Bioconversion of Hybrid Poplar to Ethanoland Co-Products Using an OrganosolvFractionation Process: Optimization ofProcess 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

1Department 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.ca2Lignol Innovations Corp., 3650 Wesbrook Mall, Vancouver, British Columbia,Canada V6S 2L23Graduate School of Energy Science, KyotoUniversity, 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 extractionwith hot aqueous ethanol has been evaluated forbioconversion of hybrid poplar to ethanol. The processresulted in fractionation of poplar chips into a cellulose-rich solids fraction, an ethanol organosolv lignin(EOL) fraction, and a water-soluble fraction containinghemicellulosic sugars, sugar breakdown products,degraded lignin, and other components. The influenceof four independent process variables (temperature, time,catalyst dose, and ethanol concentration) on productyields was analyzed over a broad range using a smallcomposite design and response surface methodology.Center point conditions for the composite design (1808C,60 min, 1.25% H2SO4, and 60% ethanol), yielded a solidsfraction containing �88% of the cellulose present in theuntreatedpoplar.Approximately 82%of the total cellulosein the untreated poplar was recovered as monomericglucose after hydrolysis of the solids fraction for 24 husing a low enzyme loading (20 filter paper units ofcellulase/g cellulose); �85% was recovered after 48 hhydrolysis. Total recovery of xylose (soluble and insolu-ble) was equivalent to �72% of the xylose present inuntreated wood. Approximately 74% of the lignin inuntreated wood was recovered as EOL. Other cookingconditions resulted in either similar or inferior productyields although the distribution of components betweenthe various fractions differed markedly. Data analysisgenerated regression models that describe processresponses for any combination of the four variables.� 2006 Wiley Periodicals, Inc.

Keywords: bioconversion; bioethanol; biorefinery; orga-nosolv; lignocellulose; hybrid poplar

INTRODUCTION

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 today’s 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 surroundingmatrix 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.

Correspondence to: X. Pan

Contract grant sponsors: Natural Sciences and Engineering Research

Council of Canada (NSERC); Natural Resources Canada (NRCan);

BIOCAP Canada

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, 4–8 inches in diameter, were

chipped using aMitts andMerrill knife mill (Saginaw,MI) to

pass a 14inch 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

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) wereinjected 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 1208Cfor 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 afterpassing 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 2L 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 thewashes 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

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

DOI 10.1002/bit

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 andwaterwasheswere 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 andAppliedChemists (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

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: 26–94 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 17.1–17.5) 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 atCenter Point Conditions

Statistical analysis of data from five replicate samples

produced under center point conditions (#s 17.1–17.5;

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. 4–6).

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

Figure 1. Flowchart of the laboratory-scale ethanol organosolv process.

Pan et al.: Organosolv Fractionation for Bioconversion 853

Biotechnology and Bioengineering. DOI 10.1002/bit

Tab

leI.

Massbalancesfororganosolv

pretreatm

entofpoplarunder

arangeofprocessingconditions.

Varablesb

Solidsc

Rejectsc

EOLc

Water-solublefractionc

Conditiona

Tt

SC

Total

KL

AL

glu

xyl

man

AL

glu

xyl

man

gal

ara

furfural

HMF

Wood

100.00

20.95

2.30

48.95

17.85

3.88

1165

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

2195

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

3165

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

4195

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

5165

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

6195

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

7165

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

8195

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

9155

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

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

aWoodisuntreatedfeedstock.#s1–17arethecompleteexperim

entalmatrixof21conditions;#s17.1–17.5arereplicatedcenterpointconditions.Values

forcondition#18arecalculatedusingEquations2–17

(TableII)atT¼1908C

,t¼

70min,S¼1.4%,andC¼60%.

bT,cookingtemperature

(8C);t,time(m

in)atthecookingtemperature;S,sulfuricacid

(%,w/w

oven-dried

wood);C,ethanolconcentration(%

,v/v).

cAlldata

are

yieldsofcomponents(g)per

100g(oven-dried

weight)untreatedpoplarchips.

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

DOI 10.1002/bit

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

conditionswas�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

thewater-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 fromhybrid poplar (PEP) at center point conditions (1808C, 60min, 1.25%H2SO4, and 50%ethanol)with

enzymes loading of 20 FPU (20.9 mg protein) cellulase and 40 IU (5.7mg protein) b-glucosidase/g cellulose. Hydrolysis of a solids fraction prepared by steamexplosion of Douglas fir (SEDF), oxygen-delignified SEDF (O-SEDF), and mixed softwoods ethanol pulp (MSEP) is shown for comparison.

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

Pan et al.: Organosolv Fractionation for Bioconversion 855

Biotechnology and Bioengineering. DOI 10.1002/bit

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

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 17.1–17.5; 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 observedwithin 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%

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.

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

DOI 10.1002/bit

(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 ofmixed 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

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

insolubleþ 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

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 (%).

Pan et al.: Organosolv Fractionation for Bioconversion 857

Biotechnology and Bioengineering. DOI 10.1002/bit

changing single process parameters, while others are fixed,

using response surface methodology (Figs. 4–6).

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).

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 productionwasminimal 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 thendeclined.Asdiscussed

above, sugar levels in the water-soluble fraction represent the

net result of hydrolysis and degradation reactions.

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 (%).

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

DOI 10.1002/bit

Increasing the ethanol concentration above the 50% center

point value produced a small gain in the yield of solids and a

small decline in reject production (Fig. 4). Delignification of

the solids and recovery of EOL were maximal, and the

production of rejects minimal, at �65%. Delignification is

the combined result of lignin degradation and lignin

solubilization: lower ethanol concentrations result in higher

water chemical activity that promote acid-catalyzed cleavage

of a- and b-ether linkages in the lignin (McDonough, 1993)

whereas high ethanol concentrations increase solubilization

of the lignin (Ni andHu, 1995; Sarkanen, 1990). In contrast to

lignin, the recoveries of cellulose (glucose) and residual

hemicellulose (xylose and mannose) in the solids continued

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

furfural and HMF. A similar stabilizing effect to that at high

ethanol concentration has been reported for organosolv

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

water chemical activity at high ethanol concentration

probably suppressed the hydrolysis of carbohydrate.

In summary, center point conditions are close to optimum

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-

productsmay also have significant economic implications. In

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

inhibit fermentation. Therefore, lignocellulose pretreatment

optimization involves a compromise between these factors.

Also, it is currently unclear whether targeting hemicellulose

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

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:

Y ¼ a0 þXk

i¼1

aiXiþXk

i¼1

aiiX2i þ

Xk�1

i¼1

Xk

j¼1

aijXiXj ð1Þ

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

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

Xis are the independent variables (temperature, time,Tab

leII.

Equationsforprocess

responses.

Response

aEquationb

Equationno.

R2

Totalsolids

�2539.29þ18.97364Tþ5.394907tþ

808.615Sþ4.242763C�0.035089T2�0.020675Tt�

2.551333TS�0.021344TC�1.187289tS�100.8928S2

20.9667

EOL

�272.116þ2.569489Tþ51.82181S�0.153372C�0.007105T2þ0.000313Tt�

0.243333TSþ0.008278TC�0.010175C2

30.9836

Rejects

40.9943�0.107144T�0.061483t�

6.839843S�0.28213Cþ0.002151C2

40.9541

KLin

solids

�152.387þ0.982352Tþ65.82606Sþ0.83784C�0.365TS�0.008028TCþ0.004236C2

50.8955

ALin

solids

�20.6118þ0.141785Tþ0.114933tþ

7.251006Sþ0.013341C�0.000202T2�0.000458Tt�

0.024333TS�0.000217TC�0.027595tS�0.613336S2þ0.000278C2

60.9735

Glu

insolids

�1808.02þ13.84639Tþ5.692375tþ

563.9703Sþ0.247147C�0.025864T2�0.02935Tt�

1.851333TS�82.14118S2

70.9135

Xylin

solids

�281.774þ2.025777Tþ1.525606tþ

97.4672Sþ0.034896C�0.003534T2�0.006667Tt�

0.326667TS�0.267905tS�10.53833S2þ0.000671C2

80.9843

Man

insolids

�118.739þ0.920677Tþ0.506054tþ

36.96629Sþ0.029624C�0.001674T2�0.002833Tt�

0.138TS�4.97941S2

90.9417

ALin

WS

�3.0099þ0.027314Tþ1.926562Sþ0.015614Cþ0.000056t2

10

0.8565

Glu

inWS

45.39307�0.238858T�0.182048t�

37.48341Sþ0.001079Ttþ

0.169667TSþ3.967278S2

11

0.8882

Xylin

WS

�292.159þ3.346597Tþ0.508627tþ

23.27865S�1.214107C�0.009797T2�0.003362Ttþ

0.007217TCþ0.002368tC

�7.178128S2�0.002726C2

12

0.9812

Man

inWS

�23.5586þ0.32709T�0.037549t�

4.736008S�0.090677C�0.001044T2þ0.025TSþ0.000561TCþ0.036417tS�0.000367C2

13

0.9871

Galin

WS

�7.49947þ0.08964Tþ0.015399tþ

0.714732S�0.043256C�0.000264T2�0.000108Ttþ

0.000211TCþ0.000086tC

�0.243036S2

14

0.9675

Ara

inWS

�3.20539þ0.042585Tþ0.009027tþ

0.028973S�0.018052C�0.000141T2�0.000054Ttþ

0.002333TSþ0.000106TC�0.140799S2

15

0.9777

Furin

WS

54.62532�0.586839T�0.159323t�

12.05638Sþ0.199876Cþ0.001652T2þ0.000971Ttþ

0.0519TS�0.001428TCþ0.00008t2�0.000229tC

þ1.258737S2þ

0.015827SCþ0.000287C2

16

0.9996

HMFin

WS

21.96658�0.2544T�0.048618t�

2.390281Sþ0.082767Cþ0.000732T2þ0.000292Ttþ

0.014667TS�0.000497TC

17

0.9862

aKL,Klasonlignin,AL,acid-solublelignin;WS,water-solublefraction;glu,glucose;xyl,xylose;man,mannose;gal,galactose;ara,arabinose;fur,furfural;HMF,hydroxymethylfurfural.

bT,cookingtemperature

(8C);t,time(m

in)atthecookingtemperature;S,sulfuricacid

(%,w/w

oven-dried

wood);C,ethanolconcentration(%

,v/v).

Pan et al.: Organosolv Fractionation for Bioconversion 859

Biotechnology and Bioengineering. DOI 10.1002/bit

catalyst dose, and ethanol concentration), the Xi, X2i , 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 equations determined for each response

are listed in Table II (Equations 2–17).

These equations can be used to calculate the responses to

all combinations of variables within the range of the small

composite design. To validate this model, the calculated

responses at center point conditions were compared with the

experimental data from five replicate pretreatment experi-

ments (#s 17.1–17.5, Table I), as shown in Table III. Some

of the calculated responses (e.g., acid-soluble lignin recovery

in the solids fraction, reject yield, glucose, and HMF in the

water-soluble) differed significantly from experimental

values (variance �� 5%). However, all other calculated

responses were in good agreement with experimental values.

As discussed above, some improvement in process yields

can be expected from small increases in the severities of all

process parameters above center point conditions (1808C; 60min; 1.25%H2SO4; 50% ethanol). As an example, calculated

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

point conditions but the recovery of cellulose (glucose) is

slightly increased to �48 g/100 g wood. A significant

increase (�30%) in the recovery of EOL is also calculated

from the model but the recovery of hemicellulose sugars is

reduced. Although these calculated responses were not

verified experimentally, they suggest that only marginal

improvements in yields can be anticipated by fine-tuning

center point conditions.

CONCLUSION

Response surface methodology indicates that center point

conditions (cooking temperature, cooking time, catalyst

dose, and ethanol concentration) chosen for the small

composite design are close to optimal for glucose recovery

in the solids fraction prepared by ethanol organosolv

pretreatment. Glucose recovery is a critical parameter for

pretreatment optimization aimed at maximizing production

of fermentable sugars. Center point conditions yielded a

solids fraction containing �88% of the cellulose present in

the untreated wood. Total recovery of xylose (monomeric

and oligomeric), the most abundant pentose sugar, was

equivalent to �72% of the xylose present in the untreated

wood. Approximately 74% of the lignin was recovered as a

precipitate following ethanol extraction.

Further process optimization for bioconversion purposes

requires more detailed evaluation of the susceptibility of

solids to enzymatic hydrolysis and the fermentation of

resultant sugars. Approximately 82% of the total cellulose in

the untreated poplar was recovered as monomeric glucose

after enzymatic hydrolysis of the solids fraction; �85% was

recovered as glucose after 48 h hydrolysis. Hydrolysis was

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

methanol organosolv pretreated aspen (P. tremuloides) and

black cottonwood (P. trichcarpa) (Chum et al., 1988). These

data support the conclusion that organosolv pretreatment of

poplar species, using ethanol or methanol plus acid catalyst,

results in high yields of glucan and high recoveries

of monomeric glucose following enzymatic hydrolysis,

despite earlier indications to the contrary (Holtzapple and

Humphrey, 1984).

Hydrolysis of solids prepared under other cooking con-

ditions remains to be investigated. Numerous factors, which

were not evaluated in the present study, influence hydrolysis.

These include physical properties of cellulose fibers (e.g.,

surface area, porosity, and crystallinity) and the chemical and

physical characteristics of residual lignin and hemicellulose

in the solids. It is conceivable that some cooking conditions

yield solids in which lower cellulose yields are more than

compensated by improved hydrolysis. The effect of pretreat-

ment conditions on sugar fermentation and on the quality

of potential co-products, such as lignin and extractives, also

requires evaluation. Results presented above provide a plat-

form for further investigation.

NOMENCLATURE

AL acid-soluble lignin

ara arabinose

C concentration of aqueous ethanol, % (v/v)

EOL ethanol organosolv lignin

IU international unit (b-glucosidase activity)FPU filter paper unit (cellulase activity)

fur furfural

gal galactose

glu glucose

Table III. Process responses: comparison between observed and

calculated values.

Response

Observed

valueaCalculated

valuebVariancec,

%

Pulp yield, % of wood 52.72 49.55 6.0

Klason lignin, % of wood 5.89 4.81 18.3

Acid soluble lignin,% ofwood 0.30 0.28 6.7

Glucose, % of wood 43.15 43.32 �0.4

Xylose, % of wood 3.34 3.09 7.5

Mannose, % of wood 1.49 1.37 8.1

EOL yield, % of wood 15.53 15.00 3.4

Reject yield, % of wood 1.29 0.74 42.6

Water-solubles, % of wood

Acid soluble lignin 5.22 5.16 1.1

Glucose 0.55 0.65 18.2

Galactose 0.33 0.33 0.0

Arabinose 0.22 0.22 0.0

Xylose 9.37 9.44 �0.7

Mannose 1.29 1.27 1.6

Furfural 0.47 0.47 0.0

HMF 0.10 0.13 �30.0

aObserved value: average of five replicates at center point conditions(#s 17.1–17.5; Table I).

bCalculated value: calculated using equations 2–17 at center pointconditions (T¼ 1808C, t¼ 60 min, S¼ 1.25%, and C¼ 50%).

cVariance¼ (observed value�calculated value)/observed value� 100).

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, 8CWS water-soluble fraction

xyl xylose

We thank Novozymes for providing enzyme samples.

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