Ind. Eng. Chem. Res. 2007, 46, 2609-2617
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Pretreatment of Lodgepole Pine Killed by Mountain Pine Beetle Using the Ethanol Organosolv Process: Fractionation and Process Optimization Xuejun Pan,*,†,‡ Dan Xie,‡ Richard W. Yu,‡ Dexter Lam,‡ and Jack N. Saddler‡ Department of Biological Systems Engineering, UniVersity of Wisconsin-Madison, 460 Henry Mall, Madison, Wisconsin 53706, and Department of Wood Science, UniVersity of British Columbia, 2424 Main Mall, VancouVer, BC, V6T 1Z4 Canada
Lodgepole pine (Pinus contorta) killed by mountain pine beetle (Dendroctonus ponderosae) (MPB-LPP) was evaluated for bioconversion to ethanol using the ethanol organosolv process. The pretreatment was optimized using an experimental matrix designed with response surface methodology. It was found that MPBLPP was easy to pretreat and delignify, but gave low yields of substrate and carbohydrate as a result of excessive hydrolysis and subsequent decomposition of cellulose and hemicellulose during the pretreatment. The center-point conditions (170 °C, 60 min, 1.1% H2SO4 and 65% ethanol) were close to the optimum for the recovery of glucose and ethanol organosolv lignin. At the center-point conditions, ∼75% of the cellulose present in the untreated wood was recovered in the substrate fraction, and approximately 79% of the lignin in the wood was recovered as ethanol organosolv lignin (EOL). The combined recovery of carbohydrate in the substrate and water-soluble fractions was ∼83% glucose, ∼46% mannose, ∼53% xylose, ∼78% galactose, and ∼55% arabinose. The lost carbohydrate was decomposed to furfural, hydroxymethylfurfural, and levulinic and formic acids. The substrate generated at center-point conditions from MPB-LPP was readily digestible. Cellulose-to-glucose conversion yields of ∼93% and ∼97% were achieved within 24 and 48 h, respectively, with 20 FPU of cellulase/g of cellulose. 1. Introduction Lodgepole pine (LPP, Pinus contorta) is the most commercially important tree species in British Columbia (BC), Canada, covering 14.9 of 60 million ha of forested land in BC.1 However, the current outbreak of mountain pine beetle (MPB, Dendroctonus ponderosae) in BC has infested about 7 million ha of LPP and is still accelerating. It is forecasted that 80% of LPP will be killed by MPB by the middle of the next decade.1 Although the beetle infestation does not significantly affect the physical strength of the wood, the fungi associated with MPB produce melanin and cause a blue to black discoloration of sapwood, thus reducing its value in the market.2 Furthermore, the beetle-killed trees are easy for decay fungi to colonize, further reducing the quality and value of the wood.3 In addition, leaving killed trees untouched increases the risk of wild fire. However, large-scale salvage operation will produce an abundance of stained wood that exceeds the normal market ability for consumption, and therefore, new markets and uses in addition to timber and pulp have to be found. Biorefining of lignocellulosic biomass to produce fuels, chemicals, and materials is believed to be a potential alternative to the current dependence on petroleum oil.4,5 The bioconversion of biomass to ethanol through a “carbohydrate platform” is of current interest. A typical bioconversion process consists of pretreatment, saccharification, fermentation, and ethanol distillation steps. Several bioconversion schemes have been proposed, including steam explosion, ammonia fiber explosion (AFEX), dilute acid or hot water treatment, and the organosolv process.6-9 A primary technical-economic challenge in all lignocellulosicsto-ethanol bioconversion processes is the development of costeffective pretreatment methods. The main objective of a * To whom correspondence should be addressed. Tel.: 608-2624951. Fax: 608-262-1228. E-mail:
[email protected]. † University of Wisconsin-Madison. ‡ University of British Columbia.
pretreatment is to produce a cellulose-rich substrate that is susceptible to enzymatic hydrolysis (i.e., to provide fast conversion of cellulose to glucose at low enzyme loading).10 In addition, good recovery of hemicellulosic sugars and isolation of high-quality lignin are important. Development of high-value coproducts from hemicellulose and lignin is critical to improving the economics of bioconversion processes. Among the pretreatment technologies currently being evaluated is an ethanol organosolv process. The process was originally developed as a chemical process for fractionating lignocellulosics using organic solvents11 and was later adapted as a pulping method.12 The Alcell process further refines ethanol organosolv pulping as an alternative to Kraft pulping of hardwoods to produce high-quality fibers.13,14 The organosolv process has not been developed significantly for softwoods, however, because of the higher lignin content and difference in lignin structure.15 Historically, the organosolv process has been investigated largely from the perspective of pulp production,16-19 but it is not wellstudied as a pretreatment/biorefining tool for the bioconversion of lignocellulosic biomass. Recently, the pretreatment of hardwood hybrid poplar using the ethanol organosolv process was investigated in detail from the angles of the recovery of carbohydrate and lignin, the enzymatic hydrolyzability of the substrates, and the effects of process variables on substrate characteristics.20,21 The ethanol organosolv lignin from the poplar was evaluated as an antioxidant.22 The efficacy of ethanol organosolv pretreatment for mixed softwoods (spruce, pine, and Douglas fir) was demonstrated in our previous research as well.6 The process produced a substrate with superior enzymatic digestibility over those pretreated by alternative processes and a particularly high-quality lignin fraction with potential applications.23-25 However, the ethanol organosolv pretreatment of softwood has not been optimized, and the process mass balance data are not available. The objective of the present research was to further investigate the ethanol organosolv pretreatment of softwood using mountain-
10.1021/ie061576l CCC: $37.00 © 2007 American Chemical Society Published on Web 03/14/2007
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Table 1. Chemical Composition of Untreated MPB-LPP
a
component
contenta
ash extractives (water followed by ethanol) Klason lignin acid-soluble lignin carbohydrate (as monosaccharide) glucose mannose xylose galactose arabinose carbohydrate (as polysaccharide) glucan mannan xylan galactan arabinan
0.26 ( 0.01 4.66 ( 0.21 24.79 ( 0.09 0.29 ( 0.00 50.46 ( 0.25 13.09 ( 0.24 7.21 ( 0.04 2.22 ( 0.01 1.42 ( 0.00 45.42 ( 0.22 11.78 ( 0.21 6.34 ( 0.03 2.00 ( 0.01 1.25 ( 0.00
Content reported as % (w/w) in oven-dried MPB-LPP chips.
pine-beetle-killed lodgepole pine (MPB-LPP) as a feedstock. In addition, the compositional and morphological differences between beetle-killed wood and healthy wood might impact the pretreatment, i.e., the infested sapwood has lower lignin, carbohydrate, and extractives contents but increased permeability compared to sound sapwood.26 The study described herein used response surface methodology to optimize the ethanol organosolv pretreatment of MPB-LPP. The effects of process parameters on the yields and distributions of cellulose, hemicellulose, and lignin in the fractions generated during the pretreatment were examined, and the process mass balance and enzymatic hydrolysis of the resulting substrate were investigated. Mathematical equations were regressed to quantitatively predict the effects of the process variables on the fractionation and substrate characteristics. 2. Materials and Methods Feedstock Preparation. Lodgepole pine trees infested by mountain pine beetle (MPB-LPP) at the gray phase (dead tree) were harvested at Burns Lake (53°96′43′′ N, 600°58′20′′ W), Prince George (50°48′25′′ N, 594°76′47′′ W), and Quesnel (49°50′98′′ N, 587°08′01′′ W), BC, Canada. Seven trees without heart rot (one from Burns Lake, two from Prince George, and four from Quesnel) were used in the present research. The average age of the trees was 99 ( 25 years. Two sections, one from the top part and another from the bottom part, were collected from each trunk. After being debarked and air-dried, the tree trunks were chipped using a custom-designed chipper. The chips were then screened using a plate screen; the fraction larger than 2.5 × 2.5 cm and smaller than 5.0 × 5.0 cm, approximately 0.5 cm thick, was collected as the feedstock for ethanol organosolv pretreatment. A sample of the chips was ground using a Wiley mill, and the fraction passing 40-mesh was collected for chemical analysis. The chemical composition of the wood is summarized in Table 1. Ethanol Organosolv Pretreatment. A flowchart of the laboratory-scale ethanol organosolv process was schematically described before.20 In brief, MPB-LPP chips were pretreated in aqueous ethanol with sulfuric acid as a catalyst using a custom-built, four-vessel (2 L each) rotating digester made by Aurora Products Ltd. (Savona, BC, Canada). A 200-g (ovendried weight) batch of chips was pretreated in each vessel. Vessels were opened after being cooled to room temperature in a water bath. Spent liquor (i.e., aqueous ethanol in the vessel) was sampled immediately for determination of furfural, hydroxymethylfurfural (HMF), and formic and levulinic acids. The
substrate (i.e., defiberized solid fraction) and spent liquor were then separated using nylon cloth. The substrate was washed three times (300 mL each) with warm (60 °C) aqueous ethanol having the same concentration as the pretreatment liquor. The washes were combined with the spent liquor. The substrate was then washed three times with water at 60 °C, and the washes were discarded. The washed substrate was homogenized in a standard British disintegrator for 5 min and passed through a laboratory flat screen with 0.008-in. (0.203-mm) slits (Voith, Inc., Appleton, WI) to remove rejects (i.e., non-defiberized woodchips and knots). The yields of rejects and screened substrate were determined. The screened substrate was stored at 4 °C for analysis and hydrolysis. The spent liquor and the ethanol washes were combined and mixed with three volumes of water to precipitate the dissolved lignin. The lignin precipitate, henceforth denoted as ethanol organosolv lignin (EOL), was collected on Whatman No. 1 filter paper, washed thoroughly with water, and air-dried. The filtrate and the water washes were combined to give a water-soluble fraction containing monomeric and oligomeric saccharides, depolymerized lignin, and compounds derived from saccharides. Analytical Procedures. Oven-dried weights were determined by drying to constant weight at 105 °C in a convection oven. Ash of the wood powder was determined according to TAPPI (Technical Association of Pulp and Paper Industry) standard method T211 om-93. Extractives of the wood powder were determined according to the procedure of TAPPI standard method T264 cm-97 using ethanol and water as solvents. Klason lignin of the wood powder and substrates was determined according to TAPPI standard method T-222 om-98. The hydrolysate from Klason lignin determination was retained for analysis of monosaccharides and acid-soluble lignin. Acidsoluble lignin was determined by UV absorbance at 205 nm according to the method described by Dence.27 Monosaccharides were determined using a DX-500 HPLC system (Dionex, Sunnyvale, CA) equipped with an AS3500 autosampler, a GP40 gradient pump, an anion-exchange column (Dionex CarboPac PA1), and an ED40 electrochemical detector. The column was eluted with deionized water at a flow rate of 1 mL/min. Aliquots (20 µL) were injected after being passed through a 0.45-µm nylon syringe filter (Chromatographic Specialties Inc., Brockville, ON, Canada). Optimization of baseline stability and detector sensitivity was achieved by postcolumn addition of 0.2 M NaOH. The column was reconditioned using 1 M NaOH after each analysis. Monosaccharides were quantified with reference to saccharide standards. The saccharide standards were autoclaved at 120 °C for 1 h prior to analysis to compensate for possible decomposition caused by heating involved in Klason lignin determination. Furfural and HMF were determined using a Dionex Summit HPLC system equipped with a P680 pump, an ASI-100 autosampler, and a PDA100 photodiode array detector. A LiChrospher 5RP18 column (Varian, Palo Alto, CA) was used at 60 °C with an eluent flow rate of 0.5 mL/min. A gradient of (A) 7.4 mM H3PO4, (B) acetonitrile, and (C) a mixture of 7.4 mM H3PO4, methanol, and acetonitrile (4:3:3, v/v) was applied as follows: 0-20 min, from 95% A and 5% C to 50% A and 50% C; 20-24 min, from 50% A and 50% C to 100% C; 24-25 min, 100% C; 25-26 min, from 100% C to 100% B; 26-27 min, 100% B; 27-28 min, from 100% B to 95% A and 5% C; 28-38 min, 95% A and 5% C. Appropriately diluted aliquots (20 µL) were injected after being passed through a 0.45-µm PTFE syringe filter (Chromatographic Specialties Inc.). Furfural and HMF were determined by absorbance at 280 nm. Formic and levulinic acids were quantified using an
Ind. Eng. Chem. Res., Vol. 46, No. 8, 2007 2611 Table 2. Experimental Matrix and Results for Ethanol Organosolv Pretreatment of MPB-LPP variablesb no.a
T
t
S
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 avg 17-21 SD 17-21
160 180 160 180 160 180 160 180 153 187 170 170 170 170 170 170 170 170 170 170 170
50 50 70 70 50 50 70 70 60 60 43 77 60 60 60 60 60 60 60 60 60
0.90 0.90 0.90 0.90 1.30 1.30 1.30 1.30 1.10 1.10 1.10 1.10 0.76 1.44 1.10 1.10 1.10 1.10 1.10 1.10 1.10
water-soluble componentsc C
subc
75 75 55 55 55 55 75 75 65 65 65 65 65 65 48 82 65 65 65 65 65
26.47 35.37 11.12 37.73 9.91 38.53 41.53 31.23 7.15 27.58 43.04 40.75 41.56 39.24 33.51 39.81 41.48 41.73 40.27 41.72 41.26 41.29 0.61
KL in sub (%)
gluc
rejc
EOLc
AL
glu
xyl
man
gal
ara
17.56 4.98 22.84 11.22 22.88 12.17 13.24 7.92 20.91 11.15 12.75 8.80 13.26 8.21 22.80 10.08 10.34 10.61 8.94 10.23 10.34 10.09 0.66
20.51 35.04 8.50 35.22 7.59 35.01 35.72 29.54 5.09 25.18 37.51 36.69 36.72 36.91 25.51 35.09 38.30 37.48 37.11 37.41 37.87 37.63 0.46
29.87 0.75 44.93 0.04 43.44 0.29 3.89 0.05 52.79 0.06 2.26 0.46 6.13 0.66 15.22 1.63 1.22 0.93 1.14 1.24 0.93 1.09 0.16
14.07 21.95 11.31 19.30 11.30 18.40 18.00 24.96 12.33 23.41 18.40 20.17 17.86 20.51 11.76 20.08 19.67 20.24 19.03 19.30 19.61 19.57 0.45
3.55 7.65 3.87 6.31 2.93 5.43 3.98 3.01 4.55 6.06 4.25 6.46 3.82 5.45 4.22 5.15 4.69 4.66 4.71 5.07 4.69 4.76 0.17
1.79 5.31 2.91 6.94 2.64 4.63 3.68 5.25 3.04 5.36 5.30 3.42 2.79 4.24 5.41 4.85 4.95 4.73 3.84 3.06 4.36 4.19 0.76
3.73 2.60 3.82 1.60 4.59 0.94 3.81 0.32 3.06 0.25 2.57 2.78 4.30 1.99 3.75 3.78 3.39 3.28 3.32 2.72 3.44 3.23 0.29
6.64 4.96 6.72 3.56 8.18 2.50 6.30 1.49 5.26 1.03 4.49 5.16 7.65 3.72 6.18 6.07 5.53 5.25 5.47 4.84 5.75 5.37 0.34
2.13 1.42 2.18 1.35 2.47 0.91 2.12 0.46 2.06 0.27 1.70 1.56 2.27 1.29 2.03 1.81 1.74 1.80 1.70 1.53 1.90 1.73 0.14
1.26 0.60 1.18 0.50 1.38 0.25 1.04 0.06 1.15 0.11 0.74 0.69 1.20 0.49 0.98 0.87 0.83 0.79 0.79 0.63 0.85 0.78 0.09
a Numbers 1-21 are the complete experimental matrix of 21 conditions. Numbers 17-21 are replicated center-point conditions. b T, temperature (°C); t, time (min) at that temperature; S, sulfuric acid (%, w/w, oven-dried wood); C, ethanol concentration, (%, v/v). c All data are yields of components (g) per 100 g (oven-dried weight) of untreated MPB-LPP chips. d Abbreviations: SD, standard deviation; sub, substrate; KL, Klason lignin; glu, glucose; rej, rejects; EOL, ethanol organosolv lignin; AL, acid-soluble lignin; xyl, xylose; man, mannose; gal, galactose; ara, arabinose.
Allilance 2695 HPLC system (Waters, Milford, MA) equipped with an AD20 absorbance detector (Dionex, Sunnyvale, CA), a SUPELCO (Bellefonte, PA) SUPELCOGEL C610H column, and a SUPELGUARD C601H guard column. The column temperature was 30 °C, and the mobile phase was 0.1% H3PO4 at a flow rate of 0.5 mL/min. Sample (5 µL) was injected onto the column after being filtered through a 0.45-µm nylon syringe filter as described above. The viscosity (degree of polymerization) of the cellulose solution was measured according to TAPPI standard method T230 om-99, as described before.21 Enzymatic Hydrolysis. Commercial cellulase (Spezyme CP) and β-glucosidase (Novozym 188) were provided by Genencor International Inc. (Rochester, NY) and Novozymes (Franklinton, NC), respectively. Cellulase activity was determined using the filter paper assay recommended by the International Union of Pure and Applied Chemists28 and is expressed in terms of filter paper units (FPUs). β-Glucosidase activity was determined using p-nitrophenyl-β-D-glucoside as the substrate, as previously described,29 and is expressed in terms of International Units (IUs). Protein was determined using Bio-Rad Protein Assay (Method of Bradford).30 Cellulase was supplemented with β-glucosidase (1:2 FPU/IU) to avoid product inhibition caused by cellobiose accumulation. Batch hydrolysis was conducted at 2% consistency (cellulose, w/v) in 50 mM acetate buffer, pH 4.8, with 0.004% tetracycline as an antibiotic. Cellulase was used at a loading of 20 FPU (21 mg of total protein)/g of cellulose with a supplementation of β-glucosidase at a loading of 40 IU (5.7 mg of total protein)/g of cellulose. The reaction mixture (100 mL) was incubated at 150 rpm, 50 °C, in a rotary shaker and sampled periodically for glucose determination. The glucose was quantified using HPLC, as described above, with the exception that the saccharic standards were not autoclaved. Hydrolysis data are averages from duplicate experiments. Experimental Design and Data Analysis. Pretreatment conditions [temperature, time, catalyst (H2SO4) dosage, and ethanol concentration] were optimized by response surface
methodology using a small Hartley composite design,31 as described in detail in our previous report.20 The complete experimental matrix is shown in Table 2. The selection of the conditions in Table 2 was based on the results of preliminary experiments (see Result and Discussion). Data were analyzed using Statistical Analysis System (SAS, SAS Institute Inc., Cary, NC). All data reported are the averages of at least duplicate experiments. 3. Results and Discussion Preliminary Experiments for Selecting Process Conditions. As shown in Table 1, MPB-LPP has the typical chemical composition of softwood. Compared to hybrid poplar (hardwood),20 MPB-LPP has more total lignin (more Klason lignin but less acid-soluble lignin) and a comparable amount of total carbohydrate but with a different composition. MPB-LPP has significantly more mannan and substantially less xylan than poplar. Previous research has indicated that the set of conditions (180 °C, 60 min, 1.25% H2SO4, 50% ethanol) is close to the optimum for poplar in the recovery of cellulose, hemicellulose, and lignin.20 In general, softwood requires more severe conditions for delignification than hardwood because softwood contains more lignin and, in particular, more guaiacyl units that tend to condense. Therefore, the pretreatment of MPB-LPP was started from more severe conditions than those used for poplar. As shown in Table 3, test 1 was conducted at 185 °C, 80 min, 1.5% H2SO4, and 60% ethanol. The substrate yield was only ∼35%, but residual lignin was high (∼27% Klason lignin). Increasing H2SO4 to 2.34% resulted in an additional reduction in substrate yield and increase in Klason lignin (test 2), implying the removal of more carbohydrate than lignin. It was noted that the substrates from tests 1 and 2 had no rejects and residual hemicellulose. However, a significant amount of acid-soluble lignin was observed in the water-soluble fraction as a result of the extensive destruction of lignin. These results suggest that the conditions used in tests 1 and 2 were too severe for MPBLPP. When the temperature was lowered to 170 °C (test 3), the
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Table 3. Results of Preliminary Pretreatment Experiments pretreatment conditionsa
fraction yieldb (% of wood)
substrate compositionb (%)
test no.
T
t
S
C
pulp
rej
EOL
AL in WS
KL
glu
xyl
man
1 2 3 4 5 6 7 8 9 10
185 185 170 170 170 170 170 160 160 190
80 80 80 80 40 80 60 60 100 120
1.50 2.34 1.50 1.20 1.20 1.20 1.00 1.00 1.00 2.50
60 60 60 60 60 70 70 70 70 60
35.10 29.30 38.80 40.30 42.55 38.52 40.75 14.06 38.82 28.63
0.00 0.00 0.15 0.58 3.14 0.22 2.24 39.05 8.56 0.00
19.20 18.22 20.90 19.20 17.10 22.00 21.25 15.20 18.13 16.93
5.95 5.37 4.85 4.27 4.32 5.17 4.61 3.74 4.49 8.03
27.42 31.99 10.55 11.62 14.19 7.40 9.21 18.43 13.93 63.65
81.49 77.53 97.49 93.73 83.73 96.14 91.09 76.42 89.16 42.97
ND ND ND ND 0.97 1.30 1.02 2.34 2.77 ND
ND ND ND ND 1.05 1.40 1.12 2.87 3.04 ND
a T, temperature (°C); t, time (min) at that temperature; S, sulfuric acid (%, w/w, oven-dried wood); C, ethanol concentration, (%, v/v). b Abbreviations: rej, rejects; EOL, ethanol organosolv lignin; AL, acid-soluble lignin; WS, water-soluble fraction; KL, Klason lignin; glu, glucose; xyl, xylose; man, mannose; ND, not detected.
substrate yield increased, and the Klason lignin decreased significantly. The reduction of catalyst dosage to 1.2% H2SO4 (test 4) resulted in additional improvement in substrate yield but a slight increase in Klason lignin. Still, no hemicellulose was detected in the substrates of tests 3 and 4. Compared to test 4, a 40-min reduction of reaction time in test 5 increased the substrate yield, but this yield increase was primarily from the increase in Klason lignin, not the reservation of carbohydrate. On the other hand, increasing the ethanol concentration by 10% (test 6) reduced the Klason lignin, suggesting that the dissolution of lignin from wood chips was enhanced. Further reducing the severity of test 6 by decreasing the reaction time by 20 min and the catalyst by 0.2% improved neither the recovery of carbohydrate nor the delignification (test 7). Lowering the temperature by 10 °C (test 8) resulted in a very high rejects (non-defiberized wood chips) rate of ∼39%, indicating a failure of defiberization. Extension of the reaction time by 40 min (test 9) did reduce the rejects rate, thus improving the pulp yield, but did not improve delignification (i.e., Klason lignin was still high). The MPB-LPP was also pretreated at extremely severe conditions of 190 °C, 120 min, 2.5% H2SO4, and 60% ethanol (test 10). The resulting substrate (
