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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
DOI 10.1002/bit
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).
Conditiona T t S C Total KL AL glu xyl man Rejectsc EOLc AL glu xyl man gal ara furfural HMF
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% the pulp, EOL, and water soluble fractions at center point
of the total lignin remained associated with the solids. Most conditions was 116% of the amount present in the untreated
of the lignin was dissolved during aqueous ethanol extraction wood. Part of the apparent excess can be explained by
and was recovered as a precipitate after dilution with water extensive depolymerization of lignin during organosolv
but a portion of lignin remained in the water-soluble fraction extraction. Furthermore, the value for acid-soluble lignin in
(Fig. 2). The precipitated ethanol organosolv lignin (EOL) the water-soluble fraction is probably inflated by the presence
accounted for approximately 74% of the Klason lignin in extractive components (e.g., lignans, tannins, and related
untreated wood. The total amount of lignin recovered from 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.
removed prior to compositional analysis of the untreated and furfural, degradation products of hexoses and pentoses,
wood. respectively (Wenzl, 1970). However, the total amounts of
Approximately 88% of the total glucan in untreated wood HMF and furfural detected account for only a small fraction
was recovered in the solids fraction (calculated from data in of the unrecovered carbohydrate, suggesting further degra-
Fig. 2). The small amount of glucose present in the water- dation of monosaccharides to unidentified products (Harris
soluble fraction (1% of total) probably includes material et al., 1963; Sjostrom, 1992). It should also be noted that the
derived from glucomannan. The occurrence of xylose weight contribution of methyl glucoronic acid and acetate
(19% of total) and mannose (38% of total) in the solids side groups was not determined in these analyses.
fraction indicates the presence of some residual polymeric
hemicellulose. However, most of the recovered hemicellu-
Enzymatic Hydrolysis of Solids Fraction
losic sugars (53% total xylose, 33% of total mannose,
85% of total arabinose, and 87% of total galactose) were Enzymatic hydrolysis of solids prepared at center point
found in the water-soluble fraction. The amounts of conditions is shown in Figure 3. Data are the average for five
oligomeric and monomeric sugars in the water-soluble replicated solids samples (#s 17.117.5; Table I) hydrolyzed
fraction were determined by analysis before and after at 2% consistency (g cellulose/100 mL liquid) in separate
incubation in 3% H2SO4 under conditions which hydrolyze reactions using cellulase (Celluclast) and b-glucosidase
all oligomeric sugars to monomers. At center point condi- (Novozym 188) at 20 FPU (20.9 mg protein) and 40 IU
tions, 58% of the water-soluble xylose, and 71% of the (5.7 mg protein) per g cellulose, respectively.
water-soluble mannose was oligomeric. Substantial conversion of the poplar solids (containing
The extent of xylose recovery in the combined solids and 11.2% w/w average residual lignin) was observed within 24 h
water-soluble fractions (72%) indicates significant degra- (93% hydrolysis of the glucan in the solids fraction
dation of this component. Substantial losses of other hydrolyzed to glucose). When the hydrolysis was extended
monosaccharides were also observed, such that the total to 48 h, the conversion of cellulose to glucose reached 97%
recovery of carbohydrate (solids plus water-soluble fractions (Fig. 3). As discussed above, approximately 88% of the
excluding the reject fraction) was 84%. All of the major original cellulose was recovered in the solids fraction.
monosaccharides found in lignocellulose undergo thermal Therefore, the net conversion of cellulose (i.e., glucan in
decomposition under acidic conditions (Wenzl, 1970). The untreated wood) to glucose in the hydrolyzate was 82%
water-soluble fraction contained significant levels of HMF (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 center point conditions (52.72 1.17%) (Fig. 2; Table I).
fraction containing 42% residual lignin, prepared by SO2- Likewise, none of the conditions resulted in a higher recovery
catalyzed steam explosion of softwood (Douglas-fir), of cellulose in the solids (43.16 1.67 g; 88% of total
showed <30% at 24 h and 32% at 48 h hydrolysis of glucose in untreated wood), or a higher recovery of
cellulose to glucose, and 75% at 24 h and 90% at 48 h insoluble soluble xylose (12.73 0.27 g; 72% of total
hydrolysis after further delignification by oxygen bleaching xylose in untreated wood).
(Fig. 3), respectively. The rate of hydrolysis of the poplar All other sets of experimental conditions examined in the
solids was slower during the first 12 h than that of a solids small composite design resulted in yields of total solids,
fraction with a similar lignin content (9.2%) prepared by cellulose recoveries, and xylose recoveries that were
organosolv pretreatment of mixed softwood (Pan et al., 2005) comparable (e.g., condition #s 7 and 16) or inferior to those
but the extents of conversion at 24 and 48 h were higher given by the center point conditions. Nevertheless, examina-
(Fig. 3). The mechanistic basis for this difference in tion of differences in other process responses (e.g., solids
hydrolysis kinetics is presently unclear. delignification, solubilization of hemicellulose, and produc-
tion of sugar degradation products) indicates the potential for
fine-tuning process performance beyond those achieved at
Optimization of Sugar Recovery
center point conditions. For example, solids produced under
Mass balance data for other processing conditions, chosen conditions # 7 and #16 produced solids fractions containing
according to the Hartley small composite design, are less lignin and a water-soluble fraction containing less
presented in Table I. The yields and compositions of the furfural and HMF, than center point conditions, and a lower
solids, EOL, water-soluble, and reject fractions showed proportion of recovered hemicellulose was solubilized in
marked differences as cooking conditions were varied within both cases.
the experimental range. However, none of the conditions Further insights into the potential for optimizing process
tested produced a significantly higher yield of solids than the performance are gained from modeling the effects of
changing single process parameters, while others are fixed, Most responses showed a linear time-dependence (Figs. 4
using response surface methodology (Figs. 46). 6). Solids and ethanol-soluble lignin yields increased with
The predicted yields of solids and EOL increase slightly longer cooking time, while the amount of rejects fell (Fig. 4).
above 1808C to a maximum at 1958C (Fig. 4). The recovery Higher levels of acid-soluble lignin in the water-soluble
of glucose in the solids fraction is highest (Fig. 5), and the fraction following extended cooking times indicate increased
production of rejects lowest, at 1958C (Fig. 4). Reduced lignin degradation (Fig. 6). Levels of residual hemicellulosic
solids yield above 1958C is attributed largely to accelerated sugars in the solids and water-soluble fractions were
solubilization and degradation of hemicellulosic sugars. This generally insensitive to cooking time (Figs. 5 and 6). Levels
explanation is consistent with the general decline in levels of of furfural and HMF were proportional to cooking time.
xylose, mannose, arabinose, and galactose in the solids and Evidently, sugar levels in the water-soluble fraction are the
water-soluble fractions (Figs. 5 and 6), accompanied by net result of both hydrolysis and degradation during cooking.
increasing amounts of furfural, HMF in the water-soluble The yield of solids was maximal between 1.25% and 1.5%
fraction (Fig. 6), as the cooking temperature is increased H2SO4; reject production was minimal at 1.5% (Fig. 4). The
above 1808C. yield of EOL increased linearly up to 1.65%. The effect of
The slight increase in reject production above 1958C increased catalyst dose on solids composition was similar to
(Fig. 4) is probably due to promotion of lignin condensation that seen at higher temperatures (Fig. 5). Solubilization of
reactions leading to reduced defibration. This explanation is hemicellulosic sugars was markedly increased at higher
consistent with the net rise in Klason lignin content of the H2SO4 concentrations (Fig. 6). Recoveries of oligomeric
solids fraction as temperature is increased (Fig. 5). However, xylose and mannose levels in the water-soluble fraction
high temperatures also appear to increase lignin degradation, increased up to 1.5% H2SO4 and then declined. As discussed
as shown by elevated levels of acid-soluble lignin in the above, sugar levels in the water-soluble fraction represent the
water-soluble fraction (Fig. 6). 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
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
Regression Modeling
0.015827SC 0.000287C2
Table II. Equations for process responses.
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
b
a
DOI 10.1002/bit
HMF hydroxymethylfurfural Kurabi A, Berlin A, Gilkes N, Kilburn D, Bura R, Robinson J, Markov A,
KL Klason lignin Gusakov A, Okunev O, Sinitsyn A, et al. 2005. Enzymatic hydrolysis of
man mannose steam-exploded and ethanol organosolv-pretreated Douglas-fir by novel
S catalyst (concentrated sulfuric acid) dosage, % on wood chip (w/w) and commercial fungal cellulases. Appl Biochem Biotechnol 121124:
t time at maximum temperature, min 219230.
T maximum temperature, 8C Lehnen R, Saake B, Nimz HH. 2001. Furfural and hydroxymethylfurfural as
WS water-soluble fraction by-products of FORMACELL pulping. Holzforschung 55(2):199204.
xyl xylose Liu Y, Carriero S, Pye K, Argyropoulos DS. 2000. A comparison of the
structural changes occurring in lignin during Alcell and kraft pulping of
hardwoods and softwoods. Lignin: historical, biological, and materials
We thank Novozymes for providing enzyme samples. perspectives. p 447464.
Lora JH, Goyal GC, Raskin M. 1993. Characterization of residual lignins
after Alcell Pulping, Beijing. p 327336.
Lynd LR, Wyman CE, Gerngross TU. 1999. Biocommodity enginering.
Biotechnol Progr 15:777793.
References Lynd LR, Weimer PJ, van Zyl WH, Pretorius IS. 2002. Microbial cellulose
utilization: Fundamentals and biotechnology. Microbiol Mol Biol Rev
Boocock DGB, Balatinecz JJ. 1992. Alcell lignin modification and use in 66:506577.
adhesives. Abstr Pap Am Chem Soc 203:106. McDonough TJ. 1993. The chemistry of organosolv delignification. TAPPI J
Bradford MM. 1976. Rapid and sensitive method for quantitation of 76(8):186193.
microgram quantities of protein utilizing principle of protein-dye Mosier N, Wyman C, Dale B, Elander R, Lee YY, Holtzapple M, Ladisch M.
binding. Anal Biochem 72(12):248254. 2005. Features of promising technologies for pretreatment of ligno-
Cavaleri MA, Gilmore DW, Mozaffari M, Rosen CJ, Halbach TR. 2004. cellulosic biomass. Bioresour Technol 96:673686.
Hybrid poplar and forest soil response to municipal and industrial Myers RH, Montgomery DC. 2002. Response surface methodology: Process
by-products. J Environ Qual 33:10551061. and product optimization using designed experiments. New York:
Chum HL, Johnson DK, Black S, Baker J, Grohman K, Sarkanen KV, J. Wiley. 798p.
Wallace K, Schroeder HA. 1988. Organosolv pretreatment for Ni Y, Hu Q. 1995. Alcell1 lignin solubility in ethanol-water mixtures. J Appl
enzymatic hydrolysis of poplars: 1. Enzyme hydrolysis of cellulosic Polym Sci 57(12):14411446.
residues. Biotechnol Bioeng 31:643649. Ni Y, van Heiningen ARP. 1996. Novel ozone bleaching of Alcell1 pulp.
Dence CW. 1992. The determination of lignin. In: Lin SY, Dence CW, Abstr Pap Am Chem Soc 211:117.
editors. Methods in lignin chemistry. Berlin: Springer-Verlag. p 33 Ni Y, van Heiningen ARP. 1997. TCF bleaching for the Alcell1 process
61. including oxygen delignification. Pulp Pap Canada 98(11):3841.
Diaz MJ, Alfaro A, Garcia MM, Engenio ME, Ariza J, Lopez F. 2004. Pan XJ, Arato C, Gilkes N, Gregg D, Mabee W, Pye K, Xiao ZZ, Zhang X,
Ethanol pulping from tagasaste (Chamaecytisus proliferus L.F. ssp. Saddler J. 2005. Biorefining of softwoods using ethanol organosolv
palmensis). A new promising source for cellulose pulp. Ind Eng Chem pulping: Preliminary evaluation of process streams for manufacture of
Res 43(8):18751881. fuel-grade ethanol and co-products. Biotechnol Bioeng 90(4):473481.
Ghose TK. 1987. Measurement of cellulase activities. Pure Appl Chem Pye EK, Lora JH. 1991. The Alcell process, a proven alternative to kraft
59:257268. pulping. TAPPI J 74(3):113118.
Gilarranz MA, Oliet M, Rodriguez F, Tijero J. 1998. Ethanol-water pulping: Sarkanen KV. 1990. Chemistry of solvent pulping. Tappi J 73(10):215219.
Cooking variables optimization. Can J Chem Eng 76(2):253260. Sjostrom E. 1992. Wood chemistry. Fundamentals and application. San
Harris JF, Saeman JF, Locke EG. 1963. Wood as a chemical raw material. In: Diego: Academic Press, Inc. 230p.
Browning BL, editor. The chemistry of wood. New York: Interscience Stockburger P. 1993. An overview of near-commercial and commercial
Publishers. p 535585. solvent-based pulping processes. Tappi J 76(6):7174.
Hepditch MM, Thring RW. 1997. Alkaline cupric oxide and nitrobenzene Thring RW, Vanderlaan MN, Griffin SL. 1997. Polyurethanes from Alcell1
oxidation of Alcell1 lignin. Can J Chem Eng 75(6):11081114. lignin. Biomass Bioenerg 13(3):125132.
Holtzapple MT, Humphrey AE. 1984. The effect of organosolv pretreatment Wenzl HFJ. 1970. The chemical technology of wood. New York: Academic
on the enzymatic-hydrolysis of poplar. Biotechnol Bioeng 26(7):670 Press. 174p.
676. Williamson PN. 1988. Repaps Alcell processNew demonstration facility
Jimenez L, Perez I, Garcia JC, Lopez F, Ariza J. 2004. The influence of the shows how pulpmills can be cheaper. Sven Papperstidn 91(7):2123.
ethanol pulping of wheat straw and of the beating of pulp on the resulting Wood TM, Bhat M. 1988. Methods for measuring cellulase activities. In:
paper sheets. Wood Sci Technol 38(2):127137. Colowick SP, Kaplan NO, editors. Methods in enzymology, biomass
Kamm B, Kamm M. 2004. Principles of biorefineries. Appl Microbiol (part A, cellulose and hemicellulose), Vol. 160. New York: Academic
Biotechnol 64:137145. Press, Inc. p 87112.
Kubo S, Kadla JF. 2004. Poly(ethylene oxide)/organosolv lignin blends: Wyman CE, Dale BE, Elander RT, Holtzapple M, Ladisch MR, Lee YY.
Relationship between thermal properties, chemical structure, and blend 2005. Coordinated development of leading biomass pretreatment
behavior. Macromolecules 37(18):69046911. technologies. Biores Technol 96(18):19591966.