlin, 1992; pp 301–321. (26) Kim, H.; Ralph, J.; Akiyama, T. Solution-state 2D NMR of ball milled plant cell-wall gels in DMSO-d6. Bioenergy Res. 2008, 1, 56−66.

461

(27) Rencoret, J.; Marques, G.; Gutiérrez, A.; Nieto, L.; Jiménez-Barbero, J.; Martínez, A.T.;

462

del Río, J.C. Isolation and structural characterization of the milled wood lignin from

463

Paulownia fortune wood. Ind. Crops Prod. 2009, 30, 137−143.

464

(28) Overend, R.P.; Johnson, K.G. Lignin carbohydrate complexes from

465

poplarwood-isolation and enzymatic degradation. ACS Symp. Ser. 1991, 460, 270–287.

466

(29) Lu, F.; Ralph, J. Lignin. In Cereal straw as a resource for sustainable biomaterials and

467

biofuels: Chemistry, Extractives, Lignins, Hemicelluloses and Cellulose; Sun, R.C. Ed.;

468

Elsevier Press: Amsterdam, 2010; pp 169–200.

469

(30) Ikeda, T.; Holtman, K.; John, F.K.; Chang, H.M.; Jameel, H. Studies on the effect of

470

ball milling on lignin structure using a modified DFRC method. J. Agric. Food Chem.

471

2001, 50, 129–135.

472

(31) Balakshin, M.Y.; Capanema, E.A.; Chang, H.M. Recent advances in the isolation and

473

analysis of lignins and lignin–carbohydrate complexes. In: Characterization of

474

Lignocellulosics; Hu, T. Ed.; Blackwell: Oxford, UK, 2008; pp 148–170.

475

(32) Capanema, E.A.; Balakshin, M.; Katahira, R.; Chang, H.M.; Jameel, H. How well do

476

MWL and CEL preparations represent the whole hardwood lignin? J. Wood Chem.

477

Techn. 2015, 35, 17–26. 22


Page 22 of 33

Page 23 of 33

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

478


(33) Salamanca, E.F.; Kaneko, N.; Katagiri, S.; Nagayama, Y. Nutrient dynamics and

479

lignocellulose degradation in decomposing Quercus serrata leaf litter. Ecolog. Res.

480

1998, 13, 199–210.

481

(34) Sarkanen, K.V.; Hergert, H.L. Classification and distribution. In Lignins: Occurrence,

482

Formation, Structure and Reactions; Sarkanen, K.L., Ludwig, C.H., Eds.;

483

Wiley-Interscience: New York, 1971; pp 43–94.

484

(35) Johansson, M.B.; Kogel, I., Zech, W. Changes in the lignin fraction of spruce and pine

485

needle litter during decomposition as studied by some chemical methods. Soil Biol.

486

Biochem. 1986, 18, 611–619.

487

(36) Jin, Z.F.; Akiyama, T.; Chung, B.Y.; Matsumoto, Y.; Iiyama, K.; Watanabe, S. Changes

488

in lignin content of leaf litters during mulching. Phytochemistry 2003, 64, 1023–1031.

489 490

(37) Kim, H.; Ralph, J. Solution-state 2D NMR of ball-milled plant cell wall gels in DMSO-d6/pyridine-d5. Org. Biomol. Chem. 2010, 8, 576−591.

491

(38) Gu, F.; Wu, W.J.; Wang, Z.G.; Toyoma, Y.; Jin, Y.C. Effect of complete dissolution in

492

LiCl/DMSO on the isolation and characteristics of lignin from wheat straw internode.

493

Ind. Crop. Prod. 2015, 74, 703–711.

494

(39) Chang, C.L.; Wang, G.J.; Zhang, L.J.; Tsai, W.J.; Chen, R.Y.; Wu, Y.C.; Kuo, Y.H.

495

Cardiovascular protective flavolignans and flavonoids from Calamus quiquesetinervius.

496

Phytochemistry 2010, 71, 271−279.

497

(40) del Río, J.C.; Prinsen, J.; Rencoret, J.; Nieto, L.; Jiménez-Barbero, J.; Ralph, J.;

498

Martínez, A.T.; Gutiérrez, A. Structural characterization of the lignin in the cortex and

499

pith of elephant grass (Pennisetum purpureum) Stems. J. Agric. Food Chem. 2012, 60,

500

3619–3634.

501 502 503 504 505 506

(41) Koshijima, T.; Watanabe, T. Association between lignin and carbohydrates in wood and other plant tissues. Forestry 2004, 77, 175–176. (42) Park, H.S.; Lim, J.H.; Kim, H.J.; Choi, H.J.; Lee, I.S. Antioxidant flavone glycosides from the leaves of Sasa borealis. Arch. Pharmacal Res. 2007, 30, 161–166. (43) Li, X.; Ximenes, E.; Kim, Y.; Slininger, M.; Meilan, R.; Ladisch, M.; Chapple, C. Lignin monomer composition affects Arabidopsis cell-wall degradability after liquid

23



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

507 508

hot water pretreatment. Biotechnol. Biofuels 2010, 3, 27–34. (44) Min, D.Y.; Yang, C.M.; Shi, R. The elucidation of the lignin structure effect on the

509

cellulase-mediated saccharification by genetic engineering poplars (Populus nigra L.

510

and Populus maximowiczii A.). Biomass Bioenergy 2013, 58, 52–57.

511

(45) Li, M.; Si, S.; Hao, B.; Zha, Y.; Wan, C.; Hong, S.; Kang, Y.; Jia, J.; Zhang, J.; Li, M.;

512

Zhao, C.; Tu, Y.; Zhou, S.; Peng, L. Mild alkali-pretreatment effectively extracts

513

guaiacyl-rich lignin for high lignocellulose digestibility coupled with largely

514

diminishing yeast fermentation inhibitors in Miscanthus. Bioresour. Technol. 2014, 169,

515

447–454.

516

(46) Xu, N.; Zhang, W.; Ren, S.; Liu, F.; Zhao, C.; Liao, H.; Xu, Z.; Huang, J.; Li, Q.; Tu, Y.

517

Hemicelluloses negatively affect lignocellulose crystallinity for high biomass

518

digestibility under NaOH and H2SO4 pretreatments in Miscanthus. Biotechnol. Biofuels

519

2012, 5, 58.

520

(47) Jiang, B.; Wang, W.X.; Gu, F.; Cao, T.Y.; Jin, Y.C. Comparison of the substrate

521

enzymatic digestibility and lignin structure of wheat straw stems and leaves pretreated

522

by green liquor. Bioresour. Technol. 2016, 199, 181–187.

523

(48) Nakagame, S.; Chandra, R.P.; Kadla, J.F.; Saddler, J.N. Enhancing the enzymatic

524

hydrolysis of lignocellulosic biomass by increasing the carboxylic acid content of the

525

associated lignin. Biotechnol. Bioeng. 2011, 108, 538–548.

526 527

(49) Santos, R.B.; Capanema, E.A.; Balakshin, M.Y.; Chang, H.M.; Jameel, H. Lignin structural variation in hardwood species. J. Agric. Food Chem. 2012, 60, 4923–4930.

528

(50) Capanema, E.A.; Balakshin, M.Y.; Katahira, R.; Chang, H.M.; Jameel, H. Structural

529

variations in hardwood lignins. Proc. 14th Intern. Symp. Wood Fibre Pulping Chem.,

530

CD-ROM. Durban, South Africa, 2007.

531

(51) Karmanov, A.P.; Derkacheva, O.Y. Application of Fourier transform infrared

532

spectroscopy for the study of lignins of herbaceous plants. Russ. J. Bioorg. Chem. 2013,

533

39, 677–685.

534

(52) Watkins, D.; Nuruddin, M.; Mahesh H.; Alfred T.N.; Jeelani, S. Extraction and 24


Page 24 of 33

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535

characterization of lignin from different biomass resources. J. Mater. Res. Technol.

536

2015, 4, 26–32.

537 538 539

(53) Mansouri, N.E.E.; Yuan, Q.; Huang, F. Characterization of alkaline lignins for use in phenol-formaldehyde and epoxy resins. Bioresources 2011, 3, 2647–2662. (54) Joffres, B.; Lorentz, C.; Vidalie, M.; Laurenti, D.; Quoineaud, A.A.; Charon, N.;

540

Daudin, A.; Quignard, A.; Geantet, C. Catalytic hydroconversion of a wheat straw soda

541

lignin: Characterization of the products and the lignin residue. Appl. Catal. B 2014,

542

145, 167–176.

543

(55) Li, H.Q.; Qu, Y.S.; Xu, J. Microwave-assisted conversion of lignin. In Production of

544

Biofuels and Chemicals with Microwave; Fang, Z., Smith, R.L., Qi, X.H., Eds.;

545

Springer: Berlin, 2015; pp 61–82.

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547

Comparison of the structural characteristics of cellulolytic enzyme lignin preparations

548

isolated from wheat straw stem and leaf

549 550

Bo Jiang, Tingyue Cao, Feng Gu, Wenjuan Wu, Yongcan Jin*

551 552

TOC graphic

553 554

555

Synopsis: The different structures of stem and leaf lignins in herbaceous plants influence

556

the saccharification efficiency and lead to different biorefinery processes.

557 558

26


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559


Figures and Tables

560

561 562

Figure 1. Isolation procedure of cellulolytic enzyme lignin from wheat straw stem and leaf.

563

27



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564 565

Figure 2. The effects of ball milling hours on the yield of extractable lignin on the basis of

566

lignin in raw materials.

567

28


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568 569

Figure 3. The signals of 2D HSQC NMR spectra in side chain (δC/δH 50−90/2.5−6.0) and

570

aromatic regions (δC/δH 90−150/6.0−8.0) of SCEL and LCEL.

571

29



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

OMe R

HO

O

O O γ

HO

β O 4'

α

γ

OMe

HO

OMe

O α'

5'

γ β

HO

β O 4'

α

α

β' γ'

β

O

OMe

α O

OMe O

O

OMe

O

O A

OMe

A’

OMe

B

C

OMe

O

H

γ OH

α β

HO γ

O 1' α'

O

γ

β

α

OAr

OMe

OMe

OMe

O

O

O

OH

O F

I

OH

J

R

OH

α

FA

O

PCA

OMe

OH

α

α

α

8 9 HO 7 O

OMe O

β

α

OH

OMe

O

O

γ β

α

α

γ' β'

O

O γ

β

MeO

OMe O

MeO

OMe O

6

O

5 10

4

3' O 2' 4' 1' 2 6' 5' OMe 3

OH O

572

G

S

S’

H

T

573

Figure 4. Main structures present in the lignins of wheat straw: (A) β–O–4’ alkyl-aryl

574

ethers; (A′) β–O–4’ alkyl-aryl ethers with acylated γ-OH; (B) phenylcoumarans; (C)

575

resinols; (F) spirodienones; (I) cinnamyl alcohol end-groups; (J) cinnamyl aldehyde

576

end-groups; (FA) ferulates; (PCA) p-coumarates; (G) guaiacyl units; (S) syringyl units; (H)

577

p-hydroxyphenyl units; (T) tricin units connected with lignin polymer through β–O–4’

578

linkage.

579

30


Page 30 of 33

Page 31 of 33

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


580

Table 1. Mass balance of original, ball-milled, enzyme hydrolyzed straw samples and CEL

581

preparations (%). Data are the mean of two measurements. Samplesa

Carbohydrate

Lignin

Ash

SRb

2.1 ± 0.1

7.6 ± 0.3

100

22.3 ± 0.3

0.8 ± 0.1

3.0 ± 0.0

42.4

0.7 ± 0.0

19.6 ± 0.0

0.5 ± 0.1

3.0 ± 0.0

33.2

2.9 ± 0.0

0.6 ± 0.0

18.9 ± 0.0

0.4 ± 0.0

2.8 ± 0.1

30.7

0.2 ± 0.0

1.1 ± 0.0

0.2 ± 0.0

14.8 ± 0.2

0.2 ± 0.0

0.0 ± 0.0

16.6

Leaf

35.1 ± 0.1

22.7 ± 0.1

4.8 ± 0.3

14.2 ± 0.5

2.4 ± 0.0

11.3 ± 0.5

100

EML-2h

2.6 ± 0.1

3.6 ± 0.0

0.8 ± 0.0

11.8 ± 0.2

0.7 ± 0.0

6.7 ± 0.0

30.7

EML-4h

0.9 ± 0.1

1.6 ± 0.0

0.4 ± 0.0

10.7 ± 0.1

0.5 ± 0.0

6.5 ± 0.0

25.9

EML-6h

0.7 ± 0.1

1.6 ± 0.1

0.3 ± 0.0

10.1 ± 0.0

0.5 ± 0.1

6.2 ± 0.1

21.3

LCEL

0.1 ± 0.0

0.6 ± 0.0

0.1 ± 0.0

6.9 ± 0.1

0.1 ± 0.0

0.1 ± 0.0

7.6

Glucan

Xylan

Arabinan

KL

ASL

Stem

40.7 ± 0.6

22.4 ± 1.0

2.8 ± 0.1

21.7 ± 0.3

EMS-2h

5.7 ± 0.0

5.6 ± 0.1

0.9 ± 0.0

EMS-4h

1.9 ± 0.1

3.4 ± 0.1

EMS-6h

1.7 ± 0.2

SCEL

582

a

583

respectively; EMS: enzymatic residue of the ball-milled stem (2–6 h); EML: enzymatic residue of the

584

ball-milled leaf (2–6 h); SCEL: CEL isolated from stem; LCEL: CEL isolated from leaf.

585

b

The content of benzene-ethanol extractives from wheat straw stem and leaf was 4.6% and 6.5%,

SR: solid recovery on the basis of starting material.

586

31



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Page 32 of 33

587

Table 2. The yields and ratios of nitrobenzene oxidation (NBO) and ozonation products of

588

lignin in wheat straw stem and leaf. Data are the mean of two measurements. Samplesa

Nitrobenzene oxidation

Ozonation

Yield (mmol/g-lignin)

S/V/H

Stem

2.23 ± 0.05

MS

b

Yield (mmol/g-lignin)

E/T

41/47/12

0.84 ± 0.07

1.58 ± 0.08

2.20 ± 0.02

40/48/12

0.78 ± 0.03

1.53 ± 0.02

EMS

84.5 ± 0.7

2.33 ± 0.08

45/45/10

0.83 ± 0.03

1.53 ± 0.02

SCEL

63.0 ± 0.9

2.67 ± 0.10

51/40/9

0.77 ± 0.09

1.51 ± 0.00

Leaf

1.60 ± 0.03

29/59/12

0.33 ± 0.04

1.29 ± 0.03

ML

1.50 ± 0.03

29/58/13

0.33 ± 0.02

1.23 ± 0.10

1.42 ± 0.06

30/58/12

0.36 ± 0.03

1.23 ± 0.10

EML 589

Lignin yield (%)

67.5 ± 0.5

LCEL 42.2 ± 0.7 2.18 ± 0.06 41/51/8 0.46 ± 0.12 1.18 ± 0.03 a MS: 4 h ball-milled stem; ML: 4 h ball-milled leaf; EMS: enzymatic residue of MS; EML: enzymatic

590

residue of ML; SCEL: CEL isolated from stem; LCEL: CEL isolated from leaf.

591

b

592

p-hydroxybenzoic acid.

S = syringaldehyde + syringic acid; V = vanillin + vanillic acid; H = p-hydroxybenzaldehyde +

593

32


Page 33 of 33

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594

Table 3. Structural characteristics (lignin interunit linkages, aromatic units and S/G ratio,

595

p-coumarate/ferulate, tricin) from integration of C–H correlation peaks in the HSQC

596

spectra of the SCEL and LCEL. SCEL

LCEL

β–O–4’ substructures (A/A’)

64

56

β–5’ phenylcoumaran substructures (B)

2

5

β–β’ resinol substructures (C)

Comparison of the Structural Characteristics of Cellulolytic Enzyme

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