Survey of Lignin-Structure Changes and Depolymerization during

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A Survey of Lignin-Structure Changes and Depolymerization during Ionic Liquid Pretreatment Tanmoy Dutta, Nancy G. Isern, Jian Sun, Eileen Wang, Sarah Hull, John Robert Cort, Blake A. Simmons, and Seema Singh ACS Sustainable Chem. Eng., Just Accepted Manuscript • DOI: 10.1021/ acssuschemeng.7b02123 • Publication Date (Web): 06 Sep 2017 Downloaded from http://pubs.acs.org on September 10, 2017

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ACS Sustainable Chemistry & Engineering

A Survey of Lignin-Structure Changes and Depolymerization during Ionic Liquid Pretreatment Tanmoy Dutta,a,b Nancy G. Isern,c, d Jian Sun,a,b Eileen Wang,a Sarah Hull,a John R. Cort,c,d Blake A. Simmons,a,e Seema Singha,b* a

Deconstruction Division, Joint BioEnergy Institute, 5885 Hollis Street, Emeryville, California 94608, United States. b

Biological and Engineering Sciences Center, Sandia National Laboratories, 7011 East Avenue, Livermore, California 94551, United States. c Environmental Molecular Sciences Laboratory and Biological Sciences Division, Pacific Northwest National Laboratory, Richland, Washington 99352, USA. d

Earth and Biological Sciences Directorate, Pacific Northwest National Laboratory, Richland, Washington 99352, USA

e

Biological Systems and Engineering Division, 1 Cyclotron Road, Berkeley, California 94720, United States. * Corresponding author: E-mail: [email protected].

ABSTRACT A detailed study of chemical changes in lignin structure during the ionic liquid (IL) pretreatment process is not only pivotal for understanding and overcoming biomass recalcitrance during IL pretreatment, but also is necessary for designing new routes for lignin valorization. Chemical changes in lignin were systematically studied as a function of pretreatment temperature, time and type of IL used. Kraft lignin was used as the lignin source and common pretreatment conditions were employed using three different ILs of varying chemical structure in terms of acidic or basic character. The chemical changes in the lignin structure due to IL pretreatment processes were monitored using 1H-13C HSQC NMR, 31P NMR, elemental analysis, GPC, FT-IR, and the depolymerized products were

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analyzed using GC-MS. Although pretreatment in acidic IL, triethylammonium hydrogensulfate ([TEA][HSO4]) results in maximum decrease in β-aryl ether bond, maximum dehydration and recondensation pathways were also evident, with the net process showing a minimum decrease in the molecular weight of regenerated lignin. However, 1-ethyl-3-methylimidazolium acetate ([C2C1Im][OAc]) pretreatment yields a smaller decrease in the β-aryl ether content along with minimum evidence of recondensation, resulting in the maximum decrease in the molecular weight. Cholinium lysinate ([Ch][Lys]) pretreatment shows an intermediate result, with moderate depolymerization, dehydration and recondensation observed. The depolymerization products after IL pretreatment are found to be a function of the pretreatment temperature and the specific chemical nature of the IL used. At higher pretreatment temperature, [Ch][Lys] pretreatment yields guaiacol, [TEA][HSO4] yields guaiacylacetone, and [C2C1Im][OAc] yields both guaiacol and guaiacylacetone as major products. These results clearly indicate that the changes in lignin structure as well as the depolymerized product profile depend on the pretreatment conditions and the nature of the ILs. The insight gained on lignin structure changes and possible depolymerized products during IL pretreatment process would help future lignin valorization efforts in a potential IL-based lignocellulosic biorefinery. KEYWORDS: Lignin, Lignin depolymerization, Ionic liquid, Pretreatment, Cholinium Lysinate.


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INTRODUCTION Lignocellulosic biomass is considered to be a sustainable and renewable resource for the production of fuels and chemicals.1 It is primarily composed of three major biopolymers, namely, cellulose, hemicellulose and lignin. Although lignin constitutes 15-30 wt% of biomass and is the second most abundant biopolymer on Earth after cellulose, in terms of proposed biorefinery configurations it is largely underutilized.2-4 In recent years, lignin valorization has attracted a significant interest in the scientific community.3, 5-7 Lignin is an amorphous, heterogeneous aromatic polymer comprised of three phenylpropanoid monomers, namely p-coumaryl alcohol (H), coniferyl alcohol (G), and sinapyl alcohol (S) linked by C-C or C-O bonds. The common lignin interunit linkages include β-O-4’, 55’, β-5’, 4-O-5’, β-1, dibenzodioxocin, β-β’, and α-O-4’ (Figure 1). Although the relative proportions differ of these linkages depend on the origin of the lignin, the β-O-4’ bond is the major inter-unit linkage linkage in lignin, irrespective of the source.8 For both the biofuel and pulp and paper industries, carbohydrate polymers are the main focus and lignin is traditionally separated from the biomass via different pretreatment methods. Ionic liquid (IL) pretreatment of lignocellulosic biomass has been demonstrated to be one of the most efficient pretreatment methods to reduce biomass recalcitrance and to effectively fractionate biomass components.9-12 Amongst the various factors affecting IL pretreatment efficiency, the ability of the IL to disrupt the lignincarbohydrate interlinked structure and thereby enable lignin removal was found to be the important factors for most of the efficient ILs.9 In order to effectively valorize lignin, it is important to understand lignin transformation during the pretreatment process. Previous literature reports confirm that changes in the chemical structure of lignin occur during the



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IL pretreatment process.13-16 However, few studies to date have focused on providing a detailed understanding of the chemical transformations of lignin during the IL pretreatment process; furthermore, most of them are focused on imidazolium-based ILs.16-24 George et al. reported a systematic study on changes in macromolecular structure of technical lignin during thermal treatment with imidazolium-based ILs having different anions. These studies show significant reductions of lignin molecular weight during the pretreatment process.19 Sun et al. carried out an extensive study on the chemical transformation of alkaline lignin under a broad range of pretreatment conditions using the IL 1-ethyl-3-methylimidazolium acetate ([C2C1Im][OAc]) and reported dissociation of the β-O-4’ linkage and degradation of β–β’ and β-5’ linkages, primarily at higher pretreatment temperatures.16 Sathitsuksanoh et al. characterized different lignin streams isolated during the pre-treatment of wheat straw, Miscanthus and pine with [C2C1Im][OAc] using solution-state two-dimensional (2D) NMR and size exclusion chromatography (SEC). The results suggested that the lignin isolated from the pretreatment of three different feedstocks were depolymerized.13 Since the inception of ILbased pretreatment technology, various types of ILs such as acidic, protic, bionic IL, etc. have been studied and many of them have excellent pretreatment efficiencies.9 However, a comparative analysis of the transformations of lignin that occur during the ILpretreatment process as a function of IL is lacking. In this work, we aim to understand the chemical transformation of lignin during the IL pretreatment process as a function of the IL used. We have employed kraft lignin (KL) as a lignin source and used three different ILs: [C2C1Im][OAc], cholinium lysinate ([Ch][Lys]), and triethylammonium hydrogensulfate ([TEA][HSO4]). KL is one of the


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most common commercial lignin sources. Apart from KL’s easy availability, the exploration of chemical changes induced during IL pretreatment process using a relatively inert lignin will provide a baseline for future studies with other lignin sources. [C2C1Im][OAc] is the most studied IL to date for the pretreatment of lignocellulosic biomass and is considered as a benchmark system. [Ch][Lys], often termed as a renewable IL or “bionic liquid”, both the cation and anion are derived from amino acids; it has been demonstrated to have excellent pretreatment efficiencies.25-30 Lastly, [TEA][HSO4] is a protic IL and attractive due to its low cost.31 Apart from the structural differences, the acidic or basic characteristics associated with these three ILs are also very different; [Ch][Lys] is strongly basic,11 [TEA][HSO4] is acidic,32 and [C2C1Im][OAc] is near neutral (Table S1). Apart from the variable chemical nature of ILs, we also seek to understand the effect of the pretreatment conditions on the lignin structure and therefore conducted the study using two common pretreatment temperatures and variable pretreatment times. The lignin streams (Figure 2) were characterized using two-dimensional (2-D) 13C-1H Heteronuclear Single Quantum Coherence (HSQC) and 1D

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P Nuclear Magnetic Resonance (NMR) methods to probe relative changes in the

lignin interunit linkages, Gel Permeation Chromatography (GPC) for determining the relative changes in the hydrodyanamic volume or molecular weight, Fourier Transform Infrared Spectroscopy (FT-IR), elemental analysis for probing change in chemical structure, and Gas Chromatography–Mass Spectrometry (GC-MS) for identifying the monomeric lignin depolymerization products formed during the pretreatment process. These results provide a deep insight about the chemical changes in lignin structure as a function of IL and pretreatment conditions.



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MATERIAL AND METHODS CHEMICALS AND MATERIALS Kraft lignin (KL) was purchased from Sigma-Aldrich (Batch number: 1001044421). All other chemicals were reagent grade and purchased from Sigma-Aldrich (St. Louis, MO), unless otherwise noted. [C2C1Im][OAc] was purchased from BASF with a 98% purity (lot no. 11-0005) and used as received. [Ch][Lys] and [TEA][HSO4] were synthesized following previously described literature procedures.33-34 IL PRETREATMENT KL (0.5 g) was mixed with IL (4.50 g) in a glass pressure tube with a magnetic stirrer and pretreated in an oil bath at an array of times and temperatures under air. After pretreatment, the slurry was cooled to room temperature and diluted with 10 mL deionized (DI) water. The lignin was isolated (L1) by adjusting the pH of the solution to 2-3 using 6 N HCl and the solution was kept in a 4 °C refrigerator overnight to ensure total precipitation. The content was transferred to a 50 mL Falcon tube with an additional 20 mL DI water and centrifuged at high speed (8000 rpm) for 15 minutes to separate solids. The clear supernatant liquid was carefully aspirated and the resulting solid lignin was additionally washed with DI water and centrifuged (each 35 mL) five times. The regenerated solid lignin (L1) was then freeze dried. To the first wash (supernatant liquid), 10 mL ethyl acetate (EA) was added and mixed vigorously. Then the organic layer was separated and the supernatant liquid was washed with additional 10 mL of EA. The combined organic layer was then dried over anhydrous magnesium sulfate and solvent was removed to isolate the monomeric lignin depolymerization products (L2). CHARACTERIZATION OF LIGNIN


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The molecular weight distribution of lignin was investigated using gel permeation chromatography (GPC). The lignin was acetylated with pyridine and acetic anhydride following a previously published procedure.35 The acetylated lignin was dissolved in tetrahydrofuran (THF) with a concentration of 1 g/L. GPC analysis was performed using a Tosoh Ecosec HLC-8320 GPC equipped with a Refractive Index (RI) and Diode array detector (DAD) detector. Separation was achieved with an Agilent PLgel 5 µm Mixed-D column at 35 °C using a mobile phase of THF at a flow rate of 1.0 mL/min. The GPC standards, which contained polystyrene ranging from 162 to 29,150 Da, were purchased from Agilent and used for calibration. Absorbance of materials eluting from the column was detected at 280 nm (UV). NMR samples were prepared by dissolving 50 mg in 600 µL DMSO-d6. Twodimensional 1H-13C HSQC (adiabatic HSQC, hsqcad.c) spectra were acquired at 298K on a 600 MHz Varian Direct Drive (VNMRS) spectrometer equipped with a Varian triple resonance salt-tolerant cold probe. Spectra were collected in States-TPPI mode using 6.1 µs and 14.5 µs nonselective 90 degree pulses and 12 ppm and 166 ppm spectral widths for 1H and 13C respectively, 2048 complex points, 256 increments (Varian parameter ni) to give 81 Hz (0.65 ppm) digital resolution in the indirect dimension, 32 transients, and a recycle delay of 1.5 seconds. The KL and IL-pretreated lignin samples were analyzed by quantitative

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P NMR using published procedures.36 An accurately weighed amount of

dried lignin sample (40−45 mg) was dissolved in 500 µL of anhydrous pyridine/CDCl3 mixture (1.6:1, v/v). A total of 200 µL of an endo-N-hydroxy-5- norbornene-2,3dicarboximide solution (internal standard; 9.23 mg/mL) and 50 µL of a chromium(III) acetylacetonate solution (relaxation reagent; 5.6 mg/mL) were added to the above



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pyridine/CDCl3 solution. Finally, 100 µL of phosphitylating reagent II (2-chloro-4,4,5,5tetramethyl-1,2,3- dioxaphospholane) was added and transferred into NMR tube for analysis. One-dimensional 31P spectra were acquired at 298K on a 500 MHz Varian Inova spectrometer equipped with a Nalorac HCNP triple resonance probe, a 15.6 µs nonselective 90 degree pulse, 286 ppm spectral width, 64k complex points, 1024 transients, 25 s recycle delay, and WALTZ 1H decoupling during the acquisition period. Chemical shifts were referenced to the DMSO peak (δC/δH: 39.50/2.50 ppm). Spectra were processed using MestReNova 9.0 (Mestrelab Research, Spain), using cosine bell apodization and 2X zero filling, and referenced to residual protonated DMSO (for 1H and 13

C) or indirectly to phosphoric acid (for 31P). Identification of chemical compounds in depolymerization products was carried out

using an Agilent 6890N gas chromatography (GC) equipped with Agilent 5973N mass spectrometry (MS). The compounds were separated using an Agilent DB-5MS (30 m × 0.25 mm × 0.25 µm) capillary column. 1.0 µl of the sample with a concentration of 1 g/L in EA was injected into the GC at an inlet temperature of 280 °C and was operated in a split mode (split flow of 30 mL/min, split ratio = 30). Helium was used as a carrier gas with a constant flow rate of 1 mL/min. The temperature of the GC was held at 50 °C for 5 min, was then increased at a rate 5 °C/min up to 300 °C and was held at this temperature for 5 min. The MS was used until the end of GC run with a solvent delay of 7.0 min. The ion source was maintained at a temperature of 280 °C and the MS was operated in scan mode. The instrument control and data processing were carried out using the installed Agilent ChemStation software, to identify each peak NIST MS library was used. The


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monomeric lignin depolymerization products were quantified using calibration curves constructed from the standards. Elemental analysis was performed on a CHNS 2400 Perkin Elmer series II analyzer. The oxygen content was obtained by subtracting the contents of other elements. The C900 formula and degree of unsaturation (DU) were calculated, on the basis of the contents of various elements.

RESULTS AND DISCUSSION THEORETICAL SURVEY OF LIGNIN INTERUNIT LINKAGES The presence of various types of interunit linkages in lignin and the considerable variation in relative proportions of these linkages depending on the type or source of the lignin creates a complex heterogeneous structural network. The structural complexity of lignin poses a challenge for lignin depolymerization studies. Theoretical investigations of lignin linkages have been an area of research. These theoretical studies were conducted on lignin model compounds mimicking the interunit linkages present.37-41 The calculated bond dissociation energies (BDE) of different lignin bonds provide an idea about the lignin depolymerization chemistry. Lignin interunit linkages can be classified into two broad categories, namely, ether (CO) and carbon-carbon (C-C) covalent linkages. In a particular substructure, the ether linkages typically have lower BDEs than carbon-carbon linkages. Parthasarathi et al. did a theoretical study involving 65 different lignin model compounds and reported their BDEs. The results show that α-O-4’ followed by β-O-4’ is the weakest among various ether linkages and β-1 followed by β-5’ are the strongest among various C-C linkages.37 Beckham et al., Younker et al., and Elder et al. did thorough theoretical studies of lignin



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linkages using different model compounds; a summary of BDEs from these studies of different covalent bonds in various lignin substructures (Figure 1) are listed in Table 1.3742

Although BDEs depend also on the substitutions present and can vary in an actual

macromolecular lignin structure, this simple comparison gives a general idea about the relative bond strengths of major lignin inter-unit linkages. CHARACTERIZATION OF REGENERATED LIGNIN (L1) 2D-HSQC NMR ANALYSIS The changes in the chemical structure of the regenerated lignin (L1) in different IL pretreatment conditions were analyzed using

1

H-13C HSQC NMR (Figure S1-4,

Supporting Information). A comparison of untreated lignin (KL) with regenerated lignin provides an insight into the changes in the chemical structure in lignin as a function of pretreatment conditions. Table 2 tabulates different pretreatment conditions and the changes in the interunit linkages and Figure 1 depicts the chemical structures of the interunit linkages. The peaks were assigned following literature protocols.43-45 The HSQC analysis of lignin samples indicate that initial lignin is primarily comprised of guaiacyl (G) units with a minor contribution of syringyl (S) units identified by the cross peaks at δC/δH: 110.9/6.98 (G2), 114.7/6.74 (G5), 119.0/6.80 (G6), and 104.0/6.72 (S2,6) respectively. The HSQC analysis also shows distinct β-O-4’ aryl ether (A), β–β’ (resinol, B), and β-5’ (phenylcoumaran, C) interunit linkages. The β-O-4’ aryl ether (A) was identified by the cross peaks at δC/δH: 71.8/4.88 (Aα), 83.6/4.32 (Aβ(G/H)) and 59.559.7/3.40-3.63 (Aγ(G/H)), whereas β–β’ (resinol, B), β-5’ (phenylcoumaran, C) interunit linkages were identified by cross peaks at δC/δH 84.8/4.69 (Bα), 86.7/5.49 (Cα), 81.0/5.10 (Dα), 53.5/3.10 (Bβ), 53.4/3.49 (Cβ) respectively. The quantitative evaluation of interunit


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linkages was performed using an internal standard that represents aromatic C9 units in the lignin. The C2-H2 position of the G-unit and C2,6-H2,6 positions of S-unit are considered as stable. The total amount of C9 units present in the lignin can be estimated by the sum of half of C2,6-H2,6 signal of S-unit plus C2-H2 signal of G-unit. The relative amounts of interunit linkages are estimated from the volume integrals of the Aα, Bα, and Cα correlations and expressed as a fraction of 100 aromatic C9 units; B-unit integrals are divided by 2 as a resinol unit contains two C/H pairs per unit. In the untreated lignin (KL) amongst all the interunit linkages the β-O-4’ linkage dominates with 12/100Ar apart from that other interunit linkages like β–β’ (3/100Ar), and β-5’ (5/100Ar) linkages were also identified. The HSQC analysis of the pretreated lignin clearly demonstrated the decrease in the Aα (β-O-4’) content, which could be a manifestation of depolymerization, dehydration or recondensation pathways involving β-O-4’ subunit of lignin during IL-pretreatment. The effect of pretreatment temperature on the chemical structure of lignin was first explored. KL was pretreated in two common pretreatment temperatures, 140 °C and 160 °C for 1 h.29, 46 For all three ILs studied, higher pretreatment temperature favors a decrease in β-O4’ content. Between these three ILs, the acidic IL ([TEA][HSO4]) shows the maximum temperature effect. A 25% increase in the β-O-4’ degradation is observed for [TEA][HSO4], as compared to 8% for [C2C1Im][OAc] and [Ch][Lys] by increasing the pretreatment temperature from 140 to 160 °C. At 140 °C, for all three IL pretreated lignin samples, no changes were observed for β–5’ linkages, but at 160 °C, a slight β–β’ degradation was observed. To determine the effect of time, pretreatment experiments were carried out at 160 °C for 1, 2, and 3 hrs, respectively, which are common biomass



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IL pretreatment conditions. All three ILs show a high β-O-4’ bond degradation rate in the first hour which then levels off with an increase in pretreatment time. The effect of the type of IL on the chemical changes in the lignin structure during pretreatment shows some interesting trends. Amongst various lignin inter-unit linkages, a decrease in the β-O-4’ content was observed for all three ILs. [Ch][Lys] and [C2C1Im][OAc] show comparable levels of β-O-4’ bond degradation. The acidic IL ([TEA][HSO4]) was found to be most effective, with β-O-4’ bond degradation 2-3 fold higher than that for the other two ILs. The ILs show a different trend for the change in ββ’ inter-unit content after pretreatment. Both [Ch][Lys] and [C2C1Im][OAc] pretreatment were found to be more effective in decreasing the β-β’ content than the acidic IL. The dibenzodioxocin linkage was more or less unchanged during the pretreatment with all three ILs. From the relative changes in the different inter-unit linkages of lignin during the pretreatment in three different ILs, it is evident that, [Ch][Lys] and [C2C1Im][OAc] behave similarly and that these two ILs differ significantly from [TEA][HSO4]. QUANTITATIVE 31P NMR Quantitative 31P NMR analysis of phosphitylated lignin is a useful tool for estimating the amounts and distributions of different hydroxyl (alcohol) groups such as aliphatic hydroxyl, condensed and non-condensed phenolic hydroxyls and carboxylic acid groups in lignin.47 Figure S5 and Table 3 show the quantitative 31P spectra and relative amounts of different hydroxyl groups of KL and lignin isolated after IL pretreatment at 160 °C for 3 hrs in the three different ILs. The aliphatic hydroxyl content was found to be lower for all three IL pretreated lignin samples as compared to KL. The highest reduction was observed for [TEA][HSO4]


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pretreated lignin (~62%), and lowest observed for [C2C1Im][OAc] pretreatment (~4%). This reduction in the aliphatic hydroxyl group content can be attributed to factors such as loss of the γ-methylol group as formaldehyde, oxidation of Cα-OH groups to ketones, and loss of whole side chains to form β-1 linkages.48 This result corroborates the β-O-4’ degradation trend observed in HSQC NMR measurements presented above. Both condensed and non-condensed phenolic hydroxyl content was found to be higher for T160-3 as compared to KL and lignins generated from [C2C1Im][OAc] and [Ch][Lys] pretreatment. The increase in the total phenolic hydroxyl groups in [TEA][HSO4] pretreated lignin as compared to the KL may suggest the acid-catalyzed cleavage of aryl ether bonds during IL pretreatment. As compared to KL, [Ch][Lys]-pretreated lignin shows a reduction in aliphatic hydroxyl groups, and the total amount of phenolic hydroxyl groups was also found to be lower. This observation may suggest that in contrast to [TEA][HSO4] pretreatment, [Ch][Lys] pretreatment leads to less aryl ether cleavage. The [C2C1Im][OAc] pretreated lignin demonstrates very little change in terms of the aliphatic and phenolic hydroxyl content as compared to KL, suggesting a minor chemical change during the pretreatment process. The total phenolic to aliphatic hydroxyl content ratio in the case of [C2C1Im][OAc] pretreated lignin was found to be similar, and lower for [Ch][Lys] pretreated lignin, as compared to the KL. However, [TEA][HSO4] pretreatment shows an increase in the ratio of total phenolic- to aliphatic-hydroxyl content. This high aliphatic to total phenolic hydroxyl ratio and increase in the condensed phenolic hydroxyl content indicate high dissociation of phenolic ether linkages and subsequent higher recondensation via non-classical linkages, ether bonds and C-C coupling during the [TEA][HSO4]-pretreatment process.49 The carboxyl content was



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found to be higher in case of [C2C1Im][OAc]-pretreated lignin and lower in case of [TEA][HSO4] and [Ch][Lys] pretreated lignin as compared to KL. MOLECULAR WEIGHT ANALYSIS A depolymerization reaction via dissociation of β-O-4’ interunit linkage is expected to result in a decrease in the observed molecular weight, and any recondensation reactions would contribute to an increase in the molecular weight of lignin. At any point in the IL pretreatment process, depending on the conditions, competing depolymerization and recondensation reaction pathways could occur simultaneously and their net effect will be reflected in the lignin molecular weight. The regenerated lignin after pretreatment (L1) was characterized by GPC to obtain the number average molecular weight (Mn), weight average molecular weight (Mw), and polydispersity index (PDI). The results are tabulated in Table 4 and Figure S6. Because polystyrene is a random coil polymer, it is not an ideal reference for lignin; the molecular weight numbers give a rough estimation for comparisons between untreated and pretreated lignin.50 From the GPC elugram depicted in Figure S6, the main peak shows a gradual shift to the lower molecular weight region, with the largest shift observed for [C2C1Im][OAc] and lowest for [TEA][HSO4] pretreatment. Although all three IL pretreated lignins show some lower molecular weight peaks, the main peak remains unchanged, indicating no major change in molecular weight. The starting KL shows Mw of 1347 Da and a PDI of 2.67. The molecular weights of L1 after pretreatment in three different ILs at 160 °C for 3 hrs gives an idea about the net change in the lignin molecular weight during pretreatment. The Mw of L1 from [C2C1Im][OAc] pretreatment shows a minimum value of 830 Da, whereas, [TEA][HSO4]


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pretreatment shows the highest Mw of 1200 Da and [Ch][Lys] shows an intermediate value of 979 Da. From the GPC results, it is evident that the hydrodynamic volume of the lignin after [TEA][HSO4] pretreatment is highest, and [C2C1Im][OAc] pretreatment is the lowest. The higher Mw of L1 in spite of having higher β-O-4’ dissociation, suggests possible recondensation pathways during the [TEA][HSO4] pretreatment process. FT-IR CHARACTERIZATION OF LIGNIN The KL and the acid precipitated lignin (L1) after pretreatment with three different ILs at the most severe pretreatment condition (160 °C for 3 hrs) were characterized by FT-IR spectroscopy (Figure 3). Table 4 summarizes the band assignments of the lignin samples. The band assignments were carried out according to previously reported literature.51-52 As depicted in Figure 3, the FT-IR of all lignin show bands corresponding to predominately G-units. The three pretreated lignin samples shows similar spectral features to the initial KL, indicating that no major change in the main chemical structure of the lignin backbone occurs. However, changes in the relative intensity of the bands were observed, indicating relative changes in the functional groups during the IL pretreatment process. All lignin samples exhibit a broad band centred around 3430 cm-1 corresponding to the OH stretching, and peaks centered at 2938 and 2856 cm-1 corresponding to C-H stretching of methyl and methylene groups and the methyl group of methoxyl, respectively. Various peaks characteristic to aryl ring stretching, aromatic skeleton vibration, aromatic C-H deformation were observed and listed in Table 5. During the IL pretreatment new bands near 1704 and 1680 cm-1, which were not distinct in KL, become prominent, The band centered at 1704 cm-1 can be assigned to carbonyl stretching in un-conjugated ketones, carbonyls and in ester groups, whereas the



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1680 cm-1 band is due to carbonyl stretching of conjugated carbonyl and carboxyl groups. The pretreated lignin also shows increased intensities for 1270 and 1226 cm-1 bands, which are associated with C=O stretches. The increase in intensity of these peaks may arise from the oxidation of lignin (or formation of Hibbert ketones) during pretreatment. As evident in the Figure 3, these peaks are most prominent for the [TEA][HSO4] pretreatment and least for [Ch][Lys] pretreatment. The data suggests that [TEA][HSO4] pretreatment generates both unconjugated and conjugated carbonyl groups due to oxidation of lignin. Also a reduction in intensity of 1600 cm-1 band, assigned to aromatic skeletal vibration, was observed after IL pretreatment. This effect was more prominent for [TEA][HSO4] and [Ch][Lys] pretreatments. The intensity reduction of this band is believed to be a result of condensation reactions and/or splitting of aliphatic side chains of lignin.53-54 ELEMENTAL ANALYSIS To get more insight into the chemical changes of the lignin streams, elemental analysis on the lignin streams pretreated with three different ILs at the most severe pretreatment condition (160 °C for 3 hrs) was performed and compared with that of KL. The elemental composition and C900 formula along with the degree of unsaturation are listed in Table 6. The KL control shows 4.02% sulphur due to the nature of the lignin (Kraft process) and the %S decreases with IL pretreatment process. A similar decrease in sulfur content of KL after pretreatment was also observed in a few recent reports.55-56 It is also reported that the low molecular weight fractions of KL have higher sulphur content as compared to the high molecular weight fractions.56 Thus, removal of lower molecular weight fractions of lignin with high sulphur content during the pretreatment process could


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account for the observed reduction in the %S. The loss in the sulphur content could also arise from the loss of inorganic sulphur present in KL as contaminant. However, the %N in the starting KL was very negligible (< 0.15%) and the lignin isolated after pretreatment shows increased amount of %N (Table S1), which may be due to residual IL in the lignin streams.15 Although increased amount of %N could also be a manifestation of the reaction between reactive intermediates (e.g. imidazole carbene) formed during the thermal decomposition of ILs and lignin.57 In this work the increase in the %N was considered solely due to residual ILs and was subtracted from the composition accordingly. The degree of unsaturation (DU) in the KL was found to be 371, which was increased in all the lignin streams isolated after IL pretreatment, implicating an increase in the unsaturated bonds. Amongst the three pretreated lignin streams, lignin pretreated with [Ch][Lys] at 160 °C for 3 hrs (C-160-3, Table 6) demonstrated lowest DU and lignin pretreated with [TEA][HSO4] at 160 °C for 3 hrs (T-160-3, Table 6) shows highest DU. The HSQC NMR analysis of these lignin streams show similar trends for the β-O-4’ degradation pattern, which can give rise to formation of unsaturated double bonds.

CHARACTERIZATION OF EA SOLUBLE DEPOLYMERIZED LIGNIN (L2) EA SOLUBLE DEPOLYMERIZED LIGNIN (L2) The lignin-depolymerized products were extracted from the IL reaction mixture using EA, and the yields are listed in Table 1 and depicted in Figure 4. It is worth noting that some of the depolymerized lignin oligomers or monomeric lignin depolymerization products have different solubility in different ILs and can preferentially extracted by EA



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or remain in the IL-solution and not be extracted. However, the EA extractable ligninderived compounds from these three different reaction mixtures give a relative measure of lignin depolymerization during the IL pretreatment process. As depicted in Figure 4, amongst three ILs, highest yield of L1 was observed for [C2C1Im][OAc] pretreatment and lowest was observed for [TEA][HSO4] pretreatment. This data could be easily correlated with the GPC data obtained for L1, demonstrating the regenerated lignin size was highest for [TEA][HSO4] pretreatment and lowest for [C2C1Im][OAc] pretreatment. No distinct or convincing effect of pretreatment condition (either temperature or time) on the L2yield was observed for [C2C1Im][OAc] and [TEA][HSO4] pretreatment. However a steady increase in the L2 yield with increasing pretreatment severity was observed for [Ch][Lys] pretreatment. GC-MS CHARACTERIZATION OF THE DEPOLYMERIZED PRODUCTS The oil obtained from the pretreatment process (L2) was characterized by GC-MS and depolymerized products were identified using the NIST mass spectral library. The yields of the major monomeric lignin depolymerization products are depicted in Figure 5. Apart from the calibrated monomeric products; the EA-extracted oil is composed of many other aromatic monomeric products, listed in Table S2. Nearly 18 aromatic compounds were identified and most of them are phenolic in nature. Some of the identified aromatic monomeric lignin depolymerization products also contain functional groups such as aldehyde, ketone, and carboxylic acids. Among the aromatic phenolic monomeric lignin depolymerization products, phenol, guaiacol, 4-ethylguaiacol, 2-methoxy-4-vinylphenol, syringnol, vanillin, isoeugenol, 2-methoxy-4-propylphenol, acetovanillone, methyl vanillate, guaiacylacetone, eugenol, and 3-allyl-6-methoxyphenol were identified. In all


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three IL pretreated EA-extracted oils guaiacol, vanillin, guaiacylacetone, acetovanillone were identified as major products and their relative yields differ as a function of nature of IL, pretreatment temperature and time. The identified products are mostly guaiacyl derived, along with a minor amount of syringyl derived phenolics. This observation is consistent with the HSQC and FT-IR analysis, which shows that the KL consists of predominately G-units. The GC-MS results indicate that the depolymerized products depend on the pretreatment temperature and time. At lower pretreatment temperature of 140 °C, vanillin yield was highest for [Ch][Lys] and [TEA][HSO4] pretreatment. At 160 °C, the vanillin yield for [Ch][Lys] pretreatment is reduced most amongst all three ILs and guaiacol becomes the major identified depolymerized product, with the yield increasing with pretreatment time. The vanillin yield further decreases and guaiacol yield increases with increasing pretreatment time for [Ch][Lys] pretreatment. However, in the case of [TEA][HSO4] pretreatment at 160 °C, guaiacylacetone becomes the major identified depolymerized product. [C2C1Im][OAc] pretreatment at 160 °C results approximately similar yields of guaiacol and guaiacylacetone.

PLAUSIBLE PATHWAYS FOR CHEMICAL CHANGES IN LIGNIN NMR, GPC and FT-IR and elemental analysis results suggest that the chemical reactivity of three ILs towards KL is different and therefore the chemical changes in the lignin backbone induced by three ILs are also different. Previous literature reports confirm that thermal treatment of lignin in IL can induce both depolymerization and recondensation reactions.15,

55

In an IL pretreatment process, where both depolymerization and



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recondensation pathways are operative, the final chemical structure and size of the pretreated/regenerated lignin is therefore a net effect of these two pathways. The exact mechanism of these reactions is very complex and not clearly understood. Several studies on the chemical transformation of β-ether linkages in lignin under acidic conditions are reported in the literature.15,

58

Depolymerization reaction pathways in acidic ILs have

been studied in depth recently using model compounds.59-62 Kubo et al. reported when treated with imidazolium-based ILs at 120 °C, guaiacylglycerol-β-guaiacyl ether (GGE, lignin model dimer containing β-O-4’ linkage) could be converted to corresponding glycerol type enol-ether (III-type, Figure. 6A) compounds. The authors also reported that the enol-ether formed as a primary compound and could be isolated as a stable product. However, the decomposition rate and the secondary decomposition products vary, depending on the nature of IL used.63 More recently Hallet and Welton et al. carried out a detailed mechanistic study on hydrolysis of β-O-4’ interunit linkage using different lignin model compounds in hydrogen sulphate (HSO4)-based acidic ILs and proposed a pathway that proceeds via dehydration reaction followed by formation of enol-ether and consequent hydrolysis to yield guaiacol and Hibbert ketones.62 The authors also reported the rate of ether cleavage increases with the acidity of the IL and the initial dehydration step is the rate-determining step of the reaction.62 A proposed general depolymerization and recondensation mechanism in acidic IL is depicted in Figure 6A. The depolymerization pathway is suggested to proceed via protonation of the hydroxyl group attached to the α-carbon of the β-ether subunit (I) followed by dehydration to generate a stabilized benzylic carbocation (II). Then the carbocation can proceed via enol ether (III) type intermediate followed by hydrolysis to yield oligomeric/monomeric phenols (V) and


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Hibbert ketones type structures (IV). It is suggested that the recondensation reaction commonly proceed via formation of a covalent bonds between electron-rich positions of aromatic rings of a subunit and α-carbons of the benzylic carbocation (II) to form a diphenylmethane structure (VI)

64

or a coupling between electron-rich positions of

aromatic rings of a subunit and the electrophilic positions of aromatic rings of the benzylic carbocation (II) to form a biphenyl type structure (VII).65 In an alternate pathway (not depicted in Figure 6), the benzylic carbocation (II) can lose formaldehyde molecule (CH2O) to yield α-β unsaturated compound, which then hydrolysed to yield guaiacol and carbonyl compounds.

It has been suggested that in acidic ILs the

decomposition pathway depicted in Figure 6A is favoured when the IL anion interacts with γ-OH group and with out interacting anion, it goes via the pathway with elimination of formaldehyde molecule. 66 The HSQC analysis of L1 after [TEA][HSO4] pretreatment indicates significant reduction of the β-ether linkage. The increase in the aliphatic hydroxyl and subsequent increase in the phenolic hydroxyl content shown by

31

P-NMR analysis suggest a high

level of degradation. This observation is also corroborated by the increase in the intensity of the C=O stretch observed in FT-IR, indicating more aldehyde/ketone functional groups. The GC-MS analysis of L2 also shows that more aldehyde/ketone monomeric products form during the [TEA][HSO4] pretreatment. The higher DU observed by elemental analysis suggests formation of more unsaturated linkages during the pretreatment, indicating formation of dehydrated (α-β unsaturation) β-ether substructures or formation of carbonyl functional groups (as corroborated by FT-IR). As shown in Figure 6A, the possible benzylic cation generated during acidic IL pretreatment process



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can undergo recondensation reactions producing thermodynamically stable C-C bonds. Increase in the condensed phenolic hydroxyl content observed by

31

P-NMR, along with

relatively lower reduction of molecular weight observed by GPC are indicative of the higher recondensation rate during [TEA][HSO4] pretreatment process. Under alkaline conditions, the β-ether linkage of lignin can undergo the chemical reactions depicted in Figure 6B. The phenolic β-ether moiety (I, R=H) under alkaline conditions can form a quinone methide-type intermediate (VIII), which then undergoes depolymerisation and/or recondensation reactions. Very few lignin depolymerisation studies with basic ionic liquids are available in literature.

In a recent study with

cholinium-based ILs including [Ch][Lys], Zong et al. reported decrease in the β-O-4’ content, along with demethoxylation and dehydration (α-β unsaturation) reactions. The authors also reported recondensation pathways mostly via formation of β-β’ and β-5’ linkages.55 The HSQC results obtained with [Ch][Lys]-pretreatment also show reduction of the βO-4’ content, but contrary to the earlier report no increase in the β-β’ and β-5’ content was observed.55 The relatively severe pretreatment conditions used in the current study may be a possible cause of the deviation as compared to the previous report.55 The decrease in the aliphatic hydroxyl content by

31

P analysis and increase in the DU by elemental

analysis supports the dehydration (α-β unsaturation) mechanism during the [Ch][Lys]pretreatment process. The β-O-4’ depolymerization is generally associated with formation of phenolic compounds. In case of

[Ch][Lys]-pretreatment, a decrease in the total

phenolic hydroxyl content of the regenerated lignin (L1) suggests possible recondensation pathways yielding higher molecular weight compounds, whereas the higher yield of the


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phenolic monomeric lignin depolymerization products (e.g. guaiacol) in L2 suggests β-O4’ depolymerization.63 As a cumulative effect the relative molecular weight of L1 was found to be intermediate amongst three ILs. The [C2C1Im][OAc]-pretreatment was reported earlier to yield regenerated lignin with reduced β-O-4’, β-5’, and β-β’ content and lower relative molecular weight. It was also reported that [C2C1Im][OAc]-pretreatment selectively degrades G-type lignin fragments and that recondensation occurs preferentially at S-units than G-units.16 Our study confirms a decrease in β-O-4’ and β-β’ content with highest decrease in the molecular weight of the regenerated lignin. The similar aliphatic and phenolic hydroxyl content observed in

[C2C1Im][OAc]-pretreated L1 as compared to KL suggests structurally

similar lignin backbones, although increase in DU suggests some dehydration. The similar condensed phenolic hydroxyl content and reduction of molecular weight indicate fewer recondensation reactions. As KL consists mainly of G-units, this result corroborates with the previous report.16

CONCLUSION In conclusion, this study illustrates the changes of KL structure during the pretreatment with three ILs of different acid/base properties and functionalities in varying pretreatment conditions along with characterization of depolymerized products. The IL-pretreatment mainly proceeds via depolymerization, dehydration, and recondensation pathways. The physical and chemical structure of the regenerated lignin is the net product of these three



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pathways. Although the protic acidic IL ([TEA][HSO4]) demonstrated maximum β-O-4’ depolymerization, the dehydration and recondensation pathways are also maximum to result in lowest decrease in the molecular weight. [C2C1Im][OAc]-pretreatment results in minimum β-O-4’ depolymerization, the highest monomeric lignin depolymerization products yield albeit with the most structural diversity of the lignin derived monomeric products leading to lower yields of some of the more commonly sought compounds (e.g. vanillin and acetovanillinone), and lowest molecular weight of L1 suggests fewer recondensation reactions. At severe pretreatment conditions (high temperature and longer time),

[Ch][Lys]

produced

mainly

guaiacol,

whereas

[TEA][HSO4]

yields

guaiacylacetone as a major product and [C2C1Im][OAc]-pretreatment yields nearly equivalent amount of guaiacylacetone and guaiacol. Although KL is a highly recalcitrant and chemically inert lignin as compared to other possible reported sources, KL gives a base line for this type of depolymerization study with varying pretreatment conditions.67 More native sources of lignin, such as milled wood lignin (MWL), enzymatically mild acidolysis lignin (EMAL) and lignin from various biomass origins will be investigated in future studies. In addition, this study provides an overall exploration of various reactions of lignin during the IL pretreatment process. The depolymerization product studies give a general idea about potential lignin valorization. A further detailed mechanistic insight of lignin transformation in these ILs using a more relevant lignin source is warranted and is currently under investigation.

SUPPORTING INFORMATION


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1

H-13C HSQC NMR spectra,

31

P NMR spectra and integration ranges, GPC elugram,

Identified compounds, chemical structure, and retention time (min) using GC-MS. pH of aqueous IL solution, Elemental analysis results,

ACKNOWLEDGEMENT This work conducted by the Joint BioEnergy Institute was supported by the Office of Science, Office of Biological and Environmental Research of the U.S. Department of Energy under contract no. DE-AC02-05CH11231. A portion of the research was performed using EMSL, a DOE Office of Science User Facility sponsored by the Office of Biological and Environmental Research located at Pacific Northwest National Laboratory. The United States Government retains and the publisher, by accepting the article for publication, acknowledges that the United States Government retains a nonexclusive, paid-up, irrevocable, world-wide license to publish or reproduce the published form of this manuscript, or allow others to do so, for United States Government purposes.

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Figures and Tables

Figure 1. Common lignin interunit linkages; (A) β-O-4’, (B) β-5’, (C) β–β’, (D) dibenzodioxocin, (E) 4-O-5’, and (F) 5-5’ linkage and structures of three ILs.



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Figure 2. Work flow diagram of different lignin streams in IL pretreatment of KL.


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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


Figure 3. FT-IR spectrum of KL and regenerated lignin (L1) at 160 °C for 3 hrs.



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Figure 4. Yield (%) of EA soluble depolymerized lignin (L2) as a function of pretreatment conditions (℃ ℃-h) and IL categories.


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Figure 5. Monomeric lignin depolymerization product yields (g/kg lignin) of L2 as a function of pretreatment conditions and IL categories.



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OH

OH

(A)

OH O

O OCH3

O OCH 3

OH

OCH 3

OCH 3

OCH3

OCH3

OR

OR

(II)

(III)

OR (I)

OR OH

OCH 3

HO

HO

O H 3CO

+

O OCH 3

O

O OCH 3

+

OCH 3 (oligomeric/ OR Hibbert Ketones monomeric phenols) (V) (IV)

OCH 3 OR (VII)

OCH 3 OR

(VI)

OCH3 OH

Recondensation Products

Depolymerization Products Depolymerization

(B) OH O OCH 3

OH

OH O

-HCHO

OCH 3 OCH3

(I)

OR

O OCH 3 OCH 3

OCH 3 (VIII)

OH

O

(IX)

O

Recondensation OCH3

Figure 6. Proposed lignin reaction pathways under (A) acidic and (B) alkaline conditions.


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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


Table 1. Bond dissociation energies of common lignin interunit linkages. Substructure

Bond type

BDE (kcal/mol)

β-O-4’

Cβ-O-C4’

54-72

Cα-Cβ

75-80

Cα-O-C4’

50-56

Cα-Cβ

54-63

Cα-O/ Cγ-O

68/79

Cα-Cβ/Cβ-Cβ’

67/81

4-O-5’

C4-O-C5’

78-83

5-5’

C5-C5’

115-118

β-5’

β-β’



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Table 2. Relative amounts of interunit linkages in L1 determined using 1H-13C HSQC NMR spectra and yields of L2 in different processing conditions.

Lignin

a

IL

T (°C)a

Lignin Interunit Linkage c

t L2 (hr)b Yield(%)

β-O-4’

β-5’

β-β’

KL

-

-

-

-

12

5

3

E-140-1

[C2C1Im][OAc]

140

1

8.4

12

5

3

E-160-1

[C2C1Im][OAc]

160

1

8.4

11

5

2

E-160-2

[C2C1Im][OAc]

160

2

8.0

10

5

2

E-160-3

[C2C1Im][OAc]

160

3

6.7

10

5

2

C-140-1

[Ch][Lys]

140

1

2.6

12

5

3

C-160-1

[Ch][Lys]

160

1

3.5

11

5

2

C-160-2

[Ch][Lys]

160

2

3.9

11

5

2

C-160-3

[Ch][Lys]

160

3

4.8

10

5

2

T-140-1

[TEA][HSO4]

140

1

2.1

10

5

3

T-160-1

[TEA][HSO4]

160

1

2.0

7

5

2

T-160-2

[TEA][HSO4]

160

2

2.0

5

5

2

T-160-3

[TEA][HSO4]

160

3

1.8

5

5

2

Prossing temperature; b processing time; c abundances of different interunit linkages are

expressed as per 100 aromatic units.


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Table 3. Quantitative estimation of hydroxyl groups in KL and different lignin streams by quantitative 31P NMR. Aliphatic-

Condensed

Non-

Total

Carboxylic

OH

phenolic-

condensed

Phenolic-OH

acid

OH

phenolic OH

KL

3.44

2.37

1.32

3.69

0.38

E-160-3

3.31

2.24

1.39

3.63

0.44

C-160-3

2.58

1.63

1.08

2.71

0.26

T-160-3

1.29

2.73

1.61

4.34

0.17



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Table 4. Molecular weight results of KL and regenerated lignin (L1) after pretreatment at 160 °C for 3 hrs. L1

Mn (Da)

Mw (Da)

PDI

KL

505

1347

2.67

E-160-3

319

830

2.60

C-160-3

383

979

2.56

T-160-3

646

1200

1.86


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Table 5. FT-IR band assignments for lignin described in Figure 5. Number

Wavenumber

Assignment

1

3430

O-H stretching, H-bonded

2

2938

C-H stretching CH3 and CH2 groups

3

1704

C=O stretching in unconjugated ketones, carbonyl,

4

1680

C=O stretching in conjugated carbonyl, carboxyl

5

1600

Aryl ring stretching, symmetric

6

1513

Aryl ring stretching, asymmetric

7

1466

C-H deformation, asymmetric

8

1428

Aromatic skeletal vibration combined with C-H in

9

1270

Guaiacyl ring and C=O stretching

10

1226

C-C, C-O and C=O stretching

11

1142

Aromatic C-H in plane deformation

12

1085

C-O deformation, secondary alcohol and aliphatic ether

13

1035

Aromatic C-H in plane deformation

14

864

Guaiacyl ring C-H deformation out of plane

15

742

Guaiacyl ring skeletal deformation, substituent groups



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Table 6. Elemental analysis of KL and regenerated lignin (L1) at 160 °C for 3 hrs. Lignin

C (%)

H (%)

O (%)

S (%)

C900 formula

DU

KL

48.79

4.82

42.22

4.02

C900H1059O585S28

371

E-160-3

53.78

4.97

24.72

2.63

C900H990O309S16

406

C-160-3

49.79

4.67

26.99

2.55

C900H1006O367S17

398

T-160-3

52.47

4.71

24.00

1.02

C900H962O309S7

420


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TOC/Abstract Graphic.

SYNOPSIS. The chemical and physical structure changes of lignin as well as the depolymerization product yield and profile during pretreatment were found to depend on the chemical nature of the ionic liquid (IL).


Survey of Lignin-Structure Changes and Depolymerization during

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