Comparing the Recalcitrance of Eucalyptus, Pine,and Switchgrass Using Ionic Liquid and DiluteAcid Pretreatments

Chenlin Li & Lan Sun & Blake A. Simmons & Seema Singh

Published online: 27 May 2012# Springer Science+Business Media, LLC 2012

Abstract Pine, eucalyptus, and switchgrass were evalu-ated for the production of fermentable sugars via ionicliquid and dilute acid pretreatments and subsequent en-zymatic hydrolysis. The results show that among thethree feedstocks, switchgrass has the highest sugaryields and faster hydrolysis rates for both pretreatmenttechnologies by achieving 48 % (dilute acid) and 96 %(ionic liquid) sugar yields after 24 h. Of the two woodspecies, eucalyptus has a higher and faster sugarrecovery after ionic liquid pretreatment than pine(93 vs. 62 % in 24 h) under 160 °C for 3 h with[C2mim][OAc]. Pretreatment of pine and eucalyptus isobserved to be ineffective under 1.2 % dilute acidcondition and 160 °C for 15 min, indicating that furtherenhancement of reaction temperature or acid concentra-tion is necessary to increase the digestibility of pre-treated materials. Raman spectroscopy data show thatthe extent of lignin depolymerization that occurs duringpretreatment also varies for the three different feed-stocks. Under similar hemicellulose removal conditions,lignin removal in ionic liquid pretreatment can helpimprove cellulose conversion. This finding may helpexplain the observed variation in the saccharificationyields and kinetics. These results indicate that ionicliquid pretreatment not only improved saccharificationover dilute acid for all three feedstocks but also better

dealt with the differences among them, suggesting bettertolerance to feedstock variability.

Keywords Pine . Eucalyptus . Switchgrass . Ionic liquid .

Dilute acid . Enzymatic saccharification

Introduction

Among the various proposed biofuel feedstocks, ligno-cellulosic biomass, such as agricultural and forestryresidues, as well as dedicated energy crops grown onlands not suitable for food production, has been recog-nized as the most significant renewable resourceavailable worldwide that can be utilized for sustainablebiofuel production at potenially competitive prices [1,2]. Some of the leading energy crop candidates includeherbaceous species from agriculture resources, such asswitchgrass, and woody materials from forestry, such aspine and eucalyptus. As a dedicated perennial feedstock,switchgrass can be grown in various regions of theUSA with significant positive net energy yields [3]. Asthe woody biomass with other uses as well, pine andeucalyptus have been recently brought forward as po-tential bioenergy crops [2, 4]. Pine is one of the strongestshort-rotation woody feedstock candidates and has beendemonstrated in temperate-region plantations worldwide[5]. Eucalyptus, native to Australia but grown throughoutthe world, has also been grown and studied extensively inCalifornia and Florida to be targeted for energy productionand appears to be amenable to high-density cultivation inplantation farms [6] and is another strong candidate forbiofuel production [7–9].

Technology is rapidly advancing to utilizing crop bio-mass, perennial grasses, woody perennials, and forest

C. Li : L. Sun : B. A. Simmons : S. Singh (*)Deconstruction Division, Joint BioEnergy Institute,Emeryville, CA, USAe-mail: ssingh@lbl.gov

C. Li : L. Sun : B. A. Simmons : S. SinghBiomass Science and Conversion Technology Department,Sandia National Laboratories,Livermore, CA, USA

Bioenerg. Res. (2013) 6:14–23DOI 10.1007/s12155-012-9220-4

products for the production of biofules via a cellulosicplatform [4]. However, to meet the biofuel production tar-gets, a robust pretreatment technique must be developed andestablished that can handle a wide range of feedstocks withminimal impact on performance as various feedstocks mayperform differently as a function of pretreatment [10]. Dilutesulfuric acid pretreatment has been extensively studied be-cause it is generally inexpensive, convenient, and effectivefor lignocellulosic biomass [10, 11]. There have been manystudies of the pretreatment of lignocellulosic biomass withdilute acid to enhance the enzymatic digestibility for herba-ceous feedstocks such as switchgrass [12], Bermuda grass[13, 14], corn stover [15, 16], and wheat straw [17, 18].However, limited reports of the dilute acid pretreatment areavailable on woody biomass, such as hardwood, eucalyptusand aspen [8, 19–21], and softwood, such as pine [22, 23].

Recently, ionic liquid pretreatment has been shown to re-duce cellulose crystallinity and lignin content and to increasebiomass surface area [24–29]. Several studies demonstratedthat 1-ethyl-3-methylimidazolium acetate ([C2mim][OAc])can effectively pretreat switchgrass and corn stover by extract-ing a significant amount of lignin and generates a product thatis efficiently digestible [24, 28, 29]. Other recent reports havealso shown both complete dissolution and partial delignifica-tion of sugarcane, softwood, and hardwood in various ionicliquids under different process conditions [26, 27, 30].

Various pretreatments may perform differently when appliedto feedstocks due to the difference in their nature characteristics.One of the biggest challenges is the “fine tuning” of pretreat-ment for each type of feedstock, which limits the operationalwindow to handle mixed feedstocks at a biorefinery. We haveconducted a side-by-side comparison of ionic liquid and diluteacid pretreatments on switchgrass [24]. To fully and fairlycompare the effectiveness of a set of pretreatment techniqueson a range of targeted feedstocks, it is necessary to use the samesource of biomass, identical enzyme cocktails and loadinglevels, and identical analytical methods [31, 32]. In recognitionof these requirements and also as a follow-up to our earlierwork, we selected representative feedstocks for grasses, soft-woods, and hardwoods as an exploratory first step for thisapproach. Eucalyptus globuluswas selected to represent Euca-lyptus species grown worldwide, Pinus radiatawas selected asit is one of the largest pine species in the world and is alsonative to the Central Coast of California, andPanicum virgatumwas selected as it is one of the most promising herbaceousspecies. We evaluated their potential for fermentable sugarproduction through a direct comparison of ionic liquid anddilute acid pretreatments. The objectives of this paper were:(1) to conduct a robust comparison between dilute acid andionic liquid pretreatments by evaluating their performance onthe same feedstock source and (2) to study the response of thethree feedstocks to the two different pretreatment techniquesusing an identical basis of comparison.

Materials and Methods

Raw Materials

Pine (P. radiata) and eucalyptus (E. globulus) were kindlysupplied by ArborGen (Sumerville, SC, USA). Switchgrass(P. virgatum L.) was kindly supplied by Dr. Ken Vogel at theUnited States Department of Agriculture, Lincoln, NE. Allsamples were milled with a Thomas-Wiley® Mini Mill fittedwith a 40-mesh screen (model 3383-L10, Arthur H. ThomasCo., Philadelphia, PA, USA) and air-dried until the moisturewas <10 %. The samples were stored at 4 °C in a sealedplastic bag for use in all experimentation.

Cellulase (NS50013) and β-glucosidase (NS50010) weregenerously provided by Novozymes (Davis, CA). Ionicliquid, [C2mim][OAc], acetic acid, sodium acetate, sulfuricacid, hydroxylamine hydrochloride, sodium hydroxide, andthe monosaccharides including fucose, arabinose, rhamnose,galactose, mannose, xylose, glucose, glucuronic acid, andgalacturonic acid were from Sigma-Aldrich (St. Louis, MO).Acetyl bromide was from Alfa-Aesar (Ward Hill, MA).

Biomass Pretreatment and Product Recovery

Ionic Liquid Pretreatment

A 3 % (w/w) biomass solution was prepared by combining300 mg of a sample with 9.7 g [C2mim][OAc] in a 50-mLautoclave vial. The vials and the contents were heated andstirred in an oil bath at 160 °C for 3 h. All experiments wereconducted in triplicate. After a 3-h incubation, 30 mL ofdeionized water was slowly added into the stirred biomass/[C2mim][OAc] slurry for regeneration of the solubilized bio-mass. A precipitate immediately formed, and the sample wascentrifuged at 10,000×g for 10 min. The supernatant contain-ing ionic liquid was removed and the precipitate washed fourtimes with additions of water in order to ensure that excessionic liquid had been removed. The presence of ionic liquidwas tracked by measuring the absorbance (Shimadzu UV-2401) of the recovered liquid in the wavelength region from250 to 300 nm.

Dilute Acid Pretreatment

The biomass samples were presoaked in pressurized glasstubes at room temperature in 1.2 % (w/w) sulfuric acidsolution at 3 % (w/w) total solid loading for at least 4 h.The glass tubes were then heated in an oil bath at 160 °C for20 min with three replicates. The acidic slurry was filteredthrough Whatman® filter paper with a Buchner funnel. Thefiltrates were stored at 4 °C prior to sugar analysis. Therecovered solids were washed with deionized water until thepH of the washed water reached 6.0 [33]. The buffer

Bioenerg. Res. (2013) 6:14–23 15

solution used in the enzymatic saccharification (see below)was used for the final washes.

For both pretreatment processes, the recovered productwas lyophilized at −50 °C for 48 h before compositionalanalysis and saccharification.

Chemical Characterization of Three Biomass Samples

The structural carbohydrates of three feedstocks before andafter pretreatment, including glucan, xylan, arabinan, andmannan, were determined according to analytical procedureof the National Renewable Energy Laboratory using a two-step sulfuric acid hydrolysis [34, 35]. Carbohydrates wereanalyzed by high-pressure anion exchange chromatography(HPAEC) on an ICS-3000 system (Dionex, Sunnyvale, CA)equipped with an electrochemical detector and a 4×250-mmCarboPac PA20 analytical column. Elution was initiated with97.2 % (v/v) water and 2.8 % (v/v) 1 M NaOH for first 15 min,with a 20-μL injection volume. Elute concentration was thenswitched to 55.0% (v/v) water and 45.0% (v/v) 1MNaOH forthe next 20 min and returned to 97.2 % (v/v) water and 2.8 %(v/v) 1 M NaOH for the last 10 min to equilibrate the column.The flow rate was 0.5 mL/min. The monosaccharides includ-ing fucose, arabinose, rhamnose, galactose, mannose, xylose,glucose, glucuronic acid, and galacturonic acid were used asthe external standards for HPAEC and prepared at levels of0–5 mM before use. The monomeric sugar contents in thedilute acid and ionic liquid pretreatment liquors were analyzedusing the same procedure.

The lignin content of both untreated and treated sampleswas determined with a modified acetyl bromide method [36,37]. Biomass powder (5 mg) was extracted with ethanol andthen treated with 25 % (w/w) acetyl bromide in glacial aceticacid (0.2 mL). The tubes were sealed and incubated at 50 °Cfor 2 h at 1,000 rpm on an Eppendorf Thermomixer(Thermo Fisher Scientific Inc., Pittsburgh, PA). After diges-tion, the solutions were diluted with three volumes of aceticacid, and then 0.1 mL was transferred to 15-mL centrifugetubes with addition of another 0.5 mL acetic acid. Thesolutions were mixed well, and then 0.3 M sodium hydrox-ide (0.3 mL) and 0.5 M hydroxylamine hydrochloride(0.1 mL) were added. The final volume was made up to2 mL with the addition of acetic acid. The UV spectra of thesolutions were measured against a blank prepared using thesame method. The lignin content was determined from theabsorbance at 280 nm [38].

Enzymatic Saccharification

Batch enzymatic saccharification of the pretreated anduntreated biomass samples was carried out at 50 °C and150 rpm in a reciprocating shaker. All samples were dilutedto 5 g glucan per liter in a 50 mM sodium acetate buffer

(pH 4.8) supplemented with 0.08 g/L tetracycline solutionfor enzymatic hydrolysis. The total batch volume was 5 mLwith cellulase (NS50013, 70 FPU/g, commercial proteinvalue provided by Novozymes) and β-glucosidase(NS50010, 250 CBU/g, commercial protein value providedby Novozymes) concentrations of 50 and 5 mg protein pergram glucan (3.5 FPU/g glucan and 1.25 CBU/g glucan),respectively. The reaction was monitored by taking 50 μLsupernatant at specific time intervals, followed by centrifu-gation at 10,000×g for 5 min, and measuring the release ofmonomeric sugars by HPAEC. Three untreated biomasscontrols were run concurrently with all recovered samplesto eliminate potential differences in temperature history orenzyme loading [25]. The initial rates of enzymatichydrolysis are calculated based on the glucose released inthe first 30 min of hydrolysis [25]. All assays were per-formed in triplicate. Error bars show the standard deviationof triplicate measurements. The enzymatic digestibility,defined as the percentage of glucan in the pretreated bio-mass converted to glucose, and the enzymatic glucose yieldin grams per 100 g untreated biomass were determined.Total sugar recoveries, defined as the percentage of mono-meric sugars (glucose, mannose, xylose) recovered frompretreatment liquor and glucose from the enzymatic hydro-lysate on the basis of original glucan, xylan, and mannan inthe untreated biomass, were also used to compare therecalcitrance of three feedstocks.

Raman Spectroscopy

Raman spectra were measured using a LabRam HR 800(Horiba Jobin Yvon, Edison, NJ). The spectrometer isequipped with a confocal microscope and a CCD detector.The samples were photo-bleached for 10 min to eliminatethe effect from lignin autofluorescence and then excitedwith a 785-nm diode laser. A confocal pinhole diameter of200 μm and an exposure time of 60 s were used for allexperiments. All samples were scanned in triplicate. Thespectra collected were baseline-corrected and smoothedusing LabSpec software in the spectral ranges of 1,000–1,800 cm−1. OMNIC software was used to deconvolutespectra in the range of 1,220–1,700 cm−1 to analyze changesin lignin composition [39].

Results and Discussion

Chemical Composition and Solid RecoveryBefore and After Pretreatment

Previous studies have found that [C2mim][OAc] is aneffective solvent to solubilize the plant cell wall, regeneratecellulose, but reject lignin upon anti-solvent addition. The

16 Bioenerg. Res. (2013) 6:14–23

optimal conditions for switchgrass have been identified at160 °C for 3 h [40]. Although the acid pretreatment conditionsused in this study are known to produce highly enzymaticdigestible biomass from corn stover [32, 41], they have notbeen specifically optimized for the three feedstocks, i.e.,switchgrass, pine, and eucalyptus. It should be addressed thatone of the biggest challenges to realize a commercially viableprocess is the tuning of pretreatment conditions for each typeof feedstock, which actually limits the practical operationalwindow for the mixed feedstocks at biorefinery. Thus, insteadof optimizing each pretreatment type for each biomass type,this study investigated the impact of known pretreatmentconditions on all three feedstocks.

Table 1 demonstrates that the cell wall chemical compo-sitions of three feedstocks were similar in glucan content(38.2–41.7 %), but very different in xylan and lignin con-tents. The switchgrass sample has the lowest lignin content(22.1 %), but the highest xylan content (20.3 %). Eucalyptushas the highest lignin content (30.2 %) and lower xylancontent (14.3 %), whereas pine wood has similar lignincontent (29.9 %), but the lowest xylan content (12.2 %).Despite the inherent differences between the polysaccharidelinkages present in grasses and woody biomass samples,arabinan, galactan, and/or mannan, which were calculatedbased on the arabinose, galactose, and mannose contents,accounted for only a small portion of the biomass composi-tion. Mannan was not detected in switchgrass, but wasdetected in pine (5.1 %) and eucalyptus (2.6 %) [10]. Thedata indicate that switchgrass contained arabinan, galactan,and xylan, while softwood and hardwood have the addedpresence of mannan in the hemicellulose composition [10].

For three feedstocks, ionic liquid and dilute acid pretreat-ments generated similar trends for carbohydrate content afterpretreatment, but exhibited distinct differences in lignincontent. Compared with the starting materials, the diluteacid-pretreated pine, eucalyptus, and switchgrass had de-creased xylan contents (i.e., 3.7–5.6 %) and enriched glucancontents (44.7–50.4 %), which are similar to ionic liquid-pretreated samples (4.6–7.6 % of xylan and 49.5–67.6 % ofglucan). However, all three ionic liquid-pretreated feedstockshad lower levels of residual lignin (13.2–24.2 %), significant-ly different from those pretreated with dilute acid that haveincreased relative lignin contents (28.3–38.6 %). The ionicliquid-pretreated switchgrass had the highest glucan content(67.6 %) and lowest lignin content (13.2 %), followed byeucalyptus (51.6 % of glucan and 21.9 % of lignin) and pine(49.5 % of glucan and 24.2 % of lignin).

Table 1 also shows that the solid recoveries of pine (80.2 and62.8 %, respectively) and eucalyptus (70.7 and 59.9 %) underboth pretreatments are significantly higher than switchgrass(59.3 and 49.3 %), suggesting the easier dissolution of grassbiomass than the woody samples. The weight loss mainly camefrom the solubilization of components in each feedstock such T

able

1Chemical

compo

sitio

n,solid

recovery,andcompo

nent

remov

alof

threefeedstocks

from

dilute

acid

andionicliq

uidpretreatments

Feedstocks

Pretreatm

ent

metho

dsSolid

recovery

a(%

)Glucan(%

)Xylan

(%)

Arabinan(%

)Galactan(%

)Mannan(%

)Lignin(%

)

Pine

Untreated

–38

.2±1.7

12.2±0.5

2.3±0.1

2.4±0.1

5.1±0.4

29.9±0.6

Dilu

teacid

80.2±2.5

44.7±1.2(6.2±0.4)

3.7±0.4(75.8±3.1)

0.2±0.0(92.9±4.7)

0.4±0.0(86.3±5.7)

2.1±0.2(66.8±2.4)

34.4±0.4(8.0±0.5)

Ionicliq

uid

62.8±0.6

49.5±1.3(18.4±0.7)

4.7±0.4(75.6±1.3)

0.8±0.1(78.1±3.8)

1.1±0.1(71.2±2.3)

0.8±0.1(89.7±5.4)

24.2

±0.5

(49.1±0.5)

Eucalyp

tus

Untreated

–41

.7±0.8

14.3±0.6

2.0±0.2

3.2±0.2

2.6±0.2

30.2±1.1

Dilu

teacid

70.7±1.3

49.8±1.1(15.5±0.3)

5.6±0.9(72.6±0.8)

0.3±0

.0(89.1±2.4)

0.4±0.0(90.7±5.7)

1.8±0.2(51.0±3.5)

38.6±0.9(9.7±1.3)

Ionicliq

uid

59.9±0.7

51.6±1.5(22.9±0.6)

4.6±0.9(79.8±2.5)

0.9±0.1(73.1±2.9)

2.0±0.1(62.5±3.1)

0.5±0.0(88.4±4.7)

21.9±0.8(54.9±1.1)

Switchg

rass

Untreated

–39

.5±1.8

20.3±1.4

2.1±0.1

2.6±0.1

ND

22.1±0.3

Dilu

teacid

59.3±0.2

50.4±1.6(24.3±0.4)

4.5±1.8(86.8±0.9)

1.1±0.0(68.8±2.5)

2.4±0.1(45.2±3.1)

ND

28.3±0.7(24.1±0.9)

Ionicliq

uid

49.3±0.2

67.6±0.4(15.6±0.5)

7.6±0.3(81.5±2.3)

2.1±0.1(50.7±2.8)

2.1±0.1(60.2±3.5)

ND

13.2±0.8(70.6±0.8)

Valuesin

parenthesesrepresenttheremov

alof

each

compo

nent

afterpretreatmenton

thebasisof

itsoriginal

amou

ntin

theraw

biom

ass

ND

notdetected

aValuesarecalculated

from

theweigh

tof

raw

biom

ass

Bioenerg. Res. (2013) 6:14–23 17

as lignin, xylan, glucan, and other soluble extractives. The lossof glucan fraction was <24 % in all three feedstocks, but theremoval of xylan and lignin was significantly different. Calcu-lations based on the solid weight recovery show that pretreat-ment with dilute acid effectively hydrolyzed 72.6–86.8 % ofxylan in the form of soluble sugars, but removed only8.0–24.1 % of lignin for all three biomass types, with thehighest xylan and lignin extracted from switchgrass. In contrast,ionic liquid pretreatment removed comparable amounts of xy-lan, but resulted in higher lignin extraction levels. Among thethree feedstocks, switchgrass showed the highest xylan andlignin removal under dilute acid pretreatment, whereas ionicliquid pretreatment was, in general, effective on all three feed-stocks studied, but similarly with the highest xylan (81.5 %)and lignin (70.6 %) removal rates observed for switchgrass.Pine had a relatively higher resistance to both pretreatments interms of lignin removal.

Biomass Lignin Characterization by Raman Spectroscopy

Lignin is formed through free radical polymerization of thedifferently substituted phenolic monolignols (confieryl alco-hol, sinapyl alcohol, and p-coumaryl alcohol) [42]. Thesemonolignols contribute to guaiacyl, syringyl, and p-hydrox-yphenyl propane units in lignin, respectively. Softwood ligninmainly consists of guaiacyl, whereas hardwood lignin is com-posed of both guaiacyl and syringyl units; switchgrass ligninconsists of all three units [42]. Raman spectroscopy methodshave been used to characterize the chemical changes in thevarious biomass samples by monitoring the identical finger-printing peaks for lignin and carbohydrates and are especiallywell suited for a rapid and non-deconstructive qualitative andquantitative lignin characterization with a possible classifica-tion of various lignin structures [24, 43].

From the Raman spectra in this study (Fig. 1), the primarylignin feature is present in the 1,500- to 1,700-cm−1 region foruntreated biomasses, referring to lignin aromatic ring stretchmode (1,600 cm−1) [24]. Raman data of ionic liquid-regenerated pine, eucalyptus, and switchgrass clearly showsignificant breakage/modification of the aromatic ring. In thecase of the dilute acid pretreatment, only a slight decrease ofthe lignin bands was observed when compared to the ionicliquid-pretreated feedstocks.

Researchers have shown great interest to understand thecorrelation between cell wall recalcitrance and syringyl/guaiacyl ratio in the literature [24, 43]. The p-hydroxy-phenyl data are less reported mainly due to its low amountin many species (softwood and hardwood) and differenttechnical problems [24, 43]. This study is focused on usingwell-accepted parameters to characterize cell wall recalci-trance of different feedstocks under various pretreatmentconditions. Therefore, only syringyl and guaiacyl changesare presented in this paper, although H changes can be

determined by a similar Raman approach. As shown inFig. 1, two peaks, corresponding to syringyl lignin(~1,331 cm−1) and guaiacyl lignin (~1,270 cm−1), respec-tively [43], were selected to monitor the compositionalchanges in lignin using spectral deconvolution over therange 1,220–1,530 cm−1 [39]. Figure 2 illustrates thechanges of intensity from syringyl and guaiacyl peaks asthe function of pretreatment techniques in three feedstocks.For the untreated materials, in switchgrass, both syringyland guaiacyl bands have strong intensities, indicating highamounts of both units [44]. As evidenced by the strongintensity, the guaiacyl unit is the dominant lignin in pine,which is known to contain none or a very limited amount ofsyringyl units [45]. In comparison, the syringyl band ismuch stronger than the guaiacyl band for eucalyptus, whichis known to have a very high syringyl-to-guaiacyl ratio [46].It should be noted that the weak syringyl band of pine andthe partial intensity of the weak guaiacyl band of eucalyptusare nonspecific background signals generated during thespectral deconvolution process. Thus, intensity changes inthe syringyl band of pine and the guaiacyl band of

0

4000

8000

12000

16000

1000 1100 1200 1300 1400 1500 1600 1700 1800

Ionic liquidDilute acidUntreated

0

10000

20000

30000

40000

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Fig. 1 Raman spectra of pine (a), eucalyptus (b), and switchgrass (c)under various pretreatment conditions

18 Bioenerg. Res. (2013) 6:14–23

eucalyptus do not necessarily reflect the impact of pretreat-ment, and these bands were not used for comparison. Underdilute acid pretreatment, there are no obvious changes oftwo lignin units in pine and eucalyptus; however, significantremovals of guaiacyl lignin in pine (80 %) and syringyllignin (31 %) in eucalyptus are observed after ionic liquidpretreatment. In comparison, both dilute acid- and ionicliquid-pretreated switchgrass samples show significantreductions of both structures. Specifically, the removal ofsyringyl and guaiacyl units reached up to 82 and 88 % byionic liquid pretreatment and 71 and 34 % by dilute acidpretreatment, respectively.

These data are in agreement with the total lignin removaldata shown in Table 1 using the analytical chemistry method

and also suggest that ionic liquid pretreatment is very effectivetoward the depolymerization of both syringyl and guaiacylunits, although guaiacyl lignins are more strongly cross-linked and, therefore, more resistant to chemical degradationthan lignins with a high syringyl content [47]. As Raman onlyreflects the intensities of the selected major lignin bands,whereas wet chemistry analysis gives the overall lignin con-tents of samples, it should be noted that spectroscopy techni-ques are more accurate for trend analysis. These results clearlydemonstrate that ionic liquid pretreatment at these processconditions effectively weakens the van der Waals interactionbetween cell wall polymers, disrupts the covalent linkagesbetween hemicellulose and lignin [48], and generates a productwith low lignin content, but other interactions between matrixpolysaccharides, cellulose, and lignin may also be affected.

Substrate Enzymatic Digestibility and Sugar Recovery

Enzymatic hydrolysis of cellulose in untreated and pretreatedpine, eucalyptus, and switchgrass to glucose was carried outusing commercial enzyme cocktails in order to compare theirinitial kinetics and cellulose digestibility. Enzyme loadingswere normalized to the glucan present in each sample. Figure 3shows the glucose production over a 72-h time period and thecorresponding enzymatic digestibility as the function of pre-treated feedstocks. Theoretically, 1 g of cellulose upon com-plete hydrolysis produces 1.11 g of glucose [24]. The ionicliquid-pretreated pine, eucalyptus, and switchgrass exhibit sim-ilar and significantly fast saccharification rates by reaching96 % digestibility in 48 h. However, the three dilute acid-pretreated feedstocks show a different hydrolysis performance.Only in the case of switchgrass was a significantly improvedconversion observed by reaching 76 % of glucose recovery in72 h, whereas both pine and eucalyptus show much lessdigestibility. Only small amounts of xylose were generateddue to the high removal of hemicelluloses during both diluteacid and ionic liquid pretreatments.

The main mechanisms of improving enzymatic sacchar-ification for dilute acid and ionic liquid are different. Ionicliquid pretreatment significantly removes both hemicellulo-ses (75.6–81.5 %) and lignin (49.1–70.6 %) by dissolutionto weaken the van der Waals interaction between cell wallpolymers and disrupt the covalent linkages between hemi-cellulose and lignin. However, for dilute acid, significantremoval of hemicelluloses (75.8–86.8 %) is the key to breakdown the cell wall linkages because lignin is mainly con-densed by pretreatment with a relatively low removalpercentage (8.0–24.1 %) [49]. Thus, under similar hemicel-lulose removal, significant lignin reduction by ionic liquidpretreatment improves the cellulose conversion.

As described previously, lignin provides a robust linkagebetween polysaccharide chains and is a primary source ofrecalcitrance to conversion as it obstructs enzyme accessibility

0

500

1000

1500

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2500

Untreated Dilute acid Ionic liquid

Syringyl

Guaiacyl

0

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nsity

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tens

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nsity

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a

b

c

Fig. 2 Guaiacyl and syringyl lignin removal from pine (a), eucalyptus(b), and switchgrass (c) by various pretreatment methods as character-ized by Raman spectroscopy

Bioenerg. Res. (2013) 6:14–23 19

to the polysaccharides present in the plant cell walls [49, 50].Yang andWyman [51] also reported that condensed lignin canabsorb protein from aqueous solutions and that lignin removalshould improve the hydrolysis by reducing the specificadsorption of cellulase enzymes. Previous studies have shownthat cellulose hydrolysis improves with increased ligninremoval after ionic liquid pretreatment [40]. The effects oflignin removal on the enzymatic hydrolysis rate and the

24-h digestibility of all pretreated solids are further presentedin Fig. 4a, b, respectively. The data indicate that an increase inlignin removal from 8.0 to 69.3 % results in a significantenhancement in the enzymatic hydrolysis rate anddigestibility. The initial enzymatic rates of hydrolysis to glu-cose are 0.23, 0.88, and 1.11 gL−1 h−1 for ionic liquid-pretreated pine, eucalyptus, and switchgrass, respectively,which are 7.6, 24.3 and 16.8 times greater than the diluteacid-pretreated ones. The rank of digestibility from greatestto least for both pretreatment methods is: switchgrass, euca-lyptus, and pine. The ionic liquid-pretreated pine, eucalyptus,and switchgrass have digestibilities of 62, 93, and 96 % undercorresponding higher lignin removal percentages, respective-ly. In contrast, within the low lignin removal ranges, thedigestibilities of dilute acid-pretreated pine (9 %) and euca-lyptus (10 %) were very poor; only switchgrass reached up to48 %. Thus, lignin removal plays a crucial role in enhancing

0

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2.0

3.0

4.0

5.0

0 20 40 60 80

Enz

ymat

ic d

iges

tibili

ty(%

)

Glu

cose

con

cent

ratio

n (g

/L)

Time (h)

Ionic liquidDilute acidUntreated

0

20

40

60

80

100

0.0

1.0

2.0

3.0

4.0

5.0

0 20 40 60 80

Enz

ymat

ic d

iges

tibili

ty(%

)

Glu

cose

con

cent

ratio

n (g

/L)

Time (h)

Ionic liquidDilute acidUntreated

a

b

c

Fig. 3 Comparison of enzymatic saccharification profiles of pine (a),eucalyptus (b), and switchgrass (c) after dilute acid and ionic liquidpretreatments. Glucan loading05 g/L, with cellulase loading03.5 FPU/g glucan and β-glucosidase loading01.25 CBU/g glucan

0.0

0.2

0.4

0.6

0.8

1.0

1.2

8.0 9.7 22.3 49.1 54.9 69.3

Enz

ymat

ic h

ydro

lysi

s ra

te (

g gl

ucos

e/L

/h)

Lignin removal efficiency (%)

0

20

40

60

80

100

8.0 9.7 22.3 49.1 54.9 69.3

Enz

ymat

ic d

iges

tibi

lity

in 2

4 h

(%)

Lignin removal efficiency (%)

a

b

Fig. 4 Effect of lignin removal on enzymatic hydrolysis rate (a) andenzymatic digestibility (b) in 24 h for three feedstocks under twopretreatment conditions. From left to right, Dilute-acid pretreated pine,eucalyptus, switchgrass; ionic liquid-pretreated pine, eucalyptus andswitchgrass. Glucan loading05 g/L, with cellulase loading03.5 FPU/gglucan and β-glucosidase loading01.25 CBU/g glucan

20 Bioenerg. Res. (2013) 6:14–23

the digestibility of the three feedstocks studied, and the ob-served improvement after ionic liquid pretreatment isattributed to the depolymerization of syringyl and guaiacylunits, as shown by Raman spectroscopy data. These resultsindicate that [C2mim][OAc] pretreatment under such condi-tions effectively increases the sugar recovery and cellulosesaccharification of various biomass types, whereas dilute acidpretreatment at these conditions is only effective at pretreatingswitchgrass.

Table 2 further summarizes the most complete informa-tion, in terms of monomer sugar recovery, from both pre-treatment and enzymatic hydrolysis. For dilute acidpretreatment, small amounts of amorphous cellulose andthe majority of hemicelluloses were hydrolyzed into fer-mentable sugars, with xylose as the major monomeric sugarin the pretreatment liquor. Xylose yields from switchgrasswere significantly higher than those from the pine andeucalyptus for dilute acid pretreatment (17.6 g/100 g switch-grass, 78.1 % theoretical yield, vs. 9.7 g/100 g pine, 71.6 %theoretical yield, and 10.5 g/100 g eucalyptus, 66.2 % the-oretical yield, respectively). The hemicelluloses removed byionic liquid pretreatment for all three feedstocks were com-parable to those observed in the dilute acid pretreatmentprocess, but the HPAEC analysis on washes did not showthe presence of monosaccharides, which is distinct fromacid pretreatment. Although the liquors from each feedstockwere not further analyzed in this study, our previous workconfirmed that the majority of hemicelluloses and smallamounts of cellulose were effectively depolymerized intooligo- and polysaccharides forms, which can be furtherconverted into xylose and small amounts of glucose,arabinose, and galactose by trifluoroacetic acid [24, 40].This reduces the overall sugar content in the recovered solidbiomass for downstream fermentation and also complicatesthe recovery and recycling of the ionic liquid. As the result,

although ionic liquid pretreatment significantly increasedenzymatic glucose yield on all three biomasses, theenhancement of total monomer sugar recovery was notencouraging (Table 2). Especially in the case of switchgrass,less sugar yield (49.2 %) was achieved by ionic liquidpretreatment than that from dilute acid pretreatment(78.6 %). Hemicelluloses as well as lignin remains in theliquid fraction should be recovered with additional process-ing steps, which are currently under intense development atJBEI and other research labs worldwide. A solvent extrac-tion technique using boronate complexes has beendeveloped at JBEI and can extract up to 90 % of mono-and disaccharides from aqueous ionic liquid solutions [52].Furthermore, a recent study also shows that using ion ex-clusion chromatography can effectively separate concentrat-ed fermentable sugars from ionic liquid [53]. Althoughfurther processing steps could pose additional challengesto commercialization, these techniques have shown greatpotentials to deliver a concentrated solution of fermentablesugars, minimizing toxic by-products and facilitating ionicliquid cleanup and recycling.

Both pretreatment technologies show great promise in theimprovement of biomass saccharification, but ionic liquidpretreatment may offer some unique advantages over diluteacid pretreatment in terms of higher delignification, reducedprocessing time, and higher sugar yields. However, consid-ering the current high cost associated with ionic liquid andthe amount of material lost during pretreatment, decreasingthe pretreatment and enzyme costs are all necessary for theionic liquid pretreatment technology to be commercializedand cost-competitive to dilute acid in terms of technicalmaturity and scalability. The significant delignification dem-onstrated by the ionic liquid pretreatment is promising forrecovering and converting a lignin stream as a co-product.Extracting hemicellulose from the liquid fraction after

Table 2 Sugar recovery yields of three feedstocks by pretreatment and enzymatic saccharification (only monomeric sugars are included in the data)

Feedstocks Pretreatmentmethods

Pretreatment liquor Enzymaticglucose yield(g/100 g untreatedbiomass)

Totalsugarrecovery(%)a

Glucose yield(g/100 g untreatedbiomass)

Xylose yield(g/100 g untreatedbiomass)

Mannose yield(g/100 g untreatedbiomass)

Pine Dilute acid 2.2 9.7 2.3 3.8 29.9

Ionic liquid –b –b –b 30.7 49.7a

Eucalyptus Dilute acid 3.4 10.5 1.1 3.2 30.9

Ionic liquid –b –b –b 31.6 48.6a

Switchgrass Dilute acid 9.4 17.6 ND 24.0 78.6

Ionic liquid –b

–b

–b 32.6 49.2a

ND not detecteda Total sugars include monomeric sugars (glucose, mannose, xylose) recovered from pretreatment liquor and glucose from enzymatic hydrolysateb Sugars dissolved in ionic liquid liquor during pretreatment are in oligosaccharide forms

Bioenerg. Res. (2013) 6:14–23 21

pretreatment and recycling the ionic liquid are two impor-tant issues for the overall economic viability of this processthat must be addressed.

Conclusion

The present work takes a major step toward providing arobust comparative framework between two pretreatmentscoupled with enzymatic saccharification in terms of theirperformance of converting pine, eucalyptus, and switch-grass to fermentable sugars for biofuel production. Signifi-cantly enhanced saccharification kinetics and sugar yieldwere observed for all three biomasses after ionic liquidpretreatment using [C2mim][OAc], whereas dilute acid pre-treatment was found to improve the saccharification of onlyswitchgrass, with a minimal impact on pine and eucalyptus.Both hemicellulose and lignin removals were attributed tothe enhancement of saccharification. There is a direct cor-relation between lignin removal and enzymatic saccharifi-cation of the pretreated feedstocks, suggesting that ionicliquid pretreatment works effectively toward the depolymer-ization and extraction of both guaiacyl and syringyl lignin.These results suggest that ionic liquid pretreatment mayoffer unique advantages when compared to the dilute acidpretreatment process for a wide range of biomass feed-stocks, but extracting hemicellulose from ionic liquid pre-treatment is necessary to maximize the fermentable sugarrecovery for downstream processing.

Acknowledgments The authors thank Dr. Henrik V. Scheller andDr. Ning Sun for reviewing this manuscript. This work was partof the DOE Joint BioEnergy Institute (http://www.jbei.org)supported by the U.S. Department of Energy, Office of Science,Office of Biological and Environmental Research through contractDE-AC02-05CH11231 between Lawrence Berkeley NationalLaboratory and the U.S. Department of Energy.

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