A statistical approach for the identification of cellulolytic enzyme

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A statistical approach for the identification of cellulolytic enzyme inhibitors using switchgrass dilute acid prehydrolyzates as a model system Angele Djioleu, and Danielle Julie Carrier ACS Sustainable Chem. Eng., Just Accepted Manuscript • DOI: 10.1021/ acssuschemeng.7b03686 • Publication Date (Web): 16 Jan 2018 Downloaded from http://pubs.acs.org on January 16, 2018

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ACS Sustainable Chemistry & Engineering

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A statistical approach for the identification of cellulolytic enzyme inhibitors using

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switchgrass dilute acid prehydrolyzates as a model system

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Angele Djioleu1 and Danielle Julie Carrier2*

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Abstract

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Department of Biological and Agricultural Engineering and Institute for Nanoscience and Engineering, 203 White Engineering Hall, University of Arkansas, Fayetteville, AR 72701

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Department of Biosystems Engineering and Soil Science, 2506 E.J. Chapman, The University of Tennessee, Knoxville, TN 37996 *corresponding author: [email protected]

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The identification of biomass pretreatment-generated compounds that impede

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cellulose hydrolysis is critical for improving the overall biomass saccharification process.

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The aim of this study was to correlate the identification and concentrations of switchgrass

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dilute acid pretreatment-generated compounds to cellulolytic enzyme inhibition and to tie

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this back to processing parameters. Twenty-four dilute acid prehydrolyzates were

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prepared with switchgrass at temperatures from 140°C to 180°C, processing times from

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10 to 40 min, and sulfuric acid concentrations of 0.5% or 1% (v/v). Results showed that

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all the switchgrass prehydrolyzates significantly reduced cellulolytic enzyme activities

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when assayed against model substrates. Exoglucanase was the most sensitive with its

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activity reduction, ranging from 58% to 88%; the inhibitory effect on β-glucosidase and

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the cellulase cocktail ranged from 32% to 63% and 16% to 41%, respectively.

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Polyphenolic compounds were the most detrimental pretreatment-generated products to

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the cellulolytic enzymes, especially to exoglucanase. Limited enzyme inhibition, with

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acceptable biomass digestibility were observed with pretreatment conditions

ACS Paragon Plus Environment

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corresponding to 160°C. The statistical based approach used in this study proved to be a

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valid method to assess the effect of pretreatment-generated compounds on cellulolytic

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enzyme activities, linking their generation to pretreatment processing parameters.

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Keywords: Cellulase, Betaglucosidase, Exoglucanase, Centrifugal Partition

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Chromatography, Phenolics

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Introduction

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To facilitate the enzymatic hydrolysis of its cellulosic fraction, biomass needs to

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be pretreated to overcome its inherent recalcitrance to biological and chemical attack.1-4

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Pretreatment techniques improve cellulose saccharification by loosening plant cell wall-

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derived hemicellulose and lignin fractions.1-4 Unfortunately, there are some pretreatment

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techniques, such as dilute acid, that result in the generation of by-products in the

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corresponding liquid prehydrolyzates; these by-products inhibit cellulolytic enzymes and

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consequently reduce the efficiency of the saccharification process.5-9 By-product

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generation and characterization limit the integration of feedstock pretreatment,

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saccharification, and fermentation as a one-pot integrated operation.10,11

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By-products generated during biomass pretreatment have been classified in the

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following categories: 1) aromatic compounds, such as phenolic compounds; 2) furan

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aldehydes, like furfural and hydroxymethyl furfural; and 3) aliphatic acids, such as acetic

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and formic acids.5-7 Other pretreatment by-products identified as inhibitors to cellulolytic

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enzyme activities include xylose oligomers12,13 and xylose derived phenolic

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compounds.14-17 These by-products are generated from the hydrolysis and degradation of

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the structural components in lignocellulosic biomass. Mitigation of the negative effect of

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such inhibitors often requires a detoxification step of biomass pretreatment liquor,6,7 a


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large amount of water to wash the pretreated biomass,8 or increasing dosage of

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cellulolytic enzyme during saccharification.18 Such mitigation strategies are non-

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sustainable and expensive after scale-up. Therefore, an understanding of which

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compounds impede saccharification enzymes would help design better mitigation

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strategies and increase the overall efficiency of lignocellulosic biomass processing.

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Biomass saccharification is mainly accomplished by the synergistic interactions

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among cellulolytic enzymes belonging to three distinct categories: β-1,4- endoglucanases

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(EC 3.2.1.4), β -1-4-exoglucanase (cellobiohydrolase, EC 3.2.1.91), and 1,4-β -

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glucosidase (EC 3.2.1.21).5,19 Although it is widely accepted that by-products formed

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during pretreatment inhibit cellulolytic enzyme activity, there is no clear consensus as to

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which category of compounds are the most potent inhibitors. When comparing the

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actions of compounds released during biomass pretreatment on cellulolytic enzyme

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activities, formic acid,20 acetic acid,21 monomeric sugars,22 xylose oligomers12,13 and

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phenolic compounds23-29 have been identified as potent cellulolytic enzyme inhibitors by

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reducing enzyme activity at concentrations raising from 1 to 11.5 g/L.

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In addition to determining which prehydrolyzate compounds are most inhibitory,

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there is a lack of understanding as to which of the cellulolytic enzyme activities are most

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affected. For example, polyphenolic compounds at 2 g/L were reported to mostly affect β

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–glucosidase by reducing its activity by 80%,24 whereas other reports27,28,30 identified

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exoglucanase as the most sensitive activity by showing that this activity is completely

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deactivated in the presence of polyphenolic compounds at concentrations less than 2 g/L.

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Differences in terms of the documentation of the inhibition of cellulolytic enzyme by

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pretreatment generated by-products could be attributed to the inconsistency of the


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methodology employed across studies, which is greatly affected by the origin and

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concentration of the by-products used in the studies.

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By definition, liquid prehydrolyzates are very complex mixtures, comprised of a

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multitude of compounds, of which many remain to be identified.17,31 When studying

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cellulolytic enzyme inhibition, it is common to use commercial standards or to isolate the

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targeted compounds from prehydrolyzates. The latter option is usually difficult to carry

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out due to the complex nature of prehydrolyzates, which requires sophisticated

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fractionation techniques. To develop a realistic assessment of the inhibitory effect of

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pretreatment-generated by-products on cellulolytic enzyme activities, the use of intact

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prehydrolyzate, as opposed to a synthetic solution, can be most useful.32 In a previous

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study, 24 dilute acid switchgrass prehydrolyzates were generated and pretreated materials

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were evaluated in terms of their propensity to saccharification.33 This current study

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reports on a statistical approach combined with a chromatography technique to identify

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the critical cellulolytic enzyme inhibitors generated during dilute acid pretreatment of

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switchgrass using these 24 switchgrass prehydrolyzates. Cellulase cocktail, as well as

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individual cellulolytic activities, exoglucanase and β –glucosidase, were tested, as these

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enzymes are key players in the cellulase cocktail.34 As saccharified biomass becomes

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substrate to various bio-derived processing operations, coupled to the need of

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consolidating unit operations for achieving low processing costs, the development of a

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comprehensive understanding of the inhibitory effect of by-products contained in

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pretreatment prehydrolyzates on the saccharification step is imperative.


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Materials and Methods

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Liquid prehydrolyzates

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Liquid prehydrolyzates were prepared by pretreating switchgrass with dilute sulfuric acid, using 24 different combinations of temperature, time and acid

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concentration, as previously outlined.33 All 24 pretreatments conditions are listed in

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Table 1. After pretreatment, the pH of the prehydrolyzates was adjusted to 4.8 ± 0.03

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using 5 N ammonium hydroxide (Sigma-Aldrich, St Louis, MO), to eliminate any pH

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effect during enzyme inhibition assays. Adjusted prehydrolyzates were filtered through

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0.45 µm PTFE membranes attached to a syringe filter (VWR International, Radnor, PA).

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Filtered prehydrolyzates (P1 to P24) were immediately stored at -20°C until they were

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used for characterization or for enzyme inhibition assays.

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Centrifugal partition chromatography (CPC)

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Solvent preparation

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HPLC-grade butanol and methanol, along with filtered water from a Direct-Q

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system (Millipore, Billerica, MA) with 18.2 MΩ resistivity, were used to prepare a

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biphasic solvent system.35 All solvents were mixed in a 2-L separatory funnel at a ratio of

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5:1:4 (v/v/v). The mixture was allowed to separate for at least 2 h into: 1) an upper

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organic rich phase and 2) a bottom aqueous phase. Each phase was carefully collected in

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1-L glass bottles; the aqueous phase was used as the stationary phase while the organic

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phase was the mobile phase during the CPC fractionation experiments.

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Sample preparation

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Liquid prehydrolyzate (P15) prepared at 160°C, 30 min, and 1% H2SO4 was dried

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under low vacuum and no heat with a Savant SpeedVac Concentrator SPD 1010 (Thermo


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Scientific, Ashville, NC) set at 7 Torr. The CPC sample was prepared by mixing 2 g of

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dry P15 with 5 mL of each CPC solvent phases. The CPC sample was filtered through a 5

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µm PTFE membrane and stored at -20 ºC until the CPC fractionation experiments.

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CPC fractionation

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CPC sample was fractionated using a bench scale SCPC-250 system from Armen

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Instruments (Saint-Avé, France), equipped with a prep-scale HPLC pump and controlled

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by Trilution® software (Gilson, Middleton, WI). Fractionation was based on previous

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methods.30 In brief, the stationary phase was loaded at 10 mL/min in the rotor, spinning at

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500 RPM for 30 min. The rotor speed was then increased to 2300 RPM, at which point

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the mobile phase was introduced in the rotor at 8 mL/min until equilibrium between the

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two immiscible phases was observed. At equilibrium, 110 mL of stationary phase was

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present in the 250-mL capacity rotor. Sample was injected through a 10-mL sample loop

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after equilibrium. The fractionation process lasted 105 min and fraction collection with a

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Foxy R1 (Teledyne Isco, Lincoln, NE) started 30 min after sample injection. Eluent was

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monitored with a UV detector set at 280 and 300 nm. All of the resulting fractions were

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dried in the SpeedVac and then reconstituted with Millipore water for further analysis.

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Characterization of liquid prehydrolyzates and CPC fractions

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Monosaccharides, furans, and aliphatic acids in liquid samples were analyzed

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with a High Pressure Liquid Chromatography (HPLC).36 Total phenolic (TP) compounds

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in liquid samples were determined as gallic acid equivalent (GAE), using the Folin-

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Ciocalteau (F-C) assay.30 Sample were diluted to 1 g/L of solid content prior to the F-C

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assay. Gallic acid standards were prepared at concentrations between 0.05 g/L and 1.25

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g/L to establish a standard curve. Monomeric phenolic compounds in liquid samples were


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identified with an Acquity Ultra Performance Liquid Chromatography (UPLC).30

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Enzyme activities

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Cellulase and β-glucosidase activities were determined per the standards of the

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International Union of Pure and Applied Chemistry (IUPAC).37 Accellerase® 1500, a

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cellulose hydrolysis enzyme cocktail, generously donated by Dupont Industrial

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Biosciences (Cedar Rapids, IA), was utilized for this investigation. Cellulase cocktail

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activity on filter paper was determined to be 25 FPU/mL. β-glucosidase was obtained

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from Megazyme (Wicklow, Ireland) and its activity on cellobiose was determined to be

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103 CBU/mL.

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Exoglucanase enzyme produced by Trichoderma reesei was purchased from

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Infinite Enzyme (Jonesboro, AR). Its activity on 4-methylumbelliferyl β-D-cellobioside

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(MUC) was 154 U/mL, as determined by the manufacturer. Exoglucanase activity was

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defined by the amount of enzyme necessary to release one micromole of 4-

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methylumbelliferyl (MU) per min,38 which is the fluorescent substrate released during the

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hydrolytic cleavage of MUC, purchased from Sigma-Aldrich (St. Louis, MO).

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Enzyme inhibition

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P1 to P24 and the CPC fractions (total of six) were individually used as treatments

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in enzyme inhibition assays. The experimental control consisted of assay in buffer. Assay

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blanks consisted of substrate in solvent without any enzyme, and enzyme blank was

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enzyme in buffer. Glucose released in all enzymatic assays was measured with a glucose

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analyzer, YSI 2900 (YSI Inc., Yellow Springs, OH).


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Cellulase

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Assay samples were prepared by mixing microcrystalline cellulose powder

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(MCC) from MP Biomedicals (Solon, OH) with assay solvent, and cellulase enzyme (15

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FPU/g cellulose powder) in a 13 x 100 mm glass tube, such that a 2% cellulose solution

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was obtained. Assay solvent was either 50 mM citrate buffer (pH 4.8) or filtered liquid

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prehydrolyzates. All samples and blanks were incubated for 60 min in a water bath set at

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50°C. The reaction was stopped by immersing the samples in boiling water for 5 min and

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then in iced water before being analyzed for glucose. The glucose released from the

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hydrolysis of MCC was calculated by subtracting the amount of glucose in blanks from

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glucose in enzyme assay.

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β-glucosidase

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Cellobiose solutions of 15 mM were prepared by dissolving cellobiose (Sigma-

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Aldrich, St Louis, MO) in 50 mM citrate buffer, liquid prehydrolyzates, or CPC fractions.

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β-glucosidase enzyme was added to the cellobiose solution at a dosage of 40 CBU/g

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cellobiose. All samples and blanks were incubated for 30 min in a water bath set at 50°C.

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The assay reaction was stopped and analyzed for glucose in a similar manner as in the

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cellulase assay.

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Exoglucanase

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A 0.005 M substrate stock solution was prepared by dissolving 25 mg of MUC in

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1 mL of dimethyl sulfoxide (Sigma-Aldrich, St Louis, MO) and 9 mL of 50 mM acetate

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buffer (Sigma-Aldrich, St Louis, MO) (pH = 5.0). Substrate solution (25 µL), diluted

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enzyme (8 µL) and assay solvent (92 µL) were mixed in a flat-bottom 96-wells

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microplate with lid (Corning®, Radnor, PA). The assay solvent was either 50 mM acetate


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buffer or treatments. Diluted enzyme was obtained by diluting the original enzyme

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solution (from the manufacturer) by a factor of 20X. The 96-well microplate was covered

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with a plate lid and incubated at 50°C in a water bath for 60 min. The reaction was

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stopped by mixing 25 µL of assay with 225 µL of 0.2 M sodium carbonate solution in a

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flat-bottom 96-well black reading plates (FluoroNunc™, Fischer Scientific, Pittsburg,

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PA). Assay fluorescence was analyzed with a Synergy HT (BioTek Instruments,

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Winooski, VT) micro-well plate reader (excitation 360 nm and emission 460 nm).

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Fluorescence values of MUC hydrolysis were determined by subtracting fluorescence

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obtained for blank readings from those of the samples.

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Statistical analysis

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Enzyme activity inhibition experiments were performed in duplicate. Statistical

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analysis, such as ANOVA, Dunnett’s control test or Student’ t-test, was executed in JMP

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Pro 11 (SAS Institute). The significance for all the analysis was established for P-values

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< 0.05.

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Results and Discussion

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Effect of switchgrass prehydrolyzates on activity of cellulolytic enzyme

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Cellulase

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A total of 24 liquid prehydrolyzates were obtained from dilute acid pretreatment

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of Panicum virgatum L. var. Alamo (switchgrass); compositional analysis of the

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feedstock showed contents of 36% glucan, 24% xylan, 6% arabinan, and 22% lignin.33

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The composition of the 24 liquid prehydrolyzates (P1 to P24) is presented in Table 1. The

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range of compound concentrations detected in the 24 prehydrolyzates was used to

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statistically determine their effect on the cellulase system. The cellulase activity assay


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was conducted on microcrystalline cellulose powder (MCC) with citrate buffer as the

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control, or switchgrass prehydrolyzates as treatments. An analysis of variance (ANOVA)

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and a Dunnett’s control test determined that all the prehydrolyzates significantly inhibited

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cellulase activity as the glucose released from hydrolysis of MCC in assays treated with

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prehydrolyzate was significantly lower than glucose from the control. The degree of

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inhibition from each treatment was calculated as follows: !"ℎ$%$&$'" % =

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+,-.'/01 − +,-.'/03 ×100 (1) +,-.'/01

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where GlucoseC = glucose formed in control assay and Glucosep = glucose formed in

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treatment assay.

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Figure 1A presents cellulase inhibition percentage as a function of the tested

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prehydrolyzates, where treatments displaying similar letters were not statistically

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different, as determined by a Student’s t-test. Cellulase activity was reduced by at least

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16%, where the highest degree of inhibition (41%) was obtained with P20. Of the tested

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prehydrolyzates, only P20 (41%), P4 (32.5%), P14 (23.5%), and P23 (5.7%) were found

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to have significantly different inhibition effects on the cellulase system. Most of the

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treatments affected cellulase activity in a statistically similar manner (P > 0.05).

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Using the composition and the inhibition percentage of P20, P4, P14, and P23, a

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regression analysis, validated by ANOVA, showed that there were no significant (P >

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0.05) correlations between the percentage inhibition and the concentrations of the

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identified prehydrolyzate compounds. Such lack of correlation could possibly indicate

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that key inhibitory compounds to the cellulase system remain to be identified or that

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inhibition was the result of synergistic action of prehydrolyzate compounds. The

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establishment of correlations between degradation products present in biomass


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prehydrolyzate and cellulase inhibition is directly related to the capacity of identifying

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these products.31 The use of authentic prehydrolyzates, as opposed to synthetic solutions,

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highlights the likelihood that cellulase inhibition is the result of the interaction of

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pretreatment generated compounds, many of which remain unidentified, as it has been

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previously observed.21,22

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β-glucosidase

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Similar to the cellulose system, statistical analysis highlighted that β-glucosidase

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activity was significantly inhibited by the tested liquid prehydrolyzates. Figure 1B

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presents inhibition percentages from treatments compared with Student’s t-test;

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treatments not connected by similar letters were significantly different. The highest

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inhibition effect, 62.5%, was observed with P19, while P23 displayed the lowest

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inhibition, 31.8%. Significant differences were observed from P19, P17, P22, P10, and

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P23 prehydrolyzates with inhibition calculated as 62.5%, 55.6%, 46.3%, 39.0% and

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31.8%, respectively.

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Prehydrolyzates P19, P17, P22, P10, and P23 were further analyzed to determine

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if any linear correlation could be traced between β-glucosidase inhibition and

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pehydrolyzate compound concentrations. The statistically significant (P < 0.05)

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correlations between the prehydrolyzate compound concentrations and the percentage

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inhibition of β-glucosidase are shown in Figure 2; the strength of the linear correlations

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was measured with the Pearson correlation coefficient, and its squared value (R2) is

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reported. The significance of the Pearson coefficient was validated through ANOVA

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analysis. A strong, significant positive linear correlation between β-glucosidase inhibition

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and the concentrations of glucose (Figure 2A), acetic acid (Figure 2B), HMF (Figure 2C)


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and furfural (Figure 2D) was observed. Inhibition of the β-glucosidase system increased

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with rising glucose, acetic acid, HMF, and furfural concentrations. Glucose and HMF

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concentrations showed the strongest correlation with β-glucosidase inhibition with R2 of

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0.94, 0.92, respectively. R-squared values for acetic acid and furfural were 0.89 and 0.86,

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respectively. From the correlation analysis, it can then be inferred that compounds

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identified in Figure 2 could be strong indicators of β-glucosidase inhibition. Additionally,

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these compounds have been shown to be β-glucosidase inhibitors, using commercial

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standard at similar concentration as in this study.20,27,39

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Exoglucanase

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The inhibitory action of the tested prehydrolyzates on the exoglucanase system

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was determined as previously described for the cellulase and β-glucosidase systems,

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using equation 1, where calculations were made based on differences in fluorescence.

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Figure 1C presents exoglucanase inhibition as a function of the tested prehydrolyzates;

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treatments connected with identical letters were not statistically different.

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When compared to β-glucosidase and cellulase, the exoglucanase system was the

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most sensitive to the inhibition produced by the tested prehydrolyzates. The highest

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inhibition, 88.2%, was obtained with P1. The inhibition percentage produced by P1 was

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statistically different to that of P20 and P19, which had the highest inhibitory effect on

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cellulase and β-glucosidase, respectively. On the other hand, the lowest inhibitory effect

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of 57.8% to the exoglucanase system was observed from P23 (180°C, 30 min, 1%

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H2SO4), which also yielded the lowest inhibitory effect for cellulase and β-glucosidase

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systems. Unfortunately, the pretreatment conditions associated with P23 were not

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favorable to saccharification, as the amount of glucan in the pretreated biomass was less


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than 5% of the total solid content.33 The seven prehydrolyzates with distinct significantly

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different inhibition effects on exoglucanase were: P1 (88.2%), P2 (83.0%), P3 (78.8%),

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P6 (74.0%), P5 (70.2%), P8 (63.1%), and P23 (57.8%).

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As presented in Figure 3, correlation analysis using the prehydrolyzates P1, P2,

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P3, P5, P6, P8, and P23 showed that phenolic compounds (Figure 3A), acetic acid

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(Figure 3B), glucose, (Figure 3C) and furfural (Furfural 3D) displayed a significant linear

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relationship with exoglucanase inhibition. However, only phenolic compounds and acetic

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acid had a positive linear correlation (R2 = 0.7) with exoglucanase inhibition, while

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glucose and furfural displayed a negative correlation. Results in Figure 3A and 3B

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indicated that phenolic compounds and acetic acid could possibly be good indicators of

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exoglucanase inhibition. Phenolic compounds inhibition to the cellulase system has been

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well documented.14-17,23-26 Such inhibition has been attributed to the irreversible binding

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of exoglucanase to phenolic compounds, which have been reported to form a physical

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barrier by depositing on the biomass surface after dilute acid pretreatment.4,27,30,40

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Contrary to this study, furfural, HMF and formic acid have been reported as potent

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exoglucanase inhibitors;27,30 however, no significant correlation between exoglucanase

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inhibition with formic acid and HMF was observed in this work. Interestingly, a negative

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correlation between furfural and exoglucanase inhibition was noted, indicating that

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instead of being an inhibitor, furfural may be part of a by-product system in the

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prehydrolyzate that prevents the impediment of exoglucanase activity. Further research is

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needed to clarify such shielding effect or synergistic interactions among pretreatment

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generation by-products.

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Centrifugal partition chromatography (CPC)

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The correlation analysis performed above suggested that prehydrolyzates

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contained compounds that inhibited cellulolytic enzymes. Prehydrolyzate P15 was

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fractionated by CPC fractionation and the resulting fractions were tested against

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cellulolytic enzyme activity in an attempt to elucidate which compounds or group of

306

compounds were responsible for inhibition. Prehydrolyzate P15 was selected because its

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composition affected all three enzyme activities, where cellulase, β-glucosidase, and

308

exoglucanase activities were decreased by 35.0% ± 0.0, 56.0% ± 0.0, and 68.5% ± 0.0

309

respectively, as shown in Figure 1. Pretreatment conditions used to generate P15 (160ºC,

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30 min, and 1% H2SO4) yielded pretreated biomass with a glucan content of 50.2% and a

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digestibility of 86.1 ± 7.9%, corresponding to realistic saccharification yields.33

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CPC fractionation, with the described solvent system, has been previously

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reported to separate compounds derived from hot water pretreatment of rice straw.30 CPC

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fractionation yielded six major fractions, F1 to F6; their composition and order of elution

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from the CPC separation process are shown in Table 2 and supporting document, Figure

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S1, respectively. Phenolic compounds, present in P15, eluted within the first 10 min of

317

the fractionation process and represented 10.38% and 7.02% of the solid content of

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fractions F1 and F2, respectively. The difference between F1 and F2 was that the

319

concentrations of the identified phenolic compounds were significantly lower in F2 as

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compared to those of F1. Monomeric sugars and aliphatic acids co-eluted from the 39th

321

min to the 53th min of the fractionation process, and were grouped as F3 and F4. Given

322

that the monomeric sugars and aliphatic acid co-eluted in fractions F3 and F4, it was not

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possible to individually test for their effect on cellulolytic enzyme activity. Fraction F5,


14

Page 15 of 33 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


324

which mostly contained arabinose was collected between the 54th and 60th min of the

325

process. Fraction F6, collected between 61 min and 74 min, only showed traces of xylose

326

and arabinose; however, its total solids concentration was 99.7 g/L and, as such, was kept

327

as a separate fraction. The furan compounds, originally present in the liquid

328

prehydrolyzate, were not recovered after the CPC process, and this was most likely due to

329

their evaporation during sample preparation. It is important to note that except for

330

fraction F3, identified compounds in the fractions represented at most 6% of the total

331

solid content of the corresponding fraction, as illustrated in Table 2. Known compounds

332

in F3 constituted 35% of the solid content. More advanced analytic techniques are needed

333

to identify other possible products that were present in P15, such as aldehyde, ketone,

334

aromatic, and aliphatic compounds.31

335

Effect of CPC fractions on cellulolytic enzyme

336

β-glucosidase

337

Individual CPC fractions, prepared at solid concentrations of 25 g/L, were used as

338

treatments in the β-glucosidase inhibition assay using 15 mM of cellobiose, as presented

339

in Figure 4A. Control experiments consisted of cellobiose hydrolysis in citrate buffer.

340

Comparison of treatments with a Student’s t-test showed that glucose released from

341

hydrolysis of cellobiose in assays treated with F2, F3, F4, F5 and F6 were significantly

342

lower than those of the control, as indicated by different letters. Fraction F1, which

343

contained more phenolic compounds than the other fractions, did not significantly affect

344

β-glucosidase activity, indicating that phenolics compounds were not critical for

345

inhibition. Moreover, inhibitory effect from F2 was not significantly different than what

346

was observed for F5 and F6, in which only traces of phenolic compounds were detected.


15


Page 16 of 33

347

This insensitivity of β-glucosidase to the phenolic-rich fraction F1 supported the lack of

348

correlation between phenolic compound concentration in prehydrolyzate and inhibition of

349

β-glucosidase reported earlier in this study. Such insensitivity to phenolic compounds has

350

been attributed to the origin of the enzyme system24,25 when it was determined that β-

351

glucosidase from Aspergillus niger was 10X less sensitive to phenolic compounds than

352

the ones from Trichoderma reesei.

353

The fractions F3 to F6 yielded statistically similar inhibitory effects of 56%, 56%,

354

50% and 48%, respectively. Inhibition of β-glucosidase by sugar-rich fractions F3 to F5

355

corroborated the earlier results where strong positive correlation (R2 = 0.93) was

356

observed between glucose concentration and β-glucosidase inhibition. The composition

357

of F6 was not totally elucidated; only traces of xylose and arabinose were detected in F6,

358

yet this fraction inhibited β-glucosidase in a statistically similar manner to that of F3, F4

359

and F5. It is more than likely that unidentified compounds in F6 could be attributed to its

360

observed β-glucosidase inhibition. Although β-glucosidase activity was impeded by CPC

361

fractions, it was observed that when β-glucosidase was treated with F4 at a solid

362

concentration of 15 g/L, cellobiose hydrolysis was complete after a 180-min incubation

363

period, showing that β-glucosidase activity was not completely shut down by F4 (Figure

364

S2).

365

Exoglucanase

366

Fractions F1 to F6, prepared at a concentration of 25 g/L, were tested for their

367

inhibitory effect on the exoglucanase system, using MUC as a substrate. The control

368

assay was performed in 50 mM acetate buffer. As indicated in Figure 4B, all CPC

369

fractions significantly reduced exoglucanase activity, which was correlated by the fact


16

Page 17 of 33 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


370

that fluorescence intensities from CPC fractions were lower than the intensity obtained

371

from the control. Fractions F1 and F2 had the highest inhibitory effect on exoglucanase

372

activity by decreasing MUC hydrolysis by 94.7% and 90.9%, respectively. Such

373

inhibition was attributed to unidentified polyphenolic compounds because only traces of

374

monomeric phenol were detected in F2 and yet this fraction had statistically similar effect

375

on exoglucanase activity as F1, which content more monophenols. Moreover,

376

monophenols identified in this study have been shown to stimulate cellulase activity at

377

concentrations above 2 g/L.41

378

The inhibitory effect of the polyphenolic compounds on exoglucanase activity

379

was further investigated overtime by using fraction F1. It was observed that the

380

hydrolysis rate of MUC in acetate buffer was double than the one in F1, prepared at a 4

381

g/L solid concentration, when the hydrolysis assay was conducted for 24 h (Figure S4).

382

These results confirmed the complete deactivation of exoglucanase activity by

383

polyphenolic compounds described in the literature27,30,40 and reinforced the need to

384

focus on exoglucanase when designing resistant polyphenolic-inhibition cellulase

385

enzyme.42 Although the sugar-rich fractions F3 to F5 also inhibited exoglucanase

386

activity, it is important to note that such inhibition may be caused by other unidentified

387

products present in these fractions. This possibility is highlighted with fraction F6, which

388

also inhibited exoglucanase activity, but had less than 3% of its solid content identified,

389

indicating the presence of unidentified compounds that are deleterious to the process.

390

Dilute acid pretreatment of switchgrass produced compounds that are inhibitory to

391

cellulolytic enzyme systems. Of the compounds identified in the prehydrolyzates tested,

392

polyphenolic compounds were determined to be most detrimental to cellulolytic enzyme


17


Page 18 of 33

393

activities, especially to exoglucanase. However, it was observed that inhibition was also

394

the result of the action of many other unidentified compounds present in switchgrass

395

prehydrolyzates. This study demonstrated that a better characterization of the

396

prehydrolyzate would facilitate the identification of key prehydrolyzate compounds that

397

could indicate involvement of inhibition of cellulolytic enzyme; this could be carried out

398

possibly without isolations of these compounds from the liquid prehydrolyzate.

399

Obtaining information on which prehydrolyzate compounds are related to inhibition

400

would be beneficial to the overall biorefinery operation for the design of detoxification

401

strategies and of robust enzyme system. Obtaining this information without having to

402

isolate and fractionate the compounds in play would avoid laborious and expensive

403

procedures. In a similar vein, the manipulation of controllable pretreatment parameters

404

may prove to be a better approach for the generation of liquid prehydrolyzate with

405

limited overall inhibitory effect, while preventing glucan degradation and promoting

406

digestibility. Identification of key inhibitory compounds and pretreatment conditions that

407

lead to their generation could facilitate the integration of pretreatment and enzymatic

408

saccharification steps, bypassing or minimizing the detoxification and washing steps.

409

Such an approach has been successfully implememted with ionic liquid pretreatment in

410

an integrated one-pot system where simultaneous saccharification performed in liquid

411

prehydrolyzate resulted in ethanol yield comparable to the one obtained from a traditional

412

biochemical conversion process.10,11 The data presented in Table 3, which lists

413

pretreatment conditions used in this study, the glucan content and digestibility of the

414

pretreated switchgrass,33 as well as the inhibitory effects of the ensuing pretreatment

415

liquors on the cellulolytic enzyme activities, show that pretreatment conditions at 160°C


18

Page 19 of 33 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


416

with acid sulfuric acid less than 1% (v/v) and processing time lower than 40 min would

417

minimize the effect of soluble cellulolytic enzyme inhibitors while producing

418

pretreatment biomass with an average glucan content of 54% and digestibility of 78%. It

419

is important to note that the above processing conditions are tentative and most likely

420

dependent on the feedstock. Future research that would examine the actual effect of

421

pretreatment conditions on enzyme saccharification and carbohydrate fermentation in a

422

one-pot integrated system, using dilute acid pretreatment or any other leading

423

pretreatment technique is needed.

424

Acknowledgments

425

The authors thank National Science Foundation award number 0822275, United

426

States Department of Energy award number GO88036, and the Department of Biological

427

and Agricultural Engineering, University of Arkansas, Fayetteville for their gracious

428

financial support. The authors also acknowledge the Plant Powered Production (P3)

429

Center. P3 was funded through the RII: Arkansas ASSET Initiatives (AR EPSCoR) I

430

(EPS-0701890) and II (EPS-1003970) by the National Science Foundation and the

431

Arkansas Science and Technology Center. The authors thank Dr. Edgar Clausen for proof

432

reading the manuscript.

433 434 435

References

436 437 438 439 440 441 442

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Page 24 of 33

Table 1: Composition of dilute acid prehydrolyzate of switchgrass obtained at different combinations of temperature, time, and sulfuric acid concentration Samples

Temp (°C)

Time (min)

Acid Conc. (%V)

pH

Xylose (g/L)

Glucose (g/L)

Acetic Acid (g/L)

Formic Acid (g/L)

HMF (g/L)

Furfural (g/L)

TP (g/L)

P1

140

10

0.5

4.76

19.58

1.70

4.07

0.03

0.11

0.42

1.14

P2

140

20

0.5

4.85

19.71

1.87

4.50

1.97

0.12

0.68

1.22

P3

140

30

0.5

4.72

19.25

2.15

4.21

2.24

0.12

0.81

1.15

P4

140

40

0.5

4.75

14.44

1.78

3.40

2.07

0.11

0.68

0.77

P5

140

10

1

4.71

15.88

1.67

3.93

1.66

0.12

0.82

0.85

P6

140

20

1

4.79

21.94

1.89

4.34

1.45

0.12

0.85

1.00

P7

140

30

1

4.79

18.29

2.35

4.77

1.98

0.14

1.32

0.95

P8

140

40

1

4.77

20.27

2.61

2.72

3.66

0.14

1.65

0.85

P9

160

10

0.5

4.80

13.29

2.91

3.45

2.47

0.18

1.26

1.26

P10

160

20

0.5

4.75

3.85

4.37

3.04

2.12

0.21

2.45

0.82

P11

160

30

0.5

4.80

11.06

4.01

8.51

5.48

0.28

2.06

1.39

P12

160

40

0.5

4.78

5.99

2.41

2.03

1.55

0.22

1.74

1.00

P13

160

10

1

4.75

9.56

4.99

7.94

4.31

0.24

2.42

1.29

P14

160

20

1

4.80

11.64

3.11

3.58

2.60

0.20

1.79

1.03

P15

160

30

1

4.81

4.62

6.36

6.90

6.08

0.26

3.12

1.07

P16

160

40

1

4.81

5.57

3.88

7.06

5.94

0.19

2.83

0.75

P17

180

10

0.5

4.80

1.19

8.97

8.95

4.66

0.70

3.91

1.33

P18

180

20

0.5

4.77

1.37

6.79

9.56

4.58

0.63

4.01

1.41

P19

180

30

0.5

4.79

1.29

8.58

8.95

3.56

0.83

3.83

1.36

P20

180

40

0.5

4.79

1.84

9.86

11.04

4.25

1.02

3.50

1.51

P21

180

10

1

4.79

1.56

8.27

10.33

4.88

0.66

3.36

1.57

P22

180

20

1

4.79

1.33

5.97

7.08

3.77

0.51

2.07

1.35

P23

180

30

1

4.79

1.05

2.88

2.72

1.53

0.26

1.07

0.90

P24

180

40

1

4.76

0.00

2.92

10.35

4.46

0.33

1.68

1.77

Temp = temperature; conc = concentration, HMF = hydroxymethylfurfural; TP = total phenolic compounds as gallic acid equivalent


24

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


Table 2: Composition of dilute sulfuric acid switchgrass prehydrolyzate fractions as percentage of total solid content. Fractions were obtained by centrifugal partition chromatography (CPC) CPC Fractions

HBA (%)

VA (%)

V (%)

SY (%)

P-C (%)

FE (%)

SA (%)

TP (%)

F1

0.36

1.05

0.31

0.25

0.11

0.12

0.35

10.38

0.16

0.08

0.14

0.1

0.08

7.02

F2

GL (%)

XY (%)

AR (%)

FA (%)

AA (%)

1.7

5.44

1.44

F3

2.5

24.34

6.93

F4

1.31

2.74

0.92

F5

1.47

1.58

5.23

F6

1.48

0.37

0.03

1.68

HBA = 4-Hydroxybenzoic Acid, VA = Vanillic Acid, V = Vanillin, SY = Syringaldehyde, P-C = P-Coumaric, FE = Ferulic Acid, SA= Salicylic Acid, TP = Total Phenolic as gallic acid equivalent; GL = Glucose, XY = Xylose, AR = Arabinose, FA = Formic Acid, AA = Acetic Acid


25

ACS Sustainable Chemistry & Engineering 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

Page 26 of 33

Table 3: Switchgrass dilute acid pretreatment: conditions, pretreated biomass, inhibitory effect of prehydrolyzate liquors Pretreatment Conditions Pretreated Switchgrass Acid Glucan Temp* Time Digestibility Conc** content (%) Samples (°C) (min) (v/v) (%) P1 140 10 0.5 62.91 38.97 ± 4.16 P2 140 20 0.5 57.47 48.88 ± 6.66 P3 140 30 0.5 64.28 48.15 ± 7.52 P4 140 40 0.5 62.22 60.74 ± 2.44 P5 140 10 1 63.94 57.10 ± 5.03 P6 140 20 1 54.75 49.00 ± 6.91 P7 140 30 1 63.67 59.33 ± 4.22 P8 140 40 1 62.12 64.07 ± 2.13 P9 160 10 0.5 55.65 85.54 ± 10.84 P10 160 20 0.5 55.71 74.12 ± 7.3 P11 160 30 0.5 50.14 81.29 ± 6.74 P12 160 40 0.5 52.48 79.39 ± 3.87 P13 160 10 1 51.95 75.74 ± 3.07 P14 160 20 1 54.66 79.25 ± 7.5 P15 160 30 1 50.2 86.12 ± 7.84 P16 160 40 1 55.55 58.02 ± 5.16 P17 180 10 0.5 44.37 95.80 ± 8.28 P18 180 20 0.5 46.83 68.77 ± 6.63 P19 180 30 0.5 25.05 78.41 ± 9.00 P20 180 40 0.5 34.41 95.66 ± 4.8 P21 180 10 1 13.62 N/A P22 180 20 1 9.58 N/A P23 180 30 1 8.09 N/A P24 180 40 1 3.33 N/A * Temp = temperature; **Conc = concentration; N/A= data not available

Prehydrolyzates Inhibitory Effect Beta Cellulase glucosidase Inhibition (%) Inhibition (%) 35.01 ± 0.03 43.22 ± 0.03 35.49 ± 0.02 44.79 ± 0.00 29.82 ± 0.00 43.00 ± 0.01 32.49 ± 0.00 38.78 ± 0.01 37.32 ± 0.03 42.25 ± 0.03 33.72 ± 0.03 42.04 ± 0.03 35.68 ± 0.04 40.52 ± 0.01 37.50 ± 0.00 41.68 ± 0.00 34.60 ± 0.04 43.88 ± 0.05 34.91 ± 0.02 39.31 ± 0.00 37.80 ± 0.05 38.94 ± 0.02 24.84 ± 0.01 35.99 ± 0.02 29.74 ± 0.02 44.44 ± 0.06 23.52 ± 0.04 37.00 ± 0.00 34.07 ± 0.03 54.96 ± 0.03 29.50 ± 0.05 37.96 ± 0.05 29.62 ± 0.02 55.60 ± 0.06 23.09 ± 0.04 52.65 ± 0.07 33.88 ± 0.08 62.54 ± 0.03 40.95 ± .06 53.85 ± 0.01 29.79 ± 0.04 54.26 ± 0.04 39.57 ± 0.07 46.31 ± 0.00 15.68 ± 0.00 31.85 ± 0.03 30.97 ± 0.01 39.88 ± 0.01


Exoglucanase Inhibition (%) 88.20 ± 0.02 83.03 ± 0.00 78.78 ± 0.00 72.38 ± 0.02 70.21 ± 0.02 74.31 ± 0.02 62.60 ± 0.02 63.15 ± 0.02 78.19 ± 0.03 61.72 ± 0.02 72.88 ± 0.00 62.36 ± 0.01 77.54 ± 0.02 71.08 ± 0.00 69.74 ± 0.02 74.02 ± 0.03 75.70 ± 0.01 77.77 ± 0.00 77.91 ± 0.00 82.58 ± 0.05 84.13 ± 0.01 68.17 ± 0.00 57.84 ± 0.02 73.48 ± 0.02

26

Page 27 of 33 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


Figure captions Figure 1 Effect of switchgrass dilute acid prehydrolyzates on (A): cellulase activity, (B): β-glucosidase activity, and (C): exoglucanase activity. Prehydrolyzates were prepared at 24 different combinations of temperature (140°C, 160C, 180°C), time (10,20,30, 40 min) and H2SO4 concentration (0.5%, 1%). Data and error bars are means and standard deviation of 2 replications, respectively. Treatments not connected by the same letter are significantly different (P < 0.05). Figure 2 Linear correlation between inhibition of β-glucosidase activity and concentration of (A): glucose; (B): acetic acid; (C): hydroxymethyl furfural and (D): furfural Figure 3 Linear correlation between inhibition of exoglucanase activity and concentration of (A): phenolic compounds; (B): acetic acid; (c): glucose; and (D): furfural Figure 4 Effect of centrifugal partition chromatography (CPC) fractions of switchgrass dilute acid prehydrolyzate on (A): β-glucosidase activity and (B): exoglucanase activity. C = control, F1-F6 = CPC fractions. Data and error bars are means and standard deviation of 2 replications, respectively. Treatments not connected by the same letter are not significantly different (P < 0.05).


27


Figure 1 100.00%

(A)

90.00% 80.00%

CDEFGH

AB

DEFGHI

ABCDEF

FGHI

HI

EFGHI

ABCDEF

DEFGHI

ABC

ABCDEF

ABCDEF

ABCDEF

ABCD

ABCDE

ABCDEF

BCDEFG

GHI

30.00%

DEFGHI

40.00%

ABCDEF

50.00%

ABCDEF

Cellulase Inhibition

60.00%

A

(A)

70.00%

IJ

20.00%

J

P24

P23

P22

P21

P20

P19

P18

P17

P16

P15

P14

P13

P12

P11

P9

P10

P8

P7

P6

P5

P4

P3

100.00%

P2

0.00%

P1

10.00%

90.00%

B

FGH

EFG

EFG

GH

A B

B

EFGH

DE

DEF

DEFG

DEFG

DEFG

DEFG

40.00%

EFGH

DE

DEF

60.00% 50.00%

B BC

CD

DEFG

(B)

70.00%

DEF

β-glucosidase inhibition

80.00%

H

30.00% 20.00%

P24

P23

P22

P21

P20

P19

P18

P17

P16

P15

P14

P13

P12

P11

P9

P8

L

L

P7

P8

GHIJ

DEF

DEF

FGHI

DEFG

JK

GHIJ

B BC

K

L

LM

60.00%

HIJK

70.00%

DE

EFGH

80.00%

CD

IJK

B

DEF

(C)

A

GHIJ

90.00%

P10

100.00%

P7

P6

P5

P4

P3

P2

0.00%

P1

10.00%

Exoglucanase Inhibition

M

50.00% 40.00% 30.00% 20.00%

P24

P23

P22

P21

P20

P19

P18

P17

P16

P15

P14

P13

P12

P11

P9

P10

P6

P5

P4

P3

0.00%

P2

10.00%

P1

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

Page 28 of 33

Treatments


28

Page 29 of 33

Figure 2 70.00%

70.00%

(A)

60.00%

50.00% Inhibition

Inhibition

40.00% 30.00%

R² = 0.94

20.00%

P = 0.007

40.00% 30.00%

R² = 0.89

20.00%

P = 0.016

10.00%

10.00% 0.00%

(B)

60.00%

50.00%

0

2

4

6

8

0.00%

10

0

2

70.00%

60.00%

(C)

8

10

(D)

50.00% Inhibition

50.00% 40.00% 30.00%

R² = 0.92

20.00%

40.00% 30.00%

R² = 0.85

20.00%

P = 0.01

P = 0.024

10.00%

10.00%

0.00% 0

0.2

0.4

0.6

0.8

1

0

Hydroxymethylfurfural (g/L)

6

70.00%

60.00%

0.00%

4

Acetic acid (g/L)

Glucose (g/L)

Inhibition

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



1

2

3

4

5

Furfural (g/L)

29


Figure 3 100.00%

100.00%

(A)

60.00% R 2 = 0.73

40.00%

P = 0.015

20.00% 0.00%

60.00% R 2 = 0.71 P = 0.018

40.00% 20.00%

0.7

0.8

0.9

1

1.1

1.2

0.00%

1.3

Total phenolic (g/L)

80.00%

80.00%

Inhibition

100.00%

60.00%

R 2 = 0.69

40.00%

P = 0.033

(C)

20.00%

1.5

1.7

2

2.1

2.3

2.5

3.5

40.00%

4

4.5

5

R 2 = 0.64

2.7

2.9

3.1

0.00%

P = 0.032

(D) 0

Glucose (g/L)

3

60.00%

20.00%

1.9

2.5

Acetic acid (g/L)

100.00%

0.00%

(B)

80.00%

Inhibition

Inhibition

80.00%

Inhibition

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

Page 30 of 33

0.5

1

1.5

2

Furfural (g/L)


30

Page 31 of 33

Figure 4 3.5 3

A

A

(A)

Glucose formed (g/L)

2.5 2

B C

1.5

BC

BC

F5

F6

C

1 0.5 0 1200

Control

F1

F2

F3

F4

A

1000

Fluorescence Intensity

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


(B)

800 600 400

C

200

D

B

BC

BC

F4

F5

D

0 Control

F1

F2

F3

F6

Treatments


31



Page 32 of 33

32

Page 33 of 33

Betaglucosidase

SWITCHGRASS

DILUTE ACID

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


Prehydrolyzate

Exoglucanase

Description: The graphic presents compounds identified in dilute acid prehydrolyzate of switchgrass that are inhibitory to cellulolytic enzymes


A statistical approach for the identification of cellulolytic enzyme

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