Pretreatment of Lodgepole Pine Killed by Mountain Pine Beetle Using theEthanol Organosolv Process: Fractionation and Process Optimization

Xuejun Pan,*,†,‡ Dan Xie,‡ Richard W. Yu,‡ Dexter Lam,‡ and Jack N. Saddler‡

Department of Biological Systems Engineering, UniVersity of Wisconsin-Madison, 460 Henry Mall,Madison, Wisconsin 53706, and Department of Wood Science, UniVersity of British Columbia,2424 Main Mall, VancouVer, BC, V6T 1Z4 Canada

Lodgepole pine (Pinus contorta) killed by mountain pine beetle (Dendroctonus ponderosae) (MPB-LPP)was evaluated for bioconversion to ethanol using the ethanol organosolv process. The pretreatment wasoptimized using an experimental matrix designed with response surface methodology. It was found that MPB-LPP was easy to pretreat and delignify, but gave low yields of substrate and carbohydrate as a result ofexcessive hydrolysis and subsequent decomposition of cellulose and hemicellulose during the pretreatment.The center-point conditions (170°C, 60 min, 1.1% H2SO4 and 65% ethanol) were close to the optimum forthe recovery of glucose and ethanol organosolv lignin. At the center-point conditions,∼75% of the cellulosepresent in the untreated wood was recovered in the substrate fraction, and approximately 79% of the ligninin the wood was recovered as ethanol organosolv lignin (EOL). The combined recovery of carbohydrate inthe substrate and water-soluble fractions was∼83% glucose,∼46% mannose,∼53% xylose,∼78% galactose,and∼55% arabinose. The lost carbohydrate was decomposed to furfural, hydroxymethylfurfural, and levulinicand formic acids. The substrate generated at center-point conditions from MPB-LPP was readily digestible.Cellulose-to-glucose conversion yields of∼93% and∼97% were achieved within 24 and 48 h, respectively,with 20 FPU of cellulase/g of cellulose.

1. Introduction

Lodgepole pine (LPP,Pinus contorta) is the most com-mercially important tree species in British Columbia (BC),Canada, covering 14.9 of 60 million ha of forested land in BC.1

However, the current outbreak of mountain pine beetle (MPB,Dendroctonus ponderosae) in BC has infested about 7 millionha of LPP and is still accelerating. It is forecasted that 80% ofLPP will be killed by MPB by the middle of the next decade.1

Although the beetle infestation does not significantly affect thephysical strength of the wood, the fungi associated with MPBproduce melanin and cause a blue to black discoloration ofsapwood, thus reducing its value in the market.2 Furthermore,the beetle-killed trees are easy for decay fungi to colonize,further reducing the quality and value of the wood.3 In addition,leaving killed trees untouched increases the risk of wild fire.However, large-scale salvage operation will produce an abun-dance of stained wood that exceeds the normal market abilityfor consumption, and therefore, new markets and uses inaddition to timber and pulp have to be found.

Biorefining of lignocellulosic biomass to produce fuels,chemicals, and materials is believed to be a potential alternativeto the current dependence on petroleum oil.4,5 The bioconversionof biomass to ethanol through a “carbohydrate platform” is ofcurrent interest. A typical bioconversion process consists ofpretreatment, saccharification, fermentation, and ethanol distil-lation steps. Several bioconversion schemes have been proposed,including steam explosion, ammonia fiber explosion (AFEX),dilute acid or hot water treatment, and the organosolv process.6-9

A primary technical-economic challenge in all lignocellulosics-to-ethanol bioconversion processes is the development of cost-effective pretreatment methods. The main objective of a

pretreatment is to produce a cellulose-rich substrate that issusceptible to enzymatic hydrolysis (i.e., to provide fast conver-sion of cellulose to glucose at low enzyme loading).10 Inaddition, good recovery of hemicellulosic sugars and isolationof high-quality lignin are important. Development of high-valuecoproducts from hemicellulose and lignin is critical to improvingthe economics of bioconversion processes.

Among the pretreatment technologies currently being evalu-ated is an ethanol organosolv process. The process was originallydeveloped as a chemical process for fractionating lignocellu-losics using organic solvents11 and was later adapted as a pulpingmethod.12 The Alcell process further refines ethanol organosolvpulping as an alternative to Kraft pulping of hardwoods toproduce high-quality fibers.13,14The organosolv process has notbeen developed significantly for softwoods, however, becauseof the higher lignin content and difference in lignin structure.15

Historically, the organosolv process has been investigated largelyfrom the perspective of pulp production,16-19 but it is not well-studied as a pretreatment/biorefining tool for the bioconversionof lignocellulosic biomass.

Recently, the pretreatment of hardwood hybrid poplar usingthe ethanol organosolv process was investigated in detail fromthe angles of the recovery of carbohydrate and lignin, theenzymatic hydrolyzability of the substrates, and the effects ofprocess variables on substrate characteristics.20,21 The ethanolorganosolv lignin from the poplar was evaluated as an antioxi-dant.22 The efficacy of ethanol organosolv pretreatment formixed softwoods (spruce, pine, and Douglas fir) was demon-strated in our previous research as well.6 The process produceda substrate with superior enzymatic digestibility over thosepretreated by alternative processes and a particularly high-qualitylignin fraction with potential applications.23-25 However, theethanol organosolv pretreatment of softwood has not beenoptimized, and the process mass balance data are not available.The objective of the present research was to further investigatethe ethanol organosolv pretreatment of softwood using mountain-

* To whom correspondence should be addressed. Tel.: 608-262-4951. Fax: 608-262-1228. E-mail: xpan@wisc.edu.

† University of Wisconsin-Madison.‡ University of British Columbia.

2609Ind. Eng. Chem. Res.2007,46, 2609-2617

10.1021/ie061576l CCC: $37.00 © 2007 American Chemical SocietyPublished on Web 03/14/2007

pine-beetle-killed lodgepole pine (MPB-LPP) as a feedstock.In addition, the compositional and morphological differencesbetween beetle-killed wood and healthy wood might impact thepretreatment, i.e., the infested sapwood has lower lignin,carbohydrate, and extractives contents but increased permeabilitycompared to sound sapwood.26 The study described herein usedresponse surface methodology to optimize the ethanol organo-solv pretreatment of MPB-LPP. The effects of process param-eters on the yields and distributions of cellulose, hemicellulose,and lignin in the fractions generated during the pretreatmentwere examined, and the process mass balance and enzymatichydrolysis of the resulting substrate were investigated. Math-ematical equations were regressed to quantitatively predict theeffects of the process variables on the fractionation and substratecharacteristics.

2. Materials and Methods

Feedstock Preparation.Lodgepole pine trees infested bymountain pine beetle (MPB-LPP) at the gray phase (dead tree)were harvested at Burns Lake (53°96′43′′ N, 600°58′20′′ W),Prince George (50°48′25′′ N, 594°76′47′′ W), and Quesnel(49°50′98′′ N, 587°08′01′′ W), BC, Canada. Seven trees withoutheart rot (one from Burns Lake, two from Prince George, andfour from Quesnel) were used in the present research. Theaverage age of the trees was 99( 25 years. Two sections, onefrom the top part and another from the bottom part, werecollected from each trunk. After being debarked and air-dried,the tree trunks were chipped using a custom-designed chipper.The chips were then screened using a plate screen; the fractionlarger than 2.5× 2.5 cm and smaller than 5.0× 5.0 cm,approximately 0.5 cm thick, was collected as the feedstock forethanol organosolv pretreatment. A sample of the chips wasground using a Wiley mill, and the fraction passing 40-meshwas collected for chemical analysis. The chemical compositionof the wood is summarized in Table 1.

Ethanol Organosolv Pretreatment. A flowchart of thelaboratory-scale ethanol organosolv process was schematicallydescribed before.20 In brief, MPB-LPP chips were pretreatedin aqueous ethanol with sulfuric acid as a catalyst using acustom-built, four-vessel (2 L each) rotating digester made byAurora Products Ltd. (Savona, BC, Canada). A 200-g (oven-dried weight) batch of chips was pretreated in each vessel.Vessels were opened after being cooled to room temperaturein a water bath. Spent liquor (i.e., aqueous ethanol in the vessel)was sampled immediately for determination of furfural, hy-droxymethylfurfural (HMF), and formic and levulinic acids. The

substrate (i.e., defiberized solid fraction) and spent liquor werethen separated using nylon cloth. The substrate was washed threetimes (300 mL each) with warm (60°C) aqueous ethanol havingthe same concentration as the pretreatment liquor. The washeswere combined with the spent liquor. The substrate was thenwashed three times with water at 60°C, and the washes werediscarded. The washed substrate was homogenized in a standardBritish disintegrator for 5 min and passed through a laboratoryflat screen with 0.008-in. (0.203-mm) slits (Voith, Inc., Apple-ton, WI) to remove rejects (i.e., non-defiberized woodchips andknots). The yields of rejects and screened substrate weredetermined. The screened substrate was stored at 4°C foranalysis and hydrolysis.

The spent liquor and the ethanol washes were combined andmixed with three volumes of water to precipitate the dissolvedlignin. The lignin precipitate, henceforth denoted as ethanolorganosolv lignin (EOL), was collected on Whatman No. 1 filterpaper, washed thoroughly with water, and air-dried. The filtrateand the water washes were combined to give a water-solublefraction containing monomeric and oligomeric saccharides,depolymerized lignin, and compounds derived from saccharides.

Analytical Procedures.Oven-dried weights were determinedby drying to constant weight at 105°C in a convection oven.Ash of the wood powder was determined according to TAPPI(Technical Association of Pulp and Paper Industry) standardmethod T211 om-93. Extractives of the wood powder weredetermined according to the procedure of TAPPI standardmethod T264 cm-97 using ethanol and water as solvents. Klasonlignin of the wood powder and substrates was determinedaccording to TAPPI standard method T-222 om-98. Thehydrolysate from Klason lignin determination was retained foranalysis of monosaccharides and acid-soluble lignin. Acid-soluble lignin was determined by UV absorbance at 205 nmaccording to the method described by Dence.27 Monosaccharideswere determined using a DX-500 HPLC system (Dionex,Sunnyvale, CA) equipped with an AS3500 autosampler, a GP40gradient pump, an anion-exchange column (Dionex CarboPacPA1), and an ED40 electrochemical detector. The column waseluted with deionized water at a flow rate of 1 mL/min. Aliquots(20 µL) were injected after being passed through a 0.45-µmnylon syringe filter (Chromatographic Specialties Inc., Brock-ville, ON, Canada). Optimization of baseline stability anddetector sensitivity was achieved by postcolumn addition of 0.2M NaOH. The column was reconditioned using 1 M NaOHafter each analysis. Monosaccharides were quantified withreference to saccharide standards. The saccharide standards wereautoclaved at 120°C for 1 h prior to analysis to compensatefor possible decomposition caused by heating involved in Klasonlignin determination. Furfural and HMF were determined usinga Dionex Summit HPLC system equipped with a P680 pump,an ASI-100 autosampler, and a PDA100 photodiode arraydetector. A LiChrospher 5RP18 column (Varian, Palo Alto, CA)was used at 60°C with an eluent flow rate of 0.5 mL/min. Agradient of (A) 7.4 mM H3PO4, (B) acetonitrile, and (C) amixture of 7.4 mM H3PO4, methanol, and acetonitrile (4:3:3,v/v) was applied as follows: 0-20 min, from 95% A and 5%C to 50% A and 50% C; 20-24 min, from 50% A and 50% Cto 100% C; 24-25 min, 100% C; 25-26 min, from 100% C to100% B; 26-27 min, 100% B; 27-28 min, from 100% B to95% A and 5% C; 28-38 min, 95% A and 5% C. Appropriatelydiluted aliquots (20µL) were injected after being passed througha 0.45-µm PTFE syringe filter (Chromatographic SpecialtiesInc.). Furfural and HMF were determined by absorbance at 280nm. Formic and levulinic acids were quantified using an

Table 1. Chemical Composition of Untreated MPB-LPP

component contenta

ash 0.26( 0.01extractives (water followed by ethanol) 4.66( 0.21Klason lignin 24.79( 0.09acid-soluble lignin 0.29( 0.00carbohydrate (as monosaccharide)

glucose 50.46( 0.25mannose 13.09( 0.24xylose 7.21( 0.04galactose 2.22( 0.01arabinose 1.42( 0.00

carbohydrate (as polysaccharide)glucan 45.42( 0.22mannan 11.78( 0.21xylan 6.34( 0.03galactan 2.00( 0.01arabinan 1.25( 0.00

a Content reported as % (w/w) in oven-dried MPB-LPP chips.

2610 Ind. Eng. Chem. Res., Vol. 46, No. 8, 2007

Allilance 2695 HPLC system (Waters, Milford, MA) equippedwith an AD20 absorbance detector (Dionex, Sunnyvale, CA),a SUPELCO (Bellefonte, PA) SUPELCOGEL C610H column,and a SUPELGUARD C601H guard column. The columntemperature was 30°C, and the mobile phase was 0.1% H3PO4

at a flow rate of 0.5 mL/min. Sample (5µL) was injected ontothe column after being filtered through a 0.45-µm nylon syringefilter as described above. The viscosity (degree of polymeriza-tion) of the cellulose solution was measured according to TAPPIstandard method T230 om-99, as described before.21

Enzymatic Hydrolysis. Commercial cellulase (Spezyme CP)andâ-glucosidase (Novozym 188) were provided by GenencorInternational Inc. (Rochester, NY) and Novozymes (Franklinton,NC), respectively. Cellulase activity was determined using thefilter paper assay recommended by the International Union ofPure and Applied Chemists28 and is expressed in terms of filterpaper units (FPUs).â-Glucosidase activity was determined usingp-nitrophenyl-â-D-glucoside as the substrate, as previouslydescribed,29 and is expressed in terms of International Units(IUs). Protein was determined using Bio-Rad Protein Assay(Method of Bradford).30 Cellulase was supplemented withâ-glucosidase (1:2 FPU/IU) to avoid product inhibition causedby cellobiose accumulation.

Batch hydrolysis was conducted at 2% consistency (cellulose,w/v) in 50 mM acetate buffer, pH 4.8, with 0.004% tetracyclineas an antibiotic. Cellulase was used at a loading of 20 FPU (21mg of total protein)/g of cellulose with a supplementation ofâ-glucosidase at a loading of 40 IU (5.7 mg of total protein)/gof cellulose. The reaction mixture (100 mL) was incubated at150 rpm, 50°C, in a rotary shaker and sampled periodicallyfor glucose determination. The glucose was quantified usingHPLC, as described above, with the exception that the saccharicstandards were not autoclaved. Hydrolysis data are averagesfrom duplicate experiments.

Experimental Design and Data Analysis.Pretreatmentconditions [temperature, time, catalyst (H2SO4) dosage, andethanol concentration] were optimized by response surface

methodology using a small Hartley composite design,31 asdescribed in detail in our previous report.20 The completeexperimental matrix is shown in Table 2. The selection of theconditions in Table 2 was based on the results of preliminaryexperiments (see Result and Discussion). Data were analyzedusing Statistical Analysis System (SAS, SAS Institute Inc., Cary,NC). All data reported are the averages of at least duplicateexperiments.

3. Results and Discussion

Preliminary Experiments for Selecting Process Conditions.As shown in Table 1, MPB-LPP has the typical chemicalcomposition of softwood. Compared to hybrid poplar (hard-wood),20 MPB-LPP has more total lignin (more Klason ligninbut less acid-soluble lignin) and a comparable amount of totalcarbohydrate but with a different composition. MPB-LPP hassignificantly more mannan and substantially less xylan thanpoplar. Previous research has indicated that the set of conditions(180 °C, 60 min, 1.25% H2SO4, 50% ethanol) is close to theoptimum for poplar in the recovery of cellulose, hemicellulose,and lignin.20 In general, softwood requires more severe condi-tions for delignification than hardwood because softwoodcontains more lignin and, in particular, more guaiacyl units thattend to condense. Therefore, the pretreatment of MPB-LPP wasstarted from more severe conditions than those used for poplar.

As shown in Table 3, test 1 was conducted at 185°C, 80min, 1.5% H2SO4, and 60% ethanol. The substrate yield wasonly ∼35%, but residual lignin was high (∼27% Klason lignin).Increasing H2SO4 to 2.34% resulted in an additional reductionin substrate yield and increase in Klason lignin (test 2), implyingthe removal of more carbohydrate than lignin. It was noted thatthe substrates from tests 1 and 2 had no rejects and residualhemicellulose. However, a significant amount of acid-solublelignin was observed in the water-soluble fraction as a result ofthe extensive destruction of lignin. These results suggest thatthe conditions used in tests 1 and 2 were too severe for MPB-LPP. When the temperature was lowered to 170°C (test 3), the

Table 2. Experimental Matrix and Results for Ethanol Organosolv Pretreatment of MPB-LPP

variablesb water-soluble componentsc

no.a T t S C subc KL in sub (%) gluc rejc EOLc AL glu xyl man gal ara

1 160 50 0.90 75 26.47 17.56 20.51 29.87 14.07 3.55 1.79 3.73 6.64 2.13 1.262 180 50 0.90 75 35.37 4.98 35.04 0.75 21.95 7.65 5.31 2.60 4.96 1.42 0.603 160 70 0.90 55 11.12 22.84 8.50 44.93 11.31 3.87 2.91 3.82 6.72 2.18 1.184 180 70 0.90 55 37.73 11.22 35.22 0.04 19.30 6.31 6.94 1.60 3.56 1.35 0.505 160 50 1.30 55 9.91 22.88 7.59 43.44 11.30 2.93 2.64 4.59 8.18 2.47 1.386 180 50 1.30 55 38.53 12.17 35.01 0.29 18.40 5.43 4.63 0.94 2.50 0.91 0.257 160 70 1.30 75 41.53 13.24 35.72 3.89 18.00 3.98 3.68 3.81 6.30 2.12 1.048 180 70 1.30 75 31.23 7.92 29.54 0.05 24.96 3.01 5.25 0.32 1.49 0.46 0.069 153 60 1.10 65 7.15 20.91 5.09 52.79 12.33 4.55 3.04 3.06 5.26 2.06 1.1510 187 60 1.10 65 27.58 11.15 25.18 0.06 23.41 6.06 5.36 0.25 1.03 0.27 0.1111 170 43 1.10 65 43.04 12.75 37.51 2.26 18.40 4.25 5.30 2.57 4.49 1.70 0.7412 170 77 1.10 65 40.75 8.80 36.69 0.46 20.17 6.46 3.42 2.78 5.16 1.56 0.6913 170 60 0.76 65 41.56 13.26 36.72 6.13 17.86 3.82 2.79 4.30 7.65 2.27 1.2014 170 60 1.44 65 39.24 8.21 36.91 0.66 20.51 5.45 4.24 1.99 3.72 1.29 0.4915 170 60 1.10 48 33.51 22.80 25.51 15.22 11.76 4.22 5.41 3.75 6.18 2.03 0.9816 170 60 1.10 82 39.81 10.08 35.09 1.63 20.08 5.15 4.85 3.78 6.07 1.81 0.8717 170 60 1.10 65 41.48 10.34 38.30 1.22 19.67 4.69 4.95 3.39 5.53 1.74 0.8318 170 60 1.10 65 41.73 10.61 37.48 0.93 20.24 4.66 4.73 3.28 5.25 1.80 0.7919 170 60 1.10 65 40.27 8.94 37.11 1.14 19.03 4.71 3.84 3.32 5.47 1.70 0.7920 170 60 1.10 65 41.72 10.23 37.41 1.24 19.30 5.07 3.06 2.72 4.84 1.53 0.6321 170 60 1.10 65 41.26 10.34 37.87 0.93 19.61 4.69 4.36 3.44 5.75 1.90 0.85avg 17-21 41.29 10.09 37.63 1.09 19.57 4.76 4.19 3.23 5.37 1.73 0.78SD 17-21 0.61 0.66 0.46 0.16 0.45 0.17 0.76 0.29 0.34 0.14 0.09

a Numbers 1-21 are the complete experimental matrix of 21 conditions. Numbers 17-21 are replicated center-point conditions.b T, temperature (°C); t,time (min) at that temperature;S, sulfuric acid (%, w/w, oven-dried wood);C, ethanol concentration, (%, v/v).c All data are yields of components (g) per100 g (oven-dried weight) of untreated MPB-LPP chips.d Abbreviations: SD, standard deviation; sub, substrate; KL, Klason lignin; glu, glucose; rej, rejects;EOL, ethanol organosolv lignin; AL, acid-soluble lignin; xyl, xylose; man, mannose; gal, galactose; ara, arabinose.

Ind. Eng. Chem. Res., Vol. 46, No. 8, 20072611

substrate yield increased, and the Klason lignin decreasedsignificantly. The reduction of catalyst dosage to 1.2% H2SO4

(test 4) resulted in additional improvement in substrate yieldbut a slight increase in Klason lignin. Still, no hemicellulosewas detected in the substrates of tests 3 and 4. Compared totest 4, a 40-min reduction of reaction time in test 5 increasedthe substrate yield, but this yield increase was primarily fromthe increase in Klason lignin, not the reservation of carbohydrate.On the other hand, increasing the ethanol concentration by 10%(test 6) reduced the Klason lignin, suggesting that the dissolutionof lignin from wood chips was enhanced. Further reducing theseverity of test 6 by decreasing the reaction time by 20 minand the catalyst by 0.2% improved neither the recovery ofcarbohydrate nor the delignification (test 7). Lowering thetemperature by 10°C (test 8) resulted in a very high rejects(non-defiberized wood chips) rate of∼39%, indicating a failureof defiberization. Extension of the reaction time by 40 min (test9) did reduce the rejects rate, thus improving the pulp yield,but did not improve delignification (i.e., Klason lignin was stillhigh). The MPB-LPP was also pretreated at extremely severeconditions of 190°C, 120 min, 2.5% H2SO4, and 60% ethanol(test 10). The resulting substrate (<29% yield) was composeddominantly of lignin (∼64% Klason lignin). The reason is thatcarbohydrate (both cellulose and hemicellulose) was excessivelyhydrolyzed and subsequently converted to furfural, hydroxy-methylfurfural (HMF), and organic acids. Extensive degradationof lignin was also observed at the most severe conditions,supported by the low yield of ethanol organosolv lignin (EOL)and the high concentration of acid-soluble lignin in the water-soluble fraction. Judging from the observations and discussionabove, the center-point conditions used in the experimentalmatrix for MPB-LPP were selected as 170°C, 60 min, 1.1%H2SO4, and 65% ethanol.

Optimization of the Ethanol Organosolv Pretreatment ofMPB-LPP. To optimize the ethanol organosolv pretreatmentof MPB-LPP, an experimental matrix was designed usingresponse surface methodology, as described in the Materialsand Methods section and summarized in Table 2. The center-point conditions were those selected in the preliminary experi-ments above. The ranges of the conditions were as follows:temperature, 153-187 °C; time, 43-77 min; H2SO4, 0.76-1.44% (w/w); ethanol, 48-82% (v/v). The ratio of liquor towood was constant (7:1, v/w) in all experiments.

The effects of process variables on delignification aredemonstrated in Figures 1 (Klason lignin in substrate) and 2(yield of recovered ethanol organosolv lignin, EOL). Figure1A,B clearly shows that reaction time and catalyst (sulfuric acid)did not have substantial effect on the Klason lignin of thesubstrates, but ethanol concentration and temperature did. As

discussed elsewhere,20 delignification during the ethanol organo-solv pretreatment is a combination of depolymerization andsolubilization of lignin. Lower ethanol concentration, whichgenerates higher hydrogen ion concentration (lower pH value)at the same dosage of H2SO4, promotes acid-catalyzed cleavageof R- andâ-ether linkages in the lignin,32 whereas high ethanolconcentration increases solubilization of the lignin.33,34As shownin Figure 1A, Klason lignin in the substrate reached the lowestvalue at∼75% ethanol concentration (turning point) and thenslightly increased. The turning-point concentration was higherthan that of hybrid poplar (∼65%),20 indicating that MPB-LPPlignin requires a higher ethanol concentration for solubilization

Table 3. Results of Preliminary Pretreatment Experiments

pretreatment conditionsa fraction yieldb (% of wood) substrate compositionb (%)

test no. T t S C pulp rej EOL AL in WS KL glu xyl man

1 185 80 1.50 60 35.10 0.00 19.20 5.95 27.42 81.49 ND ND2 185 80 2.34 60 29.30 0.00 18.22 5.37 31.99 77.53 ND ND3 170 80 1.50 60 38.80 0.15 20.90 4.85 10.55 97.49 ND ND4 170 80 1.20 60 40.30 0.58 19.20 4.27 11.62 93.73 ND ND5 170 40 1.20 60 42.55 3.14 17.10 4.32 14.19 83.73 0.97 1.056 170 80 1.20 70 38.52 0.22 22.00 5.17 7.40 96.14 1.30 1.407 170 60 1.00 70 40.75 2.24 21.25 4.61 9.21 91.09 1.02 1.128 160 60 1.00 70 14.06 39.05 15.20 3.74 18.43 76.42 2.34 2.879 160 100 1.00 70 38.82 8.56 18.13 4.49 13.93 89.16 2.77 3.0410 190 120 2.50 60 28.63 0.00 16.93 8.03 63.65 42.97 ND ND

a T, temperature (°C); t, time (min) at that temperature;S, sulfuric acid (%, w/w, oven-dried wood);C, ethanol concentration, (%, v/v).b Abbreviations:rej, rejects; EOL, ethanol organosolv lignin; AL, acid-soluble lignin; WS, water-soluble fraction; KL, Klason lignin; glu, glucose; xyl, xylose; man, mannose;ND, not detected.

Figure 1. Effects of process variables on Klason lignin in the substratefraction. The fixed variables are as follows: (A) 170°C and 1.1% H2SO4,(B) 60 min and 65% ethanol.

2612 Ind. Eng. Chem. Res., Vol. 46, No. 8, 2007

than poplar lignin. The temperature showed a similar effect ondelignification (Figure 1B). The lowest Klason lignin wasobserved at∼182 °C, which is lower than the∼195 °C valuefor poplar.20 This suggests that MPB-LPP is easier to delignifythan poplar. The mechanism is unclear, although beetle infesta-tion might be a cause. When the temperature was increasedbeyond the turning point, Klason lignin in the substrateincreased. An explanation for this behavior is that carbohydrateis hydrolyzed faster than lignin at high temperature, resultingin a relative increase in Klason lignin. The effects of reactiontime and ethanol concentration on the yield of EOL areconsistent with those on Klason lignin, as shown in Figure 2A,i.e., the lignin yield was independent of time, and a maximumyield was obtained at∼75% ethanol concentration. On the otherhand, the effects of temperature and sulfuric acid on EOL yieldwere different from those on Klason lignin, as shown in Figure2B. Lignin yield increased linearly with increasing catalystcontent as a result of acid-promoted cleavage and subsequentdissolution of lignin. The lignin yield kept increasing as thetemperature rose, and no turning point was observed within therange investigated.

The effects of process parameters on the yield of substrateare shown in Figure 3. The substrate yield decreased consistentlywith the extension of the reaction time because more lignin andcarbohydrate were dissolved, as shown in Figure 3A. On theother hand, the substrate yield increased with increasing ethanolconcentration and reached a maximum at∼70% ethanol

concentration. This observation is consistent with the progressof delignification discussed with respect to Figures 1 and 2above. With the removal of lignin binding the fibers together,wood chips were defiberized, thus resulting in more substrateand less rejects. However, when the ethanol concentration washigher than the turning point, the delignification was depressed,consequently resulting in a low substrate yield and a high rejectsrate. As shown in Figure 3B, the substrate yield increased rapidlyas the temperature rose as a result of defiberization enhancedby the removal of lignin, but in contrast, it declined when thetemperature was over∼175 °C because of the excessivehydrolysis of carbohydrate. The effect of sulfuric acid onsubstrate yield was dependent on temperature. When thetemperature was lower than the turning point (∼175°C), moresulfuric acid resulted in a higher yield by promoting delignifi-cation. When the temperature was over the turning point,however, more sulfuric acid enhanced the destruction ofcarbohydrate rather than delignification, thus giving a lowsubstrate yield.

Chemical analysis of the substrates (data not shown here)indicated that only small amounts of hemicellulosic sugars weredetected in the substrates, and over 90% of xylose and mannoseand almost 100% of galactose and arabinose were removed fromthe wood chips during the pretreatment. In contrast, a significantamount of hemicellulose, particularly xylan and mannan, wasretained in the hybrid poplar substrates.20 Because the conditions

Figure 2. Effects of process variables on the yield of ethanol organosolvlignin (EOL) precipitated after the pretreatment. The fixed variables are asfollows: (A) 170 °C and 1.1% H2SO4, (B) 60 min and 65% ethanol.

Figure 3. Effects of process variables on the yield of the substrate fraction.The fixed variables are as follows: (A) 170°C and 1.1% H2SO4, (B) 60min and 65% ethanol.

Ind. Eng. Chem. Res., Vol. 46, No. 8, 20072613

used for MPB-LPP were milder than those used for hybridpoplar, as mentioned above, it appears that the hemicellulosein MPB-LPP was easier to remove (hydrolyze) than that inhybrid poplar. The effects of process parameters on therecovered hemicellulosic sugars in the water-soluble fractionare demonstrated in Figure 4. All hemicellulosic sugars exceptglucose followed the same pattern, i.e., their yields in the water-soluble fraction were less sensitive to reaction time and ethanolconcentration, but significantly dependent on temperature andcatalyst. The yields of non-glucose sugars decreased consistentlywith the increases in temperature and catalyst dosage, indicatingthat the decomposition of the sugars was promoted. On the otherhand, the yield of glucose in the water-soluble fraction increasedwhen temperature and catalyst increased or ethanol concentrationdecreased. This observation suggests that hydrolysis of celluloseto glucose is faster than the subsequent decomposition of glucoseunder such conditions.

The results of the preliminary experiments (Table 3) and thematrix experiments (Table 2) and the discussions above indicatethat the center-point set of conditions was close to the optimalset for MPB-LPP from the perspective of the yields of substrate,cellulose, and EOL. The substrates from some condition sets(e.g., numbers 7, 11, and 13 in Table 2) seemed to have similaror higher yields, but the high yields resulted from more residuallignin (Klason lignin). When comparing the recovery of cellulose(glucose in the substrates), it is clear that none of the conditionsets resulted in a higher recovery of cellulose than the center-point set (37.63( 0.46%, Table 2). Certain condition sets (e.g.,numbers 2, 8, and 10 in Table 2) resulted in higher lignin yieldsthan the center point, but the cost was lower substrate (cellulose)yields caused by excessive destruction of carbohydrate.

Mass Balance of Ethanol Organosolv Pretreatment ofMPB-LPP. Five center points (numbers 17-21 in Table 2) wereevaluated by independent experiments under the same condi-tions, and statistical analysis showed the results to be reproduc-ible. In addition, the center-point conditions were close to theoptimum, as discussed above. Therefore, analysis of the massbalance at the center point is meaningful and conclusive. Asshown in Figure 5, the yield of substrate was∼41% w/w (i.e.,41 g of substrate was generated from 100 g of MPB-LPP chips),which is significantly lower than that of hybrid poplar (∼53%)at similar lignin content (∼10% and∼11% in the substratesfrom MPB-LPP and poplar, respectively).20 Approximately 17%of the total Klason lignin in the untreated wood remained inthe substrate after the pretreatment, compared to∼27% inpoplar.20 Considering the fact that MPB-LPP had more ligninand was pretreated under milder conditions than poplar, theresults suggest the easy delignification of MPB-LPP. Themajority (∼79%) of the lignin was dissolved during thepretreatment and recovered by precipitation as EOL. The acid-soluble lignin in the water-soluble fraction represented theproducts from the extensive depolymerization of lignin.

Approximately 75% of the original glucose in the wood wasrecovered in the substrate fraction, and∼8% of the glucosewas detected in the water-soluble fraction, compared to 88%and 1%, respectively, for poplar,20 suggesting that more cellulosewas hydrolyzed from MPB-LPP than from poplar during thepretreatment. The data in Figure 5 indicate that the carbohydratein the substrate fraction was composed primarily of cellulose.Only a minority of the original hemicellulose (∼8% of thexylose and∼5% of the mannose) in the untreated wood survivedfrom the hydrolysis and remained in the substrate, values that

Figure 4. Effects of process variables on the yields of sugars recovered in water-soluble fraction. The yields are expressed as grams per 100 grams ofoven-dried wood (%). Ara, arabinose; Gal, galactose; Man, mannose; Xyl, xylose; and Glu, glucose.

2614 Ind. Eng. Chem. Res., Vol. 46, No. 8, 2007

are substantially lower than those for poplar (∼19% of thexylose and∼38% of the mannose, respectively).20 It is clearthat the recovery of less hemicellulose and cellulose was thecause of the low substrate yield for MPB-LPP. Part of thedissolved hemicellulosic sugars (∼45% of the total xylose,∼41% of the total mannose,∼55% of the total arabinose, and∼78% of the total galactose in untreated wood) were found inthe water-soluble fraction. The majority of the hemicellulosicsugars in the water-soluble fraction was in an oligomeric form,similar to the result for poplar.20

In summary, the combined recovery of sugars in the substrateand water-soluble fractions was∼83% glucose,∼46% mannose,∼53% xylose,∼78% galactose, and∼55% arabinose, indicatingsignificant loss of carbohydrate during the pretreatment. It iswell-known that monosaccharides undergo thermal de-composition under acidic conditions, generating hydroxy-methylfurfural (HMF) and furfural, derived from hexoses andpentoses, respectively.35 Further decomposition of HMF resultsin levulinic and formic acids.36 As shown in Figure 5, all ofthese compounds were detected in the water-soluble fraction.The significant amount of levulinic acid and the relatively smallamount of HMF indicate that most of the hexoses (primarilyglucose and mannose) were quickly decomposed to the finalproducts (levulinic and formic acids).

Enzymatic Hydrolysis of Ethanol Organosolv Substratefrom MPB-LPP. Enzymatic hydrolysis results for selectedsubstrates prepared in the experimental matrix are shown inFigure 6, including numbers 2, 7, 9, 15, and 20. It was observedthat the substrate generated at the center point (number 20) wasreadily digestible, and cellulose-to-glucose conversion yieldsof ∼93% and ∼97% were achieved within 24 and 48 h,respectively. The enzymatic hydrolyzability was similar to thatof hybrid poplar substrate.20 Substrate number 2 had the lowestKlason lignin (∼5%), approximately half that of number 20(∼10%), thus exhibiting a better hydrolyzability (completeconversion within 48 h). On the other hand, substrate number7 had only 3% more lignin than substrate number 20, but the

conversion yield was much lower (∼82% within 48 h). Theseresults indicate that, although residual lignin is an importantfactor influencing enzymatic hydrolysis, hydrolyzability is notproportional to lignin. This is strongly supported by the resultsof numbers 9 and 15. Substrate number 15 had significantlymore lignin (∼23%) than substrate number 7 (∼13%), but theirhydrolysis profiles were very similar. On the contrary, substratenumber 9 contained slightly less lignin (∼21%) than substratenumber 15, but the former had much worse hydrolyzability thanthe latter, i.e., the 48-h conversion of number 9 was less than35%.

Figure 5. Mass balance of laboratory-scale ethanol organosolv pretreatment of MPB-LPP at center-point conditions (170°C, 60 min, 1.1% H2SO4, and 65%ethanol).

Figure 6. Enzymatic hydrolysis of selected substrates prepared from MPB-LPP using ethanol organosolv pretreatment with enzyme loadings of 20FPU of cellulase (21 mg of total protein) and 40 IU ofâ-glucosidase (5.7mg of total protein)/g of cellulose. The conditions used to prepare thesubstrates are listed in Table 2.

Ind. Eng. Chem. Res., Vol. 46, No. 8, 20072615

To explain the above observations, other substrate charac-teristics were investigated, including crystallinity, fiber size,residual hemicellulose, and degree of polymerization (DP) ofcellulose. Although there were no significant differences incrystallinity, fiber size, and hemicellulose among the samplesabove (data not included), DP was found to be a significantfactor affecting hydrolysis. As described in the Materials andMethods section, DP was measured in the present research usingthe viscosity of cellulose. As shown in Figure 6, the viscosityof substrate number 9 (20.7 mPa S) was as almost 7 times ashigh as that of number 15 (3.1 mPa S), which was the majorcause of poor hydrolyzability of number 9. Furthermore, theviscosity difference between number 7 and number 15 explainsthe similarity of their hydrolyzabilities, even though the formerhad more lignin than the latter. In addition to its low lignincontent, the low viscosity of number 2 was another contributorto its excellent hydrolyzability. It is believed that longer cellulosechains (higher DP) form stronger networks by more extensiveinter- and intramolecular hydrogen bonding, thereby limitingthe accessibility of cellulose to the enzymes.37 On the other hand,the reduction of DP increased the number of cellulose chainends available to the action of exoglucanase in the cellulasecomplex, thus generating a high reaction rate and a high glucoseyield.38-40

Predictive Modeling. The effects of pretreatment conditionson the various process responses (e.g., yields of substrates, EOL,and hemicellulosic sugars in the water-soluble fraction andchemical compositions of the substrates) were qualitativelydiscussed above. To quantitatively predict the effects of eachprocess variable on the responses, each process response wasfitted to a second-order polynomial equation using SASsoftware, as shown in eq 1

whereY is the estimated value of the process response;k is thetotal number of independent variables (4 in this case);Xi

represents the independent variables (temperature, time, catalystdose, and ethanol concentration);Xi, Xi

2, and XiXj are termsdescribing linear, quadratic, and two-variable interaction effects,respectively;a0 is a constant; andai, aii, and aij are linear,quadratic, and interaction coefficients, respectively. The predic-tive models (eqs 2-12) developed for each response are listedin Table 4. These equations can be used to predict the responsesto all combinations of variables within the investigated range.

They are also useful tools for fine-tuning the pretreatment, asdiscussed previously.20

4. Conclusions

Process optimization using the response surface methodologyindicates that the center-point conditions (170°C, 60 min, 1.1%H2SO4, and 65% ethanol) chosen through preliminary experi-ments are close to the optimum for the recovery of glucose inthe solids fraction and ethanol organosolv lignin. At the center-point conditions,∼75% of the cellulose present in the untreatedwood was recovered in substrate fraction. The combinedrecovery of sugars in the substrate and water-soluble fractionswas ∼83% glucose,∼46% mannose,∼53% xylose,∼78%galactose, and∼55% arabinose, respectively. The sugar recoverywas significantly lower than that of hybrid poplar reportedpreviously.20 The lost carbohydrate was decomposed to furfural,HMF, levulinic and formic acids, and other products. These arepotential high-value coproducts of the organosolv pretreatment.Approximately 79% of the lignin in wood was recovered as aprecipitate (ethanol organosolv lignin) following the pretreat-ment. A substantial amount (corresponding to∼17% of the totallignin in untreated wood) of acid-soluble lignin was detectedin the water-soluble fraction. The acid-soluble lignin representsthe low-molecular-weight fraction of the lignin and will beevaluated as an antioxidant.

It was observed that MPB-LPP behaved differently thanhybrid poplar during the ethanol organosolv pretreatment.Despite its high lignin content, MPB-LPP was easier to pretreatthan poplar, requiring milder conditions for delignification. Inaddition, more cellulose and hemicellulose were hydrolyzed andsubsequently decomposed during the pretreatment, which wasthe primary cause for the low substrate yield of MPB-LPP. Aproposed explanation is that the fungi associated with the beetlesmight change the lignin and carbohydrate physically andchemically. A detailed investigation is required to understandthe effects of beetle infestation on the bioconversion of MPB-LPP.

The substrate generated from MPB-LPP at the center-pointconditions was readily digestible. Cellulose-to-glucose conver-sion yields of∼93% and∼97% were achieved within 24 and48 h, respectively, with an enzyme loading of 20 FPU ofcellulase/g of cellulose. It appears that a low degree ofpolymerization of cellulose was an important contributor to theready digestibility of the organosolv substrate. A detailedinvestigation toward a fundamental understanding of the readydigestibility of ethanol organosolv substrates will be reportedlater.

Table 4. Predictive Models for Process Responses

responsea equationb R2

sub yield -373.841+ 3.812T + 0.190t - 2.376S+ 1.387C - 0.00887T2 - 0.00239Tt - 0.088TS-0.00740TC + 0.192tS+ 0.092SC- 0.00166C2

(2) 0.9907

KL in sub 734.723- 6.839T - 2.983C + 0.0189T2 + 0.0203C2 (3) 0.8991glu in sub -60.736+ 0.630T + 0.0256t + 0.0636S+ 0.218C - 0.00147T2 - 0.000394Tt - 0.0157TS-

0.00114TC + 0.0296tS+ 0.000131tC + 0.0128SC- 0.000332C2(4) 0.9986

rejects 509.379- 4.376T - 0.552t - 49.772S- 2.106C + 0.00953T2 + 0.003Tt + 0.206TS+ 0.00776TC +5.569S2 + 0.00550C2

(5) 0.9936

EOL yield -283.062+ 2.346T + 3.823S+ 1.877C - 0.00586T2 - 0.0126C2 (6) 0.9604AL in WS -221.427+ 0.806T + 1.957t + 86.463S+ 1.271C - 0.00641Tt - 0.313TS- 0.0123tC - 0.474SC (7) 0.8719glu in WS 5.628+ 0.109T - 17.293S- 0.318C + 0.276SC (8) 0.6279xyl in WS -175.25+ 1.978T + 38.086S- 0.00538T2 - 0.237TS (9) 0.8679man in WS -241.234+ 2.726T + 56.370S- 0.00736T2 - 0.353TS (10) 0.8531gal in WS -62.404+ 0.709T + 16.839S- 0.00191T2 - 0.105TS (11) 0.9248ara in WS 8.023- 0.0379T - 0.732S (12) 0.8531

a Units (unless otherwise indicated): % of wood (w/w). Abbreviations: sub, substrate; KL, Klason lignin in substrate (% of substrate); glu, glucose; EOL,ethanol organosolv lignin; AL, acid-soluble lignin; WS, water-soluble fraction; xyl, xylose; man, mannose; gal, galactose; ara, arabinose.b T, temperature(°C); t, time (min) at that temperature;S, sulfuric acid (%, w/w, oven-dried wood);C, ethanol concentration (%, v/v).

Y ) a0 + ∑i)1

k

aiXi + ∑i)1

k

aiiXi2 + ∑

i)1

k-1

∑j)1

k

aijXiXj (1)

2616 Ind. Eng. Chem. Res., Vol. 46, No. 8, 2007

Acknowledgment

The authors thank Dr. Jae-Jin Kim and Dr. Colette Breuilfor generously providing the lodgepole pine trees used in thepresent study. The support of NSERC (Natural Sciences andEngineering Research Council of Canada), NRCan (NaturalResources Canada), and BIOCAP (Biological Capital) CanadaFoundation is gratefully acknowledged.

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ReceiVed for reView December 7, 2006ReVised manuscript receiVed February 2, 2007

AcceptedFebruary 9, 2007

IE061576L

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