ARTICLE

Facile Pretreatment of Lignocellulosic Biomass atHigh Loadings in Room Temperature Ionic Liquids

Hong Wu,1,2,3 Mauricio Mora-Pale,1,3 Jianjun Miao,1,2,3 Thomas V. Doherty,3

Robert J. Linhardt,1,2,3,4,5,6 Jonathan S. Dordick1,2,3,4,6

1Department of Chemical and Biological Engineering, Rensselaer Polytechnic Institute,

110 8th Street, Troy, New York 12180; telephone: 518-355-4062; Fax: 518-276-2207;

e-mail: dordick@rpi.edu2Center for Nanotechnology, Rensselaer Polytechnic Institute, Troy, New York3Center for Biotechnology and Interdisciplinary Studies, Rensselaer Polytechnic Institute,

Troy, New York4Department of Biology, Rensselaer Polytechnic Institute, Troy, New York5Department of Chemistry and Chemical Biology, Rensselaer Polytechnic Institute, Troy,

New York6Department of Biomedical Engineering, Rensselaer Polytechnic Institute, Troy, New York

Received 11 May 2011; revision received 27 June 2011; accepted 5 July 2011

Published online 18 July 2011 in Wiley Online Library (wileyonlinelibrary.com). DOI 10.1002/bit.23266

ABSTRACT: Ionic liquids (ILs) have emerged as attractivesolvents for lignocellulosic biomass pretreatment in theproduction of biofuels and chemical feedstocks. However,the high cost of ILs is a key deterrent to their practicalapplication. Here, we show that acetate based ILs are effec-tive in dramatically reducing the recalcitrance of corn stovertoward enzymatic polysaccharide hydrolysis even at loadingsof biomass as high as 50% by weight. Under these condi-tions, the IL serves more as a pretreatment additive ratherthan a true solvent. Pretreatment of corn stover with 1-ethyl-3-methylimidizolium acetate ([Emim] [OAc]) at 125� 58Cfor 1 h resulted in a dramatic reduction of cellulose crystal-linity (up to 52%) and extraction of lignin (up to 44%).Enzymatic hydrolysis of the IL-treated biomass was per-formed with a common commercial cellulase/xylanase fromTrichoderma reesei and a commercial b-glucosidase, andresulted in fermentable sugar yields of�80% for glucose and�50% for xylose at corn stover loadings up to 33% (w/w)and 55% and 34% for glucose and xylose, respectively, at50% (w/w) biomass loading. Similar results were observedfor the IL-facilitated pretreatment of switchgrass, poplar,and the highly recalcitrant hardwood, maple. At 4.8% (w/w)corn stover, [Emim][OAc] can be readily reused up to 10times without removal of extracted components, such aslignin, with no effect on subsequent fermentable sugaryields. A significant reduction in the amount of IL combinedwith facile recycling has the potential to enable ILs to be usedin large-scale biomass pretreatment.

Biotechnol. Bioeng. 2011;108: 2865–2875.

� 2011 Wiley Periodicals, Inc.

KEYWORDS: cellulose crystallinity; ionic liquids; lignocel-lulose; lignin; recycling

Introduction

Lignocellulosic biomass from agricultural wastes, wood, orenergy crops is considered to be the most available andrenewable resource for producing liquid fuel and othervaluable chemicals (Himmel et al., 2007; Somerville et al.,2010). The bioconversion of lignocellulose into fuels andchemicals includes feedstock pretreatment, hydrolysis ofpolysaccharide to monosaccharides, and subsequent fer-mentation. While recent improvements in single-stagehydrolysis/fermentation may lead to decreased processingcosts and increased fermentation product yields, the limitingstep remains the conversion of raw biomass into a moreusable resource. In particular, there remains a critical needto efficiently and economically break down the 3D structuralnetwork of cellulose, hemicellulose and lignin, anddeconstruct the tight hydrogen-bonded cellulose network,both of which contribute to the recalcitrance of biomass tosubsequent enzymatic polysaccharide depolymerization(Scheme 1A–C) (Lynd et al., 2008).

Several pretreatment strategies have been advanced inrecent years, including physical (da Silva et al., 2010),

Correspondence to: J.S. Dordick

Contract grant sponsor: National Natural Science Foundation of China

Contract grant number: 31071559

Contract grant sponsor: Chisso Corporation

Additional supporting information may be found in the online version of this article.

� 2011 Wiley Periodicals, Inc. Biotechnology and Bioengineering, Vol. 108, No. 12, December, 2011 2865

chemical (Binder and Raines, 2010; Kim and Holtzapple2005; Li et al., 2008), physicochemical (Kaar et al., 1998;Krishnan et al., 2010; Lau et al., 2010), hot water (Weil et al.,1997; Zeng et al., 2007), and biological (Keller et al., 2003;Sousa et al., 2009), and are reviewed extensively elsewhere(Mora-Pale et al., 2011; Sousa et al., 2009).

Recently, ionic liquids (ILs) have emerged as an attractivealternative for lignocellulose pretreatment due to theirnear unique capability of dissolving not only individualsubcomponents of lignocellulose (Fukaya et al., 2008;Swatloski et al., 2002) but even whole biomass (Fortet al., 2007; Kilpelainen et al., 2007; Sun et al., 2009). Thesolvent properties of ILs can be tailored by combininganions and cations resulting in a staggering number ofpossible ILs (estimated at over 109), and hence, there issignificant flexibility to design ILs to suit a specific biomassand optimal process parameters.

We have developed an IL-mediated pretreatment processthat selectively extracts lignin from maple wood flour and

simultaneously disrupts the crystalline structure of cellulose(Lee et al., 2009). The efficacy of such a pretreatment can bepredicted by the thermodynamic Kamlet-Taft b parameter,which is closely related to the ability of the IL to serve as ahydrogen bond acceptor; ILs with b� 1.0 support highyields of fermentable sugars following pretreatment(Doherty et al., 2010). The cation and anion of a given ILare believed to play different roles during pretreatment.Generally, the cation interacts with lignin through hydrogenbonding and p–p interactions (Scheme 1D) (Janesko, 2011)while the anion acts as a hydrogen bond acceptor,interacting with the hydroxyl group of cellulose, thusdisrupting the crystalline structure of cellulose (Scheme 1B)(Fukaya et al., 2008; Remsing et al., 2006).

While there are many advantages for IL-mediatedlignocellulose pretreatment, ILs are currently far from beingused in large-scale due to their high cost. Many variables(e.g., IL chemistry, water content, feedstock, particle size,and pretreatment temperature and time) have been

Scheme 1. Interaction of ILs with cellulose and lignin. A: The semi-crystalline structure of cellulose is a result of the hydrogen-bonding network among the polysaccharide

chains. B: Incubation with ILs disrupts cellulose crystalline structure because of the IL’s ability to accept hydrogen bonds (blue spheres indicate the cation and green spheres

indicate the anion). C: Following pretreatment amorphous cellulose can be depolymerized by cellulases. D: Interaction between lignin and [Emim] cation by p–p interactions.

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investigated to optimize the downstream fermentable yieldof biomass (Binder and Raines, 2010; Doherty et al., 2010;Kilpelainen et al., 2007; Lee et al., 2009; Pezoa et al., 2010;Singh et al., 2009; Zavrel et al., 2009; Zhang et al., 2005; Zhu,2008). However, the common assumption has been that theIL must be used in vast excess to the biomass as a true non-aqueous solvent rather than a chemical additive thatsupports transformation of biomass structure. As a result,essentially no attention has been paid to the minimumamount of IL required for an efficient pretreatment. The aimof the current study was to assess the maximum biomassloading that can be achieved while retaining high pretreat-ment efficiency for a given amount of IL. In addition, thereusability of ILs is also critical for practical application, andthus was also investigated in this work. The remarkableincrease in biomass loading coupled with IL reuse improvesthe outlook of using ILs in cost-effective pretreatment oflignocellulosic biomass.

Materials and Methods

Chemicals and Reagents

1-Ethyl-3-methylimidizolium acetate ([Emim] [OAc]), 1-butyl-3-methylimidazolium acetate ([Bmim] [OAc]), 1-butyl-3-methylimidazolium methyl sulfate ([Bmim][MeSO4]), cellulase from Trichoderma reesei, b-glucosidase(Novozyme 188, 720 IU/mL), sodium citrate, citric acid,sulfuric acid, glucose, and xylose were purchased fromSigma–Aldrich (St. Louis, MO) and used without purifica-tion. Corn stover, maple wood flour (particle size 0.25mm),poplar, and switchgrass were graciously donated by P. J.Murphy Forest Products (Montvale, NJ). Corn stover,poplar, and switchgrass were ground with blender andscreened through sieves (Sieves No.: 4, 10, 18 and 35,Humboldt MFG Co., Norridge, IL) to obtain fractions of1.00–2.00mm for pretreatment. The measurements ofbiomass water content and cellulase activity were performedaccording to NREL standard procedures (Adney and Baker,2008; Ehrman, 1994). Cellulase activity in filter paper units(FPU) from T. reesei was 82 FPU/mL. The water content ofcorn stover, maple wood flour, poplar, and switchgrass wasmeasured to be 7.7%, 5.0%, 2.4%, and 6.3%, respectively.

Biomass Pretreatment

A specific amount of biomass was added into 5.0 g ILpreheated to 125� 58C in a round bottom flask. The flaskwas then placed in an oil bath and heated at 125� 58C for1 h. After heating, 20mL deionized water was added into thepretreated slurry and the undissolved solid fraction wasseparated from the IL by vacuum filtration. The recoveredsolid fraction was washed extensively with water to removeIL and dried at 1058C overnight for compositional analysis

and enzymatic depolymerization. All pretreatments wereconducted in duplicate.

Enzymatic Hydrolysis

Enzymatic hydrolysis reactions were carried out in a 20mLvial at 508C placed on a rotary shaker at 200 rpm. Thereaction mixtures contained 10mL of 50mM pH 4.8 citratebuffer, 100mg biomass based on dry weight, 4.9 FPUcellulase from T. reesei and 43.2 IU Novozyme 188. Aliquots(200mL) were removed at 0.5, 1, 1.5, 4, 10, and 24 h andplaced in a 958C block heater for 5min to quench theenzymatic reactions. Glucose and xylose were quantified byHPLC (Shimadzu model LC-Ai; Kyoto, Japan) equippedwith a refractive index (RI) detector and a HPX-87P column(300� 7.8mm2, Bio-Rad, Hercules, CA) operated at 858C.The mobile phase consisted of deionized water at a flow rateof 0.6mL/min. Glucose and xylose yields were calculated ateach time point as follows:

Sugar yieldi ¼Released sugarðtÞi

Sugar content of untreated biomassi(1)

where i represents either glucose or xylose. Initial hydrolysisrates were calculated from linear regression of slopes of theinitial 1.5 h of the reaction (Doherty et al., 2010).

Compositional Analysis of Biomass

The compositional analysis of biomass before and afterpretreatment was determined according to NREL/TP-510-42618 (Sluiter et al., 2008). The glucose and xylose producedwere analyzed by HPLC, as described above, except that thecolumn is HPX-87H column (300� 7.8mm2, Bio-Rad).The mobile phase consisted of 0.005M H2SO4 at a flow rateof 0.5mL/min. The acid-insoluble lignin after acidhydrolysis was measured as the mass of insoluble residueremaining at 5758C. The acid-soluble lignin was measuredby UV–Vis spectrophotometer (Shimadzu UV-2401) at320 nm with an extinction coefficient of 110 L/g cm.

Crystallinity Measurements

All treated and untreated biomass samples were analyzed bywide-angle X-ray scattering on a D8 Discovery diffractome-ter (Bruker, Madison, WI). All scans were in the range of88� 2u� 308 at a scan speed of 0.18/min and a step size of0.018. The cellulose crystallinity index (CrI) was determinedvia Equation (2):

CrI ¼P

ICrITot

� 100 (2)

where SIcr is the summation of the integrated intensity ofthe crystalline peaks and Itot is the integrated intensity of the

Wu et al.: High-Loading Biomass Pretreatment by Ionic Liquids 2867

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entire sample. The crystalline peaks correspond to (101)at 2u� 14.758, (101) at 2u� 16.58, (021) at 2u� 20.658, and(002) at 2u� 22.58. Curve fitting was used to separate theintensities of the four crystalline peaks from the amorphoussignal generated by specific biomass (Doherty et al., 2010).

FT-IR Spectroscopy

FT-IR spectroscopy was conducted using a Perkin-ElmerSpectrumOne Fourier transform infrared spectrometer. Thesolid samples were first mixed with KBr and ground to a finepowder in air by using a mortar and pestle, and then pressedinto pellets for IR transmission studies. Sample spectra wereobtained using an average 32 scans over the range between800 and 4,000 cm�1 with a spectral resolution of 2 cm�1.

Results and Discussion

ILs have been shown to dissolve both softwood andhardwood at loadings <5% (w/w) (Sun et al., 2009).However, the amount of IL required for effective reductionof biomass recalcitrance, thus improving subsequent ratesand yields of enzymatic hydrolysis, is a major concern.Higher loading of biomass, and thus lower volume of ILrequired, would be a significant step toward practicallignocellulosic pretreatment strategies, and thus is the focusof this work. Corn stover is the most abundant and availableagricultural waste source of lignocellulose in the US and,therefore, was used as the main feedstock in the currentstudy.

Pretreatment of Corn Stover With Different Loadingsand ILs

Corn stover (1–2mm size particles) was incubated in[Emim][OAc] at 125� 58C for 1 h at biomass loadings of4.8–50% (w/w) (Fig. 1). The initial rates of enzymatichydrolysis of cellulose and xylan following pretreatment andwashing away the IL decrease as corn stover loadingsincrease (Fig. 1A). Nevertheless, over a wide range ofbiomass loadings (4.8–33% w/w), the yields of glucose andxylose remain similar (�80% and �50%, respectively)(Fig. 1B). The low initial saccharification rates and sugaryields for the untreated corn stover and controls treated at125� 58C for 1 h without IL or 1218C for 1 h in deionizedwater indicate that enhanced enzymatic hydrolysis is a resultof the action of the IL. Simply heating the corn stover inwater is unable to effectively pretreat the biomass. It isnoteworthy that for loadings �23% w/w, [Emim][OAc] isonly able to wet the surface of corn stover during thepretreatment (Fig. 2), and hence at these loadings, the IL nolonger behaves as a true solvent.

The X-ray diffraction spectra (Fig. 1C) reveal substantialchanges to corn stover crystallinity (primarily reflectingchanges in component cellulose crystallinity). Untreatedcorn stover displays a strong peak at 2uU 22.58-,

corresponding to the (002) crystal plane of cellulose, aswell as a composite peak corresponding to the (101) and(101) crystal planes at 2u� 14.758 and 2u� 16.58,respectively. The intensity corresponding to the (002)plane decreases considerably at loadings �33% (w/w) ofcorn stover, and no distinct peak remains for the (101)and (101) planes. With loadings greater than 33% (w/w),a gradual restoration of peak intensities to the levels ofuntreated corn stover is observed. There is only marginalalteration in the intensity of all crystal planes for thecontrols. The high yields of enzymatic hydrolysis ofpretreated corn stover at loadings �33% (w/w) areconcomitant with a reduction on the CrI of the cellulose(Table I) and there is a strong correlation betweencellulose crystallinity and effectiveness of enzymatichydrolysis (Fig. 1D), consistent with our previous studies(Doherty et al., 2010; Lee et al., 2009).

Lignin is believed to be a major obstacle for theaccessibility of cellulose to enzymes (Zhu et al., 2008,2009). For an IL-mediated pretreatment process, IL appearsto first interact with lignin, which serves as a ‘‘glue’’ thatholds together the 3D structure of lignocellulose, and thendeconstructs the cellulose crystalline structure after somefraction of lignin is removed. Extracted lignin contentreaches >40% at loadings of �5% or less (Doherty et al.,2010; Lee et al., 2009; Li et al., 2010). At higher biomassloadings, however, it does not appear necessary to extract asmuch lignin from the biomass to achieve effectivedownstream enzymatic hydrolysis. Indeed, at 33% (w/w)corn stover loading, [Emim][OAc] removes only �15% ofthe lignin (Table I), yet results in comparable yields offermentable sugars (�80% glucose, �50% xylose) to thoseobtained at lower loadings (Fig. 1B and Figure S2 fromSupplemental information). At this high a corn stoverloading, the IL essentially forms a coating on the biomassparticles; this situation is far different from a typical solvent.For loadings above 33% (w/w), less lignin was extracted(Table I) and less disruption of cellulose crystallinity wasobtained, both of which contribute to the decrease inenzymatic hydrolysis efficiency. Another important aspectto consider is the potential inhibition of enzyme activity bylignin or potential lignin breakdown products (e.g., phenols,Scheme 1D). For example, vanillin, tannic acid, hydro-xybenzoic acids, and gallic acid are known to inhibitcellulases (Kim et al., 2011; Ximenes et al., 2010, 2011). Suchinhibition, however, is likely minimal because the polymericstructure of lignin remains intact (Pan, 2008).

The glucose and xylose yields of corn stover pretreatedwith [Bmim][MeSO4] at 1308C for 1 h were similar to thoseof untreated corn stover, and this was consistent with ourprevious work (Doherty et al., 2010). Despite the removal of�13% lignin during pretreatment, no significant decrease inthe CrI was observed and this can be attributed to the low b-value (0.60) of [Bmim][MeSO4]. These results are alsoconsistent with the recent report that increasing celluloseaccessibility is more important than removing lignin (Rollinet al., 2011). Conversely, [Bmim][OAc], which has a similar

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b parameter value to that of [Emim][OAc] (1.18 vs. 1.08)(Doherty et al., 2010), is an excellent IL for pretreatment ofcorn stover at high loading. Indeed, the initial reaction rates(Table II) and yields of enzymatic hydrolysis followingpretreatment at 33% (w/w) loading (Fig. 3A) werecomparable to those of pretreatment by [Emim][OAc].X-ray diffraction (Fig. 3B) indicates that the crystallinestructure of cellulose has been disrupted by [Bmim][OAc]leading to a decrease in the CrI compared with untreatedcorn stover (0.41 vs. 0.64). An increase in corn stoverloading to 41% (w/w) resulted in a clear decrease inpretreatment efficacy, again similar to that obtained with[Emim][OAc] (Table II).

Mechanism of IL-Facilitated Corn Stover Pretreatment

It is critical to address why ILs are capable of efficientlypretreating corn stover at such high loadings. A simpleapproach is to determine the ratio of moles of IL to moles ofglucose in cellulose. At 33% (w/w) loading the molar ratio of[Emim][OAc] to glucose in cellulose (6:1) is comparable to

the maximum number of hydrogen bonds (acceptor anddonor) for each glucose-repeating unit in cellulose(Scheme 1A and Table III). The similar stoichiometry ofthe hydroxyl protons of each glucose unit and the IL acetateions suggests that there are sufficient acetate anions contactsthe cellulose within the time period of incubating to formnew hydrogen bonds with each glucose moiety in cellulose,completely disrupting the crystalline structure of cellulose.When the concentration of IL drops below this stoichiome-try, i.e., when the loading is greater than 33% (w/w), the H-bond disruption is incomplete allowing cellulose’s structureto remain more intact, as evidenced by X-ray diffractionspectra (Fig. 1C). This hypothesis is further supported byFT-IR data (Fig. 4). The absorption from O–H stretchingvibration shifted from 3,397 cm�1 (untreated corn stover)to 3,422 cm�1 (at 50%, w/w, loading) and further to3,434 cm�1 (at 4.8% and 33%, w/w, loading), indicating theincrease of free hydroxyl group content due to the reductionof intermolecular hydrogen bonding (Bodirlau et al., 2010).The precise mechanism of the interactions between IL andcomplex lignocellulosic biomass subcomponents remains

Figure 1. Effect of loading on corn stover pretreatment. Pretreatment conditions: 125� 58C for 1 h in the [Emim][OAc]. Con1 indicates treating at 125� 58C for 1 h without IL;

Con2 indicates treating at 1218C for 1 h in the deionized water. Unt represents untreated corn stover. Enzymatic reaction conditions: 100 mg corn stover based on dry weight, 10 mL

of 50mM citrate buffer (pH 4.8) containing 4.9 FPU T. reesei cellulase and 43.2 IU Novozyme 188, 508C, 200 rpm, 24 h. A,B: Effect of loading on the initial reaction rate (A) and sugaryield (B). C: X-ray diffraction spectra. a: Untreated; b: Control 1; c: Control 2; d: 50%; e: 41%; f: 33%; g: 23%; h: 13%; i: 9.1%; j: 4.8% loading (w/w). D: Relationship between CrI and sugar

yield (See Supporting Information for SEM images of untreated and pretreated corn stover at different loadings). [Color figure can be seen in the online version of this article,

available at http://wileyonlinelibrary.com/bit]

Wu et al.: High-Loading Biomass Pretreatment by Ionic Liquids 2869

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largely unknown; hence, there is a need to achieve amolecular-level perspective to achieve a greater understand-ing of pretreatment at high loadings. Many interactionsbetween subcomponents apart from hydrogen bondsoccur in lignocellulose (e.g., hydrophobic interactionsbetween adjacent ring structures (carbon–carbon interac-tions). More detailed work in this are is now underway inour labs.

Influence of Feedstock on High-loading Pretreatment

Pretreatment performance varies with feedstock types due totheir distinct chemical and physical structural features, suchas composition of cellulose, hemicellulose, and lignin,accessible surface area, crystallinity, and degree of polymer-ization, etc. (Kumar et al., 2009; Zhu et al., 2008).Switchgrass is a leading candidate for biofuel production

Figure 2. Photographs of the pretreatment process. A,B: 4.8% (w/w) corn stover at the beginning of pretreatment (A) and after pretreatment (B). C,D: 33% (w/w) corn stover at

the beginning of pretreatment (C) and after pretreatment (D). [Color figure can be seen in the online version of this article, available at http://wileyonlinelibrary.com/bit]

Table I. Effect of IL pretreatment loading on composition of corn stover.

Loading (%, w/w)

IL pretreatmenta Composition of IL-pretreated corn stoverb

Recovered corn stover (mg)c Extracted lignin (mg)d CrI Cellulose (mg) Xylan (mg) Lignin (mg)

Untreated 250 (100%) 0 (0%) 0.64 81.5 (35.3%) 55.8 (24.2%) 39.9 (17.3%)

Con1e 233 (93.2%) 2.1 (5.2%) 0.58 80.2 (37.3%) 54.4 (25.3%) 37.9 (17.6%)

Con2f 223 (89.1%) 0.9 (2.1%) 0.56 71.8 (34.9%) 51.4 (25.0%) 39.1 (19.0%)

4.8 184 (73.5%) 17.4 (43.5%) 0.31 70.4 (41.5%) 37.1 (21.9%) 22.6 (13.3%)

9.1 365 (73.0%) 26.9 (33.7%) 0.32 142 (42.2%) 73.8 (21.9%) 52.9 (15.7%)

13 557 (74.3%) 30.2 (25.2%) 0.34 216 (42.0%) 118 (22.9%) 89.5 (17.4%)

23 1,151 (76.7%) 46.1 (19.3%) 0.38 441 (41.5%) 268 (25.2%) 193 (18.2%)

33 2,018 (80.7%) 60.1 (15.1%) 0.38 756 (40.6%) 482 (25.9%) 339 (18.2%)

41 2,870 (82.0%) 71.2 (12.7%) 0.48 1,028 (38.8%) 660 (24.9%) 487 (18.4%)

50 4,265 (85.3%) 65.8 (8.2%) 0.52 1,472 (37.4%) 941 (23.9%) 732 (18.6%)

aA specific amount of corn stover was incubated in 5.0 g [Emim][OAc] at 125� 58C for 1 h.bValues in parentheses indicate composition percentages.cValues in parentheses represent percent of corn stover recovered relative to the corresponding untreated corn stover. Note that the initial amount of corn

stover increases as the loading increases.dValues in parentheses represent extraction efficiency.ePretreated at 125� 58C for 1 h without IL.fPretreated with deionized water at 1218C for 1 h at 4.8% (w/w) loading.

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as an energy crop (Sarath et al., 2008), maple represents aparticular recalcitrant form of lignocellulose (hardwood),while poplar is a typical feedstock whose recalcitrance isintermediate between hardwood and softwood. To assesswhether high biomass loading in IL could be extended tofeedstocks other than corn stover, we subjected switchgrass,poplar, and maple to pretreatment at loadings of 33% (w/w)at 125� 58C for 1 h in [Emim][OAc]. As shown inFigure 5A, high sugar yields of glucose and xylose wereachieved, even with recalcitrant maple wood flour whoseyields of glucose and xylose increased from 8% and 3%,respectively, before pretreatment to 72% and 54%,respectively, after pretreatment. The pretreatment outcomevaries somewhat with the feedstock, presumably as a resultof differences in the composition, crystallinity, and particlesize (Table IV). While the extracted lignin content varieswith the feedstock, ranging from 10 to 19%, an apparentdecline in the CrI was observed (Table IV) as evidenced bythe X-ray diffraction spectra (Fig. 5B).

Recycle of [Emim][OAc] for Corn Stover Pretreatment

In addition to improving biomass loading, IL reuse alsorepresents an efficient way to decrease the cost of IL-mediated pretreatment. Regeneration of ILs may require aset of steps including precipitation by antisolvents such asdeionized water or alcohols followed by evaporation andadsorption of impurities (Revelli et al., 2010) that impactenergy consumption. Recently, a novel strategy to recycle ILshas been developed by Blanch and coworkers (Shill et al.,2010) wherein acetate-based ILs via a three-phase systemformed (a salt-rich aqueous phase, a solid phase rich incellulose, and an IL-rich phase that contains most of thelignin) by adding concentrated solutions of K3PO4

and K2HPO4 after complete dissolution of miscanthus.

Table II. Effect of IL nature on composition and rate of enzymatic hydrolysis of corn stovera.

ILb

IL pretreatment

Composition of IL-pretreated

corn stover

Initial reaction

rate (mg/mL/h)

Recovered corn

stover (mg)

Extracted

lignin (mg) CrI

Cellulose

(mg)

Xylan

(mg)

Lignin

(mg) Glucose Xylose

[Bmim][OAc] (33%) 2,040 (81.6%) 58.2 (14.6%) 0.41 726.8 (38.6%) 474.5 (25.2%) 340.8 (18.1%) 0.91� 0.03 0.48� 0.01

[Bmim][OAc] (41%) 2,947 (84.2%) 63.5 (11.4%) 0.50 1017.3 (37.4%) 636.5 (23.4%) 495.1 (18.2%) 0.86� 0.05 0.44� 0.02

[Bmim][MeSO4] (4.8%) 236.5 (94.6%) 5.0 (12.5%) 0.55 78.6 (36.0%) 52.8 (24.2%) 34.9 (16.0%) 0.20� 0.02 0.09� 0.01

aPretreatment and enzymatic hydrolysis conditions are similar to Figure 1 except for the IL used in the specific pretreatment. The meaning of values inparentheses is identical to Table I.

bValues in parentheses represent corn stover loading (% w/w).

Figure 3. Effect of IL on the pretreatment. The pretreatment and enzymatic

hydrolysis conditions are similar to Figure 1 except for the IL used in the specific

pretreatment. A: Effect of IL on sugar yield. B: X-ray diffraction spectra. a: [Bmim][OAc]

41% (w/w) loading; b. [Bmim][OAc] 33% (w/w) loading, c. [Bmim][MeSO4] 4.8% (w/w)

loading. [Color figure can be seen in the online version of this article, available at http://

wileyonlinelibrary.com/bit]

Table III. Molar ratio of [Emim][OAc] to glucose in cellulose for

different loading corn stover.

Loading (wt%) 4.8 9.1 13 23 33 41 50

Molar ratio of IL to glucose

in cellulose

53 26 18 8.8 5.3 3.8 2.6

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The main advantage of this process is that decreases theamount of water in the RTIL that must be evaporated, whichwould reduce energy costs. In our particular case, the IL-richphase consists of lignin, which is obtained in a single step.However, it remains critical to determine the number oftimes that an IL can be recycled using our approach.

To that end, [Emim][OAc] (20 g) was first used topretreat corn stover (4.8% w/w), and then washed withwater to remove the traces of dissolved cellulose andhemicellulose. The water was removed by evaporation undervacuum from the mixture of water and [Emim][OAc].Without further purification, the [Emim][OAc] solutionwas then reused to pretreat another batch of corn stoverresulting in the accumulation of lignin (Lee et al., 2009). Theextracted lignin content decreased from 44% in the firstbatch to 20% in the 10th batch; however, the ability of[Emim][OAc] to disrupt crystallinity of cellulose withincorn stover remained largely unaffected as evidenced bythe X-ray diffraction spectra (Fig. 6A) and further confirmedby subsequent CrI calculation (Fig. 6B). Previously, wehave shown that the efficacy of the IL-mediated pretreat-ment can be predicted through the b Kamlet-Taft parameter(Doherty et al., 2010), and more important, that bparameter is strongly affected by the nature of the anion.Then, is likely possible that b parameter of ILs remain

unaffected after each recycle beside the accumulation oflignin. Since the interaction between IL and lignin occursmainly through the cation, the anions are still available todisrupt hydrogen bonds from the semi-crystalline cellulose.There is also no significant loss in the sugar yields of cornstover pretreated with this recycled [Emim][OAc] after 10batches (Fig. 6C). The resulting solution of highlyconcentrated unmodified lignin (�24mg/mL) may be ofvalue in preparing lignin-based products (Binder et al., 2009;Kleinert and Barth, 2008; Matsushita and Yasuda, 2005;Yuan et al., 2010).

Figure 4. FT-IR spectra of corn stover before and after pretreatment.

[Color figure can be seen in the online version of this article, available at http://

wileyonlinelibrary.com/bit]

Figure 5. Effect of feedstock on the pretreatment. The pretreatment and enzy-

matic hydrolysis conditions are similar to Figure 1 except for the feedstock used in the

specific pretreatment. A: Effect of feedstock on the sugar yield. B: X-ray diffraction

spectra. a: Untreated maple wood flour; b: 33% (w/w) loading maple wood flour; c:

Untreated poplar; 33% (w/w) loading poplar; e: Untreated switchgrass; f: 33% (w/w)

loading switchgrass. [Color figure can be seen in the online version of this article,

available at http://wileyonlinelibrary.com/bit]

2872 Biotechnology and Bioengineering, Vol. 108, No. 12, December, 2011

In summary, we have demonstrated the feasibility of usingILs to efficiently pretreat lignocellulosic biomass at highloadings. It is not necessary to remove a large fraction of thelignin; instead the disruption of crystalline structure ofcellulose is critical in improving cellulose accessibility toenzymes for an effective pretreatment. [Emim][OAc] can bereused for at least 10 batch treatments without substantialloss in the effectiveness of subsequent enzymatic hydrolysis.Significant improvement of feedstock loading plus reuse

of IL strategies should contribute to the attractivenessof IL-mediated pretreatment in large-scale applications.Moreover, the high lignin concentration in the batch recyclesolution following multiple recycles may result in a facileroute to lignin isolation and further use in the generation oflignochemcials. It is envisioned that the potential of ILs forhigh loading, high-efficiency, pretreatment of lignocellulosicbiomass may stimulate the design and synthesis of new ILsfor their applications in this area.

Figure 6. Reuse of [Emim][OAc] to pretreat corn stover. A: X-ray diffraction spectra of corn stover after pretreatment. Curves a–j represents batches 1–10. B: CrI of residual

corn stover solids following pretreatment in each batch. C: Sugar yields of enzymatic hydrolysis of residual corn stover solids following pretreatment in each batch. [Color figure can

be seen in the online version of this article, available at http://wileyonlinelibrary.com/bit]

Table IV. Composition of different lignocellulosic biomass before and after pretreatmenta.

Biomassb

IL pretreatment Composition of IL-pretreated corn stover

Recovered corn stover (mg) Extracted lignin (mg) CrI Cellulose (mg) Xylan (mg) Lignin (mg)

Untreated maple wood flour 2,500 (100%) 0 (0%) 0.64 976.1 (41.1%) 465.5 (19.6%) 586.6 (24.7%)

Treated maple wood flour (33.3%) 2,070 (82.8%) 79.2 (13.5%) 0.37 835.8 (42.5%) 355.9 (18.1%) 507.4 (25.8%)

Untreated poplar 2,500 (100%) 0 (0%) 0.61 1,005 (41.2%) 478.2 (19.6%) 675.9 (27.7%)

Treated poplar (33.3%) 2,185 (87.4%) 68.1 (10.1%) 0.33 906.3 (42.5%) 400.9 (18.8%) 607.8 (28.5%)

Untreated switchgrass 2,500 (100%) 0 (0%) 0.74 775.4 (33.1%) 538.8 (23.0%) 520.0 (22.2%)

Treated switchgrass (33.3%) 2,098 (83.9%) 97.4 (18.7%) 0.43 719.3 (36.6%) 456.0 (23.2%) 422.6 (21.5%)

aPretreatment conditions are similar to Figure 1 except for the feedstock used in the specific pretreatment. Themeaning of values in parentheses is identicalto Table I.

bValues in parentheses represent biomass loading (% w/w).

Wu et al.: High-Loading Biomass Pretreatment by Ionic Liquids 2873

Biotechnology and Bioengineering

This work was supported by the National Natural Science Foundation

of China (Grant No. 31071559) and Chisso Corporation. We are

grateful to Prof. Daniel Lewis for providing access to the muffle

furnace.

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