Characterization of Lignin Streams during Bionic Liquid-Based

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Characterization of lignin streams during bionic liquidbased pretreatment from grass, hardwood and softwood Tanmoy Dutta, Gabriella Papa, Eileen Wang, Jian Sun, Nancy G. Isern, John Robert Cort, Blake A. Simmons, and Seema Singh ACS Sustainable Chem. Eng., Just Accepted Manuscript • DOI: 10.1021/ acssuschemeng.7b02991 • Publication Date (Web): 05 Jan 2018 Downloaded from http://pubs.acs.org on January 7, 2018

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ACS Sustainable Chemistry & Engineering

Characterization of lignin streams during bionic liquid-based pretreatment from grass, hardwood and softwood Tanmoy Dutta†‡, Gabriella Papa†,§, Eileen Wang†, Jian Sun†, ‡ , Nancy G. Isern ∥, John R. Cort∥, Blake A. Simmons†,§, and Seema Singh*†, ‡ †

Joint BioEnergy Institute, 5885 Hollis Street, Emeryville, California 94608, United

States. ‡

Biological and Engineering Science Center, Sandia National Laboratories, 7011 East

Avenue, Livermore, California 94551, United States. §

Biological Systems and Engineering Division, Lawrence Berkeley National Laboratory,

1 Cyclotron Road, Berkeley, California 94720, United States. ∥Earth

and Biological Sciences Directorate, Pacific Northwest National Laboratory,

Richland, Washington 99352, USA * Corresponding Author: [email protected]

ABSTRACT Delignification as a function of ionic liquid (IL) pretreatment has potential in terms of recovering and converting the fractionated lignin streams to renewable products. Renewable biogenic ionic liquids, or bionic liquids (eg. cholinium lysinate, ([Ch][Lys])), provide opportunities in terms of effective, economic and sustainable lignocellulosic biomass pretreatment. We have evaluated [Ch][Lys] pretreatment in terms of sugar and lignin yields for three different feedstocks: switchgrass, eucalyptus, and pine. Four lignin

ACS Paragon Plus Environment

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streams isolated during [Ch][Lys] pretreatment and enzymatic hydrolysis were comprehensively analyzed, tracking their changes in physical-chemical structures. We observed changes in major lignin linkages and lignin aromatics units (p-hydroxyphenyl (H), guaiacyl (G), and syringil (S)) that occurred during pretreatment. A compositional analysis of the different process streams and a comprehensive mass balance in conjunction with multiple analytical techniques (Nuclear Magnetic Resonance (NMR), Mass Spectroscopy, Gel Permeation Chromatography (GPC)) is presented. Qualitative and quantitative analyses indicates that there are significantly more lignin-carbohydrate interactions for G-rich lignin in pine. The lignin removal and extent of lignin depolymerization for switchgrass and eucalyptus were higher than pine, and follows the order of switchgrass > eucalyptus > pine. The insights gained from this work contribute to better understanding of physiochemical properties of lignin streams generated during [Ch][Lys] pretreatment, offering a starting point for lignin valorization strategies.

KEYWORDS: Ionic liquid, Bionic liquid, Biomass pretreatment, Cholinium Lysinate, Lignin,

INTRODUCTION Lignocellulosic biomass has the potential to be a significant and sustainable resource for the production of biofuels and chemicals.1 Lignocellulosic biomass is primarily composed of cellulose, hemicellulose and lignin. The polysaccharides in lignocellulose can be depolymerized into sugars that are then converted into biofuels and renewable chemicals.2-3 Lignin is a heterogeneous aromatic branched polymer, providing mechanical strength and rigidity to the cell wall. Lignin is composed of three monolignols: p-coumaryl alcohol (H), coniferyl alcohol (G), and sinapyl alcohol (S), which are interconnected heterogeneously via several types of C-C and C-O linkages.


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The common lignin interunit linkages include β-aryl ether (β-O-4’), phenylcoumaran (β5’), resinol (β-β’), dibenzodioxocin, 4-O-5’, β-1, and α-O-4’.4-5 The relative proportion of the lignin interunit linkages, relative amounts of S, G and H units, and overall lignin content can differ significantly between different genotypes and environmental conditions in which the plants are grown.6 Amongst the various lignin interunit linkages, the β-O-4’ bond is the major linkage irrespective of the source of lignin. Lignin present in hardwood consists of mostly G and S units and traces of H units;7-8 softwood lignin is composed of G units with low amounts of H units;9 lignin from grasses, however, contains comparable S and G units with a relatively higher proportion of H units than hardwood and softwood.10 Apart from the difference in the relative amounts of monolignols, the γ-hydroxyl groups of the lignin side chains are often esterified by acetate and p-coumarate (pCA) in the lignin from grass origins and by phydroxybenzoates (PB) in the case of hardwoods.6,

11

Another structural feature that

differentiates hardwood and softwood lignin from grass lignin is the nature of the lignincarbohydrate complex (LCC) linkages present. In woody biomass, the LCC is mostly composed of phenyl glycosidic, benzyl ether, and γ-ester linkages, whereas in grass it is composed of ferulate (FA) bridges that connect hemicellulose (mostly arabinoxylan) and lignin.12-13 These differences imply that an effective pretreatment technology may be dependent on the initial LCC present. Amongst the numerous biomass pretreatment technologies currently being studied, the use of certain ionic liquids (ILs) has been shown to be very effective at biomass pretreatment and delignification.14,15 Most of the top performing ILs, such as 1ethyl-3-methylimidazolium acetate ([C2C1Im][OAc]), are not biocompatible and exhibit


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toxicity to the hydrolytic enzymes and microbes used in the biomass conversion process, and require an extensive water wash process to remove the residual IL after pretreatment.16 In recent years, there have been reports of ILs derived from sustainable sources and some of them have shown impressive pretreatment efficiencies and lower toxicity. Cholinium lysinate ([Ch][Lys]), an example of a renewable IL, is reported to have excellent biomass pretreatment efficiency and successfully employed in a one-pot IL-based configuration, with improved economics.17-19 In this context strategies that enable IL recycling and reuse represent a field of intense research activity and have been proposed for developing cost-effective IL pretreatment technology.20 It has been highlighted that lignin valorization can have a significant positive impact on the economics of a biorefinery,21 and there are several ongoing efforts worldwide to improve utilization of lignin.22-23 In order to develop an effective lignin valorization strategy for the lignin generated from IL pretreatment processes, it is imperative to have a qualitative and quantitative understanding of the partitioning of lignin in the different process streams by performing a detailed mass balance analysis and structural characterization. Here we report a complete mass balance analysis of different components after [Ch][Lys] pretreatment followed by enzymatic saccharification of switchgrass, eucalyptus and pine. The lignin streams (Figure 1) generated from these three biomass feedstocks were characterized using two-dimensional (2D)

1

H-13C

Heteronuclear Single Quantum Coherence (HSQC) Nuclear Magnetic Resonance (NMR) spectroscopy method for probing relative changes in the lignin chemical structure; Gel Permeation Chromatography (GPC) for determining the relative changes in the hydrodyanamic volume or molecular weight for probing change in chemical structure;


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and Gas Chromatography–Mass Spectrometry (GC-MS) for identifying the monomeric products formed during the pretreatment process. The work aims to provide a deep insight about the partitioning of various components of the lignocellulosic biomass and the effects of [Ch][Lys] pretreatment on the structural features of lignin.

MATERIALS AND METHOD CHEMICALS AND RAW MATERIALS Switchgrass (Panicum virgatum L.), eucalyptus (Eucalyptus globulus L) and pine (Pinus radiata) samples were chosen as raw representative feedstocks for grasses, hardwood and softwood, respectively, and were handled as previously reported.24 Switchgrass (Panicum virgatum) was provided from the laboratory of Prof. Daniel Putnam at the University of California, Davis. Samples of Pinus radiata and Eucalyptus globulus were provided by Arborgen. The air-dried biomass was milled by a ThomasWiley Mini Mill fitted with a 40-mesh screen (Model 3383-L10 Arthur H. Thomas Co., Philadelphia, PA, USA) and sieved to the nominal sizes of 40–60 mesh (250–400 µm) and air-dried until the moisture was 70 wt%, trends consistent with previous studies on switchgrass. This confirmed the strong delignification ability of [Ch][Lys], previously attributed to its basicity and enhanced interaction with the lignin molecule.17 In addition, [Ch][Lys] pretreatment resulted in a significant delignification and higher xylan extraction level on grass and hardwood compared to softwood (i.e pine), this latter having relatively high resistance to [Ch][Lys] pretreatment in terms of both extent of delignification and hemicellulose removal. To investigate the effect of [Ch][Lys] on the solid fraction during enzymatic saccharification, the pretreated biomass samples were saccharified with commercial enzymes at a solid loading of 10% (w/v) equivalent to 3.8-5% (w/w) glucan loading. The yield of glucose after enzymatic hydrolysis was calculated as the amount of glucose obtained by enzymatic saccharification divided by the total maximum glucose amount available in pretreated biomass samples or untreated biomass as obtained by compositional analysis. As expected, all the [Ch][Lys] pretreated samples exhibited higher saccharification rates than the corresponding untreated samples (data not shown). [Ch][Lys] pretreated switchgrass exhibited the highest saccharification hydrolysis with cellulose digestibility reaching 93% within 72 h, whereas digestibility of both eucalyptus and pine only reached 50% and 23% respectively, over the same time interval (Figure 1). The high recalcitrance of pine is mostly due to the more condensed nature of the lignin aromatics and the higher lignin content.34 The same rank of digestibility (switchgrass > eucalyptus > pine) was previously observed by Li et al., which described the glucose yield enhancement upon pretreatment with [C2C1Im][OAc], and the least impact on pine was observed. By contrast, our


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experimental results demonstrate that eucalyptus samples after [Ch][Lys] pretreatment behaved differently from the samples treated with [C2C1Im][OAc], despite similar lignin and xylan removal percentages that were measured after both IL treatments.24 In particular, after 72h about double of the amount of glucose yield was achieved for eucalyptus with respect to this work. We attribute this difference to the more severe pretreatment conditions (3 h at 160 °C; logR0 =4.0), than those used in the current study (1 h at 140 ° C; logR0 = 3.0). The significant enhancement in the yield of sugars (i.e glucose and xylose) released for IL pretreated biomass samples when compared to untreated samples is extensively reported in literature.14 This could be ascribed to different mechanisms where lignin reduction, depolymerisation of hemicellulose and cellulose crystallinity play a crucial role by reducing the specific adsorption of cellulases.15 Our results confirmed that both lignin and xylan removal played a main role during the saccharification step indicating the key role of these two plant polymers in determining cellulose accessibility by enzymes as previously reported.35,36 Moreover differences in heteropolysaccharides content such as mannan, predominant of softwood than other species

24

also need to be

considered in further studies on the mechanisms driving high hydrolysis and changes in lignin upon [Ch][Lys] pre-treatment. EFFECT

OF

[CH][LYS]

PRETREATMENT

ON

CELLULOSE

CRYSTALLINITY X-ray diffraction studies (XRD) studies were conducted to determine the changes in the crystalline vs. non-crystalline components (i.e. amorphous cellulose, hemicellulose and lignin) found in the biomass samples, and to monitor the structural changes in these


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polymers that occur during [Ch][Lys] pretreatment. Figure S1 shows the X-ray diffractograms of the untreated and pretreated materials. The diffractograms obtained from all three untreated samples showed two major diffraction peaks at 22.5° and 15.7° 2θ, characteristic of the cellulose I polymorph that corresponds to [002] and combined [101] + [101] lattice planes, respectively. The third small peak at 34.5° ([040] lattice plane) corresponds to 1/4 of the length of one cellobiose unit and arises from ordering along the fiber direction.29, 37-38 The crystallinity indices obtained from the XRD patterns of the pretreated biomass indicated that [Ch][Lys] pretreatment had minimal impacts on cellulose. Moreover the diffractograms obtained from pretreated biomasses still retains the cellulose I polymorph, in agreement with previous report.17 All pretreated biomass showed an increase in the crystallinity index (CrI) values and the change in CrI for switchgrass was found to be the highest amongst all samples analyzed. CrI of switchgrass changes from 67% to 76% after IL pretreatment, whereas for eucalyptus and pine the changes ranged from 72% to 77% and 63% to 68% respectively. The increase in CrI of the pretreated biomass can be attributed to the removal of amorphous cell wall components such as lignin and hemicellulose.17, 39-40 LIGNIN MASS BALANCE Lignin was measured from the solids streams as Klason lignin per standard NREL analysis procedure.27 The lignin content of untreated switchgrass, eucalyptus and pine, was determined to be 25, 35 and 35 % by weight, respectively. Biomass was fractionated by the [Ch][Lys] pretreatment into two fractions: the solid fraction rich in carbohydrates and the liquid fractions containing predominantly aromatic products from lignin,


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alongside soluble sugars, which could be recovered from aqueous IL with a high yield of extraction.41 As previously mentioned, significant solubilization of lignin occurred during [Ch][Lys] pretreatment of switchgrass and eucalyptus, where more than 70% lignin removal was observed, whereas a low delignification was achieved in pine. This result corroborates the higher ability of [Ch][Lys] as compared to [C2C1Im][OAc] for lignin extraction as previously reported for switchgrass by Sun et al.17 The recovery of the solubilized lignin from the first wash is deduced from the chart illustrated in Figure 1. Both L3S from lignin precipitation and the aromatic products isolated in L3L by EA extraction were measured gravimetrically. The amount of the precipitated lignin L3S from switchgrass and eucalyptus was found to be around 86 and 80 g/kg biomass DM respectively, which is equivalent to 34 and 23% of the lignin contained in the raw materials. The yield based on theoretical lignin content recoverable from the IL liquid stream (L3) was 54 and 38% for switchgrass and eucalyptus respectively, whereas no values were reported for pine, for which no appreciable lignin precipitation occurred. In summary, out of the 16%, 21%, and 6.4% lignin present in the liquid fractions in switchgrass, eucalyptus and pine, respectively, only a small fraction was recoverable, indicating that the rest of the lignin mass remaining in the liquid fraction was in a form that cannot be recovered by acidification (i.e. it is soluble and likely low molecular weight material). The yield of lignin isolated with EA, representing lignin monomers, accounted for less than 1% of the lignin present in the starting biomass, and 2%, 1.4% and 4% for switchgrass, eucalyptus and pine respectively, based on the lignin present in the liquid stream. The values of phenolic compound production found here agreed with


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data found by Trajano et al.42 who worked on wood samples reported similar lignin cumulative release of phenolic compounds after hydrothermal pretreatment, and with those reported previously by Varanasi et al.43 on the same lignocellulosic biomass, although harsher conditions were employed ([C2C1Im][OAc] at 160°C for 6 h). These yields could be enhanced by employing catalytic hydrogenolysis or oxidationhydrogenation strategies that are currently under active investigation44 and beyond the scope of this article. Detection by GC-MS after extraction in EA showed nearly 10 kinds of lignin derived monomeric aromatic products from the IL pretreatment. MOLECULAR WEIGHT ANALYSIS OF LIGNIN The molecular weight of different lignin streams not only provides vital information about the lignin depolymerization and recondensation reactions, but also important for the valorization of lignin. The weight average molecular weight (Mw), number average molecular weight (Mn), and polydispersity index (PDI) of EMAL and different lignin streams generated during pretreatment were analyzed and are listed in Table 2, and the corresponding elugrams are depicted in Figure S2. It is important to note that the Mw, Mn values were calculated using polystyrene standards as reference and it should be noted that polystyrene is a random coil polymer and is generally not a technically precise standard for lignin but is commonly used due to the lack of a robust and commercially available alternative. Nevertheless the values of Mn and Mw reported in this work are considered in relative terms; thus they can be useful in qualitatively comparing different lignin samples.45 The elugrams of EMAL (L1) of all three biomass feedstocks showed a broad, multimodal peak with pine having highest Mw while comparable Mw was observed in switchgrass and eucalyptus.


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For switchgrass, the solubilized lignin (L3s) during the pretreatment showed lower Mw with respect to the untreated lignin (L1). In particular, a relative comparison of elugrams of L1 and L3s (Figure S2) showed, that L3s contained several sharp low molecular weight peaks, confirming that the solubilized lignin is partially depolymerized during pretreatment process. Interestingly, the lignin stream isolated after enzymatic saccharification (L4s) showed Mw slightly lower than L1. However, by comparing the elugrams it is evident that L4s retains the high Mw lignin structure after the pretreatment. The solubilized lignin stream (L3s) isolated from pretreatment of eucalyptus showed the lowest Mw amongst three biomasses (Table 2). The elugram exhibited multimodal peak with several sharp low Mw peaks, which were higher in number as compared to switchgrass, indicating that higher lignin depolymerization occurred during pretreatment of eucalyptus (Figure S2). Moreover, in contrast to switchgrass, the L4s for eucalyptus showed higher Mw than the parent L1, most probably due to presence of higher LCC. The lower sugar yield of eucalyptus as compare to switchgrass suggested its higher LCC content in L4s, which was also confirmed by compositional analysis and later by HSQC NMR analysis. Like switchgrass, the L4s of eucalyptus roughly retained the main lignin structure as compared to L1. For pine, the poor yield of L3s prevents any truly robust analysis, but it suggests that only a very small amount of lignin can be solubilized during the studied pretreatment conditions. The Mw of L4s was found to be higher than L1, due to presence of LCC, which was confirmed by compositional analysis and NMR analysis. A relative comparison of the elugrams of L1 and L4s for pine showed comparable peaks, which suggests minimal effect of pretreatment on the lignin structure.


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2D HSQC NMR ANALYSIS The raw biomass, L1, L3s, and L4s for switchgrass, eucalyptus and pine were subjected to HSQC-NMR analysis. Figure 2 shows the HSQC-NMR spectra of raw biomass samples and different lignin streams and the main structures. The HSQC spectra of lignin can be divided broadly into well-resolved aromatic (δH/δC 6.0-8.0/90-160 ppm) and aliphatic (δH/δC 2.5-6.0/50-90 ppm) regions that contain the carbohydrate and lignin (oxygenated) aliphatic signals. The carbohydrate signals have correlations in the δH/δC 2.5-5.5/60-85 ppm region, partially overlapping with the lignin side-chain signals, and well-resolved anomeric region (δH/δC 3.5-6.0/90-110 ppm). The aromatic region of the HSQC spectra is dominated by signals from the aromatic ring correlations from syringyl (S) lignin (derived from sinapyl alcohol, absent in softwood), guaiacyl (G) lignin (derived from coniferyl alcohol), and p-hydroxyphenyl (H) lignin (derived from p-coumaryl alcohol). The S unit shows distinct signals compared to the magnetically equivalent C2,6-H2,6 correlation, the G unit shows multiple signals corresponding to C2-H2, C5-H5, and C6-H6 correlations, and the H unit shows signal for magnetically equivalent C2,6-H2,6 correlation (the signal for C3,5-H3,5 correlation overlap with signal from C5-H5 correlation for G units). Oxidized α-ketone structures for S and G units (C2,6-H2,6 correlation for S’ and C2-H2 correlation for G’) were identified in few samples. Apart from S, G and H units, signals from p-coumarate (pCA), ferulate (FA), cinnamyl aldehyde (b), and cinnamyl alcohol (I) were also observed in some of the samples. The cell wall esters; hydroxycinnamic acids perticularly pCA and FA are mainly evident in the switchgrass samples. FA is reported to mainly acylate C5-OH of arabinoxylans forming ester linkages. As lignification is initiated, FA takes part in cross-


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coupling with lignin monomers and possibly dimers, which results in formation of ligninpolysaccharide cross-linking.46-48 It has been reported that in herbaceous plants (eg. wheat straw and other grasses) pCA primarily acylates lignin side chains (eg. γ-OH) via ester bonds and remain as free phenol.6, 49-51 The signals unsaturated side-chains from cinnamyl alcohol end groups and cinnamaldehyde end-groups (J) were observed in some of the woody samples. Tricin (T), a flavonoid type compound, is believed to act as a site for initiation of lignification, was detected in switchgrass lignin.52-53 The aliphatic/side-chain region provides important information about the lignin interunit linkages. The structures of different interunit linkages are depicted in Figure 2. The signals associated with methoxyl group, β-aryl ether unit (β-O-4’ unit, substructure A), phenylcoumaran unit (β-5’ unit, substructure B), resinol unit (β-β’ unit, substructure C), and dibenzodioxocin (substructure D) were assigned using previously published correlations.8,

33, 54

All HSQC spectra of raw biomass and lignin streams showed

correlations corresponding to β-O-4’ unit, β-5’ unit, β-β’ unit and each substructure gave signals associated with Cα-Hα, Cβ-Hβ, and Cγ-Hγ. Signals associated with dibenzodioxocin were observed in some samples associated with switchgrass and pine. Dibenzodioxocin subunits that can result from the 5-5 coupling of G phenolic endgroups of lignin oligomers are reported to be absent in HSQC-NMR analysis of some S-rich lignin (hardwood).8 The relative amounts of interunit linkages are estimated from the volume integrals of the Aα, Bα, Cα and Dα correlations, and expressed as a fraction of the total; the C unit integrals are divided by 2, as a resinol unit contains two C/H pairs per unit. The HSQC spectra also give some interesting signals corresponding to carbohydrates present in the different samples and can be divided broadly into two


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regions: the aliphatic or lignin side-chain region and the anomeric region. The lignin side chain region shows signals from O-acetylated xylans, namely 3-O-acetyl-β-Dxylopyranoside (X’3) and 2-O-acetyl-β-D-xylopyranoside (X’2). Signals corresponding to xylans (β-D-xylopyranoside) were observed for C2-H2 (X2), C3-H3 (X3), C4-H4 (X4), and correlations for C5-H5 (X5), which significantly overlap with unassigned signals from pentose and hexose polysaccharide units, were also observed in this region. Apart from these, signals from C4-H4 correlations of 3-O-methyl-α-D-glucuronic acid (U4) unit were observed in some samples. The anomeric region of the HSQC spectra consists of C1-H1 correlation signals. Different carbohydrate signals corresponding to xylans (X1, α-X1(R), and β-X1(R)), mannans (M1), arabinans (Ar1 and Ar1(T)), galactans (Ga1), glucans (Gl1), and glucuronic acid (U1) were observed in this region of the HSQC spectra. 2D HSQC NMR ANALYSIS: PRETREATMENT OF SWITCHGRASS The HSQC spectra of cell wall of switchgrass and different lignin streams are depicted in Figure 2. The HSQC spectra of the raw biomass gives a global idea of the native cell-wall structure and the L1 gives more detailed insight on the lignin structure. From HSQC spectra of cell wall and L1 it is clear that the β-aryl ether unit (β-O-4’ unit, substructure A) is the major lignin interunit linkage in switchgrass. The β-correlation of β-aryl ether unit was found to be distributed in to G/H (Aβ(H/G)) and S (Aβ(S)) units. Apart from the major β-aryl ether linkage, phenylcoumaran (β-5’ unit, substructure B), resinol (β-β’ unit, substructure C), and dibenzodioxocin (substructure D) interunit linkages were detected. The aromatic region of the HSQC spectrum indicates that switchgrass lignin consists of G (52%), S (35%), and H (13%) units, which represents a S/G ratio of 0.67, comparable to the value reported in recent literature.55-56 Minor amounts of oxidized (α-


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ketone) structure of S lignin (S’2,6) were also detected. pCA and FA are detected in substantial proportions, in agreement with result from prior reports.40,

55-56

Multiple

correlations identical to tricin structure were also detected. After pretreatment, the lignin streams L3s and L4s showed weaker intensities of signals assigned to the β-aryl ether unit. The β-5’ content remains more or less similar in both lignin streams, whereas a reduction of β-β’ content was observed for both lignin streams as compared to the L1. The decrease in the β-O-4’ linkages in L3s and L4s as compare to L1 may be due to the dehydration and the corresponding depolymerization of lignin during pretreatment. The reduction of β-O-4’ and β-β’ contents of lignin streams after pretreatment observed in HSQC analysis was also corroborated by the reduction of molecular weight of L3s from the GPC data. A complete reduction of dibenzodioxocin linkages was observed after IL pretreatment. Dibenzodioxocin linkages can result from the 5-5-coupling of G phenolic endgroups of oligomers that are associated with branching in lignin.57-58 Disappearance of these linkages indicates removal of lignin branches during IL pretreatment, resulting in a more linear lignin macromolecule. It was observed that the IL pretreatment reduces S/G ratio in both lignin streams. However, the number of H units was largely reduced in L3s and was approximately unchanged in L4s as compared to L1 (Figure 2, Table 2). The pCA and FA units were not detected in lignin stream L3s and were observed in reduced amounts in L4s. As pCA and FA are involved in lignification, and responsible for cross-coupling between lignin monomers and establishing LCCs, the absence or the reduction of pCA and FA content in the L3s and L4s might be associated with dissociation of LCC linkages during IL pretreatment. Interestingly the tricin substructure was absent in L3s and was detected in


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L4s. The main traits of HSQC spectra associated to lignin structures of L4s were found to be identical with the L1 and raw switchgrass spectra, despite some changes in the relative content, which indicate that the main lignin structure of the switchgrass was retained in L4s. The aliphatic region of the raw switchgrass HSQC spectra exhibits two distinct peaks X’2 and X’3, which were absent in both L3s and L4s, suggesting deacetylation of hemicellulose occurred readily during IL pretreatment. The signal associated with α-dGlcp(R)/α-X1(R) in the anomeric region was absent and was also noticeably decreased for L3S and L4s respectively. This may be due to glycosidic bond cleavage and reduction in the degree of polymerization (DP) of hemicellulose during IL pretreatment. The carbohydrate signals in the anomeric region corresponding to of xylans, arabinans, galactans, gluconic acid units, exhibited large reduction in intensity. Signals corresponding to xylans (X2, X3, X4, X5) in the aliphatic region were observed with reduced intensity in after pretreatment in both L3s and L4s.

2D HSQC NMR ANALYSIS: PRETREATMENT OF EUCALYPTUS The HSQC spectra of cell wall eucalyptus and different lignin streams are depicted in Figure 2. The HSQC spectra of cell wall and L1 indicates that the β-aryl ether unit (β-O-4’ unit, substructure A) accounts for the largest interunit linkage, and eucalyptus lignin contains the highest β-O-4’ content amongst the three biomass feedstocks studied. The β-correlation was found to be distributed in to G/H (Aβ(H/G)) and S (Aβ(S)) units. As observed in switchgrass, phenylcoumaran, resinol substructures have also been detected in eucalyptus. The absence of any detectable dibenzodioxocin interunit


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linkages, in the raw biomass or in any lignin streams indicate linear structure of eucalyptus lignin.58 Eucalyptus lignin (L1) consists of G (45%), S (52%), and H (3%) units, which represent a S/G ratio 1.18, comparable to the value reported in recent literature.59 Oxidized (α-ketone) structures of S lignin (S’2,6) and G-lignin (G’2) as well as FA and cinnamyl aldehyde (J) end groups were also detected. After pretreatment, a decrease in the β-O-4’ and β-β’ interunit linkages in L3s and L4s as compared to L1 was observed. As in switchgrass, the β-5’ content found to be lower in L3s and similar in L4s as compared to L1. In contrast, an increase in S/G ratio in both L3s and L4s along with an increase in H-content in L3s were observed. The O-acetylated xylans signals (X’2 and X’3) were absent L3s and were detected in L4s, suggesting that the deacetylation of hemicellulose occurred more readily in the L3s stream. As with switchgrass, a complete reduction and a noticeable decrease in α-d-Glcp(R)/α-X1(R) signals were observed for L3s and L4s respectively. The signal intensity of carbohydrate signals corresponding to in the anomeric regions was reduced in L3s and mostly retained in L4s stream. This indicates more effective reduction of the LCC and hemicellulose content for L3s during IL pretreatment as compared to L4s. In addition, both L3s and L4s showed a lower signal intensity corresponding to xylans (X2, X3, X4, X5), compared to that of untreated eucalyptus. Comparing the HSQC spectra of raw eucalyptus and L4s, it can be inferred that the main lignin structure of eucalyptus was retained in L4s. The presence of higher carbohydrate signals in L4s also explains the increase in the molecular size observed by GPC as compared to L1. 2D HSQC NMR ANALYSIS: PRETREATMENT OF PINE Only one lignin steam (L4s) was analyzed for pine by HSQC-NMR, due to the low


24

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yield of L3s as previously mentioned. The spectra of raw pine, EMAL (L1) and L4s are depicted in Figure 2 and the relative contents of different interunit linkages along with other structural information are listed in Table 2. Pine consists of mainly G units (89%) with a minor amount of H units (11%).54 The β-aryl ether unit is the largest interunit linkage in pine lignin and the relative amount of β-aryl ether unit was found to be lowest amongst the three plant materials. Phenylcoumaran, resinol, and didenzodioxocin represent the other interunit linkages in pine lignin. The presence of dibenzodioxocin interunit linkages in the raw biomass, as well in L1 indicated a branched lignin structure for pine. Cinnamyl alcohol (I) and cinnamyl aldehyde (J) end groups were also detected in both raw pine and L1. After IL pretreatment, there was no reasonable amount of L3S isolated from pine, furthermore the sugar yield was the lowest amongst the three plant samples, suggesting that pine lignin is the most recalcitrant in nature. The L4S lignin stream was found to be more or less identical to L1 in terms of relative amounts of different interunit linkages and structural content. The cinnamyl alcohol and cinnamyl aldehyde end groups were also retained in L4S. Comparing the xylan signals in the aliphatic region and the various carbohydrate signals in the anomeric signals of L4S with those of raw biomass, it is evident that a minimal change in the LCC linkages was induced by the IL pretreatment. In summary, compared to the untreated biomass, the lignin streams obtained after [Ch][Lys] pretreatment from three plant samples demonstrated markedly different structural changes: (1) For switchgrass and eucalyptus a reduction in interunit linkages, mainly β-O-4´ content was observed for both L3S and L4S. The highest reduction was observed for switchgrass, while pine showed no apparent change. (2) A decrease in S/G


25


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ratio was observed for switchgrass. In contrast, S/G ratio of both eucalyptus lignin streams was increased. (3) A decrease in H content was observed in L3S for switchgrass and L4S for pine, whereas a notable increase in H content was observed in both L3S and L4S for eucalyptus. (4) Deacetylation is hugely different with the three plant samples, especially in L4S. Switchgrass after [Ch]Lys] pretreatment showed complete deacetylation and while only a partial or minimal deacetylation was observed for eucalyptus and pine. (5) Hemicellulose retention in the L4S follows the order pine > eucalyptus > switchgrass.

MASS-SPECTROSCOPIC ANALYSIS LIGNIN DEPOLYMERIZED PRODUCTS GC-MS ANALYSIS: LIGNIN DEPOLYMERIZED PRODUCTS IN L3L To understand the impact of IL pretreatment on lignin depolymerization, the liquid stream after isolation of L3S was extracted with EA. The EA-extractable low molecular weight lignin depolymerized products (L3L) were characterized by GC-MS and depolymerized products were identified using the NIST mass spectral library. The yields of the major monomeric compounds are depicted in Figure 3. Apart from the calibrated monomeric products; the L3L is composed of many other aromatic monomers (Figure S3 and Table S1). Nearly 10 aromatic compounds were identified and most of them are phenolic in nature. Some of the identified aromatic monomers also contain functional groups such as aldehyde, ketone, and carboxylic acids. Among the aromatic phenolic monomers,

phenol,

guaiacol,

2-methoxy-4-vinylphenol,

syringnol,

4-

hydroxybenzaldehyde, vanillin, acetovanillone, vanillic acid, homovanillic acid, and acetosyringone were identified. For all three biomass feedtsocks, the G-derived


26

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monomers such as guaiacol, vanillin, acetovanillone, vanillic acid, homovanillic acid were identified. The S-derived monomers such as syringol, acetosyringone were detected during pretreatment of switchgrass and eucalyptus. The H-derived monomers, such as phenol and 4-hydroxybenzaldehyde (only for eucalyptus), were observed during pretreatment switchgrass and eucalyptus samples. As shown in Figure 3, the relative yield of different monomers differs depending on the nature of the biomass. For switchgrass, none of the identified monomer prevails as major product; guaiacol, syringol, vanillin, acetovalillone and acetosyringol were identified with comparable yields. However, for eucalyptus, 4-hydroxybenzaldehyde, vanillin, and acetosyringone are the major identified products. For pine vanillin and acetovanillone were the major EA soluble products. LC-MS ANALYSIS LIGNIN DERIVED MONOMERS IN THE HYDROLYSATE The process stream after enzymatic hydrolysis were subjected to LC-MS analysis for any lignin derived lower molecular weight (monomers) compounds. Various ligninderived monomers were detected in relatively minute quantities in the hydrolysate and listed in Figure S4. The monomers are aromatic compounds with primarily carboxylic acid and aldehyde functional groups. Benzoic acid was the major product for all three biomasses with the highest yield observed for switchgrass (4.5mg/100g DM) (Figure S4). Apart from benzoic acid, 4-hyroxybenzaldehyde and vanillin were detected in hydrolysates generated from all three biomass feedstocks. Although a negligible number of aromatic compounds were detected, these aromatic compounds might either result from the liberation of residual depolymerized monomer trapped in to the pretreated biomass matrix or due to enzymatic hydrolysis of LCC linkages.

CONCLUSION


27


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[Ch][Lys] pretreatment of switchgrass, eucalyptus and pine was compared in terms of enzymatic digestibility and lignin recovery. [Ch][Lys] pretreatment at 140 °C and 1h was found to be most effective on switchgrass, which generated excellent yield (>90%) of fermentable sugars, while it was least effective on pine. Appreciable sugar yields were obtained despite less severe processing conditions and higher solid loading (15% vs. 3% w/w) as compared to the previously described findings. Although no significant changes in cellulose crystallinity were observed, [Ch][Lys] pretreatment was highly effective in terms of lignin and hemicellulose removal. The trend of pretreatment efficiency, as measured by sugar yields after pretreatment and saccharification, for the three biomass feedstock samples was found to be strongly correlated with lignin removal. Pretreatment of switchgrass resulted in a maximum reduction of lignin, which was isolated at high yield from the liquid stream after pretreatment (L3S). [Ch][Lys] was found to be least able to solubilize pine lignin, where most of the lignin was retained in the solid isolated after enzymatic hydrolysis (L4S). Different analytical approaches indicate that the LCC linkages were more effectively cleaved for switchgrass and were less altered for pine. The molecular weight of the both lignin streams isolated (L3S and L4S) of switchgrass was found to be lower than the native switchgrass lignin indicating cleaved LCC linkages and probable depolymerization during the [Ch][Lys] pretreatment process. The 2D HSQC NMR study, coupled with the SEC and compositional analysis data, revealed more carbohydrate residues after enzymatic hydrolysis for both eucalyptus and pine indicating more recalcitrance, which will demand further process optimization. The detailed


28

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quantification of the different lignin streams generated during the [Ch][Lys] pretreatment process, along with the detailed characterization carried out in this study, provide a deeper fundamental understanding of the origins of biomass recalcitrance as a function of genotype and pretreatment process employed.

SUPPORTING INFORMATION Figure S1, X-Ray diffractograms and cellulose crystallinity indices (CrI); Figure S2, GPC elugrams; Figure S3, GC-MS spectra; Figure S4, LC-MS results; Table S1, identified compounds, chemical structure, and retention time (min) using GC- MS.

AUTHOR INFORMATION Corresponding Author *E-mail: [email protected] Author Contributions †‡ T.D. and G.P †,§ contributed equally to this work. Notes The authors declare no competing financial interest

ACKNOWLEDGMENTS 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. Authors are grateful to Novozymes, Franklinton, NC, USA for providing cellulose


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mixtures CTec2 and hemicellulase mixtures HTec2.


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REFERENCES: (1)

Somerville, C.; Youngs, H.; Taylor, C.; Davis, S. C.; Long, S. P., Feedstocks for

lignocellulosic biofuels. Science 2010, 329 (5993), 790-792. (2)

Tuck, C. O.; Pérez, E.; Horváth, I. T.; Sheldon, R. A.; Poliakoff, M., Valorization of

biomass: deriving more value from waste. Science 2012, 337 (6095), 695-699. (3)

Dietrich Fengel, G. W., Wood Chemistry. Ultrastructure, Reactions. Brelin New York

1984. (4)

Boerjan, W.; Ralph, J.; Baucher, M., Lignin biosynthesis. Ann. Rev. Plant Biol. 2003, 54

(1), 519-546. (5)

Rinaldi, R.; Jastrzebski, R.; Clough, M. T.; Ralph, J.; Kennema, M.; Bruijnincx, P. C. A.;

Weckhuysen, B. M., Paving the way for lignin valorisation: recent advances in bioengineering, biorefining and catalysis. Angew. Chem. Int. Ed. 2016, 55 (29), 8164-8215. (6)

del Río, J. C.; Prinsen, P.; Rencoret, J.; Nieto, L.; Jiménez-Barbero, J.; Ralph, J.;

Martínez, Á. T.; Gutiérrez, A., Structural characterization of the lignin in the cortex and pith of elephant grass (pennisetum purpureum) stems. J. Agr. Food Chem. 2012, 60 (14), 3619-3634. (7)

Capanema, E. A.; Balakshin, M. Y.; Kadla, J. F., Quantitative characterization of a

hardwood milled wood lignin by nuclear magnetic resonance spectroscopy. J. Agr. Food. Chem. 2005, 53 (25), 9639-9649. (8)

Rencoret, J.; Gutiérrez, A.; Nieto, L.; Jiménez-Barbero, J.; Faulds, C. B.; Kim, H.; Ralph,

J.; Martínez, Á. T.; del Río, J. C., Lignin composition and structure in young versus adult eucalyptus globulus plants. Plant Physiol. 2011, 155 (2), 667-682. (9)

Yue, F.; Lu, F.; Ralph, S.; Ralph, J., Identification of 4–O–5-units in softwood lignins via

definitive lignin models and nmr. Biomacromolecules 2016, 17 (6), 1909-1920. (10)

Buranov, A. U.; Mazza, G., Lignin in straw of herbaceous crops. Ind. Crop Prod. 2008,

28 (3), 237-259. (11)

Ralph, J.; Lu, F., The DFRC Method for Lignin Analysis. 6. A simple modification for

identifying natural acetates on lignins. J. Agr. Food Chem. 1998, 46 (11), 4616-4619. (12)

Balakshin, M.; Capanema, E.; Gracz, H.; Chang, H.-m.; Jameel, H., Quantification of

lignin–carbohydrate linkages with high-resolution NMR spectroscopy. Planta 2011, 233 (6), 1097-1110. (13)

Zeng, J.; Helms, G. L.; Gao, X.; Chen, S., Quantification of wheat straw lignin structure

by comprehensive nmr analysis. J. Agr. Food Chem. 2013, 61 (46), 10848-10857. (14)

Li, C.; Knierim, B.; Manisseri, C.; Arora, R.; Scheller, H. V.; Auer, M.; Vogel, K. P.;

Simmons, B. A.; Singh, S., Comparison of dilute acid and ionic liquid pretreatment of switchgrass: biomass recalcitrance, delignification and enzymatic saccharification. Bioresour. Technol. 2010, 101 (13), 4900-4906. ACS Paragon Plus Environment

31


(15)

Page 32 of 41

Dutta, T.; Shi, J.; Sun, J.; Zhang, X.; Cheng, G.; Simmons, B. A.; Singh, S., CHAPTER 3

Ionic liquid pretreatment of lignocellulosic biomass for biofuels and chemicals. In Ionic Liquids in the Biorefinery Concept: Challenges and Perspectives, The Royal Society of Chemistry: 2016, pp 65-94. (16)

Shi, J.; Gladden, J. M.; Sathitsuksanoh, N.; Kambam, P.; Sandoval, L.; Mitra, D.; Zhang,

S.; George, A.; Singer, S. W.; Simmons, B. A.; Singh, S., One-pot ionic liquid pretreatment and saccharification of switchgrass. Green Chem. 2013, 15 (9), 2579-2589. (17)

Sun, N.; Parthasarathi, R.; Socha, A. M.; Shi, J.; Zhang, S.; Stavila, V.; Sale, K. L.;

Simmons, B. A.; Singh, S., Understanding pretreatment efficacy of four cholinium and imidazolium ionic liquids by chemistry and computation. Green Chem. 2014, 16 (5), 2546-2557. (18)

Sun, J.; Konda, N. V. S. N. M.; Shi, J.; Parthasarathi, R.; Dutta, T.; Xu, F.; Scown, C. D.;

Simmons, B. A.; Singh, S., CO2 enabled process integration for the production of cellulosic ethanol using bionic liquids. Energy Environ. Sci. 2016, 9 (9), 2822-2834. (19)

Xu, F.; Sun, J.; Konda, N. V. S. N. M.; Shi, J.; Dutta, T.; Scown, C. D.; Simmons, B. A.;

Singh, S., Transforming biomass conversion with ionic liquids: process intensification and the development of a high-gravity, one-pot process for the production of cellulosic ethanol. Energy Environ. Sci. 2016, 9 (3), 1042-1049. (20)

Sun, J.; Shi, J.; Konda, N. V. S. N. M.; Campos, D.; Liu, D. J.; Nemser, S.; Shamshina,

J.; Dutta, T.; Berton, P.; Gurau, G.; Rogers, R. D.; Simmons, B. A.; Singh, S., Efficient dehydration and recovery of ionic liquid after lignocellulosic processing using pervaporation. Biotechnol. Biofuels 2017, 10. (21)

Konda, N. M.; Shi, J.; Singh, S.; Blanch, H. W.; Simmons, B. A.; Klein-Marcuschamer,

D., Understanding cost drivers and economic potential of two variants of ionic liquid pretreatment for cellulosic biofuel production. Biotechnol. Biofuels 2014, 7 (1), 86. (22)

Li, C.; Zhao, X.; Wang, A.; Huber, G. W.; Zhang, T., Catalytic transformation of lignin

for the production of chemicals and fuels. Chem. Rev. 2015, 115 (21), 11559-11624. (23)

Zakzeski, J.; Bruijnincx, P. C. A.; Jongerius, A. L.; Weckhuysen, B. M., The catalytic

valorization of lignin for the production of renewable chemicals. Chem. Rev. 2010, 110 (6), 35523599. (24)

Li, C.; Sun, L.; Simmons, B.; Singh, S., Comparing the recalcitrance of eucalyptus, pine,

and switchgrass using ionic liquid and dilute acid pretreatments. BioEnergy Res. 2013, 6 (1), 1423. (25)

Galbe, M.; Zacchi, G., Pretreatment of lignocellulosic materials for efficient bioethanol

production. In Biofuels, Olsson, L., Ed. Springer Berlin Heidelberg: Berlin, Heidelberg, 2007, pp 41-65. (26)

Wu, S.; Argyropoulos, D., An improved method for isolating lignin in high yield and

purity. J. Pulp Pap. Sci. 2003, 29. ACS Paragon Plus Environment

32

Page 33 of 41 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


(27)

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

Determination of structural carbohydrates and lignin in biomass. Technical report NREL/NP 510–42618. National Renewable Energy Laboratory Golden Colorado. 2008, 80401 - 83393. (28)

Segal, L.; Creely, J. J.; Martin, A. E.; Conrad, C. M., An empirical method for estimating

the degree of crystallinity of native cellulose using the X-Ray diffractometer. Tex. Res. J. 1959, 29 (10), 786-794. (29)

Park, S.; Baker, J. O.; Himmel, M. E.; Parilla, P. A.; Johnson, D. K., Cellulose

crystallinity index: measurement techniques and their impact on interpreting cellulase performance. Biotechnol. Biofuels 2010, 3. (30)

Lu, F.; Ralph, J., Derivatization followed by reductive cleavage (dfrc method), a new

method for lignin analysis: protocol for analysis of dfrc monomers. J. Agr. Food Chem. 1997, 45 (7), 2590-2592. (31)

Ibarra, D., Lignin modification during Eucalyptus globulus kraft pulping followed by

totally chlorine-free bleaching: a two-dimensional nuclear magnetic resonance, Fourier transform infrared, and pyrolysis-gas chromatography/mass spectrometry study. J. Agric. Food Chem. 2007, 55. (32)

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. (33)

Kim, H.; Ralph, J.; Akiyama, T., Solution-state 2D NMR of ball-milled plant cell wall

gels in dmso-d6. BioEnergy Res. 2008, 1 (1), 56-66. (34)

Torr, K. M.; Love, K. T.; Cetinkol, O. P.; Donaldson, L. A.; George, A.; Holmes, B. M.;

Simmons, B. A., The impact of ionic liquid pretreatment on the chemistry and enzymatic digestibility of Pinus radiata compression wood. Green Chem. 2012, 14 (3), 778-787. (35)

Papa, G.; Feldman, T.; Sale, K. L.; Adani, F.; Singh, S.; Simmons, B. A., Parametric

study for the optimization of ionic liquid pretreatment of corn stover. Biores. Technol. 2017, 241, 627-637. (36)

Adani, F.; Papa, G.; Schievano, A.; Cardinale, G.; D'Imporzano, G.; Tambone, F.,

Nanoscale structure of the cell wall protecting cellulose from enzyme attack. Environ. Sci. Technol. 2011, 45 (3), 1107-1113. (37)

Cheng, G.; Varanasi, P.; Arora, R.; Stavila, V.; Simmons, B. A.; Kent, M. S.; Singh, S.,

Impact of ionic liquid pretreatment conditions on cellulose crystalline structure using 1-ethyl-3methylimidazolium acetate. J. Phys. Chem. B 2012, 116 (33), 10049-10054. (38)

Mansikkamäki, P.; Lahtinen, M.; Rissanen, K., Structural changes of cellulose crystallites

induced by mercerisation in different solvent systems; determined by powder X-Ray diffraction method. Cellulose 2005, 12. (39)

Socha, A. M.; Parthasarathi, R.; Shi, J.; Pattathil, S.; Whyte, D.; Bergeron, M.; George,

A.; Tran, K.; Stavila, V.; Venkatachalam, S.; Hahn, M. G.; Simmons, B. A.; Singh, S., Efficient ACS Paragon Plus Environment

33


Page 34 of 41

biomass pretreatment using ionic liquids derived from lignin and hemicellulose. Proc. Natl. Acad. Sci. 2014, 111 (35), E3587-E3595. (40)

Parthasarathi, R.; Sun, J.; Dutta, T.; Sun, N.; Pattathil, S.; Murthy Konda, N. V. S. N.;

Peralta, A. G.; Simmons, B. A.; Singh, S., Activation of lignocellulosic biomass for higher sugar yields using aqueous ionic liquid at low severity process conditions. Biotechnol. Biofuels 2016, 9 (1), 1-13. (41)

Shi, J.; Gladden, J. M.; Sathitsuksanoh, N.; Kambam, P.; Sandoval, L.; Mitra, D.; Zhang,

S.; George, A.; Singer, S. W.; Simmons, B. A., One-pot ionic liquid pretreatment and saccharification of switchgrass. Green Chem. 2013, 15. (42)

Trajano, H. L.; Engle, N. L.; Foston, M.; Ragauskas, A. J.; Tschaplinski, T. J.; Wyman,

C. E., The fate of lignin during hydrothermal pretreatment. Biotechnol. Biofuels 2013, 6. (43)

Varanasi, P.; Singh, P.; Auer, M.; Adams, P. D.; Simmons, B. A.; Singh, S., Survey of

renewable chemicals produced from lignocellulosic biomass during ionic liquid pretreatment. Biotechnol. Biofuels 2013, 6. (44)

Kim, K. H.; Simmons, B. A.; Singh, S., Catalytic transfer hydrogenolysis of ionic liquid

processed biorefinery lignin to phenolic compounds. Green Chem. 2017, 19 (1), 215-224. (45)

Brunow, G., Methods to Reveal the Structure of Lignin. In Biopolymers Online, Wiley-

VCH Verlag GmbH & Co. KGaA: 2005. (46)

Bunzel, M.; Allerdings, E.; Ralph, J.; Steinhart, H., Cross-linking of arabinoxylans via 8-

8-coupled diferulates as demonstrated by isolation and identification of diarabinosyl 8-8(cyclic)dehydrodiferulate from maize bran. Journal of Cereal Science 2008, 47 (1), 29-40. (47)

Grabber, J. H.; Hatfield, R. D.; Ralph, J.; Zoń, J.; Amrhein, N., Ferulate cross-linking in

cell walls isolated from maize cell suspensions. Phytochemistry 1995, 40 (4), 1077-1082. (48)

Ralph, J.; Grabber, J. H.; Hatfield, R. D., Lignin-ferulate cross-links in grasses: active

incorporation of ferulate polysaccharide esters into ryegrass lignins. Carbohydr. Res. 1995, 275 (1), 167-178. (49)

del Río, J. C.; Rencoret, J.; Prinsen, P.; Martínez, Á. T.; Ralph, J.; Gutiérrez, A.,

Structural characterization of wheat straw lignin as revealed by analytical pyrolysis, 2D-NMR, and reductive cleavage methods. J. Agr. Food Chem. 2012, 60 (23), 5922-5935. (50)

Sun, R.; Sun, X. F.; Wang, S. Q.; Zhu, W.; Wang, X. Y., Ester and ether linkages

between hydroxycinnamic acids and lignins from wheat, rice, rye, and barley straws, maize stems, and fast-growing poplar wood. Ind. Crop Prod. 2002, 15 (3), 179-188. (51)

Ralph, J.; Hatfield, R. D.; Quideau, S.; Helm, R. F.; Grabber, J. H.; Jung, H.-J. G.,

Pathway of p-coumaric acid incorporation into maize lignin as revealed by NMR. J. Am. Chem. Soc. 1994, 116 (21), 9448-9456. (52)

Li, M.; Pu, Y.; Yoo, C. G.; Ragauskas, A. J., The occurrence of tricin and its derivatives

in plants. Green Chem. 2016, 18 (6), 1439-1454. ACS Paragon Plus Environment

34

Page 35 of 41 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


(53)

Lan, W.; Lu, F.; Regner, M.; Zhu, Y.; Rencoret, J.; Ralph, S. A.; Zakai, U. I.; Morreel,

K.; Boerjan, W.; Ralph, J., Tricin, a flavonoid monomer in monocot lignification. Plant Physiol. 2015, 167 (4), 1284-1295. (54)

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 (3), 576-591. (55)

Samuel, R.; Pu, Y.; Raman, B.; Ragauskas, A. J., Structural characterization and

comparison of switchgrass ball-milled lignin before and after dilute acid pretreatment. Appl. Biochem. Biotechnol. 2010, 162 (1), 62-74. (56)

Ragauskas, A., Structural characterization of lignin in wild-type versus COMT down-

regulated switchgrass. Front Energy Res. 2014, 1. (57)

Stewart, J. J.; Akiyama, T.; Chapple, C.; Ralph, J.; Mansfield, S. D., The effects on lignin

structure of overexpression of ferulate 5-hydroxylase in hybrid poplar. Plant Physiol. 2009, 150 (2), 621-635. (58)

Argyropoulos, D. S., Abundance and reactivity of dibenzodioxocins in softwood lignin.

J. Agric. Food Chem. 2002, 50. (59)

Wen, J.-L.; Sun, S.-L.; Yuan, T.-Q.; Xu, F.; Sun, R.-C., Structural elucidation of lignin

polymers of eucalyptus chips during organosolv pretreatment and extended delignification. J. Agr. Food Chem. 2013, 61 (46), 11067-11075.


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FIGURES AND TABLES Figure 1. Mass balances (% DM) for the major biomass components of (A) switchgrass, (B) eucalyptus and (C) pine during ionic liquid pretreatment (IL-PT) with [Ch][Lys] and lignin streams (L1, L2L, L2S, L3L, L4S, L4L) generated during the process.

A

100 g DM 33±0.3 g Glc. 19±0.5 g Xyl.

52 g DM Pretreated solid

L1 25±0.7 g

Enzymatic Hydrolyzate

27±0.1 g Glc.

Enzymatic hydrolysis

9±0.1 g Xyl.

IL – PT

L2 S 5.5±1.3 g

L4 L

0

5

10 15 20 25 30 35 g/ 100 g biomass

15.3 g DM Unhydrolyzed solid

Liquid

Switchgrass

Glc. Xyl.

7±0.1 g Glc.

5.3±1.7 g Glc.

11±0.3 g Xyl.

1.1±0.1 g Xyl.

L2 L 16±0.0 g

L4s 4±1 g

pH adjustment Solid

Guaicol Syringol Vanillin Acetovanillone Acetosyringone

Liquid solvent extraction

L3 S 8.6±1

L3 L = 0.33 g

B

100 g DM 27±0.5 g Glc. 12 ±0.3 g Xyl. L1 29±0.6 g

48 g DM Pretreated solid 22±0.1 g Glc. 4.4±0.1 g Xyl.

IL – PT

L2 S 8.7±1.3 g

5.5±0.1 g Glc.

Enzymatic hydrolysis L4 L

0

5 10 g/ 100 g biomass

15

6.4±1.7 g Glc.

9.1±0.2 g Xyl.

0.5±0.1 g Xyl.

L2 L 20±1.5 g

L4s 5±1 g

pH adjustment Solid

Glc. Xyl.

13.3 g DM Unhydrolyzed solid

Liquid

Eucalyptus


Liquid

Guaicol Syringol 4-hydroxybenzaldehyde Vanillin Acetovanillone Acetosyringone

solvent extraction

L3 S 7.9±0.9

L3 L = 0.30 g

C

100 g DM 34±0.2 g Glc. 16 ±0.4 g Xyl. L 31±2 g 1

84 g DM Pretreated solid 32±0.1 g Glc. 14±0.1 g Xyl.

IL – PT

L2 S 25±1.3 g

Enzymatic hydrolysis

Glc. Xyl.

0

L4 L

5 10 g/ 100 g biomass

15

54 g DM Unhydrolyzed solid

Liquid

Pine


1.9±0.0 g Glc.

21±0.4 g Glc.

2.8±0.1 g Xyl.

7±0.1 g Xyl.

L2 L 6.4±0.6 g

L4s 22±2 g

pH adjustment Solid

L3 S trace

Vanillin Acetovanillone

Liquid solvent extraction

L3 L = 0.27 g


36

Page 37 of 41

Figure 2. 2D HSQC NMR spectra of raw, EMAL (L1), and different lignin streams genarated during IL-pretreatment of switchgrass (top row), eucalyptus (middle row), and pine (bottom row). A = β-ether (β-O-4´) unit, B = phenylcoumaran (β-5´) unit, C = resinol (β-β´) unit, D = dibenzodioxocin unit, I = hydroxycinnamyl alcohol endgroup, J = hydroxycinnamaldehyde endgroup, pCA = p-coumaric acid unit, FA= ferulic acid unit, G = guaiacyl unit, G´ = benzyl-oxidized G unit, S = syringyl unit, S´ = benzyl-oxidized S unit, X = xylan unit, X´ = acetylated xylan unit, X(R) = xylan including reducing end-unit, U = uronic acid unit, Ar = arabinan unit, Ga = galactan unit. Bβ

-OMe

Cβ

Aγ

Bγ Aα

EMAL Switchgrass (L1)

Bβ

-OMe

Bα

Cα

Aβ(S)

T8

X1/X’1

G2 pCA +FA 8 8

FA2

S´2,6

Ga1

Ar1(T)

pCA3,5

G2

FA 2

Bα

βX 1(R)

Aβ(S)

Ga 1

Ar 1(T)

pCA 8+FA 8

G2

pCA7+FA7

{

G5+G6

H2,6

pCA 2,6

S: 35% G: 52% H: 13% S/G: 0.67

pCA7+FA 7

Bβ

Raw Eucalyptus

Cβ

-OMe X5

Aγ Bγ

X5

Cγ

Aα X’3

X4 X3

X’2

R1

U4

Cα

Aβ(S)

Bα

G5+G6

{

Bβ Aγ X5 Aα

Bγ

EMAL Pine (L 1)

S: 56% G: 31% H: 13% S/G: 1.80

FA7

Bβ

Cβ

L4S

Aγ

X4 X3

X5 Aα

M’2

X2

X’2

Aβ(G/H) U4

Dα

αX1(R) M’1 X’ βX1(R) 1 X1/X’1 Ga1 Ar1(T)

Cα

Bγ

Cγ

Aβ(G/H) Cα

Bα

α

X4 X3

6 X2

G2

{

{

Jβ

G 5+G6

H2,6

Iα I β

G: 89% H: 11%

Jβ

HO α"

γ"

4' β" O

1

OMe A(G)

HO MeO γ" 4' HO α" β" O 1 OMe A(S)

HO

γ

5' β

O 1 α B

4' OMe

γ

H

γ"

MeO

γ’ α O A

4'

α"O O β"

O

4"

J OMe

OMe O R O

R 6

2

2

S'

β"

OMe O

R

O

O

γ" α" β"

γ" α" β" 6 I

R

O

HO

1

2 3

H

5

OMe MeO O

O α’

β 1

S

Iα Iβ

5" 5'

6

α"

G: 93% H: 7%

β’

O

R

MeO

Jα

O

6 2 G' OMe 5

6

α" HO

2

G

G5+G6

Jα

Jα

5

O

Dβ

G’6

H 2,6

S: 61% G: 34% H: 5% S/G: 1.79

Cβ

Aγ X5

αX1(R) βX (R) M’1 X’1 1 U1 X1/X’1 Ar1 Ga1 Ar1(T)

G’2

Iα

X’1 M 1 X1/X’1 Ga 1

Ar1(T)

H 2,6

U4

Dα

Dβ

G2

G5+G6

M’ 1

G5+G 6

-OMe

Bγ

Aα

Bα

G2

Bβ

-OMe

R1 Ar1

G2

U4

A β(S)

H2,6

Cγ

Cγ

Cα

Cβ

X5

U1

S2,6

{

-OMe

X X3 2

Cα

S’ 2,6

FA 6

Jβ

U1

Bα

Jα

S: 52% G: 45% H: 3% S/G: 1.15

X4

Aβ(G/H)

X1/X’1 Ga1

Ar1(T)

Bγ

Cγ X’2

X’3

U4

G5+G6

FA6

Bα

Aα

X X4 X3 2

FA8

FA2

Aγ X5

X5

αX 1(R)

G2

G2

FA2 G’2

Cβ

-OMe

Aβ(S)

R1

S’2,6

1

M1 X 1/X’ 1 Gl1 Ga1

X’2 Aβ(G/H)

Bβ

L4S

U1 S2,6

S2,6

G5+G6

M’2

S: 26% G: 60% H: 14% S/G: 0.43

X5

Aβ(G/H) Cα

Cβ

Aγ γ

Cγ

FA7

H2,6

X5 B Aα

Bα

Ar 1(T)

Raw Pine

Bβ

-OMe

Cγ

H 2,6

Jβ

L3S

Aβ(G/H)

H2,6

G’2

S: 28% G: 66% H: 6% S/G: 0.42

H2,6

{

U1

G2

Cβ

Aγ

Bγ

Aβ(S) αX1(R) M’1 X’

S’ 2,6 G’ 2

Bβ

-OMe

Aα

X2

A β(G/H)

S2,6

EMAL Eucalyptus (L1)

Cα

Bα

X1/X´1

S2,6

G 5+G6

FA6 H2,6

Cα

X 1/X´1

pCA3,5

G5+G6

FA6 pCA2,6

T3

Aβ(G/H)

Aβ(S)

M1

{

T3

Cα

T6

S2,6

T´2,6

X5 X4 X3 X2

T8

βX1(R)

{

S´2,6

M1

T6

S2,6

T´2,6

Cβ

Aγ

Bγ X5 Cγ

Aα

A β(G/H)

Dα

L4s

Bβ -OMe

Cγ

Cγ Bα

L3s

Aα

Aβ(G/H)

Dα

Cβ

Aγ

Bγ

{

Raw Switchgrass

{

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


5

FA

2 OMe

O

R

α" 6 5

O

γ" β"

pCA

O

2 3

R

γ" OH D

C O OMe


37


Figure 3. Monomer yields (g/100g of DM) obtained from GC-MS analysis of L3L genarated from [Ch][Lys] pretreatment of switchgrass, eucalyptus, and pine. 18

50

16

45

14

40

Switchgrass Eucalyptus Pine

12

30 10 25 8 20 6

15

Ac et os yr

Ac et ov an illo

Va ni lli

be nz al de hy

4

-h yd ro xy

Sy r

in go ne

0

ne

0

n

5

de

2

in go l

10

ua ic ol

4

mg/ 100 g lignin DM

35

G

mg/100 g raw biomass DM

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

Page 38 of 41


38

Page 39 of 41 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. Chemical composition, solid recovery and lignin removal from untreated and [Ch][Lys]-pretreated switchgrass (SG), eucalyptus (EU) and Pine (P). Composition (%)

Samples Solid Recovery SG

Untreated After [Ch][Lys] After Hydrolysis

EU

52

74

15

9.9


P

Lignin Extracted

ASL

36.2±0.3 21.5±0.5

21±0.7

3.7±0.2

57.1±0.0 19.9±0.2

10.7±2

3.7±0.1

8.2±1.4

22.6±6

3.9±0.4

38.5±1.4

Xylose

30.1±0.5 14.1±0.3 29.2±0.6 5.3±0.2 48

70

13

15


Klason Lignin

Glucose

51.6±0.4 10.4±0.0 18.3±1.3 4.7±0.2 53±2.9

4.5±0.2

37.6±0.2 18.3±0.4 84

21

54

15

31.3±2.3 4.4±0.0 31±0.0

4±0.0

42.6±0.2 18.5±0.0 29.5±1.7 5.6±0.0 42.8±0.7 14.4±0.2 36.6±4.4 3.8±0.0


39


Page 40 of 41

Table 2. Molecular weight (Mn and Mw); S, G, H contents; relative amounts of interunit linkages of different lignin streams (L1, L3S and L4S) determined using 1H−13C HSQC NMR spectra Mn (Da)

Mw (Da)

PDI

S/G

S

G

H

β-O-4’ (A)

β-5’ (B)

β-β’ (C)

D

71 (56) 79 (48) 76 (44)

12 (9) 16 (10) 14 (8)

13 (10) 5 (3) 10 (6)

4 (3)

73 (62) 83 (57) 78 (58)

7 (6) 4 (2) 5 (4)

20 (17) 13 (9) 17 (13)

na na

64 (48) na 65 (47)

24 (17) na 21 (16)

7 (5) na 10 (7)

5 (4) na 4 (3)

Switchgrass L1

1007

1818

1.80

0.67

35

52

13

L3S

567

1362

2.40

0.42

28

66

6

L4S

862

1663

1.93

0.43

26

60

14

na na

Eucalyptus L1

820

1807

2.20

1.15

52

45

3

L3S

426

844

1.98

1.80

56

31

13

L4S

1357

3124

2.30

1.79

61

34

5

na

Pine L1

1378

2618

1.90

na

na

89

11

L3S

na

na

na

na

na

na

na

L4S

1396

3234

2.32

na

na

93

7

Abundances of different interunit linkages (A-D) are expressed as percentage of total interunit linkages (and as per 100 aromatic units); Mn: number average molecular weight; Mw : weight average molecular weight; PDI : polydispersity index.


40

Page 41 of 41 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


TOC/ABSTRACT GRAPHIC Synopsis Lignin streams from the whole process of bionic liquid pretreatment and saccharification were characterized and found to depend on biomass type.


41

Characterization of Lignin Streams during Bionic Liquid-Based

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