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Research Article

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Base-Catalyzed Depolymerization of Biorefinery † ‡ † † † Rui Katahira, Ashutosh Mittal, Kellene McKinney, Xiaowen Chen, Melvin P. Tucker, ‡ † David K. Johnson, and Gregg T. Beckham*, † National Bioenergy Center, National Renewable Energy Laboratory, Golden, Colorado 80401, United States ‡ Biosciences Center, National Renewable Energy Laboratory, Golden, Colorado 80401, United States

*S Supporting Information

ABSTRACT: Lignocellulosic biorefineries will produce a substantial pool of -enriched residues, which are currently slated to be burned for heat and power. Going forward, however, valorization strategies for residual solid lignin will be essential to the economic viability of modern biorefineries. To achieve these strategies, effective lignin depolymerization processes will be required that can convert specific lignin-enriched biorefinery substrates into products of sufficient value and market size. Base-catalyzed depolymeriza- tion (BCD) of lignin using sodium hydroxide and other basic media has been shown to be an effective depolymerization approach when using technical and isolated lignins relevant to the pulp and paper industry. To gain insights in the application of BCD to lignin-rich, biofuels-relevant residues, here we apply BCD with sodium hydroxide at two catalyst loadings and temperatures of 270, 300, and 330 °C for 40 min to residual biomass from typical and emerging biochemical conversion processes. We obtained mass balances for each fraction from BCD, and characterized the resulting aqueous and solid residues using gel permeation chromatography, NMR, and GC−MS. When taken together, these results indicate that a significant fraction (45−78%) of the starting lignin-rich material can be depolymerized to low molecular weight, water-soluble species. The yield of the aqueous soluble fraction depends significantly on biomass processing method used prior to BCD. Namely, dilute acid pretreatment results in lower water-soluble yields compared to biomass processing that involves no acid pretreatment. Also, we find that the BCD product selectivity can be tuned with temperature to give higher yields of methoxyphenols at lower temperature, and a higher relative content of benzenediols with a greater extent of alkylation on the aromatic rings at higher temperature. Overall, this study shows that residual, lignin-rich biomass produced from conventional and emerging biochemical conversion processes can be depolymerized with sodium hydroxide to produce significant yields of low molecular weight aromatics that potentially can be upgraded to fuels or chemicals. KEYWORDS: Dilute acid pretreatment, Biochemical conversion, Deacetylation, Kraft lignin, Lignin valorization, Lignin depolymerization

■ INTRODUCTION result, lignin valorization strategies targeting these biorefinery Lignin is a heterogeneous, alkyl-aromatic polymer formed lignin streams will become important for the economic viability and environmental sustainability of biofuels plants that utilize during plant cell wall synthesis from three phenyl-propanoid 1,2,11 monomers connected by a variety of C−O and C−C linkages. lignocellulose as a feedstock. For many decades, studies have been conducted to develop In terrestrial plants, lignin comprises 15 to 30% of the cell wall, ff and plants utilize lignin for various functions including water e ective depolymerization processes using either reductive or and nutrient transport, defense against microbial pathogens, oxidative homogeneous and heterogeneous catalysts, lignino- − and strength and rigidity of plant tissues.1 4 In Nature, lignin is lytic enzymes, whole-cell biocatalysts, and thermal depolyme- partially broken down by the action of white-rot fungi and rization. Many of the developed strategies have focused on “ ” some ligninolytic bacteria, both of which employ powerful either technical lignin streams (e.g., ball-milled, dioxane- oxidative enzymes to cleave the diverse covalent linkages in extracted lignin) or lignin from the pulp and paper industry − lignin.5 8 In a process context, lignin is currently produced (e.g., Kraft or soda lignin). The former is typically intended for “ ” worldwide in the pulp and paper industry in the form of Kraft the study of pure or isolated lignin whereas the latter is or soda lignin. From these industrial streams, vanillin and motivated by the need to valorize large, industrial scale streams lignosulfonates are the dominant products manufactured that have been in production for many decades. However, the today.1,2,9,10 Going forward, the lignocellulosic biofuels industry, initially founded primarily for the production of Received: November 6, 2015 ethanol from biomass-derived carbohydrates via fermentation, Revised: January 8, 2016 will begin to produce a vast amount of residual solid lignin. As a Published: January 12, 2016

© 2016 American Chemical Society 1474 DOI: 10.1021/acssuschemeng.5b01451 ACS Sustainable Chem. Eng. 2016, 4, 1474−1486 ACS Sustainable Chemistry & Engineering Research Article chemical and physical properties of a “lignin” stream will often As mentioned above, many approaches have been employed significantly depend on the nature of the preprocessing to study lignin depolymerization. Reductive metal catalysis has conducted to isolate or enrich the lignin. These properties, been reported as far back as the 1940s,31,32 and many recent for example, include the degree of polymerization, the identity studies have been conducted using noble metal catalysts for − and ratio of the chemical linkages (including new intermo- lignin depolymerization.2,23,33 38 Oxidative transformations of nomer linkages produced during processing), and the purity lignin have also been reasonably well studied, historically in the (related to the effects of residual carbohydrates, other plant cell context of aldehyde production, but more recently in a more − wall components, or species introduced during the conversion general context.39 42 Homogeneous alkaline catalysis has also process). The impact of processing will in turn affect the ability been a mainstay of lignin depolymerization research for quite of a given catalytic, thermal, or biological approach to some time. Indeed, soda pulping is based on the use of sodium depolymerize effectively lignin, such that a process that works hydroxide as a catalyst (oftentimes with anthraquinone as a well on a given lignin stream will likely not be universally cocatalyst) to depolymerize lignin in whole biomass.20 On effective on all lignin streams. In this vein, the application of lignin-enriched, solid substrates, the base-catalyzed depolyme- depolymerization strategies needs to be tailored to the process- rization (BCD) process has been studied by multiple groups for relevant substrate(s) of interest. over 2 decades. Thring conducted an early study using Alcell The need for tailoring lignin depolymerization strategies to a lignin with sodium hydroxide, and focused on liquid products, process is especially acute for the growing biofuels industry, which were obtained in yields up to 30%.43 Later, Shabtai, wherein lignin is currently being or slated to be burned for heat Chornet, Johnson and co-workers published a series of studies and power in most of the bioethanol plants operating or in which they describe a process employing BCD for lignin coming online in the near future.1,3 Typical biochemical depolymerization followed by catalytic hydrodeoxygenation to − conversion processes for bioethanol and advanced biofuels produce gasoline-range aromatic fuels.44 49 These studies were employ a mild thermochemical pretreatment step12,13 that is conducted primarily on pulping lignins with a few experiments intended to disrupt the cell wall architecture (and in some cases on dilute-acid pretreated substrates. A subset of the same − remove lignin14 23) to make the polysaccharides more authors developed a fractionation procedure to obtain several susceptible to enzymatic depolymerization in a second lignin-derived aromatic monomers from the BCD process deconstruction step through the use of (hemi)cellulolytic through a series of extraction, distillation, chromatography, and enzymes. The sugars produced from this two-step biomass crystallization steps.48 In an additional set of studies, Miller and deconstruction strategy are then converted to ethanol via co-workers examined a variety of bases and showed that fermentation,24 or to a range of fuels or chemicals via biological stronger bases (e.g., KOH, NaOH) are able to produce higher or chemical catalysis approaches.25,26 As recently reviewed,1 yields of low molecular weight species than weaker bases, e.g., lignin can be obtained in two primary points during LiOH.50,51 Roberts et al. published a more recent study wherein biochemical conversion, namely during the pretreatment step they used boric acid as a capping agent to inhibit and/or as a lignin-enriched residual material following repolymerization. It was not thoroughly discussed if boron polysaccharide deconstruction. As the most prevalent pretreat- was chemically incorporated into the resulting lignin after the ment approaches do not extract appreciable amounts of lignin, BCD reaction, which may cause challenges downstream in lignin depolymerization research efforts adapted to residual catalytic upgrading or in recycle of boron. Additional “capping” lignin-enriched streams are thus a major priority in lignin BCD experiments have been reported recently as well. valorization. Toledano et al. demonstrated the BCD concept on organosolv Current pretreatment approaches being used industrially lignin from olive prunings and measured the oil yield (which today mainly focus on the use of dilute-acid (e.g., sulfuric acid) was acidified to pH 1−2 after the BCD reaction and pretreatment, hydrothermal pretreatment, or ammonia and subsequently extracted with ethyl acetate from the acidified alkaline-based pretreatment approaches.12,13 Dilute acid and liquid phase), which varied between 5 and 20%.52 They hydrothermal environments have long been known to result in subsequently reported that the addition of phenol to the BCD the cleavage of prevalent C−O linkages in lignin (e.g., β-O-4 reaction was able to dramatically improve the oil yield, provided linkages) followed by formation of more recalcitrant C−C the phenol:lignin ratio was optimized.53 linkages.27 Recent work suggests that acid or hydrothermal It is noteworthy that many of the aforementioned BCD environments are able to cleave C−O linkages from the studies have been conducted to optimize the “oil” yield, which terminal ends of the lignin polymer via an unzipping is typically the organic solvent extractable material after mechanism.28 The more “condensed” structure of dilute-acid acidification. This approach is primarily motivated by the fact or hydrothermal pretreated lignin is in turn a challenging that the organic solvent extractable material will likely be substrate for subsequent depolymerization and upgrading. amenable to catalytic hydrodeoxygenation, whereas the water- More recently, pretreatment processes have been developed soluble fraction would not due to catalyst instability and without the use of acid that result in similar extents of poisoning. Acidification will also precipitate any high molecular polysaccharide deconstruction and sugar yields relative to weight species. However, the downstream lignin valorization dilute-acid pretreatment.29,30 Specifically, Chen et al. evaluated strategy will dictate the objective function of the depolymeriza- the use of deacetylation, which consists of a mild alkaline tion approach. Strategies are now emerging that are able to treatment with sodium hydroxide at 80 °C, and various forms accept heterogeneous mixtures of liquid-phase aromatic species of mechanical refining, the latter in lieu of dilute-acid as substrates for upgrading to value-added compounds based on pretreatment. The authors demonstrated that appreciable a combined biological funneling and chemical catalysis − sugar yields are attainable via this approach. Given the absence approach,54 57 such that the objective function will be to of high temperature and an acid catalyst, it is likely that the tune the selectivity to specific product slates and maximize the ligninfromthisprocessmaybemoreamenableto conversion of lignin to low molecular weight species that are depolymerization. stable at biological pH (often near ∼7).

1475 DOI: 10.1021/acssuschemeng.5b01451 ACS Sustainable Chem. Eng. 2016, 4, 1474−1486 ACS Sustainable Chemistry & Engineering Research Article

In light of the previous BCD work, and toward the ultimate washed, lignin-rich solids were collected and stored at 4 °C prior to goal of developing lignin depolymerization processes for analysis and further processing. biorefinery lignins, here we systematically evaluate BCD on Clean fractionation lignin-enriched (CF-LEF) residue from corn lignin-rich residues from three biorefinery processes namely, stover: Corn stover (10 g) in a single-phase mixture of methyl isobutyl ketone (MIBK)/acetone/H2O (11/44/44, g/g/g, 100 mL) with dilute-acid pretreatment followed by enzymatic hydrolysis sulfuric acid (0.1 M) was heated at 140 °C for 56 min. The liquid (DAP-EH), deacetylation, disk refining, and enzymatic fi fi fraction after ltration of the reaction mixture was phase-separated to hydrolysis (DDR-EH), deacetylation, twin-screw re ning, and obtain the lignin-enriched fraction (CF-LEF). Additional details of the enzymatic hydrolysis (DTSR-EH), and two model lignins: CF-LEF protocol can be found in Katahira et al.21 Kraft lignin, and the lignin-enriched fraction from a Clean Kraft lignin (KL) prepared from Norway spruce obtained from Fractionation process.21 All lignin BCD experiments were Sigma-Aldrich (Catalog #370959) was also used to evaluate the conducted in batch reactors for 40 min at either 2 or 4% NaOH efficacy of BCD on a model lignin substrate that is currently available concentration with 10 wt % solids loadings at temperatures of commercially. 270, 300, and 330 °C. We quantify the yield of liquid-phase BCD Experiments. BCD experiments were conducted in 50 mL stainless steel reactors. The experiments were conducted at three products, the molecular weight distributions of each resulting different temperatures (270, 300, and 330 °C) at two NaOH fraction (both the liquid and remaining solid fractions), and concentrations (2 and 4%) (Table 1). The autoclaves were heated fingerprint prevalent species in the liquid-phase products. Overall, this study represents a comprehensive, quantitative Table 1. BCD Experimental Conditions study of BCD applied to process-relevant, biorefinery lignin- enriched substrates and demonstrates that BCD is able to solid temp NaOH loading produce high yields of low molecular weight, aromatic species substrate (°C) (wt %) (wt %) that will likely be suitable for upgrading to value-added compounds through emerging lignin valorization pro- 270 2 10 54−57 Deacetylated Corn Stover/Disk Refined/ 410 cesses. EH residue [DDR-EH] Deacetylated Corn Stover/Twin Screw 300 2 10 ■ MATERIALS AND METHODS Refined/EH residue [DTSR-EH] Materials. fi Dilute-acid Pretreated Corn Stover/EH 410 Deacetylated/Disk re ned/Enzymatic hydrolysis residue residue [DAP-EH] from corn stover (DDR-EH):58 Corn stover was extracted with 0.1 M Kraft lignin [KL] 330 2 10 (0.4 wt %) NaOH at 80 °C for 2 h in a deacetylation step. The deacetylated corn stover was mechanically treated in a disk refiner 410 (high consistency Sprout 36-in. commercial scale double disk refiner, Corn Stover CF-Lignin Enriched Fraction 300 4 10 model 401, and Durametal 36104 fine bar pattern segments used to [CF-LEF] make up the rotating disk plates) followed by enzymatic hydrolysis. The deacetylated and disk-refined corn stover was enzymatically hydrolyzed at 15% solids loading at 50 °C for 5 days. Novozymes in a sand bath maintained at the target temperature where they remained for the desired reaction period (40 min, once the desired Cellic CTec3 (60 mg protein/g cellulose) and HTec3 (40 mg protein/ − cellulose) enzyme preparations were added to the suspension. We temperature was reached). It took 8 12 min to preheat the reactor to note that these enzyme loadings are very high to efficiently hydrolyze the desired reaction temperature. At the end of the reaction, the polysaccharides, but low enzyme loadings and high carbohydrate yields reactors were quenched to room temperature by immersion in cold have been reported with this process.59 The enzymatically hydrolyzed water. After removal from the water bath, the reactor was dried and weighed, opened to release the generated gas, and then reweighed. slurry was repeatedly washed with DI water followed by centrifugation ff (10,000 rpm) to yield the enzymatic hydrolysis residue (DDR-EH). The di erence of these two weights corresponds to the weight of the Deacetylated/Twin screw extruder refined/Enzymatic hydrolysis gas generated during the BCD experiment. Some part of the CO2 gas residue from corn stover (DTSR-EH):58 Deacetylation of corn stover generated during BCD reaction may have reacted with NaOH to yield was performed by the same manner as that for the DDR-EH substrate. Na2CO3, which would react with HCl during neutralization to The deacetylated corn stover was ground in a twin screw extruder generate CO2. Gas yields in Figure 3 do not include the fraction of the (Coperion ZSK-25 twin screw, corotating extruder) at 50 °C followed CO2 absorbed by NaOH. The workup procedure was the same for all experiments and is illustrated in Scheme 1. The reaction mixture was by enzymatic hydrolysis. Enzymatic hydrolysis was conducted at 20% fi solids loading under the same conditions as those for the DDR-EH rst neutralized with HCl to pH 7 followed by centrifugation. This substrate. The pretreated slurry was repeatedly washed with DI water resulted in the separation of low molecular weight lignin products followed by centrifugation (10,000 rpm) to yield the enzymatic hydrolysis residue (DTSR-EH). Scheme 1. BCD Procedure Used in This Study for Dilute-acid pretreated/Enzymatic hydrolysis residue from corn Separation of the Aqueous and Solid Fractions stover (DAP-EH): Pretreatment of whole corn stover was performed in a 1 ton/d continuous horizontal pretreatment reactor using procedures described previously.60 Corn stover samples were impregnated with 0.8% (w/w) sulfuric acid and pressed to approximately 45%−50% total solids and then pretreated at 160 °C for 10 min. Pretreated corn stover was neutralized with NH4OH at room temperature (22 °C) to pH 5.0. Neutralized, pretreated stover was enzymatically hydrolyzed at 20% solids loading at 50 °C for 120 h with Novozymes Cellic CTec2 solution (20 mg protein/g cellulose). At completion of enzymatic hydrolysis, the slurry underwent solid− liquid separation using a Western States Q-80 (Fairfield, OH) basket centrifuge with a 25 nm × 105 nm nominal pore size filtration cloth. The filter cake was resuspended in 10 L of DI water then recentrifuged in the same manner as the original slurry. This process was repeated until the glucose concentration in the supernatant was <1 g/L. The

1476 DOI: 10.1021/acssuschemeng.5b01451 ACS Sustainable Chem. Eng. 2016, 4, 1474−1486 ACS Sustainable Chemistry & Engineering Research Article

(supernatant−aqueous fraction) from the high molecular weight lignin to corn stover (specifically the lignin-enriched fraction, dubbed products (solid residue). CF-LEF). Gel Permeation Chromatography (GPC) Analysis. Starting The compositions of these five substrates are shown in Table substrates and each freeze-dried BCD fraction (20 mg) were acetylated 2.63 The lignin content in DAP-EH is higher than those in in a mixture of pyridine (0.5 mL) and acetic anhydride (0.5 mL) at 40 ° C for 24h with stirring. The reaction was terminated by addition of fi methanol (0.2 mL). The acetylation solvents were then evaporated Table 2. Compositional Analysis Data for ve BCD from the samples at 40 °C under a stream of nitrogen gas. The samples Substrates (wt %) were further dried in a vacuum oven at 40 °C overnight. A final drying DDR-EH DTSR-EH DAP-EH KL CF-LEF was performed under vacuum (1 Torr) at room temperature for 1 h. The dried, acetylated lignin samples were dissolved in tetrahydrofuran Lignin 52.8 30.2 67.3 93.2 80.0 (THF, Baker, HPLC grade). The dissolved samples were filtered (0.45 Glucan 14.7 22.5 13.0 0.0 0.49 μm nylon membrane syringe filters) before GPC analysis. The Xylan 14.2 11.0 1.77 0.0 0.35 acetylated samples were completely soluble in THF. GPC analysis was Galactan 1.37 0.9 0.00 0.0 0.00 performed using an Agilent HPLC with 3 GPC columns (Polymer Arabinan 2.86 2.2 0.38 0.0 0.68 × − Laboratories, 300 7.5 mm) packed with polystyrene divinylbenzene Acetate 0.41 0.3 0.80 4.0 0.25 μ 4 copolymer gel (10 m beads) having nominal pore diameters of 10 , Ash 2.16 15.8 13.3 3.34 5.04 103, and 50 Å. The eluent was THF and the flow rate 1.0 mL/min. An Total sugar 33.1 36.5 15.2 0.0 1.5 injection volume of 25 μL was used. The HPLC was attached to a diode array detector measuring absorbance at 260 nm (bandwidth 80 Total 88.5 82.8 96.5 101 86.9 nm). Retention time was converted into molecular weight (Mw)by applying a calibration curve established using polystyrene standards of DDR-EH and DTSR-EH, while the xylan content in DAP-EH known molecular weight (1 × 106 to 580 Da) plus toluene (92 Da). − is lower than those in the other two EH residues. This is Gas Chromatography Mass Spectrometry (GC MS) Anal- ff ysis. Analysis was performed on an Agilent 6890N GC equipped with because hemicellulose is e ectively hydrolyzed and removed in a 5973N MSD (Agilent Technologies, Palo Alto, CA). Sample DAP. DDR-EH has more lignin and less glucan contents than compounds were separated using a 30m × 0.25 mm × 0.25 mm HP- in DTSR-EH, suggesting that disk refining can increase the 5MS column (122−5532, Agilent). HP MSD Chemstation software cellulose surface area for enhanced enzymatic hydrolysis.59 Ash (Agilent) equipped with NIST11 database Rev. 2.0G (May 19, 2011 content in DDR-EH is much lower that in DTSR-EH and build) was used to identify the unknown compounds found within the DAP-EH. For DTSR-EH, this is due to the contamination of samples. Aqueous fractions were freeze-dried and extracted with metals from the twin extruder. DAP-EH exhibits 13.3% ash acetone. Each acetone-soluble sample was placed on an autosampler content because the original ash in corn stover might be (Agilent) and injected at a volume of 10 μL into the GC−MS − accumulated and remain in DAP-EH; the extensive washing of (Agilent). The GC MS method consisted of a front inlet temperature fi of 270 °C, MS transfer line temperature of 280 °C, and a scan range the DDR-EH substrate likely removed signi cant amounts of from 35 to 550 m/z. A starting temperature of 35 °C was held for 3 ash. min and then ramped at 15 °C/min to a temperature of 225 °C with 1 On the basis of the compositional analysis, these substrates min hold time, then continued at a ramped rate of 15 °C/min to 300 can be broadly divided into two main categories: (1) highly °C and held for 5 min. The carrier gas, He, was held at a constant flow lignin-enriched substrates (KL and CF-LEF) with lignin of 1.0 mL/min. The method resulted in a run time of 27 min for each content >80% with minimal or no carbohydrates present and sample. fi 13 (2) process-relevant, biore nery lignin-enriched substrates Solid-State NMR. High-resolution C CP/MAS NMR measure- (DDR-EH, DTSR-EH and DAP-EH) with moderate to high ments were carried out using a Bruker Avance 200 MHz spectrometer lignin content and low to medium carbohydrate (glucan and operating at 50.13 MHz at room temperature. The spinning speed was xylan) content. The objective of selecting these substrates with 7000 Hz, variable contact pulse, using a 50% ramp on the proton fl channel of 2 ms, acquisition time 32.8 ms, and delay between pulses of varying composition was to investigate the in uence of 1s. substrate composition and preprocessing on product yield Alkaline-Nitronbenzene Oxidation. DDR-EH and DAP-EH and quality (namely the yield of low molecular weight, aromatic were analyzed by alkaline-nitrobenzene oxidation.61,62 50 mg of species) that may be influenced by the presence of nonlignin substrate was suspended in 40 mL of 2 M NaOH with 2.5 mL of components (i.e., glucan, xylan, ash) during BCD. nitrobenzene. The mixture was heated at 170 °C for 2.5 h in a stainless 13C solid-state NMR spectra of the five BCD substrates are steal reactor (Parr instrument company, Illinoia, USA). After cool shown in Figure 1. DTSR-EH has less intensity in the aromatic down of the reactor, the reaction mixture was extracted with CH3Cl − fi carbon peaks region at 109 165 ppm compared to DDR-EH three times. The aqueous layer was acidi ed with 2 N HCl, and then and DAP-EH, which agrees with the lower lignin content of extracted with diethyl ether three times. The internal standard DTSR-EH provided in the compositional analysis data. DAP- () was added to the reaction products, following which − the entire mixture was silylated with BSTFA and analyzed by GC−MS. EH shows large lignin aromatic peaks (110 165 ppm) and a large methoxyl peak at 56 ppm, and these aromatic peaks looks RESULTS AND DISCUSSION quite similar to the lignin aromatic peaks in the DDR-EH. ■ Aromatic peaks from lignin are larger in DAP-EH because of Characterization of Initial Substrates. In this work, we the higher lignin content. C4/6 peaks from the crystalline and systematically evaluated BCD on lignin-rich residues from three amorphous cellulose structure clearly appear, suggesting a large biorefinery processes from corn stover: namely, dilute-acid portion of xylan was removed during the dilute-acid pretreat- pretreatment followed by enzymatic hydrolysis (DAP-EH), ment process, in line with the results in Table 1. Both KL and deacetylation, disk refining, and enzymatic hydrolysis (DDR- CF-LEF have prominent lignin peaks with very small peaks for EH), deacetylation, twin-screw refining, and enzymatic carbohydrates. hydrolysis (DTSR-EH). Two model substrates were also The molecular weight distributions of the lignin (which is the examined with the BCD process: Kraft lignin from spruce primary component that absorbs in the UV range) in the five and organosolv lignin from a clean fractionation process applied lignin-rich BCD substrates are shown in Figure 2A and their

1477 DOI: 10.1021/acssuschemeng.5b01451 ACS Sustainable Chem. Eng. 2016, 4, 1474−1486 ACS Sustainable Chemistry & Engineering Research Article

Figure 1. 13C CP-MAS solid-state NMR spectra of five substrates used in the BCD experiments. (A) DDR-EH, (B) DTSR-EH, (C) DAP-EH, (D) KL, and (E) CF-LEF.

Mw of the residual lignin. The PD of DTSR-EH lignin is 12.4 and that of DDR-EH is 7.2. DAP-EH lignin exhibits a much lower Mw of 2900 Da compared to the lignin in the other two enzymatic hydrolysis residues (Figure 2B), indicating that the ether and/or ester bonds in corn stover lignin were cleaved to a large extent during dilute-acid pretreatment. KL has relatively high Mw of 5100 Da. CF-LEF has the lowest Mw (2000 Da) with the lowest polydispersity (Figure 2B) and the largest − fi portion of low Mw species in the 200 500 Da range of the ve substrates (Figure 2A). BCD Yields. Mass balances are key to the effective evaluation of any biomass conversion technology, thus a major focus of the current study is aimed at obtaining effective mass closure during the BCD reactions. The mass yields of the aqueous fraction, solid residue, and gas obtained after BCD are shown in Figure 3. The total mass yields for various substrates obtained in all six conditions ranged from 93 to 100% for DDR- EH (Figure 3A), 89−103% for DTSR-EH (Figure 3B), 94− 102% for DAP-EH (Figure 3C), and 86−92% for KL (Figure 3D), respectively. Overall, DDR-EH and DTSR-EH exhibit higher yields of aqueous fractions and lower solid residues compared with the other three substrates. This suggests that the mechanically refined lignins are more native like and are thus more easily broken down; additionally, the lower lignin contents of the mechanically refined substrates may also lead to higher yields of liquid species resulting from the residual carbohydrates. The yield of solid residue decreased steadily with increasing BCD temperature in both 2 and 4% NaOH. This trend was Figure 2. Gel permeation chromatograms (A) and Mw and PD (B) of observed in all four substrates, except for BCD of KL at 300 °C the five substrates. in 4% NaOH (Figure 3D). The gas yield increased steadily with an increase in the reaction temperature at both NaOH weight-average Mw and polydispersity (PD) results are shown concentrations for all four substrates, except for BCD of in Figure 2B. The lignin in each substrate exhibits a unique DAP-EH at 300 °C in 4% NaOH (Figure 3C). molecular weight distribution. DTSR-EH lignin has the highest The yields of the aqueous fraction from DDR-EH exhibit − Mw of 16 100 Da with a small fraction of low Mw species (100 only very slight trends with reaction temperature with the yield 600 Da), whereas DDR-EH lignin exhibits the second highest increasing at 300 and 330 °C with 4% NaOH; with 2% NaOH, fi ff − Mw of 9600 Da, suggesting that the re ning method a ects the the yields are virtually identical at 57 60%. With DTSR-EH

1478 DOI: 10.1021/acssuschemeng.5b01451 ACS Sustainable Chem. Eng. 2016, 4, 1474−1486 ACS Sustainable Chemistry & Engineering Research Article

Figure 3. Yield of different fractions obtained after BCD from four substrate (wt %). (A) DDR-EH, (B) DTSR-EH, (C) DAP-EH, and (D) KL.

Figure 4. Gel permeation chromatograms of the BCD aqueous fraction and solid residue from four substrates (300 °C in 4% NaOH). (A) DDR-EH, (B) DTSR-EH, (C) DAP-EH, and (D) Kraft lignin.

1479 DOI: 10.1021/acssuschemeng.5b01451 ACS Sustainable Chem. Eng. 2016, 4, 1474−1486 ACS Sustainable Chemistry & Engineering Research Article

(Figure 3B), the gas yield increased (possibly due to exhibits a larger portion of products between 100 and 400 Da fi gasi cation of the carbohydrate components) and solid residue and lower overall Mw compared to the DDR-EH and DTSR- yield slightly decreased with increasing temperature. The EH aqueous fractions, which could be attributed to the lower aqueous fraction decreased at higher temperature with both 2 Mw of the original DAP-EH. The KL aqueous fraction contains − and 4% NaOH, in an opposite trend to that with DDR-EH and some quite low Mw compounds in the range of 100 250. 4% NaOH. Aqueous fraction yields from DTSR-EH with 4% Clearly, the presence of low Mw compounds in the KL aqueous NaOH are higher than those from DDR-EH, whereas the lignin fraction shows the effectiveness of BCD in depolymerizing even content in DTSR-EH (30%) is much lower than that in DDR- heavily processed lignin. It is surprising that some of the EH (53%) and the carbohydrate content of DTSR-EH is depolymerized products in the 500−2000 Da range remain slightly higher (36.5%) than that of DDR-EH (33.1%) (Table water-soluble at pH 7, which is likely advantageous for 2). These data suggest that some of the lignin and most of the upgrading these lignin depolymerization products to value- carbohydrates were solubilized in both DTSR-EH and DDR- added compounds via biological approaches.54,57 In the solid EH lignins, and it is likely the carbohydrates were converted to fractions (Figure 4), all substrates also exhibit much lower Mw carboxylic acids. than those of original substrates, and higher Mw distributions In DAP-EH, the yields of all three fractions exhibit a roughly than those in the aqueous fraction. The Mw distribution from similar trend to that of DDR-EH: namely the yields of gas and each BCD solid residue from DDR-EH, DTRS-EH, and DAP- aqueous fractions increase and the solid residues decrease with EH are similar, in the range of 860−980 Da. The KL solid increasing temperature. However, the aqueous fraction yield residue is slightly higher Mw (1200 Da) than the other three from DAP-EH was much lower than that from DDR-EH and substrates. DTSR-EH and the solid residue yield was higher. This is To illustrate the effect of temperature and NaOH loading on potentially because DAP-EH exhibits a lower initial carbohy- the resulting Mw distributions, GPC traces are shown in Figure drate content or because the lignin in DAP-EH is more 5 for the aqueous fractions and solid residues obtained from refractory. The monomeric yield from DDR-EH after alkaline- BCD of the DDR-EH residue at each temperature. At 270 °C nitrobenzene oxidation is 25.2 wt % based on the lignin content (Figure 5A), the aqueous fraction obtained in 4% NaOH of DDR-EH, which is higher than 20.6 wt % monomeric yield exhibits a slightly lower Mw distribution than that obtained in from DAP-EH, suggesting that the lignin in DAP-EH exhibits a 2% NaOH, suggesting that more lignin depolymerization more condensed structure than DDR-EH. It is likely that the occurs at higher NaOH concentration. Both chromatograms lower yield of the aqueous fraction from DAP-EH is also have bimodal peaks, with low Mw species from 200 to 350 Da because the more condensed lignin structure, generated during and higher MW species from 350 to 2000 Da. The former peak dilute-acid pretreatment, exhibits greater resistance to alkaline corresponds to monomeric and dimeric compounds and the treatment. The increase in gas yields with increasing temper- latter to oligomers. The Mw distribution for the solid residue ature suggests that some of the depolymerized compounds obtained in 4% NaOH also shows marginally lower Mw from lignin and carbohydrates were broken down to gas-phase distribution than that obtained in 2% NaOH, and both have molecules at higher temperature. The data also show, for the peaks at 700 Da. At 300 °C(Figure 5B), the aqueous fraction enzymatic hydrolysis residues (A, B, and C), the yields of the obtained in 2% NaOH contains more monomeric and dimeric aqueous fraction in 4% NaOH are higher than those in 2% compounds, whereas the aqueous fraction obtained in 4% NaOH. NaOH exhibits a larger percentage of oligomeric products. This For BCD of KL, the yields of solid fractions are much higher implies that a larger amount of the higher Mw solid residue was than the yields of aqueous fraction compared to the other three degraded to oligomers in 4% NaOH. The chromatograms of substrates, which is also likely due to either the more solid residues obtained in both 2 and 4 wt % NaOH shift to condensed structure of KL or its no carbohydrate content. lower Mw, suggesting greater thermal depolymerization of The yields of the aqueous fraction and gas increased and those lignin at a higher reaction temperature. At 330 °C(Figure 5C), of the solid residue decreased with increasing reaction the trends continue in the same fashion. In the range of 100− temperature with 2% NaOH, whereas such obvious trends in 3000 Da, the chromatograms of the aqueous fractions made the solid residue and aqueous fraction were not observed with with 2% NaOH at 330 °C are similar to those made with 4% 4% NaOH. For BCD of the CF-LEF (Figure S1 in Supporting NaOH; however, there is a small peak near 10 000−20 000 Da Information), the yields of aqueous fraction, solid residue, and with 4% NaOH suggesting that some repolymerization gas were 48, 26, and 14%, respectively. occurred in 4% NaOH. The chromatograms of solid residues GPC chromatograms are shown in Figure 4A−D for the obtained with both 2 and 4% NaOH are also similar to each aqueous fraction and solid residue obtained from four other. These GPC results clearly show that more depolyme- substrates after BCD at 300 °C in 4% NaOH. As previously rization of the DDR-EH residue to monomeric and dimeric noted, the GPC chromatograms should primarily represent the compounds occurs as the reaction temperature and NaOH aromatic, lignin-derived fraction. In all four substrates, the concentration increase. As shown in Figure 5D, higher severity aqueous fractions and solid residue have much lower Mw than BCD pretreatments tend to produce a lower Mw distribution those of original substrates. It is interesting to note that despite and polydispersity with 2 wt% NaOH. a varying Mw distribution of the original substrates in DDR-EH Additionally, it is of interest to gain insights into product fi and DTSR-EH (Figure 2), the Mw distribution of all aqueous selectivity by ngerprinting species in the BCD aqueous fractions are quite similar showing a bimodal distribution, with fractions as a function of NaOH loading and temperature. To peaks at 300 and 500 Da. The average Mw of the aqueous this end, the BCD aqueous fractions from DDR-EH were fraction from DDR-EH is 660 Da and polydispersity is 1.7. The neutralized, freeze-dried, and then extracted with acetone. Mw distribution of the aqueous fraction from DTSR-EH is Acetone was used as the extraction solvent because most of similar to that of DDR-EH with the majority of products lignin-derived monomeric compounds are soluble in acetone. − exhibiting Mw between 200 and 2000 Da, whereas DAP-EH The yields of the acetone extractives from DDR-EH were 2.7

1480 DOI: 10.1021/acssuschemeng.5b01451 ACS Sustainable Chem. Eng. 2016, 4, 1474−1486 ACS Sustainable Chemistry & Engineering Research Article

acetone extractives from DDR-EH are shown in Figure 6. Each peak was assigned and its relative abundance is summarized in Table 3. There are a small number of peaks from non-aromatic ketones, 4-methyl-4-penten-2-one, 4-methyl-3-penten-2-one, 4- hydroxy-4-methyl-2-pentanone and 2,3-dimethyl-2-cyclopent- en-1-one (peak 1−3, 6). It is known that carbohydrates present in biomass are degraded through peeling reactions in basic conditions, and a large portion of the hemicellulose is removed during alkaline treatments.64 The non-aromatic ketones (peaks 1−3, 6) are likely thus generated mainly from carbohydrates in DDR-EH. Their contents increased with increasing severity with 2% NaOH, especially 4-methyl-3-penten-2-one (peak 2), because both the relative abundance of these non-aromatic ketones and the yields of acetone extractives in 2% NaOH increased with increasing temperature. The starting substrate, DDR-EH, had 14.7% glucan and 18.4% hemicellulose (Table 2). It is likely that the carbohydrates in DDR-EH were degraded to the four pentenone products and other low Mw degradation products (i.e., most likely into carboxylic acids). Conversely, there are many peaks assigned to aromatic compounds. Phenol (peak 5), (peak 12), catechol (peak 17), syringol (peak 26), and acetosyringone (peak 33) are the main monomeric products at 270 °C based on peak area (Figures 6A-i and 6B-i). Their peak intensities decreased with increasing reaction temperature except for catechol, which exhibited a trend similar to that observed in BCD solution from hemp wherein catechol intensity reaches a maximum at 300 °C.65 The relative abundances of syringol (29.1%) and acetosyringone (14.7%) at 270 °C with 2% NaOH are much larger than those of guaiacol (6.79%) and acetovanillone (2.51%), suggesting that syringyl units are more easily released from lignin molecules than guaiacyl units under these conditions. This is likely because syringyl units have one less position in the aromatic ring where formation of carbon− carbon bond condensation structures can occur than in guaiacyl units. A similar trend for peaks 12, 26, 32, and 33 was observed at 270 °C with 4% NaOH, but the syringol content was much lower than that with 2% NaOH. The relative abundance of catechol (peak 17) content maximized at 300 °C with 2 and 4% NaOH (Table 3). Other benzenediol compounds 3-methyl- catechol (peak 22), 4-methylcatechol (peak 24), and 4- ethylcatechol (peak 28) were more prevalent at higher reaction temperature. 4-Methylcatechol and 4-ethylcatechol are the main aromatic compounds in the BCD aqueous fraction in addition to catechol, suggesting that 4-methylguaiacol (peak 16) and 4-ethylguaiacol (peak 23) were converted to 4- methylcatechol and 4-ethylcatechol, respectively, through demethylation. These phenomena were observed using model compound studies with 4-methylguaiacol and 4-ethylguaiacol (Johnson D., unpublished). Many of the species present here wouldbeamenabletohydrodeoxygenationreactionsor potentially to be fed to an organism that could funnel these heterogeneous aromatics into value-added compounds. Addi- tionally, 4-ethylguaiacol (peak 23) decreased while 4-ethyl- Figure 5. Gel permeation chromatograms of aqueous fraction and catechol and 4-ethylphenol (peak 14) increased. These results solid residue from DDR-EH after BCD with 2 and 4% NaOH at (A) suggest that demethylation of the methoxyl group and ° ° ° 270 C, (B) 300 C, and (C) 330 C, and (D) Mw and PD of each − following dehydroxylation on the aromatic ring proceeds aqueous fraction. The dashed lines in panels A C show 300 and 700 during BCD. Additional demethylation of guaiacol also yields Da. catechol (peak 17). The demethoxylation reaction was also observed during BCD of hemp and softwood.65 Vanillic acid 5.3% and 2.1−5.1% from DAP-EH. With 2% NaOH, both (peak 30) was also generated in small amounts at 270 °C, acetone extractives increased with increasing temperature, but disappearing at the higher temperatures examined. Vanillin decreased with 4% NaOH. GC-MS chromatograms of these (peak 29), acetovanillone (peak 32), and acetosyringone (peak

1481 DOI: 10.1021/acssuschemeng.5b01451 ACS Sustainable Chem. Eng. 2016, 4, 1474−1486 ACS Sustainable Chemistry & Engineering Research Article

Figure 6. GC−MS chromatograms of aqueous fractions from DDR-EH after BCD at (i) 270 °C, (ii) 300 °C, and (iii) 330 °C in (A) 2% NaOH and (B) 4% NaOH. Peak numbers are summarized in Table 3.

33) were generated in small amounts at 270 and 300 °C, but with other degradation products due to their high reactivity. disappeared at 330 °C, suggesting that aldol condensation The relative abundance of p-hydroxyphenyl (H), guaiacyl (G), between aldehydes in vanillin and ketones in acetovanillone and and syringyl (S) types are summarized in Figure 8. S-type acetosyringone may be occurring to generate dimers. products are the largest at 270 °C whereas no S-type product To characterize further the reaction products formed during was detected at 330 °C. Both S- and G-derived products BCD, the lumped peak areas of species categorized by decreased dramatically with increasing reaction temperature, functional group are summarized in Figure 7. We note that while H-type products decreased slightly. These data support anisole (peak 4), 1,2-dimethoxybenzene (veratrole, peak 10), the phenomenon of demethylation of methoxyl groups on the and 1,2,3−trimethoxybenzene (peak 25) are not included in aromatic rings, leading to catechols that can further react. the “total phenols”. The relative abundance of monomeric, In a similar fashion, the relative abundance of monomeric depolymerized compounds derived from lignin (total lignin) compounds from DTSR-EH, DAP-EH, and KL are shown in decreased from 90.0% to 67.0% with increasing reaction Tables S1−S4 and Figures S2−S7, in Supproting Information. temperatures in both 2 and 4% NaOH, whereas the relative With DTSR-EH, the abundances of total lignin, acetophenones, abundance of non-aromatic ketones increased. This suggests and acids monotonically decrease and non-aromatic ketones that lignin depolymerization was predominant and proceeds increase with increasing reaction temperature (Table S1 and rapidly under low severity BCD conditions and that conversion Figures S2, S3). The BCD products from DTSR-EH have of both the lignin and sugar-derived species accelerates at higher contents of non-aromatic ketones, compared to the higher temperature. This trend of non-aromatic ketones products from DDR-EH, reflecting DTSR-EH’s high content of indicates that residual carbohydrates in the DDR-EH substrate residual carbohydrates. The BCD products from DAP-EH were converted to carboxylic acids via peeling reactions and (Table S2 and Figures S4, S5) have relative abundances of that these acids were subsequently converted to ketones via lignin-derived and carbohydrate-derived products that are ketonization. The abundance of benzendiols such as catechol between the levels seen for DDR-EH and DTSR-EH, which also increases at higher severity, suggesting that demethylation is somewhat surprising given the low carbohydrate content of of the methoxyl group in guaiacylic compounds occurs at DAP-EH, but possibly this reflects the more condensed higher severity BCD. The abundance of acetophenones, structure of the lignin in DAP-EH due to the use of acid in aldehydes, and acids decreases at higher temperature because the pretreatment, resulting in production of lower levels of they readily undergo condensation or decarboxylation reactions monophenolic species.

1482 DOI: 10.1021/acssuschemeng.5b01451 ACS Sustainable Chem. Eng. 2016, 4, 1474−1486 ACS Sustainable Chemistry & Engineering Research Article

Table 3. Relative Abundance of Monomers Analyzed by GC−MS after Extraction of BCD Aqueous Fractions from DDR-EH with Acetone

2% NaOH 4% NaOH Peak # ID 270 °Ca 300 °Ca 330 °Ca 270 °Ca 300 °Ca 330 °Cb 1 4-methyl-4-penten-2-one 3.05 2.86 7.80 4.62 9.77 9.63 2 4-methyl-3-penten-2-one 4.62 5.84 19.5 7.32 17.1 15.6 3 4-hydroxy-4-methyl-2-pentanone 2.20 2.98 5.38 2.75 3.89 5.61 4 anisole 0.16 0.12 0.05 0.61 0.24 0 5 phenol 7.53 3.60 2.04 8.26 3.44 1.61 6 2,3-dimethyl-2-cyclopenten-1-one 0.12 0.18 0.31 0.19 0.21 0.24 7 2-methylphenol 0.08 0.16 0.22 0.14 0.21 0.33 8 acetophenone 0.02 0.10 0.05 0.01 0.01 0.42 9 2-ethylphenol 0.35 0.48 0.41 0.25 0.24 0.35 10 veratrole 0.48 0.48 0.07 0.94 0.10 0 11 p-cresol 0.08 0.56 0.81 0.42 0.71 1.70 12 guaiacol 6.79 2.00 0.26 6.36 0.73 0.24 13 2,4-dimethylphenol 0.06 0.04 0.13 0 0.09 0.59 14 4-ethylphenol 3.24 4.56 4.07 2.44 2.36 4.88 15 2-methoxy-5-methylphenol 0.04 0.21 0.11 0.09 0.13 0 16 4-methylguaiacol 0.64 0.88 0.19 0.88 0.31 0.03 17 catechol 8.02 32.9 19.0 15.7 26.2 12.9 18 4-isopropylphenol 0.20 0.39 0.44 0.16 0.20 0.66 19 3-methoxyphenol 0.44 0.38 0 0.22 0.02 0 20 3,4-dimethoxyphenol 0.39 0.29 0 0.43 0 0 21 4-propylphenol 0.04 0.38 0.69 0.08 0.21 1.23 22 3-methylcatechol 0 0 2.17 0 1.41 4.08 23 4-ethylguaiacol 3.37 2.99 0.37 2.38 0.49 0.06 24 4-methylcatechol 1.36 9.50 13.9 4.32 11.2 17.9 25 1,2,3-trimethoxybenzene 0.54 0.21 0 0.28 0 0 26 syringol 29.1 4.85 0 9.32 0.11 0 27 4-hydroxybenzaldehyde 0.27 0.23 0 0.21 0 0 28 4-ethylcatechol 3.44 16.9 20.9 8.74 16.0 19.2 29 vanillin 0.78 0.16 0 0.98 0 0 30 vanillic acid 4.38 1.48 0 2.12 0.01 0 31 4-hydroxyacetophenone 1.07 1.29 1.06 1.95 1.86 2.58 32 acetovanillone 2.51 1.20 0 3.54 1.16 0 33 acetosyringone 14.3 1.52 0 13.9 1.43 0 34 2,2′-methylenebisphenol 0.32 0.19 0.03 0.40 0.09 0.11 total 100 100 100 100 99.9 100 aThe values reported are the averages for the experiments conducted in triplicates. bIn duplicate.

Figure 8. Relative abundance of monomers of p-hydroxyphenyl type (H), guaiacyl type (G), and syringyl type (S) in aqueous fractions Figure 7. Relative abundance of monomers in aqueous fractions from − DDR-EH under 6 BCD conditions analyzed by GC−MS. “Total from DDR-EH under 6 BCD conditions analyzed by GC MS. lignin” includes all aromatic compounds. Anisole, 1,2-dimethoxyben- zene, and 1,2,3-trimethoxybenzene are not included in the “total increased dramatically at higher temperature (Table S3 and phenols”. Figures S6, S7). Given that Kraft lignin does not contain substantial carbohydrates (Table 2), this implies that With KL, the relative abundance of lignin-derived products compounds 1−2inTable 3 are also generated from lignin decreased to zero at 330 °C in 2% NaOH and at 300−330 °C and/or sugar degradation products, which were generated in 4% NaOH, whereas the abundance of non-aromatic ketones during the Kraft pulping process and contaminated in KL.

1483 DOI: 10.1021/acssuschemeng.5b01451 ACS Sustainable Chem. Eng. 2016, 4, 1474−1486 ACS Sustainable Chemistry & Engineering Research Article

Figure 9. Solid-state 13C NMR spectra of BCD solid residues under six different conditions from the four substrates.

Benzenediol, aldehydes, and acids were not detected in the yield of an aqueous soluble fraction, is likely a consequence of BCD liquor from KL, and acetophenones were the main both pretreatment method and the differing lignin and products of lignin depolymerization. As expected the BCD carbohydrate contents of the substrates. Minimal structural aqueous fraction obtained from KL only contained guaiacyl- changes in lignin and a higher carbohydrate content in a type lignin depolymerization products, as it was made from a substrate lead to higher liquid-phase yields after BCD. The softwood (Norway Spruce), which contains only guaiacyl-type lignin. resulting molecular weight distributions in all cases of the 13C-Solid-State NMR Analysis of BCD Solid Residues. aqueous fractions demonstrated a substantial amount of lignin The solid-state 13C NMR spectra of BCD solid residues from depolymerization to monomeric and oligomeric species. The four of the substrates are shown in Figure 9. In DDR-EH (A), BCD selectivity in all cases tended toward a higher content of peaks from carbohydrates and oxygenated lignin side chains phenolic monomers at low severity, shifting to higher extents of − ° (60 109 ppm) decrease and disappear at 270 C in 4% NaOH, dealkylation as severity was increased. Going forward, base- − whereas aliphatic carbon peaks (0 40 ppm) increase with catalyzed depolymerization of lignin-enriched biorefinery higher reaction severity. The methoxyl peak at 56 ppm also substrates may provide effective streams for either biological decreases and aromatic peaks in the range of 110−160 ppm increase under severe conditions. These results indicate that or catalytic upgrading strategies aimed toward lignin valor- lignin and carbohydrates in DDR-EH residues were degraded ization. followed by repolymerization to give high Mw products with high carbon content. Similar trends were observed in BCD ■ ASSOCIATED CONTENT solid residues from the other three substrates. *S Supporting Information ■ CONCLUSIONS The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acssusche- fi Base-catalyzed depolymerization was applied to ve lignin-rich meng.5b01451. substrates, and the resulting mass balances of the aqueous, insoluble, and gas fractions were quantified. In all cases, the Yields of BCD fraction from CF-LEF, Relative mass balances are near total mass closure (86−103 wt %). abundances of individual and categorized monomeric DDR-EH and DTSR-EH exhibited higher liquid yields of 56− compounds in aqueous fractions from DSTR-EH, DAP- 78 wt %, whereas DAP-EH, KL, and CF-LEF all exhibited lower EH, Kraft lignin, and CF-LEF. Relative abundances of yields of aqueous soluble fractions at all conditions. This monomers of p-hydroxyphenyl type, guaiacyl type, and difference in the susceptibility to BCD between mechanically syringyl type in the aqueous fractions from these refined substrates and acid-pretreated lignins as measured by substrates (PDF).

1484 DOI: 10.1021/acssuschemeng.5b01451 ACS Sustainable Chem. Eng. 2016, 4, 1474−1486 ACS Sustainable Chemistry & Engineering Research Article ■ AUTHOR INFORMATION Dilute Acid and Ionic Liquid Pretreatment of Switchgrass: Biomass Recalcitrance, Delignification and Enzymatic Saccharification. Bio- Corresponding Author − *G. Beckham. E-mail: [email protected]. resour. Technol. 2010, 101 (13), 4900 4906. (15) Banerjee, G.; Car, S.; Scott-Craig, J. S.; Hodge, D. B.; Walton, J. Notes D. Alkaline Peroxide Pretreatment of Corn Stover: Effects of Biomass, The authors declare no competing financial interest. Peroxide, and Enzyme Loading and Composition on Yields of Glucose and Xylose. Biotechnol. Biofuels 2011, 4, 16. ■ ACKNOWLEDGMENTS (16) Bozell, J. J.; Black, S. K.; Myers, M.; Cahill, D.; Miller, W. P.; We thank the U.S. Department of Energy (DOE) Bioenergy Park, S. Solvent Fractionation of Renewable Woody Feedstocks: Technologies Office (BETO) for funding this work. We thank Organosolv Generation of Biorefinery Process Streams for the Production of Biobased Chemicals. Biomass Bioenergy 2011, 35 (10), Erik Kuhn for supplying DAP-EH and William Michener for − GC-MS analysis. The U.S. Government retains and the 4197 4208. (17) Bozell, J. J.; O’Lenick, C. J.; Warwick, S. Biomass Fractionation publisher, by accepting the article for publication, acknowledges for the Biorefinery: Heteronuclear Multiple Quantum Coherence- that the U.S. Government retains a nonexclusive, paid up, Nuclear Magnetic Resonance Investigation of Lignin Isolated from irrevocable, worldwide license to publish or reproduce the Solvent Fractionation of Switchgrass. J. Agric. Food Chem. 2011, 59 published form of this work, or allow others to do so, for U.S. (17), 9232−9242. Government purposes. (18) Banerjee, G.; Car, S.; Liu, T. J.; Williams, D. L.; Meza, S. L.; Walton, J. D.; Hodge, D. B. 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1486 DOI: 10.1021/acssuschemeng.5b01451 ACS Sustainable Chem. Eng. 2016, 4, 1474−1486