Assessment of the Lignin-Derived Inhibition of Enzymatic Hydrolysis

Oct 13, 2016 - Xianliang QiaoChao ZhaoQianjun ShaoMuhammad Hassan. Energy & Fuels 2018 32 (5), 6022-6030. Abstract | Full Text HTML | PDF | PDF w/ ...
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Assessment of the Lignin-derived Inhibition of Enzymatic Hydrolysis by Adding Untreated and AFEX-treated Lignin Isolated from Switchgrass Qianjun Shao, and Chao Zhao Energy Fuels, Just Accepted Manuscript • DOI: 10.1021/acs.energyfuels.6b02243 • Publication Date (Web): 13 Oct 2016 Downloaded from http://pubs.acs.org on October 16, 2016

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Assessment of the Lignin-derived Inhibition of Enzymatic Hydrolysis by

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Adding Untreated and AFEX-treated Lignin Isolated from Switchgrass

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Qianjun Shao,*,† Chao Zhao‡

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Utilization, Zhejiang A&F University, Linan, Zhejiang 311300, China

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*Corresponding authors:

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Tel.: +86 574-8760-0872. E-mail: [email protected] (Q. Shao).

Faculty of Mechanical Engineering and Mechanics, Ningbo University, Ningbo, Zhejiang 315211, China School of Engineering, National Engineering Research Center of Wood-based Resource

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ABSTRACT

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Lignin, which is considered one of the most recalcitrant components, limits the enzymatic

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hydrolysis of cellulose. When ball-milled lignin from switchgrass ranged from 21.4 to 64.3

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mg/mL was added to a cellulose substrate (Avicel), the glucose release decreased from 17.4% to

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22.4% compared to the hydrolysis rate of zero lignin adding. However, the cellulose decreasing

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conversion was reduced to 8.5% with 21.4 mg/mL AFEX-treated lignin adding, as a result of the

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structural modification by AFEX pretreatment. The inhibitory effect of lignin was stronger on

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cellulose hydrolysis than on hemicellulose hydrolysis. The results showed that the free lignin

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released from biomass reduced the impact of the added lignin, and that the AFEX pretreatment

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intensity was the key factor in increasing the enzymatic efficiency. The lignin structural

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modifications by AFEX pretreatment, such as slight increases in the molecular weight, degree of

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polymerization and S/G ratio, reduced the lignin inhibition with a concomitant increase in the

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hydrolysis of cellulose.

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Keywords

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Lignin; Enzymatic hydrolysis; AFEX pretreatment; Lignin structure; Switchgrass

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1. INTRODUCTION

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Lignocellulosic biomass is an abundant renewable resource used in the production of alternative

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transportation fuels.1 Enzymatic hydrolysis is one of the key steps for producing monomeric

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sugars needed to produce ethanol. However, the plant cell walls of lignocellulosic materials have

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a strong natural resistance to microbial and enzymatic degradation, a property termed “biomass

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recalcitrance”.2 The recalcitrance of biomass is influenced by the presence of lignin, which

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interacts with carbohydrates, and limits the accessibility of these enzymatic substrates during the

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hydrolysis processes.2, 3

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Lignin is one of the three major components (cellulose, hemicellulose and lignin) in plant cell

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walls of lignocellulosic feedstocks. Among the phenyl propanoid building blocks, lignin

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macromolecules are primarily connected via carbon-carbon and carbon-oxygen bonds, with aryl

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ether bonds (such as β-O-4, α-O-4/β-5, β-β) being the most common and with guaiacyl (G),

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syringyl (S) and p-hydroxyphenyl (H) serving as the major inter-unit linkages of lignin.4 Lignin

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is considered the most recalcitrant component due to its recalcitrant nature and limiting enzyme

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accessibility to the cellulose.5

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It has been suggested that the inhibition effects of lignin on enzymatic hydrolysis can be divided

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into some categories: influence on the swelling/accessibility of cellulose, lignin nonspecifically

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adsorbing the hydrolysis enzymes and thereby reducing their availability,6 lignin carbohydrate

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complex (LCC) linkages further limiting the enzyme accessibility to cellulose,7 and lignin-

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derived phenolics strongly inhibiting enzymatic hydrolysis.8

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Switchgrass, an herbaceous energy crop using C4 photosynthesis is a potential high-yield

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lignocellulosic feedstock for the production of alcohol.9 The pretreatment of ammonia fiber

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expansion (AFEX) is an effective tool to overcome the recalcitrance and make the biomass

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matrix more accessible to enzymes, thus resulting in increased enzymatic digestion of

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switchgrass.10 The glucose yield of different varieties and harvest time of AFEX-treated

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switchgrass under near-optimal conditions typically was ranged from 44.6% to 75.2%.10

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A recent study showed that optimal pretreatment maximized lignin degradation or removal and

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minimized polysaccharide modification, resulting in improved enzymatic efficiency.11 However,

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the concentration of lignin in the biomass substrate using AFEX pretreatment was still extremely

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high, and the lignin polymer had not degraded significantly.12, 13 Consequently, the lignin during

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enzymatic digestion of AFEX was still the dominant cause of enzymatic hydrolysis inhibition,

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cellulase adsorption and decreased cellulose accessibility. Other research suggested that the

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adsorption of cellulase enzymes on lignin was likely associated with its structure, which with

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higher G units of lignin adsorbing more cellulase enzymes, particularly β-glucosidase.14 The

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condensation degree of lignin and the total OH groups increased significantly after pretreatment

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and played an important role in cellulase adsorption.6 Changes in the lignin structure such as aryl

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ether bonds, the S/G units ratio, the p-coumarate/ferulate ratio, and other ending structures, were

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identified by 2D NMR spectra and gel permeation chromatography (GPC).15 The NMR analysis

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also provided a mechanism for the cleavage of phenolic β-O-4 linkages in dilute acid

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pretreatment.16

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In this work, the structural features and properties of untreated and AFEX-treated lignin isolated

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from switchgrass were studied using GPC and 2D NMR analyses. The effects of lignin content

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and AFEX pretreatment on the enzymatic hydrolysis of Avicel and switchgrass were studied in

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order to determine the degree and mechanism of lignin inhibition by comparing the enzymatic

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digestibility of cellulose with or without lignin loading.

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2. EXPERIMENTAL SECTION

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2.1. Raw Materials and Chemicals.

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The switchgrass (Panicumvirgatum var. Kanlow), harvested in 2009, was kindly provided by

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the University of Georgia Plant Sciences Farm (Georgia).The switchgrass contained 34.58%

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glucan, 22.95% xylan, 2.94% arabinan, 23.11% Klason lignin, and 2.25% acid soluble lignin on

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average (dry weight basis). The data of chemical composition obtained for switchgrass is

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dependent on switchgrass varieties, locations and harvesting season reported in the literature, and

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the glucan, xylan and lignin are range from 27-35%, 19-24%, 17-25%, respectively.17-19 The

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milled switchgrass samples were passed through 0.45 mm screen sieve prior to storing in sealed

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plastic bags at -20 °C.

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All chemicals were reagent grade and purchased from Sigma–Aldrich or VWR International.

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The activities of Celluclast cellulase (Trichoderma reesei ATCC 26921) and beta-glucosidase

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(Novozyme 188) were determined to be 91.03 FPU/mL and 387.70 CBU/mL, respectively. The

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cellulase was mixture enzymes, and xylanase activity of Celluclast cellulase was determined to

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be 370.15 U/mL.

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2.2. Isolation of Ball-milled Lignin.

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The procedure for isolating ball-milled lignin extracted from finely milled switchgrass with

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aqueous dioxane is outlined in Figure 1. The samples were first extracted with benzene/ethanol

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(2:1, v/v) for 24 h followed by hot water (60 °C) for 24 h. These samples were then vacuum-

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dried (at 40 °C) for 72 h with P2O5. The vacuumed-dried materials were then ball milled in a 2-L

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vibratory porcelain jar filled with nitrogen at room temperature for 10 days at 96 rpm according

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to a methodology.20 A porcelain ball/biomass weight ratio of 30:1 was used. After milling, the

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switchgrass samples were extracted with p-dioxane/H2O (1.00 g : 10 mL; 96%, v/v) in a coned

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flask for 24 h in the dark (covered with foil). The samples were centrifuged and filtered to obtain

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a clear lignin solution after p-dioxane extraction. The lignin solutions were then evaporated to

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approximately 100 mL by rotary evaporation prior to freeze drying to obtain the crude lignin.

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This lignin was further purified with 90% acetic acid.

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2.3. AFEX Pretreatment.

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The method for AFEX pretreatment has previously been described.21, 22 Feedstock with known

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moisture content was weighed and placed into a Parr 4560 mini pressure reactor (300 mL) before

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charging with ammonia. The reactor temperature was increased rapidly to the desired level and

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held for the desired period of time. Ammonia was then released through the exhaust valve prior

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to removing the treated biomass from the reactor and either air-drying overnight

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(approximately12 h) in a fume hood or oven-drying at 40 °C to remove residual ammonia and

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water. Ball milled lignin was used as a raw material for AFEX-treated lignin. The treated

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samples were then stored in sealed plastic bags at -20 °C. The AFEX pretreatment conditions in

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this study were a temperature of 90 °C, water loading of 0.6 or 0.8 (g water : g DM biomass),

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residence time of 5-30 min, and ammonia loading of 1.0-5.0 (g ammonia : g DM biomass).

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2.4. Enzymatic Hydrolysis.

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Enzymatic hydrolysis of the substrates was carried out based on the NREL protocol with 1.0%

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glucan loading and a total volume of 15 mL in 20 mL screw-cap vials.23 Enzymatic hydrolysis of

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different samples was performed with cellulase and beta-glucosidase loadings of 15 FPU/g

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glucan and 64 CBU/g glucan, respectively, in a 0.05 M sodium citrate buffer (pH 4.8). To

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prevent microbial growth during enzymatic hydrolysis, antibiotic antimycotic solution was

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loaded at a concentration of 100 penicillin units, 100 µg of streptomycin, and 0.25 µg of

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amphotericin B per mL.

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Enzymatic hydrolysis for all experiments was conducted in a shaking incubator at 50 °C and 150

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rpm. Samples for monosaccharide analysis by HPLC were taken at 2, 8, 24 and 72 h after

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initiation of enzymatic hydrolysis.

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2.5. HPLC Analysis.

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Separation and quantification of monosaccharides was conducted using an HPLC equipped with

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an auto sampler and a refractive index detector (Agilent 1200 Series). The sugar contents of all

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composition analysis (acid hydrolysis) and hydrolysate samples were determined using a Bio-

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Rad Aminex HPX-87H column maintained at 65 °C with a 0.005 M sulfuric acid (0.2 µm filtered

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and degassed) as the mobile phase and a flow rate of 0.6 mL/min.

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The percent glucan conversion (i.e. digestion of glucan) was calculated as follows: % digestion = (gram of glucose×0.9)/(grams of cellulose added)×100.

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Where 0.9 is the multiplying factor used to correct the glucose reading as water molecules are

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produced upon hydrolysis of the cellulose polymer.

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The grams of glucose were calculated from the glucose concentration in each sample, and the

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grams of cellulose added were initially determined as glucose by acid analysis.

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2.6. Determination of Lignin Molecular Weight.

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The ball-milled lignin samples (20.0 mg) were placed in a small vial and vacuum dried at 40 °C

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for 24 h. Anhydrous pyridine (0.5 mL) and acetic anhydride (0.5 mL) were sequentially added,

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prior to capping and vortexing the vials and storing at room temperature for 24 h. Ethanol (5.0

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mL) was added to quench the reaction, and the vials were allowed to sit in a fume hood to dry for

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24 h. The sample was then placed in a vacuum oven at 40 °C for 3 h to remove any residual

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volatiles. The acetylated lignin sample was purified by dissolving in chloroform (1.0 mL) and

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precipitating with diethyl ether (75.0 mL). The resulting solids were collected by centrifugation

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and dried under vacuum at 40 °C for 2 h. Afterward, the acetylated lignin sample was dissolved

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in THF (1.0 mg/mL) for GPC analysis using an Agilent 1200 series liquid chromatography unit.

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GPC analyses were carried out using a UV detector on a four-column sequence of Waters

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Styragel columns (HR0.5, HR2, HR4, and HR6) at a flow rate of 1.00 mL/min. The number-

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average (Mn) and weight-average molecular weights (Mw) were calculated by the WinGPC Unity

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software relative to the universal polystyrene calibration curve.

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2.7. 2D NMR Analysis.

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2D

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structural analyses were performed using a Bruker AMX-400 spectrometer with DMSO-d6 as the

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solvent. The spectral widths for the 13C and 1H analyses were 200.0 and 11.0 ppm, respectively.

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The HSQC analysis was carried out with a standard gradient enhanced Bruker pulse sequence

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with a pulse of 90°, an acquisition time of 0.11 s, recycle delay of 1.5 s, a 1JC–H value of 145 Hz,

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acquisition of 256 complex data points and 256 scans. The NMR data were processed using the

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Top Spin 2.1 software.

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3. RESULTS AND DISCUSSION

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3.1. Influence of Lignin Relative Contributions on Lignin Inhibition of Enzymatic

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Hydrolysis.

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The microcrystalline cellulose Avicel was used as a model cellulosic substrate in this study.

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Experiments assessing the enzymatic hydrolysis of Avicel in the presence of different lignin

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concentration were carried out using enzyme loadings of 15 FPU/g glucan (cellulase) and 64

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CBU/g glucan (beta-glucosidase) with 1% glucan loading. The samples were hydrolyzed at 50

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°C and 150 rpm for periods of 2, 8, 24, and 72 h.

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Figure 2 shows the effect of varying concentrations with adding lignin of biomass substrate on

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the hydrolysis of Avicel. The lignin concentrations tested were as follows (mg of adding

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lignin/mL of cellulose substrate): 0, 21.4, 42.8, and 64.3 mg/mL. The data shows that the lignin

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concentration has a large impact on enzymatic hydrolysis, with lower lignin levels resulting in

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C–1H heteronuclear single quantum coherence (HSQC) experiments used for lignin

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high yield of sugars. A 17.4% decrease in hydrolysis was observed when 21.4 mg/mL lignin was

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added and a 22.4% decrease was observed when 64.3 mg/mL lignin was added when compared

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to the hydrolysis rate of zero lignin adding. Enzyme adsorption and cellulose surface coverage

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by lignin were the main reasons for reducing enzymatic hydrolysis. These data explain why

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biomass samples with lower lignin contents are more easily digested by enzymes. 24-26

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3.2. Influence of Lignin Modification (AFEX-treated) on Lignin Inhibition of Enzymatic

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Hydrolysis.

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Figure 3 shows a comparison of the hydrolysis rates of samples spiked with 21.4 mg/mL of

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either AFEX-treated or untreated lignin. The results show that the two different types of lignin

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have a similar effect on the enzymatic hydrolysis of Avicel. However, the AFEX pretreatment of

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lignin may reduce adsorption of the enzymes, resulting in substantially increased levels of

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hydrolysis. The decreases of Avicel spiked with either 21.4 mg/mL AFEX-treated or untreated

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lignin was 8.5% and 17.4%, respectively.

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3.3. Comparisons of the Inhibition of Glucan and Xylan Conversion with Added Lignin.

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Enzymatic digestion of lignocellulosic biomass with high sugar yields requires the synergy of

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several categories of enzymes, including hemicellulases, cellulases, and debranching enzymes of

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accessory hemicellulose.27 Figure 4a and 4b show the influence of lignin loading on the glucan

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and xylan enzymatic conversion of AFEX-treated switchgrass. The data show that the lignin

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significantly inhibits cellulose hydrolysis. However, the lignin inhibition on hemicellulose

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hydrolysis was less sensitive than on cellulose hydrolysis. The amount of glucose release

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decreased from 5.2% to 13.4% when 64.3 mg/mL lignin was added to switchgrass biomass

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hydrolysis after 72 h, whereas the decrease in the amount of xylose release was less than 4.3%.

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The lignin inhibition on high enzymatic hydrolysis was more sensitive than that on low

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enzymatic hydrolysis. The results also show that the sugar yields of enzymatic hydrolysis are

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closely related to AFEX pretreatment conditions and that a residence time of 30 min was more

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beneficial than 5 min.

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3.4. Effects of Lignin on the Enzymatic Digestibility of AFEX-treated Switchgrass.

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Figure 5 shows the effect of lignin concentrations on the enzymatic hydrolysis yields of AFEX-

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treated switchgrass under two different pretreatment conditions. The AFEX pretreatment had a

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strong effect on overcoming biomass recalcitrance and resulted in an increase in the sugar yields.

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The results show that a 1.6-fold higher glucose release and 3.0-fold higher xylose release after an

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AFEX pretreatment residence time of 30 min as compared with that of 5 min. It suggested that

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the AFEX pretreatment intensity such as residence time or pretreatment temperature was the key

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factor in increasing the enzymatic efficiency.

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Added lignin concentrations of 0, 21.4, and 64.3 mg/mL were studied. The results show a 13.4%

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glucose release decrease after a residence time of 30 min when 64.3 mg/mL lignin was added to

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AFEX-treated switchgrass and a 3.7% glucose release decrease after a residence time of 5 min

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when 21.4 mg/mL lignin was added to AFEX-treated switchgrass compared to no added lignin

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after 72 h of digestion. The data did not confirm an effect between lignin content and hydrolysis

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inhibition. These results are different from the other published results with Avicel and lignin

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removal by pretreatment.28 This discrepancy likely occurred because the free lignin released

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following enzymatic hydrolysis of the biomass reduced the impact of the added lignin, whereas

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the AFEX pretreatment is limited to lignin removal. This result further illustrates that

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pretreatments, such as AFEX, could not overcome the inhibitory effect of enzyme binding by

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lignin even though the binding levels were variable.

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3.5. Molecular Weight Analysis of Untreated and AFEX-treated Lignin.

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The number-average (Mn) and weight-average (Mw) molecular weights and the polydispersity

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values of untreated lignin and AFEX-treated lignin are listed in Table 1. The molecular weight of

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the untreated lignin were 2,013 (g/mol) and 4,006(g/mol) and the molecular weight of the

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AFEX-treated lignin were 2,329 (g/mol) and 5,112 (g/mol) for Mn and Mw, respectively. The

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data demonstrates that there was an approximately 5% increase in molecular weight due to the

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AFEX pretreatment process on ball-milled lignin, whereas the polydispersity indexes of

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untreated and AFEX-treated lignin (1.99 and 2.02) were highly close.

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The change in the molecular weights of lignin could provide important insights into the

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simultaneous fragmentation and recondensation reactions of lignin during AFEX pretreatment.

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The lignin molecular weight may depend on the competition between fragmentation and

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condensation which is contingent on the pretreatment conditions.29 The condensation reactions of

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AFEX pretreatment under these conditions were more active, leading to a condensed lignin

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structure with a higher mean molecular mass. Other researchers have reported that pretreatment

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procedures such as hydrothermal pretreatment and alkaline organosolv pretreatment had a

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tendency to reduce both the molecular weight and degree of polymerization of lignin.30, 31 We

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found that the molecular weight increased slightly and the recondensation reactions became

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dominant when the pretreatment time was extended. The same phenomena occurred to a greater

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extent following hydrothermal and ammonia percolation pretreatments.32, 33

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It can be concluded that the number of lignin polymers will reduce with the dominant type of

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recondensation reactions, thus reducing adsorption of enzymes onto lignin and improving

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hydrolysis efficiency.

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3.6. 2D HSQC NMR Analysis of Untreated and AFEX-treated Lignin.

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The assignments and integration value of quantitative

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untreated and AFEX-treated lignin were analyzed according to literatures.34-36

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spectra of lignin are summarized in Figure 6 and show both aromatic (13C/1H100-150/6.5-7.8

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ppm) and aliphatic side chain(13C/1H 50-95/2.5-6.5 ppm).The main signals in the aromatic region

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were produced by the syringyl (S), guaiacyl (G), and p-hydroxyphenyl (H) units; S, G and H

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units were clearly observed in all spectra. The S units showed prominent signals for C2,6/H2,6

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correlation at δc/δH 104.3/6.7 ppm, the H units showed prominent signal for C2,6/H2,6 correlation

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signals at δc/δH 130.0/7.5 ppm, and the G units showed different correlations for C2/H2 at δc/δH

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C and 2D HSQC NMR spectra of 13

C–1H HSQC

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111.5/7.0 ppm, C5H5 at δc/δH 115.5/6.8 ppm, and C6H6 at δc/δH 119.5/6.8 ppm as G2,G5, and G6,

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respectively.

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The analysis of NMR data was carried out by integrating the signal intensities between 150.0 and

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102.5 ppm and setting the value of six aromatic carbons after subtracting the value of two vinyl

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carbons from ferulic acid and p-coumaric acid. The methoxy group content was estimated as

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0.97 based on a relative integration range of 58.5-54.5 ppm. The structure of the lignin isolated

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from switchgrass indicated that p-coumaric and ferulic acid were incorporated into lignin

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through ester or ether inter-linkages.37 The S and G unit are mainly structures of switchgrass

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lignin. The S/G ratio for the untreated switchgrass lignin was estimated to be 0.81 from the

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integration of the contour S2/6 and G2. In comparison, the S/G ratio for the AFEX-treated lignin

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was 0.85, which indicates that the lignin structure changed slightly during the AFEX

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pretreatment process.

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The GPC and 2D NMR analyses indicated that AFEX pretreatment changed the structure of

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lignin. The recondensation and structural modifications of lignin during AFEX pretreatment

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process can reduce its enzyme adsorption properties. The same data showed a reduction by ARP-

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insoluble lignin.38

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4. CONCLUSIONS

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Based on the results from this study, lignin content and lignin structure were found to influence

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the enzymatic hydrolysis of both pure cellulose (Avicel) and switchgrass. However, the impact

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of adding lignin of high enzymatic hydrolysis is more sensitive than that of low enzymatic

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hydrolysis. The inhibitory effect of lignin was stronger on cellulose hydrolysis than on

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hemicellulose hydrolysis. Lignin structure has a significant impact on the lignin nonspecific

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binding of cellulose to lignin. Overall, the data presented in this manuscript demonstrate that the

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surface modification of lignin through AFEX pretreatment can be used to reduce inhibitory

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effect of lignin.

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AUTHOR INFORMATION

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Corresponding Author

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*Q. Shao. Tel: +86 571 8760 0872. E-mail: [email protected].

272

Notes

273

The authors declare no competing financial interests.

274

ACKNOWLEDGMENTS

275

This research was supported by funds from Pre-research Project of Research Center of Biomass

276

Resource Utilization, Zhejiang A & F University (No. 2013SWZ03), National Natural Science

277

Foundation of China (31500491), and K. C. Wong Magna Fund in Ningbo University.

278

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387 388

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Table 1 Molecular weight distribution and polydispersity of untreated and AFEX-treated

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lignin isolated from switchgrass polydispersity Mn (g/mol)

Mw (g/mol) index(Mw/Mn)

391 392

untreated lignin

2, 013

4, 006

1.99

AFEX-treated lignina

2, 329

5, 112

2.19

a: Ammonia pretreatment conditions were a temperature of 90 °C, water loading of 0.8, ammonia loading of 5.0 and residence time of 30 min.

393 394

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Figure Caption

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Figure 1. Preparation of ball-milled lignin from switchgrass.

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Figure 2. Influence of lignin loadings on the enzymatic hydrolysis of cellulose (Avicel).

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Figure 3. Influence of lignin modification (AFEX-treated) on enzymatic hydrolysis.

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Figure 4. A comparison of glucan and xylan conversion of AFEX-treated switchgrass based

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substrates at different pretreatment conditions when lignin added. a) glucan to glucose

401

conversion, b) xylan to xylose conversion.

402

AFEX pretreatment was performed at residence time of 5 min (AT05), 10 min (AT10), and 30

403

min (AT30), temperature of 90 °C, water loading of 0.8, and ammonia loading of 5.0.

404

Figure 5. A comparison of enzymatic hydrolysis of untreated and AFEX-treated switchgrass

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based substrates when lignin added.

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AFEX pretreatment was performed at residence time of 30 min (AT30) or 5 min (AT05),

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temperature of 90 °C, water loading of 0.6, and ammonia loading of 5.0. 3L= added 64.3 mg/mL

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lignin, 1L=added 21.4 mg/mL lignin.

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Figure 6. HSQC 2D NMR spectra of lignin isolated from switchgrass and AFEX-treated lignin.

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a, b) untreated lignin. c, d) AFEX-treated lignin at a temperature of 90 °C, ammonia loading of

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5.0 and residence time of 30 min.

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S:syringyl unit; G:guaiacyl unit; A: β-aryl ether (β-O-4); A-H/G: β-aryl ether (β-O-4-H/G); A-S:

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β-aryl ether (β-O-4-S); B: phenylcoumaran (β-5); F: cinnamyl alcohol; C:cellulose; X: xylan;

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PB: p-hydroxybenzoate; pCA: p-coumarate; FA: Ferulate.

415 416

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Figure 1 Dried switchgrass Wiley mill milled. Switchgrass samples Extracted with benzene/ethanol (2:1, v/v) for 24 h, and vacuum-dried (40 °C) for 72 h with P2O5. Extractive-free samples Ball milled for 10 days at 96 rpm. Switchgrass powder Extracted with 96% dioxane for 24 h and centrifuged at 10,000 rpm for 10 mins.

Solid (Residue)

Liquid (Filtrate) Evaporated the solvent and freezedried at -90 °C. Crude Lignin Purified with 90% acetic acid. Lignin

446

447 448

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Figure 2

70

Cellulose to glucose conversion (%)

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60 50 40 30

0 mg lignin/mL 21.4 mg lignin/mL 42.8 mg lignin/mL 64.3 mg lignin/mL

20 10 0 0

10

20

30

40

50

60

70

80

Hydrolysis time (h) 450 451

452

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453 454

Figure 3

70

Cellulose to glucose conversion (%)

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60 50 40 30 20

0 mg/mL lignin 21.4 mg/mL lignin 21.4 mg/mL AFEX-treated lignin

10 0 0

10

20

30

40

50

60

70

80

Hydrolysis time (h) 455

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Figure 4

a

Glucan to glucose conversion (%)

70 60 50 40 30

AT05 AT10 AT30

20 10 0 0

10

20

30

40

50

Lignin loading (mg/mL)

457

b 60

Xylan to xylose conversion (%)

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50

40

30

20

10

AT05 AT10 AT30

0 0

10

20

30

40

50

Lignin loading (mg/mL)

458

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Figure 5

Glucan-24h Glucan-72h

Xylan-24h Xylan-72h

80 70 Glucan / Xylan Conversion (%)

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60 50 40 30 20 10 0 Untreated

AT30

AT30+3L

AT05

AT05+1L

460 461 462

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Figure 6

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465 466

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