Ind. Eng. Chem. Res.

2007, 46, 2609-2617 2609

Pretreatment of Lodgepole Pine Killed by Mountain Pine Beetle Using the

Ethanol 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 was
optimized 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 of
excessive 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 for
the recovery of glucose and ethanol organosolv lignin. At the center-point conditions, ∼75% of the cellulose
present in the untreated wood was recovered in the substrate fraction, and approximately 79% of the lignin
in the wood was recovered as ethanol organosolv lignin (EOL). The combined recovery of carbohydrate in
the 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 levulinic
and 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 pretreatment is to produce a cellulose-rich substrate that is

susceptible to enzymatic hydrolysis (i.e., to provide fast conver-
Lodgepole pine (LPP, Pinus contorta) is the most com-
sion of cellulose to glucose at low enzyme loading).10 In
mercially important tree species in British Columbia (BC),
addition, good recovery of hemicellulosic sugars and isolation
Canada, covering 14.9 of 60 million ha of forested land in BC.1
of high-quality lignin are important. Development of high-value
However, the current outbreak of mountain pine beetle (MPB,
coproducts from hemicellulose and lignin is critical to improving
Dendroctonus ponderosae) in BC has infested about 7 million
the economics of bioconversion processes.
ha of LPP and is still accelerating. It is forecasted that 80% of
Among the pretreatment technologies currently being evalu-
LPP will be killed by MPB by the middle of the next decade.1
ated is an ethanol organosolv process. The process was originally
Although the beetle infestation does not significantly affect the
developed as a chemical process for fractionating lignocellu-
physical strength of the wood, the fungi associated with MPB
losics using organic solvents11 and was later adapted as a pulping
produce melanin and cause a blue to black discoloration of
method.12 The Alcell process further refines ethanol organosolv
sapwood, thus reducing its value in the market.2 Furthermore,
pulping as an alternative to Kraft pulping of hardwoods to
the beetle-killed trees are easy for decay fungi to colonize,
produce high-quality fibers.13,14 The organosolv process has not
further reducing the quality and value of the wood.3 In addition,
been developed significantly for softwoods, however, because
leaving killed trees untouched increases the risk of wild fire.
of the higher lignin content and difference in lignin structure.15
However, large-scale salvage operation will produce an abun-
Historically, the organosolv process has been investigated largely
dance of stained wood that exceeds the normal market ability
from the perspective of pulp production,16-19 but it is not well-
for consumption, and therefore, new markets and uses in
studied as a pretreatment/biorefining tool for the bioconversion
addition to timber and pulp have to be found.
of lignocellulosic biomass.
Biorefining of lignocellulosic biomass to produce fuels,
chemicals, and materials is believed to be a potential alternative Recently, the pretreatment of hardwood hybrid poplar using
to the current dependence on petroleum oil.4,5 The bioconversion the ethanol organosolv process was investigated in detail from
of biomass to ethanol through a “carbohydrate platform” is of the angles of the recovery of carbohydrate and lignin, the
current interest. A typical bioconversion process consists of enzymatic hydrolyzability of the substrates, and the effects of
pretreatment, saccharification, fermentation, and ethanol distil- process variables on substrate characteristics.20,21 The ethanol
lation steps. Several bioconversion schemes have been proposed, organosolv lignin from the poplar was evaluated as an antioxi-
including steam explosion, ammonia fiber explosion (AFEX), dant.22 The efficacy of ethanol organosolv pretreatment for
dilute acid or hot water treatment, and the organosolv process.6-9 mixed softwoods (spruce, pine, and Douglas fir) was demon-
A primary technical-economic challenge in all lignocellulosics- strated in our previous research as well.6 The process produced
to-ethanol bioconversion processes is the development of cost- a substrate with superior enzymatic digestibility over those
effective pretreatment methods. The main objective of a pretreated by alternative processes and a particularly high-quality
lignin fraction with potential applications.23-25 However, the
* To whom correspondence should be addressed. Tel.: 608-262-
ethanol organosolv pretreatment of softwood has not been
4951. Fax: 608-262-1228. E-mail: xpan@wisc.edu. optimized, and the process mass balance data are not available.
†
University of Wisconsin-Madison. The objective of the present research was to further investigate
‡ the ethanol organosolv pretreatment of softwood using mountain-
University of British Columbia.

10.1021/ie061576l CCC: $37.00 © 2007 American Chemical Society

Published on Web 03/14/2007
2610 Ind. Eng. Chem. Res., Vol. 46, No. 8, 2007
Table 1. Chemical Composition of Untreated MPB-LPP
component contenta
ash 0.26 ( 0.01
extractives (water followed by ethanol) 4.66 ( 0.21
Klason lignin 24.79 ( 0.09
acid-soluble lignin 0.29 ( 0.00
carbohydrate (as monosaccharide)
glucose 50.46 ( 0.25
mannose 13.09 ( 0.24
xylose 7.21 ( 0.04
galactose 2.22 ( 0.01
arabinose 1.42 ( 0.00
carbohydrate (as polysaccharide)
glucan 45.42 ( 0.22
mannan 11.78 ( 0.21
xylan 6.34 ( 0.03
galactan 2.00 ( 0.01
arabinan 1.25 ( 0.00
a
Content reported as % (w/w) in oven-dried MPB-LPP chips.
pine-beetle-killed lodgepole pine (MPB-LPP) as a feedstock.
In addition, the compositional and morphological differences
between beetle-killed wood and healthy wood might impact the
pretreatment, i.e., the infested sapwood has lower lignin,
carbohydrate, and extractives contents but increased permeability
compared to sound sapwood.26 The study described herein used
response 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 pretreatment
were examined, and the process mass balance and enzymatic
hydrolysis of the resulting substrate were investigated. Math-
ematical equations were regressed to quantitatively predict the
effects of the process variables on the fractionation and substrate
characteristics.
2. Materials and Methods
Feedstock Preparation. Lodgepole pine trees infested by
mountain 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 without
heart rot (one from Burns Lake, two from Prince George, and
four from Quesnel) were used in the present research. The
average age of the trees was 99 ( 25 years. Two sections, one
from the top part and another from the bottom part, were
collected 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 fraction
larger than 2.5 × 2.5 cm and smaller than 5.0 × 5.0 cm,
approximately 0.5 cm thick, was collected as the feedstock for
ethanol organosolv pretreatment. A sample of the chips was
ground using a Wiley mill, and the fraction passing 40-mesh
was collected for chemical analysis. The chemical composition
of the wood is summarized in Table 1.
Ethanol Organosolv Pretreatment. A flowchart of the
laboratory-scale ethanol organosolv process was schematically
described before.20 In brief, MPB-LPP chips were pretreated
in aqueous ethanol with sulfuric acid as a catalyst using a
custom-built, four-vessel (2 L each) rotating digester made by
Aurora 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 temperature
in 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 were
then separated using nylon cloth. The substrate was washed three
times (300 mL each) with warm (60 °C) aqueous ethanol having
the same concentration as the pretreatment liquor. The washes
were combined with the spent liquor. The substrate was then
washed three times with water at 60 °C, and the washes were
discarded. The washed substrate was homogenized in a standard
British disintegrator for 5 min and passed through a laboratory
flat screen with 0.008-in. (0.203-mm) slits (Voith, Inc., Apple-
ton, WI) to remove rejects (i.e., non-defiberized woodchips and
knots). The yields of rejects and screened substrate were
determined. The screened substrate was stored at 4 °C for
analysis and hydrolysis.
The spent liquor and the ethanol washes were combined and
mixed with three volumes of water to precipitate the dissolved
lignin. The lignin precipitate, henceforth denoted as ethanol
organosolv lignin (EOL), was collected on Whatman No. 1 filter
paper, washed thoroughly with water, and air-dried. The filtrate
and the water washes were combined to give a water-soluble
fraction containing monomeric and oligomeric saccharides,
depolymerized lignin, and compounds derived from saccharides.
Analytical Procedures. Oven-dried weights were determined
by 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) standard
method T211 om-93. Extractives of the wood powder were
determined according to the procedure of TAPPI standard
method T264 cm-97 using ethanol and water as solvents. Klason
lignin of the wood powder and substrates was determined
according to TAPPI standard method T-222 om-98. The
hydrolysate from Klason lignin determination was retained for
analysis of monosaccharides and acid-soluble lignin. Acid-
soluble lignin was determined by UV absorbance at 205 nm
according to the method described by Dence.27 Monosaccharides
were determined using a DX-500 HPLC system (Dionex,
Sunnyvale, CA) equipped with an AS3500 autosampler, a GP40
gradient pump, an anion-exchange column (Dionex CarboPac
PA1), and an ED40 electrochemical detector. The column was
eluted with deionized water at a flow rate of 1 mL/min. Aliquots
(20 µL) were injected after being passed through a 0.45-µm
nylon syringe filter (Chromatographic Specialties Inc., Brock-
ville, ON, Canada). Optimization of baseline stability and
detector sensitivity was achieved by postcolumn addition of 0.2
M NaOH. The column was reconditioned using 1 M NaOH
after each analysis. Monosaccharides were quantified with
reference to saccharide standards. The saccharide standards were
autoclaved at 120 °C for 1 h prior to analysis to compensate
for possible decomposition caused by heating involved in Klason
lignin determination. Furfural and HMF were determined using
a Dionex Summit HPLC system equipped with a P680 pump,
an ASI-100 autosampler, and a PDA100 photodiode array
detector. A LiChrospher 5RP18 column (Varian, Palo Alto, CA)
was used at 60 °C with an eluent flow rate of 0.5 mL/min. A
gradient of (A) 7.4 mM H3PO4, (B) acetonitrile, and (C) a
mixture 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% C
to 100% C; 24-25 min, 100% C; 25-26 min, from 100% C to
100% B; 26-27 min, 100% B; 27-28 min, from 100% B to
95% A and 5% C; 28-38 min, 95% A and 5% C. Appropriately
diluted aliquots (20 µL) were injected after being passed through
a 0.45-µm PTFE syringe filter (Chromatographic Specialties
Inc.). Furfural and HMF were determined by absorbance at 280
nm. Formic and levulinic acids were quantified using an
 Ind. Eng. Chem. Res., Vol. 46, No. 8, 2007 2611

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 1.26
2 180 50 0.90 75 35.37 4.98 35.04 0.75 21.95 7.65 5.31 2.60 4.96 1.42 0.60
3 160 70 0.90 55 11.12 22.84 8.50 44.93 11.31 3.87 2.91 3.82 6.72 2.18 1.18
4 180 70 0.90 55 37.73 11.22 35.22 0.04 19.30 6.31 6.94 1.60 3.56 1.35 0.50
5 160 50 1.30 55 9.91 22.88 7.59 43.44 11.30 2.93 2.64 4.59 8.18 2.47 1.38
6 180 50 1.30 55 38.53 12.17 35.01 0.29 18.40 5.43 4.63 0.94 2.50 0.91 0.25
7 160 70 1.30 75 41.53 13.24 35.72 3.89 18.00 3.98 3.68 3.81 6.30 2.12 1.04
8 180 70 1.30 75 31.23 7.92 29.54 0.05 24.96 3.01 5.25 0.32 1.49 0.46 0.06
9 153 60 1.10 65 7.15 20.91 5.09 52.79 12.33 4.55 3.04 3.06 5.26 2.06 1.15
10 187 60 1.10 65 27.58 11.15 25.18 0.06 23.41 6.06 5.36 0.25 1.03 0.27 0.11
11 170 43 1.10 65 43.04 12.75 37.51 2.26 18.40 4.25 5.30 2.57 4.49 1.70 0.74
12 170 77 1.10 65 40.75 8.80 36.69 0.46 20.17 6.46 3.42 2.78 5.16 1.56 0.69
13 170 60 0.76 65 41.56 13.26 36.72 6.13 17.86 3.82 2.79 4.30 7.65 2.27 1.20
14 170 60 1.44 65 39.24 8.21 36.91 0.66 20.51 5.45 4.24 1.99 3.72 1.29 0.49
15 170 60 1.10 48 33.51 22.80 25.51 15.22 11.76 4.22 5.41 3.75 6.18 2.03 0.98
16 170 60 1.10 82 39.81 10.08 35.09 1.63 20.08 5.15 4.85 3.78 6.07 1.81 0.87
17 170 60 1.10 65 41.48 10.34 38.30 1.22 19.67 4.69 4.95 3.39 5.53 1.74 0.83
18 170 60 1.10 65 41.73 10.61 37.48 0.93 20.24 4.66 4.73 3.28 5.25 1.80 0.79
19 170 60 1.10 65 40.27 8.94 37.11 1.14 19.03 4.71 3.84 3.32 5.47 1.70 0.79
20 170 60 1.10 65 41.72 10.23 37.41 1.24 19.30 5.07 3.06 2.72 4.84 1.53 0.63
21 170 60 1.10 65 41.26 10.34 37.87 0.93 19.61 4.69 4.36 3.44 5.75 1.90 0.85
avg 17-21 41.29 10.09 37.63 1.09 19.57 4.76 4.19 3.23 5.37 1.73 0.78
SD 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) per
100 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.

Allilance 2695 HPLC system (Waters, Milford, MA) equipped
with an AD20 absorbance detector (Dionex, Sunnyvale, CA),
a SUPELCO (Bellefonte, PA) SUPELCOGEL C610H column,
and a SUPELGUARD C601H guard column. The column
temperature was 30 °C, and the mobile phase was 0.1% H3PO4
at a flow rate of 0.5 mL/min. Sample (5 µL) was injected onto
the column after being filtered through a 0.45-µm nylon syringe
filter as described above. The viscosity (degree of polymeriza-
tion) of the cellulose solution was measured according to TAPPI
standard method T230 om-99, as described before.21
Enzymatic Hydrolysis. Commercial cellulase (Spezyme CP)
and β-glucosidase (Novozym 188) were provided by Genencor
International Inc. (Rochester, NY) and Novozymes (Franklinton,
NC), respectively. Cellulase activity was determined using the
filter paper assay recommended by the International Union of
Pure and Applied Chemists28 and is expressed in terms of filter
paper units (FPUs). β-Glucosidase activity was determined using
p-nitrophenyl-β-D-glucoside as the substrate, as previously
described,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 with
β-glucosidase (1:2 FPU/IU) to avoid product inhibition caused
by cellobiose accumulation.
Batch hydrolysis was conducted at 2% consistency (cellulose,
w/v) in 50 mM acetate buffer, pH 4.8, with 0.004% tetracycline
as an antibiotic. Cellulase was used at a loading of 20 FPU (21
mg of total protein)/g of cellulose with a supplementation of
β-glucosidase at a loading of 40 IU (5.7 mg of total protein)/g
of cellulose. The reaction mixture (100 mL) was incubated at
150 rpm, 50 °C, in a rotary shaker and sampled periodically
for glucose determination. The glucose was quantified using
HPLC, as described above, with the exception that the saccharic
standards were not autoclaved. Hydrolysis data are averages
from duplicate experiments.
Experimental Design and Data Analysis. Pretreatment
conditions [temperature, time, catalyst (H2SO4) dosage, and
ethanol concentration] were optimized by response surface
methodology using a small Hartley composite design,31 as
described in detail in our previous report.20 The complete
experimental matrix is shown in Table 2. The selection of the
conditions in Table 2 was based on the results of preliminary
experiments (see Result and Discussion). Data were analyzed
using Statistical Analysis System (SAS, SAS Institute Inc., Cary,
NC). All data reported are the averages of at least duplicate
experiments.
3. Results and Discussion
Preliminary Experiments for Selecting Process Conditions.
As shown in Table 1, MPB-LPP has the typical chemical
composition of softwood. Compared to hybrid poplar (hard-
wood),20 MPB-LPP has more total lignin (more Klason lignin
but less acid-soluble lignin) and a comparable amount of total
carbohydrate but with a different composition. MPB-LPP has
significantly more mannan and substantially less xylan than
poplar. Previous research has indicated that the set of conditions
(180 °C, 60 min, 1.25% H2SO4, 50% ethanol) is close to the
optimum for poplar in the recovery of cellulose, hemicellulose,
and lignin.20 In general, softwood requires more severe condi-
tions for delignification than hardwood because softwood
contains more lignin and, in particular, more guaiacyl units that
tend to condense. Therefore, the pretreatment of MPB-LPP was
started from more severe conditions than those used for poplar.
As shown in Table 3, test 1 was conducted at 185 °C, 80
min, 1.5% H2SO4, and 60% ethanol. The substrate yield was
only ∼35%, but residual lignin was high (∼27% Klason lignin).
Increasing H2SO4 to 2.34% resulted in an additional reduction
in substrate yield and increase in Klason lignin (test 2), implying
the removal of more carbohydrate than lignin. It was noted that
the substrates from tests 1 and 2 had no rejects and residual
hemicellulose. However, a significant amount of acid-soluble
lignin was observed in the water-soluble fraction as a result of
the extensive destruction of lignin. These results suggest that
the conditions used in tests 1 and 2 were too severe for MPB-
LPP. When the temperature was lowered to 170 °C (test 3), the
2612 Ind. Eng. Chem. Res., Vol. 46, No. 8, 2007

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 ND
2 185 80 2.34 60 29.30 0.00 18.22 5.37 31.99 77.53 ND ND
3 170 80 1.50 60 38.80 0.15 20.90 4.85 10.55 97.49 ND ND
4 170 80 1.20 60 40.30 0.58 19.20 4.27 11.62 93.73 ND ND
5 170 40 1.20 60 42.55 3.14 17.10 4.32 14.19 83.73 0.97 1.05
6 170 80 1.20 70 38.52 0.22 22.00 5.17 7.40 96.14 1.30 1.40
7 170 60 1.00 70 40.75 2.24 21.25 4.61 9.21 91.09 1.02 1.12
8 160 60 1.00 70 14.06 39.05 15.20 3.74 18.43 76.42 2.34 2.87
9 160 100 1.00 70 38.82 8.56 18.13 4.49 13.93 89.16 2.77 3.04
10 190 120 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.

substrate yield increased, and the Klason lignin decreased

significantly. The reduction of catalyst dosage to 1.2% H2SO4
(test 4) resulted in additional improvement in substrate yield
but a slight increase in Klason lignin. Still, no hemicellulose
was detected in the substrates of tests 3 and 4. Compared to
test 4, a 40-min reduction of reaction time in test 5 increased
the substrate yield, but this yield increase was primarily from
the 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 dissolution
of lignin from wood chips was enhanced. Further reducing the
severity of test 6 by decreasing the reaction time by 20 min
and the catalyst by 0.2% improved neither the recovery of
carbohydrate nor the delignification (test 7). Lowering the
temperature by 10 °C (test 8) resulted in a very high rejects
(non-defiberized wood chips) rate of ∼39%, indicating a failure
of defiberization. Extension of the reaction time by 40 min (test
9) did reduce the rejects rate, thus improving the pulp yield,
but did not improve delignification (i.e., Klason lignin was still
high). The MPB-LPP was also pretreated at extremely severe
conditions of 190 °C, 120 min, 2.5% H2SO4, and 60% ethanol
(test 10). The resulting substrate (<29% yield) was composed
dominantly of lignin (∼64% Klason lignin). The reason is that
carbohydrate (both cellulose and hemicellulose) was excessively
hydrolyzed and subsequently converted to furfural, hydroxy-
methylfurfural (HMF), and organic acids. Extensive degradation
of 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 discussion
above, the center-point conditions used in the experimental
matrix for MPB-LPP were selected as 170 °C, 60 min, 1.1%
H2SO4, and 65% ethanol.
Optimization of the Ethanol Organosolv Pretreatment of
MPB-LPP. To optimize the ethanol organosolv pretreatment Figure 1. Effects of process variables on Klason lignin in the substrate
of MPB-LPP, an experimental matrix was designed using fraction. The fixed variables are as follows: (A) 170 °C and 1.1% H2SO4,
(B) 60 min and 65% ethanol.
response surface methodology, as described in the Materials
and Methods section and summarized in Table 2. The center- discussed elsewhere,20 delignification during the ethanol organo-
point conditions were those selected in the preliminary experi- solv pretreatment is a combination of depolymerization and
ments above. The ranges of the conditions were as follows: solubilization of lignin. Lower ethanol concentration, which
temperature, 153-187 °C; time, 43-77 min; H2SO4, 0.76- generates higher hydrogen ion concentration (lower pH value)
1.44% (w/w); ethanol, 48-82% (v/v). The ratio of liquor to at the same dosage of H2SO4, promotes acid-catalyzed cleavage
wood was constant (7:1, v/w) in all experiments. of R- and β-ether linkages in the lignin,32 whereas high ethanol
The effects of process variables on delignification are concentration increases solubilization of the lignin.33,34 As shown
demonstrated in Figures 1 (Klason lignin in substrate) and 2 in Figure 1A, Klason lignin in the substrate reached the lowest
(yield of recovered ethanol organosolv lignin, EOL). Figure value at ∼75% ethanol concentration (turning point) and then
1A,B clearly shows that reaction time and catalyst (sulfuric acid) slightly increased. The turning-point concentration was higher
did not have substantial effect on the Klason lignin of the than that of hybrid poplar (∼65%),20 indicating that MPB-LPP
substrates, but ethanol concentration and temperature did. As lignin requires a higher ethanol concentration for solubilization

Figure 2. Effects of process variables on the yield of ethanol organosolv
lignin (EOL) precipitated after the pretreatment. The fixed variables are as
follows: (A) 170 °C and 1.1% H2SO4, (B) 60 min and 65% ethanol.
than poplar lignin. The temperature showed a similar effect on
delignification (Figure 1B). The lowest Klason lignin was
observed at ∼182 °C, which is lower than the ∼195 °C value
for poplar.20 This suggests that MPB-LPP is easier to delignify
than poplar. The mechanism is unclear, although beetle infesta-
tion might be a cause. When the temperature was increased
beyond the turning point, Klason lignin in the substrate
increased. An explanation for this behavior is that carbohydrate
is hydrolyzed faster than lignin at high temperature, resulting
in a relative increase in Klason lignin. The effects of reaction
time and ethanol concentration on the yield of EOL are
consistent with those on Klason lignin, as shown in Figure 2A,
i.e., the lignin yield was independent of time, and a maximum
yield was obtained at ∼75% ethanol concentration. On the other
hand, the effects of temperature and sulfuric acid on EOL yield
were different from those on Klason lignin, as shown in Figure
2B. Lignin yield increased linearly with increasing catalyst
content as a result of acid-promoted cleavage and subsequent
dissolution of lignin. The lignin yield kept increasing as the
temperature rose, and no turning point was observed within the
range investigated.
The effects of process parameters on the yield of substrate
are shown in Figure 3. The substrate yield decreased consistently
with the extension of the reaction time because more lignin and
carbohydrate were dissolved, as shown in Figure 3A. On the
other hand, the substrate yield increased with increasing ethanol
concentration and reached a maximum at ∼70% ethanol
Ind. Eng. Chem. Res., Vol. 46, No. 8, 2007 2613
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) 60
min and 65% ethanol.
concentration. This observation is consistent with the progress
of delignification discussed with respect to Figures 1 and 2
above. With the removal of lignin binding the fibers together,
wood chips were defiberized, thus resulting in more substrate
and less rejects. However, when the ethanol concentration was
higher than the turning point, the delignification was depressed,
consequently resulting in a low substrate yield and a high rejects
rate. As shown in Figure 3B, the substrate yield increased rapidly
as the temperature rose as a result of defiberization enhanced
by the removal of lignin, but in contrast, it declined when the
temperature was over ∼175 °C because of the excessive
hydrolysis of carbohydrate. The effect of sulfuric acid on
substrate yield was dependent on temperature. When the
temperature was lower than the turning point (∼175 °C), more
sulfuric 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 of
carbohydrate rather than delignification, thus giving a low
substrate yield.
Chemical analysis of the substrates (data not shown here)
indicated that only small amounts of hemicellulosic sugars were
detected in the substrates, and over 90% of xylose and mannose
and almost 100% of galactose and arabinose were removed from
the wood chips during the pretreatment. In contrast, a significant
amount of hemicellulose, particularly xylan and mannan, was
retained in the hybrid poplar substrates.20 Because the conditions
2614 Ind. Eng. Chem. Res., Vol. 46, No. 8, 2007
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 of
oven-dried wood (%). Ara, arabinose; Gal, galactose; Man, mannose; Xyl, xylose; and Glu, glucose.
used for MPB-LPP were milder than those used for hybrid
poplar, as mentioned above, it appears that the hemicellulose
in MPB-LPP was easier to remove (hydrolyze) than that in
hybrid poplar. The effects of process parameters on the
recovered hemicellulosic sugars in the water-soluble fraction
are demonstrated in Figure 4. All hemicellulosic sugars except
glucose followed the same pattern, i.e., their yields in the water-
soluble fraction were less sensitive to reaction time and ethanol
concentration, but significantly dependent on temperature and
catalyst. The yields of non-glucose sugars decreased consistently
with the increases in temperature and catalyst dosage, indicating
that the decomposition of the sugars was promoted. On the other
hand, the yield of glucose in the water-soluble fraction increased
when temperature and catalyst increased or ethanol concentration
decreased. This observation suggests that hydrolysis of cellulose
to glucose is faster than the subsequent decomposition of glucose
under such conditions.
The results of the preliminary experiments (Table 3) and the
matrix experiments (Table 2) and the discussions above indicate
that the center-point set of conditions was close to the optimal
set 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 similar
or higher yields, but the high yields resulted from more residual
lignin (Klason lignin). When comparing the recovery of cellulose
(glucose in the substrates), it is clear that none of the condition
sets 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 yields
than the center point, but the cost was lower substrate (cellulose)
yields caused by excessive destruction of carbohydrate.
Mass Balance of Ethanol Organosolv Pretreatment of
MPB-LPP. Five center points (numbers 17-21 in Table 2) were
evaluated 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 the
optimum, as discussed above. Therefore, analysis of the mass
balance at the center point is meaningful and conclusive. As
shown 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 substrates
from MPB-LPP and poplar, respectively).20 Approximately 17%
of the total Klason lignin in the untreated wood remained in
the substrate after the pretreatment, compared to ∼27% in
poplar.20 Considering the fact that MPB-LPP had more lignin
and was pretreated under milder conditions than poplar, the
results suggest the easy delignification of MPB-LPP. The
majority (∼79%) of the lignin was dissolved during the
pretreatment and recovered by precipitation as EOL. The acid-
soluble lignin in the water-soluble fraction represented the
products from the extensive depolymerization of lignin.
Approximately 75% of the original glucose in the wood was
recovered in the substrate fraction, and ∼8% of the glucose
was detected in the water-soluble fraction, compared to 88%
and 1%, respectively, for poplar,20 suggesting that more cellulose
was hydrolyzed from MPB-LPP than from poplar during the
pretreatment. The data in Figure 5 indicate that the carbohydrate
in the substrate fraction was composed primarily of cellulose.
Only a minority of the original hemicellulose (∼8% of the
xylose and ∼5% of the mannose) in the untreated wood survived
from the hydrolysis and remained in the substrate, values that
 Ind. Eng. Chem. Res., Vol. 46, No. 8, 2007 2615

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).
are substantially lower than those for poplar (∼19% of the
xylose and ∼38% of the mannose, respectively).20 It is clear
that the recovery of less hemicellulose and cellulose was the
cause of the low substrate yield for MPB-LPP. Part of the
dissolved 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 in
the water-soluble fraction. The majority of the hemicellulosic
sugars 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 substrate
and water-soluble fractions was ∼83% glucose, ∼46% mannose,
∼53% xylose, ∼78% galactose, and ∼55% arabinose, indicating
significant loss of carbohydrate during the pretreatment. It is
well-known that monosaccharides undergo thermal de-
composition under acidic conditions, generating hydroxy-
methylfurfural (HMF) and furfural, derived from hexoses and
pentoses, respectively.35 Further decomposition of HMF results
in levulinic and formic acids.36 As shown in Figure 5, all of
these compounds were detected in the water-soluble fraction.
The significant amount of levulinic acid and the relatively small
amount of HMF indicate that most of the hexoses (primarily Figure 6. Enzymatic hydrolysis of selected substrates prepared from MPB-
glucose and mannose) were quickly decomposed to the final LPP using ethanol organosolv pretreatment with enzyme loadings of 20
products (levulinic and formic acids). FPU of cellulase (21 mg of total protein) and 40 IU of β-glucosidase (5.7
mg of total protein)/g of cellulose. The conditions used to prepare the
Enzymatic Hydrolysis of Ethanol Organosolv Substrate substrates are listed in Table 2.
from MPB-LPP. Enzymatic hydrolysis results for selected
substrates prepared in the experimental matrix are shown in conversion yield was much lower (∼82% within 48 h). These
Figure 6, including numbers 2, 7, 9, 15, and 20. It was observed results indicate that, although residual lignin is an important
that the substrate generated at the center point (number 20) was factor influencing enzymatic hydrolysis, hydrolyzability is not
readily digestible, and cellulose-to-glucose conversion yields proportional to lignin. This is strongly supported by the results
of ∼93% and ∼97% were achieved within 24 and 48 h, of numbers 9 and 15. Substrate number 15 had significantly
respectively. The enzymatic hydrolyzability was similar to that more lignin (∼23%) than substrate number 7 (∼13%), but their
of hybrid poplar substrate.20 Substrate number 2 had the lowest hydrolysis profiles were very similar. On the contrary, substrate
Klason lignin (∼5%), approximately half that of number 20 number 9 contained slightly less lignin (∼21%) than substrate
(∼10%), thus exhibiting a better hydrolyzability (complete number 15, but the former had much worse hydrolyzability than
conversion within 48 h). On the other hand, substrate number the latter, i.e., the 48-h conversion of number 9 was less than
7 had only 3% more lignin than substrate number 20, but the 35%.
2616 Ind. Eng. Chem. Res., Vol. 46, No. 8, 2007

Table 4. Predictive Models for Process Responses

responsea equationb R2
sub yield -373.841 + 3.812T + 0.190t - 2.376S + 1.387C - - 0.00239Tt - 0.088TS -
0.00887T2 (2) 0.9907
0.00740TC + 0.192tS + 0.092SC - 0.00166C2
KL in sub 734.723 - 6.839T - 2.983C + 0.0189T2 + 0.0203C2 (3) 0.8991
glu in sub -60.736 + 0.630T + 0.0256t + 0.0636S + 0.218C - 0.00147T2 - 0.000394Tt - 0.0157TS - (4) 0.9986
0.00114TC + 0.0296tS + 0.000131 tC + 0.0128SC - 0.000332C2
rejects 509.379 - 4.376T - 0.552t - 49.772S - 2.106C + 0.00953T2 + 0.003Tt + 0.206TS + 0.00776TC + (5) 0.9936
5.569S2 + 0.00550C2
EOL yield -283.062 + 2.346T + 3.823S + 1.877C - 0.00586T2 - 0.0126C2 (6) 0.9604
AL in WS -221.427 + 0.806T + 1.957t + 86.463S + 1.271C - 0.00641Tt - 0.313TS - 0.0123tC - 0.474SC (7) 0.8719
glu in WS 5.628 + 0.109T - 17.293S - 0.318C + 0.276SC (8) 0.6279
xyl in WS -175.25 + 1.978T + 38.086S - 0.00538T2 - 0.237TS (9) 0.8679
man in WS -241.234 + 2.726T + 56.370S - 0.00736T2 - 0.353TS (10) 0.8531
gal in WS -62.404 + 0.709T + 16.839S - 0.00191T2 - 0.105TS (11) 0.9248
ara 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).

To explain the above observations, other substrate charac-
teristics were investigated, including crystallinity, fiber size,
residual hemicellulose, and degree of polymerization (DP) of
cellulose. Although there were no significant differences in
crystallinity, fiber size, and hemicellulose among the samples
above (data not included), DP was found to be a significant
factor affecting hydrolysis. As described in the Materials and
Methods section, DP was measured in the present research using
the viscosity of cellulose. As shown in Figure 6, the viscosity
of substrate number 9 (20.7 mPa S) was as almost 7 times as
high as that of number 15 (3.1 mPa S), which was the major
cause of poor hydrolyzability of number 9. Furthermore, the
viscosity difference between number 7 and number 15 explains
the similarity of their hydrolyzabilities, even though the former
had more lignin than the latter. In addition to its low lignin
content, the low viscosity of number 2 was another contributor
to its excellent hydrolyzability. It is believed that longer cellulose
chains (higher DP) form stronger networks by more extensive
inter- and intramolecular hydrogen bonding, thereby limiting
the accessibility of cellulose to the enzymes.37 On the other hand,
the reduction of DP increased the number of cellulose chain
ends available to the action of exoglucanase in the cellulase
complex, thus generating a high reaction rate and a high glucose
yield.38-40
Predictive Modeling. The effects of pretreatment conditions
on the various process responses (e.g., yields of substrates, EOL,
and hemicellulosic sugars in the water-soluble fraction and
chemical compositions of the substrates) were qualitatively
discussed above. To quantitatively predict the effects of each
process variable on the responses, each process response was
fitted to a second-order polynomial equation using SAS
software, as shown in eq 1
k k k-1 k might change the lignin and carbohydrate physically and
Y ) a0 + ∑ aiXi + ∑aiiXi2 + ∑ ∑aijXiXj (1)
i)1 i)1 i)1 j)1
where Y is the estimated value of the process response; k is the
total number of independent variables (4 in this case); Xi
represents the independent variables (temperature, time, catalyst
dose, and ethanol concentration); Xi, Xi2, and XiXj are terms
describing linear, quadratic, and two-variable interaction effects,
respectively; a0 is a constant; and ai, aii, and aij are linear,
quadratic, and interaction coefficients, respectively. The predic-
tive models (eqs 2-12) developed for each response are listed
in Table 4. These equations can be used to predict the responses
to all combinations of variables within the investigated range.
They are also useful tools for fine-tuning the pretreatment, as
discussed previously.20
4. Conclusions
Process optimization using the response surface methodology
indicates 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 in
the solids fraction and ethanol organosolv lignin. At the center-
point conditions, ∼75% of the cellulose present in the untreated
wood was recovered in substrate fraction. The combined
recovery of sugars in the substrate and water-soluble fractions
was ∼83% glucose, ∼46% mannose, ∼53% xylose, ∼78%
galactose, and ∼55% arabinose, respectively. The sugar recovery
was significantly lower than that of hybrid poplar reported
previously.20 The lost carbohydrate was decomposed to furfural,
HMF, levulinic and formic acids, and other products. These are
potential high-value coproducts of the organosolv pretreatment.
Approximately 79% of the lignin in wood was recovered as a
precipitate (ethanol organosolv lignin) following the pretreat-
ment. A substantial amount (corresponding to ∼17% of the total
lignin in untreated wood) of acid-soluble lignin was detected
in the water-soluble fraction. The acid-soluble lignin represents
the low-molecular-weight fraction of the lignin and will be
evaluated as an antioxidant.
It was observed that MPB-LPP behaved differently than
hybrid poplar during the ethanol organosolv pretreatment.
Despite its high lignin content, MPB-LPP was easier to pretreat
than poplar, requiring milder conditions for delignification. In
addition, more cellulose and hemicellulose were hydrolyzed and
subsequently decomposed during the pretreatment, which was
the primary cause for the low substrate yield of MPB-LPP. A
proposed explanation is that the fungi associated with the beetles
chemically. A detailed investigation is required to understand
the effects of beetle infestation on the bioconversion of MPB-
LPP.
The substrate generated from MPB-LPP at the center-point
conditions was readily digestible. Cellulose-to-glucose conver-
sion yields of ∼93% and ∼97% were achieved within 24 and
48 h, respectively, with an enzyme loading of 20 FPU of
cellulase/g of cellulose. It appears that a low degree of
polymerization of cellulose was an important contributor to the
ready digestibility of the organosolv substrate. A detailed
investigation toward a fundamental understanding of the ready
digestibility of ethanol organosolv substrates will be reported
later.

Acknowledgment
The authors thank Dr. Jae-Jin Kim and Dr. Colette Breuil
for generously providing the lodgepole pine trees used in the
present study. The support of NSERC (Natural Sciences and
Engineering Research Council of Canada), NRCan (Natural
Resources Canada), and BIOCAP (Biological Capital) Canada
Foundation is gratefully acknowledged.
Literature Cited
(1) McGarrity, K.; Hoberg, G. The beetle challenge: An overview of
the mountain pine beetle epidemic and its implications. Available at http://
www.policy.forestry.ubc.ca/issuebriefs/pinebeetle.html (accessed Nov 20,
2006).
(2) Zink, P.; Fengel, D. Studies on the Coloring Matter of Blue-Stain
Fungi. 1. General Characterization and the Associated Compounds.
Holzforschung 1988, 42, 217.
(3) Kim, J. J.; Allen, E. A.; Humble, L. M.; Breuil, C. Ophiostomatoid
and basidiomycetous fungi associated with green, red, and grey lodgepole
pines after mountain pine beetle (Dendroctonus ponderosae) infestation.
Can. J. Forest Res. 2005, 35, 274.
(4) Ragauskas, A. J.; Williams, C. K.; Davison, B. H.; Britovsek, G.;
Cairney, J.; Eckert, C. A.; Frederick, W. J.; Hallett, J. P.; Leak, D. J.; Liotta,
C. L.; Mielenz, J. R.; Murphy, R.; Templer, R.; Tschaplinski, T. The path
forward for biofuels and biomaterials. Science 2006, 311, 484.
(5) Kamm, B.; Kamm, M. Principles of biorefineries. Appl. Microbiol.
Biotechnol. 2004, 64, 137.
(6) Pan, X. J.; Arato, C.; Gilkes, N.; Gregg, D.; Mabee, W.; Pye, K.;
Xiao, Z. Z.; Zhang, X.; Saddler, J. Biorefining of softwoods using ethanol
organosolv pulping: Preliminary evaluation of process streams for manu-
facture of fuel-grade ethanol and co-products. Biotechnol. Bioeng. 2005,
90, 473.
(7) Mosier, N.; Wyman, C.; Dale, B.; Elander, R.; Lee, Y. Y.;
Holtzapple, M.; Ladisch, M. Features of promising technologies for
pretreatment of lignocellulosic biomass. Bioresour. Technol. 2005, 96, 673.
(8) Wyman, C. E.; Dale, B. E.; Elander, R. T.; Holtzapple, M.; Ladisch,
M. R.; Lee, Y. Y. Coordinated development of leading biomass pretreatment
technologies. Bioresour. Technol. 2005, 96, 1959.
(9) Brownell, H. H.; Yu, E. K. C.; Saddler, J. N. Steam-Explosion
Pretreatment of WoodsEffect of Chip Size, Acid, Moisture Content and
Pressure Drop. Biotechnol. Bioeng. 1986, 28, 792.
(10) McMillan, J. D. Pretreatment of lignocelulosic biomass. In Enzy-
matic ConVersion of Biomass for Fuels Production; Himmel, M. E., Baker,
J. O., Overend, R. P., Eds.; American Chemical Society: Washington, DC,
1994; p 292.
(11) Kleinert, T. N.; Tayenthal, K. Process of decomposing vegetable
fibrous substances for the purpose of obtaining simultaneously the cellulose
and the incrusting ingredients. U.K. Patent 357,821, 1931.
(12) Kleinert, T. N. Organosolv pulping with aqueous alcohol. Tappi J.
1974, 57, 99.
(13) Pye, E. K.; Lora, J. H. The Alcell ProcesssA Proven Alternative
to Kraft Pulping. Tappi J. 1991, 74, 113.
(14) Stockburger, P. An Overview of Near-Commercial and Commercial
Solvent-Based Pulping Processes. Tappi J. 1993, 76, 71.
(15) Yawalata, D.; Paszner, L. Anionic effect in high concentration
alcohol organosolv pulping. Holzforschung 2004, 58, 1.
(16) Gilarranz, M. A.; Oliet, M.; Rodriguez, F.; Tijero, J. Ethanol-
water pulping: Cooking variables optimization. Can. J. Chem. Eng. 1998,
76, 253.
(17) Ni, Y.; van Heiningen, A. R. P. TCF bleaching for the Alcell(R)
process including oxygen delignification. Pulp Pap. Can. 1997, 98, 38.
(18) Diaz, M. J.; Alfaro, A.; Garcia, M. M.; Engenio, M. E.; Ariza, J.;
Lopez, F. Ethanol pulping from tagasaste (Chamaecytisus proliferus L.F.
ssp. palmensis). A new promising source for cellulose pulp. Ind. Eng. Chem.
Res. 2004, 43, 1875.
(19) Jimenez, L.; Perez, I.; Garcia, J. C.; Lopez, F.; Ariza, J. The
influence of the ethanol pulping of wheat straw and of the beating of pulp
on the resulting paper sheets. Wood Sci. Technol. 2004, 38, 127.
Ind. Eng. Chem. Res., Vol. 46, No. 8, 2007 2617
(20) Pan, X. J.; Gilkes, N.; Kadla, J.; Pye, K.; Saka, S.; Gregg, D.; Ehara,
K.; Xie, D.; Lam, D.; Saddler, J. Bioconversion of hybrid poplar to ethanol
and co-products using an organosolv fractionation process: Optimization
of process yields. Biotechnol. Bioeng. 2006, 94, 851.
(21) Pan, X. J.; Xie, D.; Kang, K. Y.; Yoon, S. L.; Saddler, J. N. Effect
of organosolv ethanol pretreatment variables on physical characteristics of
hybrid poplar substrates. Appl. Biochem. Biotechnol. 2007, in press.
(22) Pan, X. J.; Kadla, J. F.; Ehara, K.; Gilkes, N.; Saddler, J. N.
Organosolv ethanol lignin from hybrid poplar as a radical scavenger:
Relationship between lignin structure, extraction conditions, and antioxidant
activity. J. Agric. Food Chem. 2006, 54, 5806.
(23) Lora, J. H.; Glasser, W. G. Recent industrial applications of
lignin: A sustainable alternative to nonrenewable materials. J. Polym.
EnViron. 2002, 10, 39.
(24) Thring, R. W.; Vanderlaan, M. N.; Griffin, S. L. Polyurethanes
from Alcell(R) lignin. Biomass Bioenergy 1997, 13, 125.
(25) Kubo, S.; Kadla, J. F. Poly(ethylene oxide)/organosolv lignin
blends: Relationship between thermal properties, chemical structure, and
blend behavior. Macromolecules 2004, 37, 6904.
(26) Woo, K. L.; Watson, P.; Mansfield, S. D. The effects of mountain
pine beetle attack on lodgepole pine wood morphology and chemistry:
Implications for wood and fiber quality. Wood Fiber Sci. 2005, 37, 112.
(27) Dence, C. W. The determination of lignin. In Methods in Lignin
Chemistry; Lin, S. Y., Dence, C. W., Eds.; Springer-Verlag: Berlin, 1992;
p 33.
(28) Ghose, T. K. Measurement of cellulase activities. Pure Appl. Chem.
1987, 59, 257.
(29) Wood, T. M.; Bhat, M. Methods for measuring cellulase activities.
In Methods in Enzymology; Colowick, S. P., Kaplan, N. O., Eds.; Academic
Press: New York, 1988; Vol. 160, Biomass (Part a, Cellulose and
Hemicellulose), p 87.
(30) Bradford, M. M. Rapid and Sensitive Method for Quantitation of
Microgram Quantities of Protein Utilizing Principle of Protein-Dye
Binding. Anal. Biochem. 1976, 72, 248.
(31) Myers, R. H.; Montgomery, D. C. Response Surface Methodol-
ogy: Process and Product Optimization Using Designed Experiments;
Wiley: New York, 2002.
(32) McDonough, T. J. The chemistry of organosolv delignification.
Tappi J. 1993, 76, 186.
(33) Ni, Y.; Hu, Q. Alcell(R) lignin solubility in ethanol-water mixtures.
J. Appl. Polym. Sci. 1995, 57, 1441.
(34) Sarkanen, K. V. Chemistry of solvent pulping. Tappi J. 1990, 73,
215.
(35) Wenzl, H. F. J. The Chemical Technology of Wood; Academic
Press: New York, 1970.
(36) Sjostrom, E. Wood Chemistry. Fundamentals and Application;
Academic Press: San Diego, CA, 1992.
(37) Puri, V. P. Effect of Crystallinity and Degree of Polymerization of
Cellulose on Enzymatic Saccharification. Biotechnol. Bioeng. 1984, 26,
1219.
(38) Valjamae, P.; Pettersson, G.; Johansson, G. Mechanism of substrate
inhibition in cellulose synergistic degradation. Eur. J. Biochem. 2001, 268,
4520.
(39) Zhang, Y. H. P.; Lynd, L. R. Toward an aggregated understanding
of enzymatic hydrolysis of cellulose: Noncomplexed cellulase systems.
Biotechnol. Bioeng. 2004, 88, 797.
(40) Martinez, J. M.; Reguant, J.; Montero, M. A.; Montane, D.; Salvado,
J.; Farriol, X. Hydrolytic pretreatment of softwood and almond shells.
Degree of polymerization and enzymatic digestibility of the cellulose
fraction. Ind. Eng. Chem. Res. 1997, 36, 688.
ReceiVed for reView December 7, 2006
ReVised manuscript receiVed February 2, 2007
Accepted February 9, 2007
IE061576L