Article pubs.acs.org/JAFC
Unraveling the Structural Modifications in Lignin of Arundo donax Linn. during Acid-Enhanced Ionic Liquid Pretreatment Tingting You,† Liming Zhang,† Siqin Guo,† Lupeng Shao,† and Feng Xu*,†,§ †
Beijing Key Laboratory of Lignocellulosic Chemistry, Beijing Forestry University, Beijing, China Key Laboratory of Pulp and Paper Science and Technology, Ministry of Education, Qilu University of Technology, Shandong, China
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S Supporting Information *
ABSTRACT: Solid acid-enhanced ionic liquid (IL) pretreatment is of paramount importance for boosting the yield of sugars from biomass cost-effectively and environmentally friendly. To unravel the chemical and supramolecular structural changes of lignin after pretreatment, IL−acid lignin (ILAL) and subsequent residual cellulolytic enzyme lignin (RCEL) were isolated from Arundo donax Linn. The structural features were compared with those of the corresponding milled wood lignin (MWL). Results indicated that the pretreatment caused loss of β-O-4′, β-β′, β-1′ linkages and formation of condensed structures in lignin. A preferential breakdown of G-type lignin may have occurred, evidenced by an increased S/G ratio revealed by 2D HSQC NMR analysis. It was determined that the depolymerization of β-O-4′ linkage, lignin recondensation, and cleavage of ferulate−lignin ether linkages took place. Moreover, a simulation module was first developed to define morphological changes in lignin based on AFM and TEM analyses. Briefly, tree branch like aggregates was destroyed to monodisperse particles. KEYWORDS: Arundo donax Linn., IL−acid pretreatment, lignin, structure modification
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INTRODUCTION Evidence from the increment of greenhouse gas concentration and depletion of fossil fuels in recent decades has stimulated interest in using a sustainable and renewable lignocellulosic biomass for biofuels, chemical materials, and biopower.1 Lignocellulosic biomass, such as agricultural residual, herbaceous and woody plants, and energy crops, predominantly comprise cellulose, hemicelluloses, and lignin. Among various biomass materials, energy crop is a potentially sustainable alternative feedstock for fuel production, such as bioconversion of sugars to bioethanol and some prospective biofuels due to high biomass productivity and low input required for their cultivation.2 A lignocellulosic biorefinery processing for ethanol production involves saccharification of carbohydrates to fermentable reducing sugars via enzymatic hydrolysis. Lignin is one of the major factors hindering the production of inexpensive bioethanol by nonproductive binding of enzymes to its surface that decrease cellulose accessibility.3 The total amount and type of lignin present within the plant cell wall will affect the efficiency.4,5 An efficient pretreatment is usually conducted to reduce lignin content and modify the structure. In the scientific literature, there are numerous studies that look for efficient pretreatments to facilitate cost-efficient conversion of biomass into biofuels.4,6,7 Examining the structural changes of lignin during pretreatment will contribute to the understanding of native recalcitrance and facilitate the design of more effective deconstruction strategies. Lignin in plants is a complex amorphous polymer and synthesized by chemical polymerization of three main precursors, p-coumaryl, coniferyl, and sinapyl alcohols.8 Each of these precursors gives rise to different types of lignin unit called p-hydroxyphenyl (H), guaiacyl (G), and syringyl (S) units, respectively.8,9 This biosynthesis process consists of © 2015 American Chemical Society
mainly radical coupling reactions and creates a unique lignin polymer in each plant species. In particular, lignin in gramineous plants mainly consists of H, G, and S in various ratios. These units are linked by several types of carbon−carbon (β-β′, β-5′, β-1′, and 5-5′) and ether bonds (β-O-4′ and α-O4′). During the lignification process, hydroxycynnamic acids such as p-coumaric and ferulic acids are incorporated into lignin of grasses, apparently making the structure of lignin more complex. It is well established that ferulic acid is ester-linked to carbohydrates and ether-linked to lignin, forming “lignin− ferulate−polysaccharide” (LFP) complexes, whereas p-coumaric acid is attached to lignin via ester bonds.10 Morphologically, lignin displays distinct nanoscale architecture. Well-defined spherical molecular shapes with diameter of 400 nm and at least four levels of supramolecular organization of artificial lignin have been visualized.11 It should be noted that molecular shapes and supramolecular organization of lignin are heavily dependent on the lignin type and the substrate surface characteristics. Ionic liquid (IL) pretreatment is considered a compelling alternative to traditional biomass pretreatment due to the green properties of ILs.12 After the pretreatment, lignin dissolved in acetone−water solution can be recovered by evaporation of acetone as byproduct. The structure of lignin in plant biomass was modified during the pretreatment.13 For instance, the 1ethyl-3-methylimidazolium acetate ([C2mim][OAc]) pretreatment caused chemical composition and supramolecular organization changes in lignin.13−18 More recently, chemical− ionic liquid combination pretreatment such as solid acid Received: Revised: Accepted: Published: 10747
August 4, 2015 November 26, 2015 December 1, 2015 December 1, 2015 DOI: 10.1021/acs.jafc.5b04831 J. Agric. Food Chem. 2015, 63, 10747−10756
Article
Journal of Agricultural and Food Chemistry
Chemical Composition. The chemical composition of raw and pretreated A. donax was analyzed using the methods developed by the National Renewable Energy Laboratory (NREL).23,25 FT-IR Spectroscopy. Infrared spectra were acquired on a Thermo Scientific Nicolet iN 10 FT-IR microscope (Thermo Nicolet Corp., Madison, WI, USA) equipped with a liquid nitrogen cooled MCT detector. Thirty-two scans were taken with a resolution of 4 cm−1 in the reflection mode. GPC. The weight-average (Mw) and number-average (Mn) molecular weights and polydispersity of lignins were determined by Agilent 1200 gel permeation chromatography (Agilent, Santa Clara, CA, USA) with a refraction index detector (RID). The column used was a 300 mm × 7.5 mm i.d., 10 μm, PL-gel Mixed-B, with a 50 mm × 7.5 mm i.d. guard column of the same material (Agilent, UK), calibrated with PL pullulan polysaccharide standards (peak average molecular weights of 783, 12200, 100 000, 1 600 000 g/mol, Polymer Laboratories Ltd., UK). Before the analysis, lignin samples were acetylated by pyridine/acetic anhydride (1:1, v/v) solution to dissolve the samples completely.26 NMR Spectroscopy. Lignins were analyzed by NMR using a Bruker Advance 400 MHz instrument equipped with a 5 mm gradient probe at room temperature. For the collection of 2D HSQC spectra, lignin sample (20 mg) was dissolved in DMSO-d6 in an NMR tube.23 Chemical shifts were referenced to the central DMSO peak (δC/δH 39.5/2.49). 31P NMR spectra were conducted as previously described.23 Lignin sample (20 mg) was dissolved in anhydrous pyridine and deuterated chloroform (1.6:1, v/v, 500 μL) under stirring. Cyclohexanol (10.85 mg/mL, 100 μL) was added as an internal standard, followed by the addition of chromium(III) acetylacetonate solution (5 mg/mL in anhydrous pyridine and deuterated chloroform 1.6:1, v/v, 100 μL) as a relaxation reagent. The mixture was reacted with 2-chloro-4,4,5,5-tetramethyl-1,3,2dioxaphospholane (phosphitylating reagent, 100 μL) for about 10 min and placed into the NMR tube for 31P NMR analysis. AFM Images. AFM imaging of lignins was conducted using a Bruker Multimode 8 instrument (USA) with a Nanoscope V controller. Both height and peak force error images were collected simultaneously in scanasyst mode (in air) with an MPP-11100 etched silicon probe with a nominal frequency of 300 kHz and a nominal spring constant of 40 N m−1. RMS roughness factors (Rq) and width of the lignin ellipsoid were estimated after image flattening and plane fitting of the height images using the software that came with the instrument. Lignins were prepared by casting extremely dilute solutions (0.1 mg mL−1 in deionized water) on freshly hydrophilic mica and allowing them to air-dry under ambient conditions. TEM Images. TEM imaging of lignins was undertaken with a JEM1010 (JEOL) TEM at an acceleration voltage of 80 kV. Dilute suspension of the lignin sample was sonicated (100 W) for 10 min in an ice−water bath, and then several drops of the sonicated suspension were deposited onto holey carbon-coated copper grids. The samples were then stained with aqueous uranyl acetate (2 wt %) for 30 s and dried overnight in a vacuum oven at 25 °C prior to imaging.
enhanced IL 1-butyl-3-methylimidazolium chloride ([C4mim]Cl) pretreatment has shown great potential as a cost-effective, recyclable, and environmentally benign novel process.19,20 Understanding the structural details of lignin byproduct after the pretreatment is essential for lignin utilization and improves the economic value of a biorefinery. However, there is currently no comprehensive and systematic evaluation of lignin modification during the acid-enhanced [C4mim]Cl pretreatment, nor do we develop a model that explains how the lignin supramolecular structure changes. In this study, we investigated the impact of [C4mim]Cl− Amberlyst pretreatment on the chemical and supramolecular structure of lignin from Arundo donax Linn. by using gel permeation chromatography (GPC), 31P nuclear magnetic resonance (NMR) spectroscopy, solution state two-dimensional heteronuclear single-quantum coherence NMR (2D HSQC NMR) spectroscopy, atomic force microscopy (AFM), and transmission electron microscopy (TEM). After the pretreatment, IL−acid lignin (ILAL) was recovered, and cellulolytic enzyme lignin (RCEL) from the pretreated biomass was isolated to further evaluate the effect of IL−acid pretreatment by 2D HSQC NMR spectroscopy. Milled wood lignin (MWL), considered as a representative of native lignin, was also isolated for comparison. Knowledge of the structural characteristics of lignin byproducts and degradation mechanism will help to evaluate the utilization of lignin and effectively improve the economics of the combined process.
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MATERIALS AND METHODS
Materials. One-year-old A. donax with an average height of 2.1 m was procured from an experimental field at Beijing Academy of Agricultural Sciences (China). The crop stem samples were milled and sieved to 60−80 mesh and exhaustively extracted with toluene/ethanol (2:1, v/v). The ionic liquid [C4mim]Cl (≥98.5%) was provided by the Lanzhou Institute of Chemical Physics of the Chinese Academy of Sciences, Lanzhou, China. Acidic resin Amberlyst 35DRY (moisture ≤ 3.0%) was purchased from the Dow Chemical Co. (Vandalia, IL, USA). All other chemicals used were purchased from the Sigma Chemical Co. (Beijing, China). Isolation of ILAL, MWL, and RCEL. Acid-enhanced IL pretreatment and ILAL isolation were carried out according to previous research.20,21 A. donax (5 g) was added to [C4mim]Cl (95 g) in a 250 mL three-neck flask that was preheated to 160 °C. The mixture was gently stirred for 1.5 h under N2 atmosphere. After the reaction had cooled to 80 °C, solid acid Amberlyst 35DRY (3 g) was added to the mixture and kept at 100 °C for 1 h. The slurry was then poured into 1000 mL of acetone−water (1:1, v/v) under vigorous stirring for the regeneration of dissolved A. donax.21 The mixture was then centrifuged. The pretreated A. donax was washed thoroughly and freeze-dried. Acid-soluble lignin (ASL) in the supernatant fluid was determined by a UV 2300 spectroscopy at a wavelength of 320 nm (Shanghai, China). After evaporation of the acetone in air, the lignin in solution was partially precipitated. ILAL was collected by filtration, then thoroughly washed with distilled water, and finally freeze-dried. The procedure and the following analysis were carried out in duplicate, and the indeterminancy of parallel results was
