Assessment of the Lignin-Derived Inhibition of Enzymatic Hydrolysis

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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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†

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‡

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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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ACS Paragon Plus Environment

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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

13

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].

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Notes

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The authors declare no competing financial interests.

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ACKNOWLEDGMENTS

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

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

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

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REFERENCES

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380

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387 388


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

390

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

396

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

397

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

398

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

399

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

400

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

405

based substrates when lignin added.

406

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

407

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

408

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

409

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

410

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

411

5.0 and residence time of 30 min.


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412

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:

413

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

414

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

415 416


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417 418 419 420 421 422 423 424 425 426 427 428 429 430 431 432 433 434 435 436 437 438 439 440 441 442 443 444 445

Energy & Fuels

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


25

Energy & Fuels

449

Figure 2

70

Cellulose to glucose conversion (%)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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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


26

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453 454

Figure 3

70

Cellulose to glucose conversion (%)

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Energy & Fuels

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


27

Energy & Fuels

456

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 (%)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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50

40

30

20

10

AT05 AT10 AT30

0 0

10

20

30

40

50

Lignin loading (mg/mL)

458


28

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459

Figure 5

Glucan-24h Glucan-72h

Xylan-24h Xylan-72h

80 70 Glucan / Xylan Conversion (%)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Energy & Fuels

60 50 40 30 20 10 0 Untreated

AT30

AT30+3L

AT05

AT05+1L

460 461 462


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Figure 6

464

465 466


30

Assessment of the Lignin-Derived Inhibition of Enzymatic Hydrolysis

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