Improving Lignin Homogeneity and Functionality via Ethanolysis for

Jan 7, 2019 - As an antioxidant material, lignin has some structural disadvantages such as heterogeneity, high molecular weight, and low functionality...
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Improving lignin homogeneity and functionality via ethanolysis for production of antioxidants Jae-Young Kim, Patrick Allan Johnston, Jae Hoon Lee, Ryan Smith, and Robert C. Brown ACS Sustainable Chem. Eng., Just Accepted Manuscript • DOI: 10.1021/ acssuschemeng.8b05769 • Publication Date (Web): 07 Jan 2019 Downloaded from http://pubs.acs.org on January 8, 2019

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Improving lignin homogeneity and functionality via ethanolysis for production of antioxidants

Jae-Young Kima, Patrick A. Johnstona, Jae Hoon Leeb, Ryan G. Smitha, Robert C. Browna*

a Bioeconomy b Department

Institute, Iowa State University, Ames, Iowa 50011, United States

of Forest Sciences and Research Institute for Agriculture and Life Science, Seoul

National University, 599 Gwanak-ro, Gwanak-gu, Seoul, 151-921, South Korea *Corresponding author. E-mail address: Jae-Young Kim: [email protected] Patrick A. Johnston: [email protected] Jae Hoon Lee: [email protected] Ryan G. Smith: [email protected] Robert C. Brown: [email protected]

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ABSTRACT

As an antioxidant material, lignin has some structural disadvantages such as heterogeneity, high molecular weight, and low functionality. We propose a simple method based on ethanolysis pretreatment (120, 160, and 200 °C for 1 h) of lignin by which the structure, especially the molecular weight distribution and phenolic hydroxyl content (Phe-OH) of lignin are improved. After reaction, over 80 wt% structurally modified lignin (solubilized lignin: SL) was recovered from raw lignin, and its homogeneity as well as Phe-OH content increased. The antioxidant activity of SLs was investigated by 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical scavenging ability. Interestingly, DPPH radical scavenging capacities of SL produced at 160 °C (IC50 = 0.016 mg/mL) was close to commercial antioxidants such as ferulic acid (IC50 = 0.010 mg/mL) and guaiacol (IC50 = 0.013 mg/mL), suggesting that ethanolysis pretreatment was effective in modifying the structure of lignin for use as antioxidant. We also performed experiments with lignin model compounds, which revealed that antioxidant activities of lignin were not only affected by homogeneity and functionality but also other structural characteristics including H-, G-, and S-type composition and side chain characteristics.

Keywords: Lignin, Ethanolysis, Depolymerization, Phenolic hydroxyl, Molecular weight, Antioxidant activity

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Introduction Lignin is an amorphous macromolecule consisting of three kinds of monolignols (pcoumaryl, coniferyl and sinapyl alcohols) with diverse covalent bonds including α-O-4, β-O4, β-5, β-β and biphenyl.1 Lignin is the most plentiful aromatic polymer in nature with many attractive features such as high carbon content, thermal stability, functionality, biodegradability, and antioxidant activity.2 These properties help make lignin attractive for production of valueadded products. Antioxidants are compounds that combine and stabilize free radicals generated by the presence of oxygen. They find wide application in the food, medical and chemical industries.3 Several

synthetic

antioxidants

including

butylated

hydroxytoluene

(BHT),

butyl

hydroxyanisole (BHA), and propyl gallate (PG) have commanded a main share of the present antioxidant market. Recently, natural antioxidants are finding favor owing to their nontoxicity.4 Many previous studies have investigated lignin as natural antioxidant because this phenolic polymer has suitable functionality for this purpose including phenolic hydroxyl (PheOH), methoxyl (OMe), and carboxyl group.2,

5-7

However, direct utilization of lignin is

challenging because of its structural heterogeneity and complexity, which results in broad molecular weight and low dissolubility in organic solvents.8 Previous studies have reported that higher antioxidative efficiency requires lignin to have greater amounts of Phe-OH and OMe functionality, lower amounts of aliphatic hydroxyl groups (Aliph-OH), lower molecular weight, and narrow polydispersity index.9-11 Some researchers have tried to enhance lignin antioxidant activity by using solvent fractionation, nanoscale preparation and blending with plastic materials.3,

12-14

Additionally, a natural, effective, and inexpensive antioxidant was

developed by Liu et al. who used blending method for preparation of lignin and quercetin mixture.15 3

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Partial depolymerization is one approach to producing lignin fragments (solubilized lignin: SL) with high homogeneity as well as functionality via selective degradation of C-O and condensed C-C bonds. Previous work by Xin et al. on partial lignin depolymerization through mild hydrogenolysis (140 to 200 °C) using 2.0 MPa hydrogen gas in the presence of Raney nickel catalyst was successful in decreasing molecular weight and increasing Phe-OH content of lignin depolymerization products.16 Yang et al. carried out selective degradation of the βO-4 linkage in the presence of concentrated lithium bromide (LiBr) and acid catalyst to obtain low molecular weight lignin.17

Relatively few studies have investigated SL as a source of

antioxidants. Li et al. suggested biological pretreatment of lignin with laccase, which could effectively enhance its antioxidant performance by increasing Phe-OH content and reducing molecular weight but this process required long reaction time (6 h) and enzyme.18 Zhao et al. observed the hydrogenolysis performed with formic acid and Pd/C as the catalyst significantly improved the antioxidant activity of lignin.19 We explore improving the homogeneity and functionality of lignin depolymerization products using a simple ethanolysis process under relatively low temperatures without metal or acid/base catalysts. We employed ethanol as solvent because it can dissolve a large fraction of lignin, resulting in a more homogenous reaction environment. In addition, ethanol can facilitate direct C-O bonds cleavages and inhibit repolymerization reaction.20,

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Structural

characteristics of lignin after ethanolysis were investigated by gel permeation chromatography (GPC), phosphorus-31 nuclear magnetic resonance (31P-NMR), and pyrolysis–gas chromatography/mass spectrometry (Py-GC/MS). The antioxidant property of lignin was determined by 2,2-diphenyl-1-picrylhydrazyl free radical (DPPH) scavenging assay. We found that the antioxidant properties of SLs remarkably improved as comparing with that of raw lignin due to the positive effects of increased Phe-OH and homogeneity. Additionally, 4

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compositional differences in H-, G- and S-type compounds as well as side chain features of lignin also affected its antioxidant activity.

Experimental Materials Protobind 1000 soda lignin (GreenValue SA, Switzerland) obtained from soda pulping of sarkanda grass and wheat straw was used in this study. It produced from soda pulping with sodium hydroxide as the cooking reagent in sulfur-free condition. Thus, sulfur-free soda lignin is advantageous for thermochemical conversion process because it does not generate sulfur compounds harmful to the environment. Soda lignin was washed with 0.1 N nitric acid (HNO3) at room temperature for 5 min to remove inorganic metals, followed by rinsed with sufficient deionized water, filtered and then dried in vacuum oven at 30 °C for 24 h. Inductively Coupled Plasma-Optical Emission Spectrometer (ICP-OES) (Optima 8000, PerkinElmer) was performed on as-received soda lignin to determine sodium (0.228 wt%), potassium (0.170 wt%), and calcium (0.014 wt%) content. These values decreased to 0.035 wt%, 0.024 wt%, and 0.003 wt%, respectively, after acid washing. All solvents (HPLC grade) were purchased from Fisher Scientific, and other chemicals were obtained from Sigma Aldrich.

Ethanolysis pretreatment of lignin Ethanolysis of prepared raw lignin was performed in mini-reactors (316SS, Swagelok) with total volume of 2.5 mL. Fifty milligrams of prepared raw lignin and 1.25 mL of ethanol were placed in a mini-reactor. The reactor was tightly sealed and introduced into a fluidized sand bath (Techne Industrial Bed 51) heated to the desired reaction temperature (120, 160, or 200 °C) for 60 min after which the reactor was rapidly quenched in cold water to room 5

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temperature. The cooled reactor was opened and insoluble fraction (solid) and SL (liquid) were recovered. Four milliliters of ethanol was used to rinse the inside of the reactor and combined with recovered products to improve mass recovery. The insoluble fraction was separated by centrifugation (3500 RPM for 20 min) and dried at 105 °C for 24 h. SL was recovered from liquid product after evaporation of solvent in vacuum oven at 40 °C for 48 h. The yield of each product was determined as follows.

Solubilized lignin yield (wt%) = Insoluble fraction yield (wt%) =

𝑊𝑒𝑖𝑔ℎ𝑡 𝑜𝑓 𝑟𝑒𝑐𝑜𝑣𝑒𝑟𝑒𝑑 𝑙𝑖𝑔𝑛𝑖𝑛 (𝑚𝑔) 𝑊𝑒𝑖𝑔ℎ𝑡 𝑜𝑓 𝑟𝑎𝑤 𝑙𝑖𝑔𝑛𝑖𝑛 (𝑚𝑔) 𝑊𝑒𝑖𝑔ℎ𝑡 𝑜𝑓 𝑠𝑜𝑙𝑖𝑑 𝑓𝑟𝑎𝑐𝑡𝑖𝑜𝑛 (𝑚𝑔) 𝑊𝑒𝑖𝑔ℎ𝑡 𝑜𝑓 𝑟𝑎𝑤 𝑙𝑖𝑔𝑛𝑖𝑛 (𝑔)

(1)

× 100

× 100

(2)

Lignin characterization The AVIII-600 NMR spectrometer with narrow bore 14.1 tesla superconducting magnet and an Avance III spectrometer console from Bruker Corporation (Billerica, MA and Karlsruhe, Germany) was used for

31P-NMR

analysis. In sample preparation, 2-chloro-4,4,5,5,-

tetramethyl-1,3,2-dioxaphospholane (TMDP) was used for lignin phosphorylation with the use of cyclohexanol as an internal standard.22 The detailed NMR operating condition was described in our prior study.23 The carbon (C), hydrogen (H), nitrogen (N), and sulfur (S) content of lignin and products were determined by Elementar elemental analyzer (vario MICRO cube). Oxygen content was determined by subtraction. The molecular weight information of SLs were investigated using gel permeation chromatography (GPC) analysis. All SL samples were acetylated with 1:1 v/v acetic anhydride and pyridine at 70 °C for 6 h prior to GPC analyses. Approximately 3 mg of sample was dissolved in 1 mL THF and then filtered using a syringe filter. Filtrate was introduced to Dionex 6

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Ultimate 3000 HPLC system equipped with two GPC columns (3 μm, 100 Å, 300 × 7.5 mm; PLgel, Agilent) connected in series. Six polystyrene standards (Agilent EasiVial, PL2010-0400) with different molecular weights ranging from 162-50,000 Da were used for calibration. The effluents were detected by a Shodex refractive index detector and a diode-array detector. A coil-type CDS Pyroprobe 5000 (CDS Analytical Inc., Oxford, PA, USA) equipped with GC-MS/FID (Agilent Technologies 7890A/Agilent Technologies 5975A, USA) was used to trace structural changes resulting from ethanolysis of lignin. Lignin sample was pyrolyzed at 600 °C for 20 s in an inert condition. DB-5 capillary column (30 m×0.25 mm ID×0.25 μm film thickness) with a split ratio of 1:100 was used for product separation. The detailed operating condition for GC-MS/FID and quantification method was described in our earlier study.24,25

Determination of antioxidant activity of solubilized lignin The 2,2-diphenyl-1-picrylhydrazyl free radical (DPPH) scavenging assay of SL was determined using modified methodologies established by previous researchers.3, 5 SL samples were dissolved in methanol at five different concentrations ranging from 0.0067 to 0.067 mg/mL was combined with 1.5 mL of DPPH/methanol solution (0.026 mg/mL) at room temperature for 30 min in the dark. Several lignin model compounds were also dissolved in methanol and combined with DPPH/methanol solution for determining their antioxidant activities (166.7 μmol/mL phenol, 33.3 μmol/mL guaiacol, 4-methylguaiacol, 4-ethylguaiacol, 4-vinylguaiacol, eugenol, isoeugenol, syringol, and 83.3 μmol/mL vanillin). The commercial antioxidants ferulic acid and guaiacol were utilized as controls.5 The absorbance at 515 nm of the mixture was measured using a Cary 60 UV-VIS spectrophotometer (Agilent Technologies, Sant Clara, CA, USA). The DPPH free radical scavenging activities (RSA) of SL was calculated by following equation: 7

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RSA (%) = (1 ―

𝐴𝑠 ― 𝐴𝑏 𝐴𝑐

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(3)

) × 100

where As is the absorbance of mixture of SL and DPPH solution; Ab is the absorbance of mixture of lignin sample and methanol; Ac is the absorbance of DPPH solution. The half maximal inhibitory concentration (IC50) values were further calculated on the basis of the RSA (%) under different concentrations. Reaction rate was investigated by the absorbance measurement at 515 nm in the dark. In this case, 0.013 mg/mL samples were selectively measured. Samples were sealed during absorbance measurements, which were conducted over 1000 min at 20 min intervals.

Results and discussion Structural and chemical characteristics of solubilized lignin Figure 1 shows the yield of SL and insoluble fraction from raw lignin as a function of reaction temperatures. The yield of SL at 25 °C, which represents the solubility of raw lignin in ethanol at ambient conditions, was 44.9 wt%. The yield of SL gradually increased with reaction temperature from 77.9 wt% (120 °C) to 84.6 wt% (200 °C). We have previously reported lignin decomposition into small molecular weight fragments of low polydispersity index (high homogeneity) via fragmentation of β-O-4 and β−β linkages for solvolysis at temperatures between 200 °C and 350 °C over noble metal catalysts.26, 27 Elemental composition and C9 formula of SLs were investigated and these results are presented in Table 1. With respect to elemental composition, carbon content slightly increased while oxygen content decreased after ethanolysis but there was no specific trend as a function of reaction temperature. Previous studies reported that SL of low molecular weight and narrow polydispersity index had advantageous for its high antioxidant activity.6, 18, 28 In this study, the weight average 8

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molecular weight (Mw), number average molecular weight (Mn), and polydispersity index (Mw/Mn, PDI) of SL were measured by GPC instrument. Figure 2 shows GPC profiles of raw lignin and SL produced at various temperatures. The GPC curve of raw lignin shows bimodal peaks near 2500 g/mol and 10000 g/mol. The calculated Mw, Mn, and PDI of raw lignin was 4050 Da, 1290, Da, and 3.14, respectively as given in Table 2. After ethanolysis, the GPC curve shifted to lower molecular weights and the peak around 10000 g/mol significantly decreased for all of SLs, indicating significant lignin depolymerization even under mild processing conditions. The Mw of SL ranged from 1770 Da (120 °C) to 2140 Da (200 °C), which were 43.4% to 52.8 % level of that of raw lignin (4050 Da). It meant SLs consisted of lignin fragments with relatively uniform molecular weight, suggesting that homogeneity of SLs improved after ethanolysis pretreatment. Interestingly, both the Mw and PDI tended to increase with increasing reaction temperature. 31P-NMR

analysis was performed to determine the Phe-OH, Aliph-OH, and carboxyl

content of SL. As shown in Figure S1, all SL samples contained hydroxyl (released from aliphatic, syringyl, guaiacyl, and p-hydroxyphenyl) and carboxyl functionalities. The amount of each functional group was calculated by integration of the corresponding spectra with a standard area. As shown in Figure 3, Phe-OH content increased from 2.57 mmol/g of raw lignin to 3.24 mmol/g of SL produced at 200 °C with increasing reaction temperature while AliphOH as well as carboxyl content decreased. Thus, aryl-ether bond cleavage to produce Phe-OH was promoted by increasing temperature.23, 29 The decrease in Aliph-OH and carboxyl content is likely due to the combination of dehydration and hydrogenation or hydrodeoxygenation from hydrogen donated by ethanol.30 A plausible pathway for ethanolysis is summarized in Figure 4, using a simplified lignin structures for clarity. According to previous studies, Phe-OH plays an crucial role in free radical scavenging activity because the antioxidant capacity of lignin 9

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determined by its ability to form phenoxyl radicals by H atoms donation and the stability of the formed free radicals.3, 6, 7, 18, 31, 32 Another hydroxyl group, Aliph-OH, did not impact to the DPPH inhibitory, but a large amount of Aliph-OH would play a deleterious role in antioxidant activity as reported previously.6, 7 Meanwhile, carboxyl groups have been previously supposed to responsible for increasing antioxidant properties.33 Overall, the results from GPC and 31P-NMR indicate that ethanolysis of lignin under mild temperature condition increases homogeneity as well as Phe-OH content of SL suggesting the product has higher antioxidant activity than untreated lignin. Py-GC/MS is an efficient analytical instrument that can provide structural information on SL, especially with regards to the ratio of H-, G-, and S-type phenolic compounds.34 It has several analytical advantages such as ability to use small samples, ease of sample preparation, and rapid analysis.35 We expect ethanolysis will produce major structural changes in SLs.36, 37 In this study, raw lignin and SLs were pyrolyzed at 600 °C for 20 s and the products analyzed by on-line GC/MS. A total of 28 pyrolysis products were identified, which were classified on the basis of structure (Table 3). These products were divided into H-, G- and S-type based on substitution in aromatic ring. The yields for these compounds are presented in Table S1. Figure 5 (a) shows yield of H-, G- and S-type phenolic products as a function of ethanolysis temperature. As shown in Figure 5 (a), ethanolysis increased the yield of volatile products from Py-GC/MS. The most noticeable change was increase in G- and S-type compounds after ethanolysis, especially in SL produced at 160 °C. According to previous studies, OMe in orthoposition significantly increased antioxidant activity of phenolic compounds because the presence of o-methoxylgroups decreased the Phe-OH bond dissociation enthalpy (BDE).9, 10 The possible pathways for lignin reaction with DPPH were displayed in Figure 6. Firstly, lignin donated a hydrogen from Phe-OH to capture DPPH molecules. After then, OMe could stabilize 10

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Phe-OH radical, which explained the reason that OMe enhanced the antioxidant activity of lignin. Additionally, DPPH free radical molecules might be stabilized by combining with aryl radical to form electron pair. In addition to substitution in aromatic rings, side chains also affect antioxidant activity.9, 10, 38

For in-depth evaluation of the effect of side chains, we divided products into four

categories: phenols without a side chain (C6); phenols with a saturated side chain (SP); phenols with an unsaturated side chain (UP); and phenols with an oxygenated functional group (OP). We did not include all H-type compounds because their contribution to antioxidant activities are known to be negligible. Figure 5 (b) shows changes in side chains known to occur during ethanolysis. Dizhbite et al. reported that a double bond between the outermost carbons of the side chain can enhance radical scavenger capacity.9 Besides, a side chain with CH2 in the α-position was also found to have antioxidant activity.10 It can be inferred from prior studies that UP structures in SL had positive effect on its antioxidant activity in the DPPH test. In addition, SP structure in SL had also positive effect on antioxidant activity because electron-donating capability of the alkyl groups in the para position could better stabilize the phenoxyl radical than C6 compounds, reducing the BDE of the Phe-OH bond.39, 40 On the other hand, the oxygen-containing side chain (OP structure) has previously been thought to have a negative influence on antioxidant activity of SL.10 As presented in Figure 5 (b), all types of side chains increased after ethanolysis, and this tendency was most conspicuous in SL produced at 160 °C.

Antioxidant activities of solubilized lignin DPPH is a stable radical molecules and

its absorption band near 515 nm disappears in

the presence of an antioxidant.3 Normally, the free radical scavenging capacity of DPPH is 11

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associated with its hydrogen donating ability.41 The antioxidant activities of raw lignin, SLs, commercial ferulic acid and guaiacol, known antioxidants, are presented as DPPH RSA (%).5 The UV-VIS spectrum showed that the highest peak (at 515 nm) of DPPH solution was significantly decreased by adding SLs and commercial antioxidants (Figure S2 (a)). In decreasing order, the most effective antioxidants were guaiacol, ferulic acid, SL produced at 160 °C, 200 °C, 120 °C, and raw lignin. According to previous study, RSA (%) of ferulic acid (27.3 %) was similar comparing with that of synthetic antioxidant BHA (29.2 %).42 Additionally, it was reported that guaiacol had the antiradical power of 4 determined by DPPH method, which was similar value as compared with BHA (4.17) and BHT (4.20).43 It meant that both ferulic acid and guaiacol could be used as controls for comparison. Figure 7 (a) shows the DPPH scavenging curves of the SLs as a function of solution concentration. All SL samples had higher RSA (%) values than raw lignin at the same loading concentration. As previously suggested, ethanolysis likely increased the homogeneity and Phe-OH content of the SL while decreasing Aliph-OH content and molecular weight. Table 4 quantitatively lists the calculated IC50 of SL samples and the commercial antioxidants. IC50 value means the concentration of antioxidant that is required for 50 % DPPH stabilization, and can be used as an indicator of the antioxidant activity. As shown in Table 4, except for the commercial antioxidants, SL produced at 160 °C showed the highest RSA (%) with the lowest IC50 value 0.016 mg/mL followed by 200 °C and 120 °C with IC50 values of 0.024 mg/mL and 0.027 mg/mL, respectively. These were 35.6 – 60 % of the IC50 value for raw lignin (0.045 mg/mL). Furthermore, it was noticeable that the DPPH radical scavenging capacities of SL produced at 160 °C (IC50 = 0.016 mg/mL) was close to the control ferulic acid (IC50 = 0.010 mg/mL) and guaiacol (IC50 = 0.013 mg/mL), suggesting that ethanolysis has potential for producing commercial antioxidants from lignin. 12

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It is unclear why the antioxidant activity of SL produced at 160 °C was greater than for SL produced at lower or high temperatures. In the view of homogeneity of lignin, SL produced at 120 °C had the lowest Mw value as well as PDI. On the other hand, SL produced at 200 °C had the highest Phe-OH content among the SL products. Antioxidant activity of SL might depend on not only homogeneity and functionality but other structural characteristics. For example, compositional difference among H-, G-, and S-type phenolics could possibly explain this result. As previously mentioned, SL from 160 °C produced the most G- and S-type pyrolysis compounds among three SL products. We compared DPPH free radical scavenging capacity of three lignin model compounds (phenol, guaiacol, and syringol) which were representative phenols of H-, G-, and S-type compound (Figure S2 (b)). The DPPH free radical scavenging capacity was highest in syringol (S), followed by guaiacol (G), and phenol (H). In addition, differences in side chain composition could account for this result. We investigated the effect of side chains in guaiacol on its DPPH free radical scavenging capacity by comparing several lignin model compounds (guaiacol, 4-methylguaiacol, 4-ethylguaiacol, 4-vinylguaiacol, eugenol, isoeugenol, and vanillin) (Figures S2 (b)). The results of this experiment showed that G-type compounds with an alkyl group at the para position (SP compounds: 4-methylguaiacol and 4-ethylguaiacol) had higher antioxidant capacity than guaiacol, which is consistent with previous results.44 The presence of an unsaturated side chain at the para position of guaiacol (UP compounds: 4-vinylguaiacol, eugenol and isoeugenol) also increased antioxidant activity, which supports previous studies.9, 10 On the other hand, OP compounds (vanillin) showed considerably decreased DPPH free radical scavenging capacity compared to guaiacol. The results of model compound experiments demonstrated that antioxidant activity of SL was well in line with its monomeric composition. Based on these results, the highest antioxidant activity of SL produced at 160 °C could be explained by 13

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significant increase in C6 (G+S type compounds), UP, and SP compounds that would offset the increase in OP compounds after ethanolysis. Figure 7 (b) displays increases in RSA (%) vs time for SL products at a concentration of 0.013 mg/mL. The commercial antioxidants (ferulic acid and guaiacol) rapidly reached equilibrium after 200 min. On the other hand, raw lignin as well as SLs showed more gradual increases in RSA (%) with time, and did not attain equilibrium during the experiment. According to a previous study, DPPH radical scavenging reaction is initiated by abstraction of a hydrogen from Phe-OH.3 While commercial antioxidants had specific BDE value (ferulic acid: 84.3 kcal/mol and guaiacol: 83.6 kcal/mol), SL consisted of various monomeric phenols with different BDE values (Phe-OH bond), resulting in this tendency.38, 45 Interestingly, RSA (%) value of SL from 160 °C tended to be higher than guaiacol after 920 min, suggesting that the antioxidant activity of SL would be equal to or higher than that of a commercial antioxidant for sufficiently long reaction times.

Conclusions Mild temperature ethanolysis of lignin produced SL from soda lignin with enhanced antioxidant activity. Due to the positive effects of Phe-OH and molecular weight distribution, antioxidant properties of all SL products improved compared with the lignin from which they were produced. Among ethanolysis products, SL-160 °C presented outstanding RSA (%) value close to commercial antioxidants (ferulic acid and guaiacol). Differences in RSA (%) values of SL-160 °C might be attributable to differences in G- and S-type phenolic compounds and side chain features among the SL products. These results suggest that ethanolysis has the potential for producing antioxidants from lignin, offering a new pathway for valorizing this important co-product of biomass deconstruction. 14

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Abbreviations C6: Phenols without a side chain SP: Phenols with a saturated side chain UP: Phenols with an unsaturated side chain OP: Phenols with an oxygenated functional group

Acknowledgement This research was supported by Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education (NRF2017R1A6A3A03003031) and the Bioeconomy Institute at Iowa State University.

Supporting Information 31P-NMR

spectra, UV–VIS spectrum, Pyrolysis-GC/MS analysis

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performance of lignin and quercetin mixtures. ACS Sustainable Chem. Eng. 2017, 5 (9), 8424-8428, DOI 10.1021/acssuschemeng.7b02282. 16. Xin, J.; Zhang, P.; Wolcott, M. P.; Zhang, X.; Zhang, J., Partial depolymerization of enzymolysis lignin via mild hydrogenolysis over Raney Nickel. Bioresour. Technol. 2014, 155, 422-426, DOI 10.1016/j.biortech.2013.12.092. 17. Yang, X.; Li, N.; Lin, X.; Pan, X.; Zhou, Y., Selective cleavage of the aryl ether bonds in lignin for depolymerization by acidic lithium bromide molten salt hydrate under mild conditions. J. Agric. Food Chem. 2016, 64 (44), 8379-8387, DOI 10.1021/acs.jafc.6b03807. 18. Li, Z.; Zhang, J.; Qin, L.; Ge, Y., Enhancing antioxidant performance of lignin by enzymatic treatment with laccase. ACS Sustainable Chem. Eng. 2018, 6 (2), 2591-2595, DOI 10.1021/acssuschemeng.7b04070. 19. Zhao, L.; Ouyang, X.; Ma, G.; Qian, Y.; Qiu, X.; Ruan, T., Improving antioxidant activity of lignin by hydrogenolysis. Ind. Crops Prod. 2018, 125, 228-235, DOI 10.1016/j.indcrop.2018.09.002. 20. Song, Q.; Wang, F.; Cai, J.; Wang, Y.; Zhang, J.; Yu, W.; Xu, J., Lignin depolymerization (LDP) in alcohol over nickel-based catalysts via a fragmentation– hydrogenolysis process. Energy Environ. Sci. 2013, 6 (3), 994-1007, DOI 10.1039/c2ee23741e. 21. Ye, Y.; Zhang, Y.; Fan, J.; Chang, J., Novel method for production of phenolics by combining lignin extraction with lignin depolymerization in aqueous ethanol. Ind. Eng. Chem. Res. 2011, 51 (1), 103-110, DOI 10.1021/ie202118d. 22. Pu, Y.; Cao, S.; Ragauskas, A. J., Application of quantitative 31P NMR in biomass lignin and biofuel precursors characterization. Energy Environ. Sci. 2011, 4 (9), 31543166, DOI 10.1039/c1ee01201k. 23. Kim, J.-Y.; Park, S. Y.; Lee, J. H.; Choi, I.-G.; Choi, J. W., Sequential solvent fractionation of lignin for selective production of monoaromatics by Ru catalyzed ethanolysis. RSC Adv. 2017, 7 (84), 53117-53125.24, DOI 10.1039/c7ra11541e. 24. Kim, J.-Y.; Heo, S.; Choi, J. W., Effects of phenolic hydroxyl functionality on lignin pyrolysis over zeolite catalyst. Fuel 2018, 232, 81-89, DOI 10.1016/j.fuel.2018.05.133. 25. Kim, J.-Y.; Lee, J. H.; Park, J.; Kim, J. K.; An, D.; Song, I. K.; Choi, J. W., Catalytic pyrolysis of lignin over HZSM-5 catalysts: Effect of various parameters on the production of aromatic hydrocarbon. J. Anal. Appl. Pyrolysis 2015, 114, 273-280, DOI 10.1016/j.jaap.2015.06.007. 26. Kim, J.-Y.; Park, J.; Kim, U.-J.; Choi, J. W., Conversion of lignin to phenol-rich oil fraction under supercritical alcohols in the presence of metal catalysts. Energy Fuels 2015, 29 (8), 5154-5163, DOI 10.1021/acs.energyfuels.5b01055. 27. Kim, J.-Y.; Oh, S.; Hwang, H.; Cho, T.-s.; Choi, I.-G.; Choi, J. W., Effects of various reaction parameters on solvolytical depolymerization of lignin in sub-and supercritical ethanol. Chemosphere 2013, 93 (9), 1755-1764, DOI 10.1016/j.chemosphere.2013.06.003. 28. García, A.; Toledano, A.; Andrés, M. Á.; Labidi, J., Study of the antioxidant capacity of Miscanthus sinensis lignins. Process Biochem. 2010, 45 (6), 935-940, DOI 10.1016/j.procbio.2010.02.015. 29. El Hage, R.; Brosse, N.; Sannigrahi, P.; Ragauskas, A., Effects of process severity on the chemical structure of Miscanthus ethanol organosolv lignin. Polym. Degrad. Stab. 2010, 95 (6), 997-1003, DOI 10.1016/j.polymdegradstab.2010.03.012. 17

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30. Kleinert, M.; Gasson, J. R.; Barth, T., Optimizing solvolysis conditions for integrated depolymerisation and hydrodeoxygenation of lignin to produce liquid biofuel. J. Anal. Appl. Pyrolysis 2009, 85 (1-2), 108-117, DOI 10.1016/j.jaap.2008.09.019. 31. Arshanitsa, A.; Ponomarenko, J.; Dizhbite, T.; Andersone, A.; Gosselink, R. J.; van der Putten, J.; Lauberts, M.; Telysheva, G., Fractionation of technical lignins as a tool for improvement of their antioxidant properties. J. Anal. Appl. Pyrolysis 2013, 103, 78-85, DOI 10.1016/j.jaap.2012.12.023. 32. Sun, S.-L.; Wen, J.-L.; Ma, M.-G.; Sun, R.-C.; Jones, G. L., Structural features and antioxidant activities of degraded lignins from steam exploded bamboo stem. Ind. Crops Prod. 2014, 56, 128-136, DOI 10.1016/j.indcrop.2014.02.031. 33. Aadil, K. R.; Barapatre, A.; Sahu, S.; Jha, H.; Tiwary, B. N., Free radical scavenging activity and reducing power of Acacia nilotica wood lignin. Int. J. Biol. Macromol. 2014, 67, 220-227, DOI 10.1016/j.ijbiomac.2014.03.040. 34. Marques, A. V.; Pereira, H., Lignin monomeric composition of corks from the barks of Betula pendula, Quercus suber and Quercus cerris determined by Py–GC–MS/FID. J. Anal. Appl. Pyrolysis 2013, 100, 88-94, DOI 10.1016/j.jaap.2012.12.001. 35. Rodrigues, J.; Meier, D.; Faix, O.; Pereira, H., Determination of tree to tree variation in syringyl/guaiacyl ratio of Eucalyptus globulus wood lignin by analytical pyrolysis. J. Anal. Appl. Pyrolysis 1999, 48 (2), 121-128, DOI 10.1016/s0165-2370(98)00134-x. 36. Ohra-aho, T.; Gomes, F.; Colodette, J.; Tamminen, T., S/G ratio and lignin structure among Eucalyptus hybrids determined by Py-GC/MS and nitrobenzene oxidation. J. Anal. Appl. Pyrolysis 2013, 101, 166-171, DOI 10.1016/j.jaap.2013.01.015. 37. Kim, J.-Y.; Hwang, H.; Park, J.; Oh, S.; Choi, J. W., Predicting structural change of lignin macromolecules before and after heat treatment using the pyrolysis-GC/MS technique. J. Anal. Appl. Pyrolysis 2014, 110, 305-312, DOI 10.1016/j.jaap.2014.09.020. 38. Nenadis, N.; Zhang, H.-Y.; Tsimidou, M. Z., Structure− antioxidant activity relationship of ferulic acid derivatives: Effect of carbon side chain characteristic groups. J. Agric. Food Chem. 2003, 51 (7), 1874-1879, DOI 10.1021/jf0261452. 39. Wright, J. S.; Johnson, E. R.; DiLabio, G. A., Predicting the activity of phenolic antioxidants: theoretical method, analysis of substituent effects, and application to major families of antioxidants. J. Am. Chem. Soc. 2001, 123 (6), 1173-1183, DOI 10.1021/ja002455u. 40. Kajiyama, T.; Ohkatsu, Y., Effect of para-substituents of phenolic antioxidants. Polym. Degrad. Stab. 2001, 71 (3), 445-452, DOI 10.1016/s0141-3910(00)00196-8. 41. Molyneux, P., The use of the stable free radical diphenylpicrylhydrazyl (DPPH) for estimating antioxidant activity. Songklanakarin J. Sci. Technol. 2004, 26 (2), 211-219. 42. Kikuzaki, H.; Hisamoto, M.; Hirose, K.; Akiyama, K.; Taniguchi, H., Antioxidant properties of ferulic acid and its related compounds. J. Agric. Food Chem. 2002, 50 (7), 2161-2168, DOI 10.1021/jf011348w. 43. Brand-Williams, W.; Cuvelier, M.-E.; Berset, C., Use of a free radical method to evaluate antioxidant activity. LWT 1995, 28 (1), 25-30, DOI 10.1016/s00236438(95)80008-5. 44. Bortolomeazzi, R.; Sebastianutto, N.; Toniolo, R.; Pizzariello, A., Comparative evaluation of the antioxidant capacity of smoke flavouring phenols by crocin bleaching inhibition, DPPH radical scavenging and oxidation potential. Food Chem. 2007, 100 (4), 1481-1489, DOI 10.1016/j.foodchem.2005.11.039. 45. Verma, A. M.; Kishore, N., DFT analyses of reaction pathways and temperature effects on various guaiacol conversion reactions in gas phase environment. ChemistrySelect 18

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2016, 1 (19), 6196-6205, DOI 10.1002/slct.201601139.

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Table 1. Elemental composition and C9 formula of solubilized lignins Elemental composition (wt%) C H N S Oa Raw lignin 61.5 5.2 0.7 1.0 31.6 SL-120 °C 62.6 5.1 0.5 1.0 30.8 SL-160 °C 63.1 5.1 0.5 1.0 30.3 SL-200 °C 61.7 5.1 0.5 0.9 31.8 a Calculated by difference Sample

C9 formula C9H9.03O3.48N0.09S0.06 C9H8.45O3.33N0.06S0.05 C9H8.45O3.25N0.06S0.05 C9H8.45O3.49N0.06S0.05

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Table 2. The GPC information of solubilized lignins Sample

Mw (Da)

Mn (Da)

PDI (Mw/Mn)

Raw lignin SL-120 °C SL-160 °C SL-200 °C

4050 1770 2030 2140

1290 850 880 920

3.14 2.08 2.31 2.33

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Table 3. List of GC/MS detectable compounds released from pyrolysis of solubilized lignins at 600 °C No. 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

Compounds Toluene Styrene Phenol o-Cresol p-Cresol Guaiacol 3,5-Dimethylphenol 4-Ethylphenol 4-Methoxy-3-methylphenol Creosol Catechol 4-Vinylphenol 2-Ethyl-5-methylphenol 3-Methoxycatechol 3-Methylcatechol 4-Ethylguaiacol 4-Methylcatechol p-Isopropenylphenol 4-Vinylguaiacol Syringol 4-Propylguaiacol Vanillin 4-Methylsyringol trans-Isoeugenol Acetoguaiacone Syringaldehyde Methoxyeugenol Acetosyringone

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Type Aromatic/Others Aromatic/Others C6/H SP/H SP/H C6/G SP/H SP/H SP/G SP/G C6/G UP/H SP/H C6/S SP/G SP/G SP/G UP/H UP/G C6/S SP/G OP/G SP/S UP/G OP/G OP/S UP/S UP/S

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Table 4. IC50 values for the DPPH radical scavenging activity of solubilized lignins Sample

IC50 value (mg/mL)

Raw lignin SL-120 °C SL-160 °C SL-200 °C Ferulic acid Guaiacol

0.045 0.027 0.016 0.024 0.010 0.013

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Figure 1. The yield of solubilized lignin and insoluble fraction from raw lignin as a function of ethanolysis temperature

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700 Raw lignin SL-120 ℃ SL-160 ℃ SL-200 ℃

600 500

dW/dlogM

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

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400 300 200 100 0 100

1000

1E4

Log (Mw)

Figure 2. GPC profiles of solubilized lignins (All peaks are nomalized based on sample weight)

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Figure 3. The content of functional groups of solubilized lignins from 31P-NMR results

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OH HO

O O

OH OCH3

H3CO

OH O HO

Ethanolysis

OCH3

OH OCH3

H3CO

O

OCH3 O

Figure 4. A plausible pathways for lignin depolymerization during ethanolysis

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(a) 120 100

Yield (mg/g)

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

Sum of H-unit Sum of G-unit Sum of S-unit Others

H-type

G-type

S-type

R1

R1

R1

OH

OH

R2

R3

R2 OH

80 60 40

15.8

19.8

30.6

32.3

17.2

16.3

25.1

20.5

40.1

39.3

19.1

19.5

20 0

Raw lignin SL-120 °C SL-160 °C SL-200 °C

Lignin

(b)

35 30 25

Sum of C6 Sum of SP Sum of UP Sum of OP

20 15 10 5 0

Raw lignin SL-120 °C SL-160 °C SL-200 °C

Lignin

Figure 5. The yield of (a) H-, G-, and S-type pyrolysis products and (b) C6, SP, UP, and OP type pyrolysis products from solubilized lignins at 600 °C

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Lignin

DPPH DPPH-H

OCH3

OCH3

OCH3

O

O

OH

Lignin

Lignin

Lignin

Lignin

Lignin

Lignin DPPH OCH3

OCH3

H3CO O

O



DPPH

O

OCH3 O

Figure 6. A plausible reaction mechanism between lignin and DPPH free radical

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(a)

90 80 70

RSA (%)

60 50 Raw lignin SL-120 °C SL-160 °C SL-200 °C Ferulic acid Guaiacol

40 30 20 10

0 0.00 0.01 0.02 0.03 0.04 0.05 0.06 0.07 0.08

Concentration (mg/mL)

(b)

1.0 0.9 0.8 0.7

RSA (%)

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

0.6 0.5

Raw lignin SL-120 °C SL-160 °C SL-200 °C Ferulic acid Guaiacol

0.4 0.3 0.2 0.1 0.0

0

200

400

600

800

1000

Time (min)

Figure 7. (a) DPPH free radical scavenging capacity of solubilized lignins (ferulic acid and guaiacol were used as positive control) and (b) rate of DPPH free radical scavenging for solubilized lignins at sample concentration of 0.013 mg/mL 30

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TABLE OF CONTENTS (TOC) GRAPHIC

Synopsis. Ethanolysis pretreatment can improve antioxidant activity of lignin by changing its structural features.

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