Bioconversion of Hybrid Poplar to Ethanol

and Co-Products Using an Organosolv

Fractionation Process: Optimization of
Process Yields

Xuejun Pan,1 Neil Gilkes,1 John Kadla,1 Kendall Pye,2 Shiro Saka,3 David Gregg,1
Katsunobu Ehara,3 Dan Xie,1 Dexter Lam,1 Jack Saddler1
1
Department of Wood Science, University of British Columbia, 2424 Main Mall,
Vancouver, BC, Canada V6T 1Z4; telephone: 604-822-6775; fax: 604-822-9104;
e-mail: xuepan@forestry.ubc.ca
2
Lignol Innovations Corp., 3650 Wesbrook Mall, Vancouver, British Columbia,
Canada V6S 2L2
3
Graduate School of Energy Science, Kyoto University, Yoshida Hon-machi, Sakyo-ku,
Kyoto 606-8501, Japan
Received 11 August 2005; accepted 14 February 2006

Published online 7 March 2006 in Wiley InterScience (www.interscience.wiley.com). DOI: 10.1002/bit.20905

Abstract: An organosolv process involving extraction INTRODUCTION

with hot aqueous ethanol has been evaluated for
bioconversion of hybrid poplar to ethanol. The process
resulted in fractionation of poplar chips into a cellulose-
rich solids fraction, an ethanol organosolv lignin
(EOL) fraction, and a water-soluble fraction containing
hemicellulosic sugars, sugar breakdown products,
degraded lignin, and other components. The influence
of four independent process variables (temperature, time,
catalyst dose, and ethanol concentration) on product
yields was analyzed over a broad range using a small
composite design and response surface methodology.
Center point conditions for the composite design (1808C,
60 min, 1.25% H2SO4, and 60% ethanol), yielded a solids
fraction containing
88% of the cellulose present in the
untreated poplar. Approximately 82% of the total cellulose
in the untreated poplar was recovered as monomeric
glucose after hydrolysis of the solids fraction for 24 h
using a low enzyme loading (20 filter paper units of
cellulase/g cellulose);
85% was recovered after 48 h
hydrolysis. Total recovery of xylose (soluble and insolu-
ble) was equivalent to
72% of the xylose present in
untreated wood. Approximately 74% of the lignin in
untreated wood was recovered as EOL. Other cooking
conditions resulted in either similar or inferior product
yields although the distribution of components between
the various fractions differed markedly. Data analysis
generated regression models that describe process
responses for any combination of the four variables.
2006 Wiley Periodicals, Inc.
Keywords: bioconversion; bioethanol; biorefinery; orga-
nosolv; lignocellulose; hybrid poplar
Correspondence to: X. Pan
Contract grant sponsors: Natural Sciences and Engineering Research
Council of Canada (NSERC); Natural Resources Canada (NRCan);
BIOCAP Canada
The development of biorefineries to produce fuels and
commodity chemicals from lignocellulosic biomass is
viewed as a potential alternative to current reliance on non-
renewable resources. The so-called sugar platform,
involving enzymatic hydrolysis of the cellulose component
to glucose, followed by fermentation to fuel-grade ethanol, is
a focus of current attention, but ultimately lignocellulose
biorefineries could produce a wider range of products, much
like todays petroleum refineries (Kamm and Kamm, 2004;
Lynd et al., 1999). Several bioconversion schemes have been
demonstrated in laboratory or pilot-scale experiments but
full-scale commercial bioconversion of lignocellulosic
biomass has yet to be realized.
One of the primary challenges for process commercializa-
tion is the development of cost-effective pretreatment
technologies for lignocellulosic feedstocks (Lynd et al.,
2002; Mosier et al., 2005; Wyman et al., 2005). Pretreatment
is necessary to increase the accessibility of cellulose in
lignocellulosic biomass to facilitate enzymatic hydrolysis.
Unlike traditional sources of fermentable sugar, such as
starch and sucrose, the cellulose component of lignocellulose
is a structural polymer and is protected against enzymatic
attack by the surrounding matrix of lignin and hemicellulose.
Among the pretreatment technologies currently being
evaluated for woody feedstocks is an ethanol organosolv
process that was originally designed to produce clean biofuel
for turbine generators and later developed by the Canadian
pulp and paper industry into the Alcell1 pulping process for
hardwoods (Pye and Lora, 1991; Stockburger, 1993;
Williamson, 1988). Historically, the organosolv process has
been investigated largely from the perspective of paper

2006 Wiley Periodicals, Inc.

production (Diaz et al., 2004; Gilarranz et al., 1998; Jimenez
et al., 2004; Ni and van Heiningen, 1996, 1997), but several
studies have also involved analysis of the lignin fraction
extracted during pulping (Hepditch and Thring, 1997; Liu
et al., 2000; Lora et al., 1993), including demonstration of its
potential for manufacture of various industrial co-products
such as adhesives or biodegradable polymers (Boocock and
Balatinecz, 1992; Kubo and Kadla, 2004; Thring et al.,
1997). Generation of high-quality lignin and other poten-
tially valuable co-products (e.g., furfural and acetic acid)
from woody feedstocks may be critical to otherwise
marginally cost-effective biorefinery schemes for fuel
ethanol production (Pan et al., 2005). However, the ethanol
organosolv process has not been extensively studied as a
pretreatment for lignocellulose bioconversion. In particular,
the effects of process parameters on the fractionation of the
major components in wood and on the mass balance of
the process have not been investigated in detail, although the
efficacy of ethanol organosolv pretreatment for particularly
recalcitrant feedstocks was demonstrated in a preliminary
study on mixed softwoods (spruce, pine, and Douglas-fir)
using substrates prepared in the pilot plant facility operated
by Lignol Innovations Corp. in Vancouver, Canada (Pan
et al., 2005).
The study described below employs response surface
methodology to examine the effects of various process
parameters on the yield and distribution of cellulose,
hemicellulose, and lignin in the fractions generated during
ethanol organosolv pretreatment of hybrid poplar. Enzymatic
digestion of the cellulose fraction produced in high yield
using one favorable combination of parameters is described
as a prelude to more detailed investigation of enzymatic
hydrolysis and fermentation.
MATERIALS AND METHODS
Feedstock Preparation
A commercially available hybrid poplar clone, NM-6
(Populus nigra  P. maximowiczii), was cultivated in Alex-
andria, MN and provided by the U.S. Department of
Agriculture. NM-6 is an industry standard used in hydrid
poplar research (Cavaleri et al., 2004). Approximately thirty
delimbed and debarked trees, 48 inches in diameter, were
chipped using a Mitts and Merrill knife mill (Saginaw, MI) to
pass a 14 inch round screen. The milled feedstock was then
thoroughly mixed by a cone-and-quarter method, subdivided
into 5 gallon pails, and stored at 208C before further
processing.
Analytical Procedures
Oven-dried weights were determined by drying to constant
weight at 1058C in a convection oven. Ash was determined
according to TAPPI standard method T211 om-93. Extrac-
tives were determined according to the ASTM Standard Test
Method E1690-01. Klason lignin was determined according
852 Biotechnology and Bioengineering, Vol. 94, No. 5, August 5, 2006
to the TAPPI standard method T-222. The hydrolyzate from
this determination was retained for analysis of monosacchar-
ides and acid-soluble lignin. Acid-soluble lignin was
determined from absorbance at 205 nm (Dence, 1992).
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 CarboPacTM PA1) and an ED40 electro-
chemical detector. The column was eluted with deionized
water at a flow rate of 1 mL/min. Aliquots (20(mL) were
injected after passing through a 0.45 mm nylon syringe filter
(Chromatographic Specialties, Inc., Brockville, Ont.,
Canada). Optimization of baseline stability and detector
sensitivity was achieved by postcolumn addition of 0.2M
NaOH. The column was re-conditioned using 1M NaOH
after each analysis. Monosaccharides were quantified with
reference to standards. Standards were autoclaved at 1208C
for 1 h prior to analysis to compensate for destruction during
heating.
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 608C with an eluent flow rate of 0.5 mL/min. A
gradient of 7.4 mM H3PO4 (A), acetonitrile (B), and a
mixture of 7.4 mM H3PO4, methanol and acetonitrile (4:3:3,
v/v) (C) was applied as follows: 0 ! 20 min, 95% A and
5% C ! 50% A and 50% C; 20 ! 24 min, 50% A and 50%
C ! 100% C; 24 ! 25 min, 100% C; 25 ! 26 min, 100%
C ! 100% B; 26 ! 27 min, 100% B; 27 ! 28 min,
100% B ! 95% A and 5% C; 28 ! 38 min, 95% A and 5%
C. Appropriately diluted aliquots (20 mL) were injected after
passing through a 0.45 mm PTFE syringe filter (Chromato-
graphic Specialties, Inc.). Furfural and HMF were deter-
mined from absorbance at 280 nm.
Ethanol Organosolv Pretreatment
A flowchart summarizing ethanol organosolv pretreatment
(process development scale) is shown in Figure 1. Hybrid
poplar chips were cooked in aqueous ethanol, with sulfuric
acid as catalyst, in a custom-built, four-vessel, rotating
digester made by Aurora Products Ltd. (Savona, BC,
Canada). A 200 g (oven-dried weight) batch of chips was
cooked in each 2 L vessel. After cooking, vessels were cooled
to room temperature in a water bath and the spent liquor (i.e.,
aqueous ethanol extract) was sampled immediately for
determination of furfural and HMF. Pulp and liquor were
then separated using nylon mesh. The pulp was washed
three times with 300 mL aqueous ethanol with the same
concentration of cooking liquor at 608C and the washes
combined with the spent liquor. The pulp was then washed
three times with water at 608C and the washes discarded. The
washed pulp was homogenized in a standard British
disintegrator for 5 min and passed through a laboratory
flat screen with 0.008 inch (0.203 mm) slits (Voith,
Inc., Appleton, WI) to remove rejects (non-defiberized
DOI 10.1002/bit

Figure 1. Flowchart of the laboratory-scale ethanol organosolv process.
woodchips and knots). The screened pulp comprises the
solids fraction.
The spent liquor and ethanol washes were combined and
mixed with three volumes of water to precipitate the
dissolved lignin. The lignin precipitate, henceforth described
as ethanol organosolv lignin (EOL), was collected on
Whatman No.1 filter paper, washed thoroughly with water,
and air-dried. The filtrate and water washes were combined to
give a water-soluble fraction containing monomeric and
oligomeric hemicellulosic sugars, depolymerized lignin, and
other unidentified components.
Enzymatic Hydrolysis
Commercial cellulase and b-glucosidase preparations (Cel-
luclast and Novozym 188, respectively) were provided by
Novozymes. Cellulase activity was determined using the
filter paper assay recommended by the International Union of
Pure and Applied Chemists (Ghose, 1987) and is expressed in
filter paper units (FPUs). b-Glucosidase activity was
determined using p-nitrophenyl-b-D-glucoside as substrate,
as previously described (Wood and Bhat, 1988) and is
expressed in International Units (IUs). Protein was deter-
mined using Bio-Rad Protein Assay (Method of Bradford)
(Bradford, 1976). Celluclast was supplemented with b-
glucosidase (1 FPU:2 IU) to avoid product inhibition caused
by cellobiose accumulation.
Batch hydrolysis reactions contained solids (2% cellulose
w/v) in 50 mM acetate buffer, pH 4.8, with 0.004%
tetracycline as antibiotic. Celluclast was used at a loading
Pan et al.: Organosolv Fractionation for Bioconversion
of 20 FPU (21 mg total protein)/g cellulose in the solids;
Novozym 188 was used at a loading of 40 IU (
6 mg total
protein)/g cellulose. Reaction mixtures (100 mL) were
incubated at 150 rpm, 458C, in a rotary shaker and sampled
periodically for glucose determination, as described above.
Hydrolysis data are averages from duplicate experiments.
Data Analysis
Data was analyzed using Statistical Analysis System (SAS)
V9 for Windows (SAS Institute, Inc., Cary, NC).
RESULTS AND DISCUSSION
A previous investigations of the ethanol organosolv process
for lignocellulose bioconversion used mixed softwood
feedstock pretreated in a three-stage pilot plant facility at
Lignol Innovations Corp. A diagram of the Lignol process
was presented earlier (Pan et al., 2005). In the experiments
described below (see Fig. 1 for flowchart), hybrid poplar was
pretreated using a four-vessel custom-built digester in order
to expedite production of samples under a broad range of
processing conditions.
The influence of four process variables (cooking tempera-
ture, cooking time, acid catalyst dose, and ethanol concen-
tration) on the yields and compositions of the process streams
was investigated by response surface methodology using a
small Hartley composite design (Myers and Montgomery,
2002). The composite design comprised 21 sets of condi-
tions, including eight factorial points, eight star points, and
five center points, as summarized in Table I. The range of
conditions examined was as follows: temperature: 155
2058C; time: 2694 min; H2SO4: 0.83%1.67% w/w;
ethanol: 25%75% (v/v). The ratio of liquor to wood was
constant (7:1 v/w) in all experiments. Center point (Table I,
#s 17.117.5) conditions (1808C; 60 min; 1.25% H2SO4;
50% ethanol) were selected on the basis of results from
preliminary tests (data not shown) and prior experience with
softwood feedstocks (Kurabi et al., 2005).
Mass Balance of Organosolv Pretreatment at
Center Point Conditions
Statistical analysis of data from five replicate samples
produced under center point conditions (#s 17.117.5;
Table I) shows that the response to pretreatment was
reproducible (Fig. 2). In contrast, alteration of process
conditions within the prescribed range resulted in large
differences in the yields and compositions of the process
streams relative to center point values, as discussed below
(Table I; Figs. 46).
Defibration of the pulp was essentially complete at center
point conditions, with only
1% of the original wood chips
recovered as rejects during screening. Fractionation of the
major lignocellulose components was clearly demonstrated.
The total yield of solids (i.e., screened pulp) was
53% w/w.
The solids fraction contained
11.5% w/w lignin, compared
853
Biotechnology and Bioengineering. DOI 10.1002/bit
 854
Table I. Mass balances for organosolv pretreatment of poplar under a range of processing conditions.

Varablesb Solidsc Water-soluble fractionc

Conditiona T t S C Total KL AL glu xyl man Rejectsc EOLc AL glu xyl man gal ara furfural HMF

Wood 100.00 20.95 2.30 48.95 17.85 3.88

1 165 40 1.00 65 4.37 0.81 0.09 2.37 0.76 0.16 85.38 5.70 4.68 0.01 0.65 0.00 0.13 0.20 0.012 0.004
2 195 40 1.00 65 51.62 4.72 0.49 41.09 5.92 2.31 6.80 16.95 5.07 0.02 5.54 0.24 0.25 0.16 0.193 0.057
3 165 80 1.00 35 13.82 3.90 0.17 8.86 1.02 0.41 53.90 4.95 4.65 0.29 8.01 0.88 0.30 0.22 0.142 0.028
4 195 80 1.00 35 55.47 13.91 0.49 43.05 1.52 0.54 4.45 4.35 4.73 0.63 2.37 0.74 0.10 0.02 2.774 0.879
5 165 40 1.50 35 23.53 6.20 0.22 16.51 1.11 0.48 37.48 6.25 5.17 0.71 10.05 1.47 0.35 0.26 0.147 0.028
6 195 40 1.50 35 51.72 11.86 0.45 40.54 0.71 0.24 2.40 6.45 5.95 2.30 6.81 1.58 0.26 0.16 2.392 0.748
7 165 80 1.50 65 52.49 3.57 0.35 43.27 4.15 1.77 0.46 17.65 5.55 0.38 8.42 0.99 0.31 0.24 0.177 0.031
8 195 80 1.50 65 36.66 0.88 0.11 36.61 0.41 0.15 0.01 20.90 6.70 4.23 7.64 1.73 0.28 0.17 2.302 0.655
9 155 60 1.25 50 4.67 0.95 0.08 2.69 0.61 0.15 70.49 7.10 4.13 0.02 3.06 0.24 0.21 0.22 0.017 0.004
10 205 60 1.25 50 47.35 6.09 0.26 40.77 1.17 0.35 0.15 15.75 6.03 1.20 3.33 0.95 0.15 0.05 3.001 1.139
11 180 26 1.25 50 37.04 4.96 0.38 27.79 2.83 1.17 22.34 13.45 5.76 0.34 8.85 0.96 0.34 0.26 0.093 0.019
12 180 94 1.25 50 51.61 5.45 0.29 42.42 2.91 1.04 0.62 15.70 6.08 0.84 10.37 1.63 0.37 0.22 1.007 0.224
13 180 60 0.83 50 15.84 2.84 0.20 10.27 1.52 0.53 49.92 12.35 4.71 0.07 5.91 0.36 0.26 0.17 0.168 0.039
14 180 60 1.67 50 45.17 3.59 0.18 37.06 1.03 0.34 0.09 17.15 6.10 2.14 10.39 1.99 0.35 0.23 1.196 0.252

Biotechnology and Bioengineering, Vol. 94, No. 5, August 5, 2006

15 180 60 1.25 25 35.74 11.06 0.42 21.91 0.99 0.37 27.41 1.70 4.55 1.08 8.98 1.69 0.32 0.19 1.186 0.278
16 180 60 1.25 75 55.96 4.11 0.53 43.46 6.13 2.36 1.64 17.25 5.56 0.02 6.39 0.36 0.33 0.24 0.099 0.027
17.1 180 60 1.25 50 52.47 5.93 0.31 42.67 3.43 1.62 2.13 15.60 5.00 0.55 9.52 1.28 0.34 0.23 0.428 0.084
17.2 180 60 1.25 50 53.90 6.74 0.31 43.45 3.40 1.61 0.74 14.75 5.15 0.53 9.35 1.28 0.33 0.22 0.458 0.091
17.3 180 60 1.25 50 53.50 6.41 0.32 43.39 3.41 1.54 1.49 15.20 5.08 0.50 9.21 1.22 0.33 0.21 0.453 0.094
17.4 180 60 1.25 50 52.87 5.69 0.29 43.54 3.27 1.35 1.19 15.95 5.41 0.58 9.30 1.31 0.33 0.21 0.519 0.105
17.5 180 60 1.25 50 50.88 4.69 0.27 42.68 3.20 1.33 0.88 16.15 5.45 0.60 9.47 1.35 0.33 0.22 0.487 0.098
18 52.49 3.31 0.09 48.15 0.82 0.57 0.00 20.12 6.09 2.05 8.99 1.49 0.34 0.22 1.42 0.39
a
Wood is untreated feedstock. #s 117 are the complete experimental matrix of 21 conditions; #s 17.117.5 are replicated center point conditions. Values for condition #18 are calculated using Equations 217
(Table II) at T 1908C, t 70 min, S 1.4%, and C 60%.
b
T, cooking temperature (8C); t, time (min) at the cooking 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) untreated poplar chips.

DOI 10.1002/bit
Figure 2. Mass balance of laboratory-scale ethanol organosolv process of poplar at center point conditions (1808C, 60 min, 1.25% H2SO4, and 50% ethanol).

to
23.3% w/w in the untreated wood. Approximately 27%
of the total lignin remained associated with the solids. Most
of the lignin was dissolved during aqueous ethanol extraction
and was recovered as a precipitate after dilution with water
but a portion of lignin remained in the water-soluble fraction
(Fig. 2). The precipitated ethanol organosolv lignin (EOL)
accounted for approximately 74% of the Klason lignin in
untreated wood. The total amount of lignin recovered from
the pulp, EOL, and water soluble fractions at center point
conditions was
116% of the amount present in the untreated
wood. Part of the apparent excess can be explained by
extensive depolymerization of lignin during organosolv
extraction. Furthermore, the value for acid-soluble lignin in
the water-soluble fraction is probably inflated by the presence
extractive components (e.g., lignans, tannins, and related
phenolic compounds); these components are routinely

Figure 3. Enzymatic hydrolysis of solids prepared from hybrid poplar (PEP) at center point conditions (1808C, 60 min, 1.25% H2SO4, and 50% ethanol) with
enzymes loading of 20 FPU (20.9 mg protein) cellulase and 40 IU (5.7 mg protein) b-glucosidase/g cellulose. Hydrolysis of a solids fraction prepared by steam
explosion of Douglas fir (SEDF), oxygen-delignified SEDF (O-SEDF), and mixed softwoods ethanol pulp (MSEP) is shown for comparison.

Pan et al.: Organosolv Fractionation for Bioconversion 855

Biotechnology and Bioengineering. DOI 10.1002/bit

Figure 4. Effect of process variables on yields of solids, ethanol organosolv lignin (EOL) and rejects. Yields (%) are expressed as g per 100 g oven-dried
wood.
removed prior to compositional analysis of the untreated
wood.
Approximately 88% of the total glucan in untreated wood
was recovered in the solids fraction (calculated from data in
Fig. 2). The small amount of glucose present in the water-
soluble fraction (
1% of total) probably includes material
derived from glucomannan. The occurrence of xylose
(
19% of total) and mannose (
38% of total) in the solids
fraction indicates the presence of some residual polymeric
hemicellulose. However, most of the recovered hemicellu-
losic sugars (
53% total xylose,
33% of total mannose,

85% of total arabinose, and
87% of total galactose) were
found in the water-soluble fraction. The amounts of
oligomeric and monomeric sugars in the water-soluble
fraction were determined by analysis before and after
incubation in 3% H2SO4 under conditions which hydrolyze
all oligomeric sugars to monomers. At center point condi-
tions,
58% of the water-soluble xylose, and
71% of the
water-soluble mannose was oligomeric.
The extent of xylose recovery in the combined solids and
water-soluble fractions (
72%) indicates significant degra-
dation of this component. Substantial losses of other
monosaccharides were also observed, such that the total
recovery of carbohydrate (solids plus water-soluble fractions
excluding the reject fraction) was
84%. All of the major
monosaccharides found in lignocellulose undergo thermal
decomposition under acidic conditions (Wenzl, 1970). The
water-soluble fraction contained significant levels of HMF
856 Biotechnology and Bioengineering, Vol. 94, No. 5, August 5, 2006
and furfural, degradation products of hexoses and pentoses,
respectively (Wenzl, 1970). However, the total amounts of
HMF and furfural detected account for only a small fraction
of the unrecovered carbohydrate, suggesting further degra-
dation of monosaccharides to unidentified products (Harris
et al., 1963; Sjostrom, 1992). It should also be noted that the
weight contribution of methyl glucoronic acid and acetate
side groups was not determined in these analyses.
Enzymatic Hydrolysis of Solids Fraction
Enzymatic hydrolysis of solids prepared at center point
conditions is shown in Figure 3. Data are the average for five
replicated solids samples (#s 17.117.5; Table I) hydrolyzed
at 2% consistency (g cellulose/100 mL liquid) in separate
reactions using cellulase (Celluclast) and b-glucosidase
(Novozym 188) at 20 FPU (20.9 mg protein) and 40 IU
(5.7 mg protein) per g cellulose, respectively.
Substantial conversion of the poplar solids (containing
11.2% w/w average residual lignin) was observed within 24 h
(
93% hydrolysis of the glucan in the solids fraction
hydrolyzed to glucose). When the hydrolysis was extended
to 48 h, the conversion of cellulose to glucose reached
97%
(Fig. 3). As discussed above, approximately 88% of the
original cellulose was recovered in the solids fraction.
Therefore, the net conversion of cellulose (i.e., glucan in
untreated wood) to glucose in the hydrolyzate was
82%
(88%  93%) of the theoretical conversion at 24 h or
85%
DOI 10.1002/bit
 Figure 5. Effect of process variables on yields of components in the solids fraction. Yields are expressed as g per 100 g oven-dried wood (%).
(88%  97%) at 48 h, respectively. In comparison, a solids
fraction containing
42% residual lignin, prepared by SO2-
catalyzed steam explosion of softwood (Douglas-fir),
showed <30% at 24 h and
32% at 48 h hydrolysis of
cellulose to glucose, and
75% at 24 h and
90% at 48 h
hydrolysis after further delignification by oxygen bleaching
(Fig. 3), respectively. The rate of hydrolysis of the poplar
solids was slower during the first 12 h than that of a solids
fraction with a similar lignin content (9.2%) prepared by
organosolv pretreatment of mixed softwood (Pan et al., 2005)
but the extents of conversion at 24 and 48 h were higher
(Fig. 3). The mechanistic basis for this difference in
hydrolysis kinetics is presently unclear.
Optimization of Sugar Recovery
Mass balance data for other processing conditions, chosen
according to the Hartley small composite design, are
presented in Table I. The yields and compositions of the
solids, EOL, water-soluble, and reject fractions showed
marked differences as cooking conditions were varied within
the experimental range. However, none of the conditions
tested produced a significantly higher yield of solids than the
Pan et al.: Organosolv Fractionation for Bioconversion
center point conditions (52.72  1.17%) (Fig. 2; Table I).
Likewise, none of the conditions resulted in a higher recovery
of cellulose in the solids (43.16  1.67 g;
88% of total
glucose in untreated wood), or a higher recovery of
insoluble soluble xylose (12.73  0.27 g;
72% of total
xylose in untreated wood).
All other sets of experimental conditions examined in the
small composite design resulted in yields of total solids,
cellulose recoveries, and xylose recoveries that were
comparable (e.g., condition #s 7 and 16) or inferior to those
given by the center point conditions. Nevertheless, examina-
tion of differences in other process responses (e.g., solids
delignification, solubilization of hemicellulose, and produc-
tion of sugar degradation products) indicates the potential for
fine-tuning process performance beyond those achieved at
center point conditions. For example, solids produced under
conditions # 7 and #16 produced solids fractions containing
less lignin and a water-soluble fraction containing less
furfural and HMF, than center point conditions, and a lower
proportion of recovered hemicellulose was solubilized in
both cases.
Further insights into the potential for optimizing process
performance are gained from modeling the effects of
857
Biotechnology and Bioengineering. DOI 10.1002/bit
Figure 6. Effect of process variables on yields of components in the water-soluble fraction. Yields are expressed as g per 100 g oven-dried wood (%).
changing single process parameters, while others are fixed,
using response surface methodology (Figs. 46).
The predicted yields of solids and EOL increase slightly
above 1808C to a maximum at 1958C (Fig. 4). The recovery
of glucose in the solids fraction is highest (Fig. 5), and the
production of rejects lowest, at 1958C (Fig. 4). Reduced
solids yield above 1958C is attributed largely to accelerated
solubilization and degradation of hemicellulosic sugars. This
explanation is consistent with the general decline in levels of
xylose, mannose, arabinose, and galactose in the solids and
water-soluble fractions (Figs. 5 and 6), accompanied by
increasing amounts of furfural, HMF in the water-soluble
fraction (Fig. 6), as the cooking temperature is increased
above 1808C.
The slight increase in reject production above 1958C
(Fig. 4) is probably due to promotion of lignin condensation
reactions leading to reduced defibration. This explanation is
consistent with the net rise in Klason lignin content of the
solids fraction as temperature is increased (Fig. 5). However,
high temperatures also appear to increase lignin degradation,
as shown by elevated levels of acid-soluble lignin in the
water-soluble fraction (Fig. 6).
858 Biotechnology and Bioengineering, Vol. 94, No. 5, August 5, 2006
Most responses showed a linear time-dependence (Figs. 4
6). Solids and ethanol-soluble lignin yields increased with
longer cooking time, while the amount of rejects fell (Fig. 4).
Higher levels of acid-soluble lignin in the water-soluble
fraction following extended cooking times indicate increased
lignin degradation (Fig. 6). Levels of residual hemicellulosic
sugars in the solids and water-soluble fractions were
generally insensitive to cooking time (Figs. 5 and 6). Levels
of furfural and HMF were proportional to cooking time.
Evidently, sugar levels in the water-soluble fraction are the
net result of both hydrolysis and degradation during cooking.
The yield of solids was maximal between 1.25% and 1.5%
H2SO4; reject production was minimal at
1.5% (Fig. 4). The
yield of EOL increased linearly up to 1.65%. The effect of
increased catalyst dose on solids composition was similar to
that seen at higher temperatures (Fig. 5). Solubilization of
hemicellulosic sugars was markedly increased at higher
H2SO4 concentrations (Fig. 6). Recoveries of oligomeric
xylose and mannose levels in the water-soluble fraction
increased up to
1.5% H2SO4 and then declined. As discussed
above, sugar levels in the water-soluble fraction represent the
net result of hydrolysis and degradation reactions.
DOI 10.1002/bit
 Increasing the ethanol concentration above the 50% center

0.9667
0.9836
0.9541
0.8955
0.9735
0.9135
0.9843
0.9417
0.8565
0.8882
0.9812
0.9871
0.9675
0.9777
0.9996

0.9862
R2
point value produced a small gain in the yield of solids and a
small decline in reject production (Fig. 4). Delignification of
Equation no. the solids and recovery of EOL were maximal, and the
production of rejects minimal, at
65%. Delignification is

10
11
12
13
14
15
16

17
2
3
4
5
6
7
8
9
the combined result of lignin degradation and lignin

KL, Klason lignin, AL, acid-soluble lignin; WS, water-soluble fraction; glu, glucose; xyl, xylose; man, mannose; gal, galactose; ara, arabinose; fur, furfural; HMF, hydroxymethylfurfural.
solubilization: lower ethanol concentrations result in higher
water chemical activity that promote acid-catalyzed cleavage
20.6118 0.141785T 0.114933t 7.251006S 0.013341C  0.000202T2  0.000458Tt  0.024333TS  0.000217TC  0.027595tS  0.613336S2 0.000278C2 of a- and b-ether linkages in the lignin (McDonough, 1993)

54.62532  0.586839T  0.159323t  12.05638S 0.199876C 0.001652T2 0.000971Tt 0.0519TS  0.001428TC 0.00008t2  0.000229tC 1.258737S2
whereas high ethanol concentrations increase solubilization
of the lignin (Ni and Hu, 1995; Sarkanen, 1990). In contrast to
lignin, the recoveries of cellulose (glucose) and residual

292.159 3.346597T 0.508627t 23.27865S  1.214107C  0.009797T2  0.003362Tt 0.007217TC 0.002368tC  7.178128S2  0.002726C2
hemicellulose (xylose and mannose) in the solids continued
2539.29 18.97364T 5.394907t 808.615S 4.242763C  0.035089T2  0.020675Tt  2.551333TS  0.021344TC  1.187289tS  100.8928S2

281.774 2.025777T 1.525606t 97.4672S 0.034896C  0.003534T2  0.006667Tt  0.326667TS  0.267905tS  10.53833S2 0.000671C2

to increase up to 75% ethanol, as reflected in reduced

recoveries of xylose and mannose (particularly as mono-
mers) in the water-soluble fraction and lower production of

T, cooking temperature (8C); t, time (min) at the cooking temperature; S, sulfuric acid (%, w/w oven-dried wood); C, ethanol concentration (%, v/v).
3.20539 0.042585T 0.009027t 0.028973S  0.018052C  0.000141T2  0.000054Tt 0.002333TS 0.000106TC  0.140799S2
furfural and HMF. A similar stabilizing effect to that at high
7.49947 0.08964T 0.015399t 0.714732S  0.043256C  0.000264T2  0.000108Tt 0.000211TC 0.000086tC  0.243036S2

ethanol concentration has been reported for organosolv

23.5586 0.32709T  0.037549t  4.736008S  0.090677C  0.001044T2 0.025TS 0.000561TC 0.036417tS  0.000367C2

acetic/formic acid pulping (Lehnen et al., 2001). The lower

water chemical activity at high ethanol concentration
272.116 2.569489T 51.82181S  0.153372C  0.007105T2 0.000313Tt  0.243333TS 0.008278TC  0.010175C2

probably suppressed the hydrolysis of carbohydrate.

1808.02 13.84639T 5.692375t 563.9703S 0.247147C  0.025864T2  0.02935Tt  1.851333TS  82.14118S2

21.96658  0.2544T  0.048618t  2.390281S 0.082767C 0.000732T2 0.000292Tt 0.014667TS  0.000497TC

In summary, center point conditions are close to optimum

118.739 0.920677T 0.506054t 36.96629S 0.029624C  0.001674T2  0.002833Tt  0.138TS  4.97941S2

in terms of solids yield, cellulose recovery in the solids, and

total recoveries of hemicellulosic sugars, but incremental
improvements in these parameters are predicted as a result of
small increases in process severity. However, optimization of
pretreatment in terms of maximizing yields of fermentable
sugars requires more detailed investigation. Yields of co-
45.39307  0.238858T  0.182048t  37.48341S 0.001079Tt 0.169667TS 3.967278S2

products may also have significant economic implications. In

152.387 0.982352T 65.82606S 0.83784C  0.365TS  0.008028TC 0.004236C2
Equationb

general, pretreatment of lignocellulosic biomass at higher

severities improves enzymatic digestibility of the cellulose
component but increases sugar degradation, thereby dimin-
ishing cellulose yield; in turn, sugar degradation products
40.9943  0.107144T  0.061483t  6.839843S  0.28213C 0.002151C2

inhibit fermentation. Therefore, lignocellulose pretreatment

optimization involves a compromise between these factors.
Also, it is currently unclear whether targeting hemicellulose
 3.0099 0.027314T 1.926562S 0.015614C 0.000056t2

sugars to the water-soluble fraction during pretreatment

offers advantages over retaining these components in the
solids. The presence of hemicellulose in the solids is thought
to limit cellulose hydrolysis by steric interference but the
benefit of supplementing cellulase preparations with hemi-
cellulase remains to be evaluated.

Regression Modeling
0.015827SC 0.000287C2
Table II. Equations for process responses.

To model the effect of each process variable on the various

process responses (yield of solids, EOL, and water-soluble
fraction, or recoveries of a specific lignocellulose component
within each fraction), each process response was fitted to a
second-order polynomial equation using SAS software:
X
k X
k k1 X
X k
Y a0 a i Xi aii Xi2 aij Xi Xj 1
i1 i1 i1 j1
Man in solids
Glu in solids
Xyl in solids
KL in solids
AL in solids

HMF in WS
Total solids

Man in WS
Glu in WS
Xyl in WS

Ara in WS
Gal in WS

Fur in WS
Responsea

AL in WS

where Y is the estimated value of the process response, k is

Rejects

the total number of independent variables (four in this case),

EOL

b
a

Xis are the independent variables (temperature, time,

Pan et al.: Organosolv Fractionation for Bioconversion 859

Biotechnology and Bioengineering. DOI 10.1002/bit

Table III. Process responses: comparison between observed and CONCLUSION
calculated values.
Response surface methodology indicates that center point
Observed Calculated Variancec, conditions (cooking temperature, cooking time, catalyst
Response valuea valueb %
dose, and ethanol concentration) chosen for the small
Pulp yield, % of wood 52.72 49.55 6.0 composite design are close to optimal for glucose recovery
Klason lignin, % of wood 5.89 4.81 18.3 in the solids fraction prepared by ethanol organosolv
Acid soluble lignin, % of wood 0.30 0.28 6.7
pretreatment. Glucose recovery is a critical parameter for
Glucose, % of wood 43.15 43.32 0.4
Xylose, % of wood 3.34 3.09 7.5 pretreatment optimization aimed at maximizing production
Mannose, % of wood 1.49 1.37 8.1 of fermentable sugars. Center point conditions yielded a
EOL yield, % of wood 15.53 15.00 3.4 solids fraction containing
88% of the cellulose present in
Reject yield, % of wood 1.29 0.74 42.6 the untreated wood. Total recovery of xylose (monomeric
Water-solubles, % of wood and oligomeric), the most abundant pentose sugar, was
Acid soluble lignin 5.22 5.16 1.1
Glucose 0.55 0.65 18.2 equivalent to
72% of the xylose present in the untreated
Galactose 0.33 0.33 0.0 wood. Approximately 74% of the lignin was recovered as a
Arabinose 0.22 0.22 0.0 precipitate following ethanol extraction.
Xylose 9.37 9.44 0.7 Further process optimization for bioconversion purposes
Mannose 1.29 1.27 1.6
requires more detailed evaluation of the susceptibility of
Furfural 0.47 0.47 0.0
HMF 0.10 0.13 30.0
solids to enzymatic hydrolysis and the fermentation of
resultant sugars. Approximately 82% of the total cellulose in
a
Observed value: average of five replicates at center point conditions the untreated poplar was recovered as monomeric glucose
(#s 17.117.5; Table I).
b
Calculated value: calculated using equations 217 at center point after enzymatic hydrolysis of the solids fraction;
85% was
conditions (T 1808C, t 60 min, S 1.25%, and C 50%). recovered as glucose after 48 h hydrolysis. Hydrolysis was
c
Variance (observed valuecalculated value)/observed value  100). performed using a low enzyme loading (20 FPU cellulase/g
cellulose). This result is similar to maximum glucose
recoveries of 75% and 88% following 100 h hydrolysis of
catalyst dose, and ethanol concentration), the Xi, Xi2 , and
methanol organosolv pretreated aspen (P. tremuloides) and
XiXj are terms describing linear, quadratic and two-variable
black cottonwood (P. trichcarpa) (Chum et al., 1988). These
interaction effects, respectively, a0 is a constant and ai, aii,
data support the conclusion that organosolv pretreatment of
and aij are linear, quadratic, and interaction coefficients,
poplar species, using ethanol or methanol plus acid catalyst,
respectively. The equations determined for each response
results in high yields of glucan and high recoveries
are listed in Table II (Equations 217).
of monomeric glucose following enzymatic hydrolysis,
These equations can be used to calculate the responses to
despite earlier indications to the contrary (Holtzapple and
all combinations of variables within the range of the small
Humphrey, 1984).
composite design. To validate this model, the calculated
Hydrolysis of solids prepared under other cooking con-
responses at center point conditions were compared with the
ditions remains to be investigated. Numerous factors, which
experimental data from five replicate pretreatment experi-
were not evaluated in the present study, influence hydrolysis.
ments (#s 17.117.5, Table I), as shown in Table III. Some
These include physical properties of cellulose fibers (e.g.,
of the calculated responses (e.g., acid-soluble lignin recovery
surface area, porosity, and crystallinity) and the chemical and
in the solids fraction, reject yield, glucose, and HMF in the
physical characteristics of residual lignin and hemicellulose
water-soluble) differed significantly from experimental
in the solids. It is conceivable that some cooking conditions
values (variance   5%). However, all other calculated
yield solids in which lower cellulose yields are more than
responses were in good agreement with experimental values.
compensated by improved hydrolysis. The effect of pretreat-
As discussed above, some improvement in process yields
ment conditions on sugar fermentation and on the quality
can be expected from small increases in the severities of all
of potential co-products, such as lignin and extractives, also
process parameters above center point conditions (1808C; 60
requires evaluation. Results presented above provide a plat-
min; 1.25% H2SO4; 50% ethanol). As an example, calculated
form for further investigation.
responses for 1908C; 70 min; 1.4% H2SO4; and 60% ethanol
are presented in Table I. Under these conditions, the
calculated yield of solids (
52%) is similar to that for center NOMENCLATURE
point conditions but the recovery of cellulose (glucose) is AL acid-soluble lignin
slightly increased to
48 g/100 g wood. A significant ara arabinose
increase (
30%) in the recovery of EOL is also calculated C concentration of aqueous ethanol, % (v/v)
from the model but the recovery of hemicellulose sugars is EOL ethanol organosolv lignin
reduced. Although these calculated responses were not IU international unit (b-glucosidase activity)
FPU filter paper unit (cellulase activity)
verified experimentally, they suggest that only marginal fur furfural
improvements in yields can be anticipated by fine-tuning gal galactose
center point conditions. glu glucose

860 Biotechnology and Bioengineering, Vol. 94, No. 5, August 5, 2006

DOI 10.1002/bit
HMF hydroxymethylfurfural
KL Klason lignin
man mannose
S catalyst (concentrated sulfuric acid) dosage, % on wood chip (w/w)
t time at maximum temperature, min
T maximum temperature, 8C
WS water-soluble fraction
xyl xylose
We thank Novozymes for providing enzyme samples.
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