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nanofibrillated by continuous extrusion process II: Effect of hot-compressed water treatment
Bioresource Technology 101 (2010) 9645–9649
Contents lists available at ScienceDirect
Bioresource Technology journal homepage: www.elsevier.com/locate/biortech
Enzymatic saccharification of woody biomass micro/nanofibrillated by continuous extrusion process II: Effect of hot-compressed water treatment Seung-Hwan Lee a,*, Seiichi Inoue a, Yoshikuni Teramoto b, Takashi Endo a a b
Biomass Technology Research Center, National Institute of Advanced Industrial Science and Technology (AIST), 3-11-32, Kagamiyama, Higashihiroshima, Hiroshima 739 0046, Japan Division of Forest and Biomaterials Science, Graduate School of Agriculture, Kyoto University, Kyoto 606 8502, Japan
a r t i c l e
i n f o
Article history: Received 31 May 2010 Received in revised form 15 July 2010 Accepted 15 July 2010 Available online 27 July 2010 Keywords: Extrusion Bioethanol Enzymatic saccharification Hot-compressed water treatment Fibrillation
a b s t r a c t An extrusion process involving a twin-screw extruder was used for the micro/nanofibrillation of Douglas fir and Eucalyptus treated with hot-compressed water (HCW). Partial removal of hemicellulose and lignin by HCW treatment effectively improved the fibrillation by extrusion. Only HCW treatment produced glucose less than 5 weight percent (wt.%) in Douglas fir in a temperature range of 140–180 °C by enzymatic hydrolysis. Glucose production yields of 18 and 26 wt.% were obtained by HCW treatment at 170 and 180 °C, respectively, in Eucalyptus. Use of extrusion after HCW treatment drastically improved monosaccharide production yield in both woods. In the case of Douglas fir, the obtained values were 5 times higher than those obtained by HCW treatment alone. Total monosaccharide production yields were higher in Eucalyptus than in Douglas fir. The extruded production had a fine fibrous morphology on a sub-micro/nanoscopic scale. This result shows the great potential of the extrusion process after HCW treatment as a cost-effective pretreatment for enzymatic saccharification of woody biomass. Ó 2010 Elsevier Ltd. All rights reserved.
1. Introduction Extensive research and development programs in the area of renewable bioenergy have been initiated in many countries. As part of this initiative, researchers have been encouraged to develop technologies for the production of bioethanol from lignocellulosic biomass. According to the U.S. Department of Energy, Office of the Biomass Program (Office of the Biomass Program, 2010), a reliable biofuels market of at least 7.5 billion gallons will be available by 2010 (Himmel et al., 2007). The European Union has also envisioned that by 2030, one-fourth of the EU’s transportation fuel requirement will be met by biofuels (Biofuels Research Advisory Council, 2006). It is expected that this change will significantly decrease the EU’s dependence on imported fossil fuels. It has been reported that bioethanol from lignocellulosic resources is much less petroleum-intensive than gasoline and can contribute to reducing greenhouse gas emissions (Farrel et al., 2007). However, in order to realize the large-scale use of bioethanol as a liquid fuel, new technology needs to be developed to allow for more efficient utilization of lignocellulose. Mechanical size reduction as a pretreatment for enzymatic saccharification of lignocellulosic biomass is known as one of the most energy-intensive processes (Himmel et al., 2007; Mani et al., 2004). Some research has been conducted to enhance the economical * Corresponding author. Tel./fax: +81 82 420 8278. E-mail addresses: [email protected], [email protected] (S.-H. Lee). 0960-8524/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.biortech.2010.07.068
feasibility of this process along with its effectiveness to increase enzymatic accessibility (Lee et al., 2008, 2009, 2010; Zhu et al., 2009, 2010). Recently, Zhu et al. (2010) reported on the energy consumption of the post-chemical pretreatment size-reduction method; they investigated the effects of chemical treatment and disk-milling conditions on energy consumption for size reduction and the efficiency of enzymatic cellulose saccharification of lodgepole pine wood. They treated lodgepole pine with 3 different methods, namely, hot-water, dilute-acid, and sulfite treatment, and milled the treated product using a disk mill. They found that this combined method decreased size-reduction energy consumption by 20–80%, depending on the conditions. In an earlier study, they also found that chemical pretreatment not only increased cellulose conversion but also reduced mechanical milling energy consumption (Zhu et al., 2009). Thermomechanical disk milling was more energy intensive than high-pressure thermomechanical disk milling, but the former was more effective than the latter in accomplishing enzymatic saccharification. Our research group has reported that mechanical size reduction by ball and disk milling of hot-compressed water (HCW)-treated wood at mild temperatures (below 180 °C) can reduce energy consumption, which then reduces the amount of water required for HCW treatment and enzyme loading for saccharification (Hideno et al., 2009; Inoue et al., 2008; Lee et al., 2009, 2010). HCW treatment can partially remove hemicellulose and lignin from the cell wall supramolecular structure to produce nanoscopic pores between cellulose microfibrils, facilitating mechanical fibrillation.
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This process prevents the formation of fermentation inhibitors such as furaldehydes and hydroxylmethyl furan. In particular, development of new pretreatment technology has also been attempted to achieve high cost feasibility by utilizing a batch-type kneader with combination-available twin-screw elements and a twin-screw extruder (Lee et al., 2008, 2009). A batch-type kneader with combination-available twin-screw elements was used in a pre-experiment for the utilization of a twin-screw extruder. The fibrillated product showed sub-micro/ nanoscopic morphology, which increased the surface area of cellulose. However, use of mechanical kneading alone appears to have some limitations in exposing cellulose for complete enzymatic saccharification. It was found that cooking of the fibrillated products using hot water at 130 °C under 0.25 MPa can be an option to overcome this limitation. Furthermore, a continuous extrusion process using additives with cellulose affinity was employed as a practical and economical method (Lee et al., 2008). As described in this report, this process has great potential for large-scale production with various advantages: highly efficient pulverization by application of high shearing force, high throughputs, and excellent temperature control (Dale et al., 1999; Pilli et al., 2005; Thymi et al., 2005). Based on the experimental results of our previous studies, this study was undertaken in an attempt to improve the enzymatic saccharification yield of Douglas fir and Eucalyptus by using an HCW treatment extrusion process. 2. Experimental 2.1. Materials Douglas fir and Eucalyptus wood chips were purchased from Oji Paper Co., Ltd. (Tokyo, Japan) and cutter-milled under 3 mm using a milling cutter (Masuko Sangyo Co., Ltd., Japan). All chemicals were purchased from commercial sources and used without further purification. Acremonium cellulose (Meiji Seika Co., Ltd., Japan), Novozyme 188 (cellobiase from Aspergillus niger, Sigma– Aldrich Japan, Japan), and Optimash BG (Multifect xylanase, Genencor Kyowa Inc. Japan) were used for the saccharification of the fibrillated product. The Meicelase (Meiji Seika Co., Ltd., Japan) from the fungus Trichoderma viride was also used for cellulose hydrolysis in both woods. 2.2. Chemical composition determination Chemical composition was determined as follows. The holocellulose, a-cellulose, and lignin content were determined as the residue obtained after delignification by NaClO2, the residue insoluble in 17.5% aqueous NaOH solution (as determined by TAPPI-203), and the residue insoluble in 72% aqueous sulfuric acid solution, respectively. The dry-basis content of holocellulose, a-cellulose, and Klason lignin in the starting materials were calculated from their respective weights. The a-cellulose content was quantified as cellulose content. The hemicellulose content was determined by subtracting the a-cellulose content from the holocellulose content. The dry-basis contents of a-cellulose, hemicellulose, and Klason lignin were 46.8, 28.9, and 25.3 weight percent (wt.%) in Douglas fir and 42.1, 34.6, and 28.8 wt.% in Eucalyptus, respectively. 2.3. HCW treatment and extrusion HCW treatment was performed by the same method and the detailed experimental procedure is described in previous report (Lee et al., 2010). Briefly described, the used temperatures ranged from 140 °C to 180 °C and reaction time that excludes the warming-up period was 30 min. The initial pressure and the
water:starting material weight ratio were adjusted to 1 MPa of nitrogen gas and 5:1, respectively. After the treatment, the reactor was immediately soaked in a water bath maintained at room temperature. The treated products were filtrated and washed with water. The obtained water-soluble fraction was pre-condensed and dried at 40 °C in vacuo to calculate its amount. The water-insoluble residue was provided to the twin-screw extruder (2D15 W; Toyo Seiki, Tokyo, Japan). The residual product was extruded through the co-rotating twin screws 1–5 times. The barrel diameter and length/diameter (L/D) of screws were 15.4 and 17 mm respectively. Extrusion was conducted at room temperature with the rotating speed of 45–120 rpm. 2.4. Enzymatic saccharification and morphology Enzymatic hydrolysis of the extruded products was conducted using Acremonium cellulase, Novozyme 188, and Optimash BG. The substrate concentration was set to 33 mg/mL with 10 FPU of enzymes. Saccharification was carried out with 30 mL of acetate buffer (pH 5.0) at 45 °C for 72 h. The samples were stored at 18 °C and then heated at 95 °C for 15 min to deactivate the enzymes. The produced monomeric sugars were analyzed by LC-2000 Plus HPLC system (JASCO Corporation, Tokyo, Japan) equipped with a column (Aminex HPX-87, Bio-Rad Laboratories, K. K., Tokyo, Japan) at 80 °C; the eluent used was distilled water at a flow rate of 1 mL/ min. To compare the data in previous reports, hydrolysis using Meicelase was also carried out using ca. 50 mg of the fibrillated product in 17 mL of 50 mM acetate buffer (pH 5.0) containing 2 mg Meicelase (10 FPU). The morphological characteristics of the HCW-treated and the extruded products were analyzed by scanning electron microscopy (SEM) (S-4800, Hitachi Co. Tokyo, Japan). The samples were freezedried and coated with a 1 nm-thick layer by an osmium plasma coater (NEOC-AN, Meiwa Fosis, Tokyo, Japan). 3. Results and discussion HCW treatment is often called ‘‘autohydrolysis treatment” because the acetyl groups of hemicellulose and the water itself act as acids during treatment, thereby dissolving both the hemicellulose and the lignin of lignocellulosic materials. In particular, this treatment very effectively removes selectively hemicellulose without hydrolyzing cellulose at mild temperatures less than 200 °C (Ando et al., 2000; Sakaki et al., 1996 a,b). In this study, the temperature range of 140–180 °C was used to prevent cellulose degradation and formation of fermentation inhibitors seen at higher temperatures. Further, a water:starting material weight ratio of 5:1 was used to save operating energy required to heat water. Table 1 shows the amount of water-soluble fraction depending on HCW treatment temperature. Of the initial composition of hemicellulose (34.6 and 28.9 wt.% in Eucalyptus and Douglas fir, respectively), about half was removed by HCW at 180 °C. In our previous report, partial removal of hemicellulose and lignin by HCW treatment in the same temperature range was discussed. Experimental
Table 1 Amount of water-soluble fraction (wt.%) obtained according to temperature of hotcompressed water (HCW) treatment. Sample
Douglas fir Eucalyptus a
Lee et al., 2010.
HCW temperature (°C) 140
160
170
180
5.3 2.9a
11.3 14.3a
13.4 14.5
14.7 15.8
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results using Eucalyptus showed that most of the fraction was found to be xylooligosaccharides and only a small amount of lignin (<2.4 wt.%) was removed in a manner that was not dependent on HCW treatment temperature (Lee et al., 2010). It is also reported that removal of the water-soluble fraction can generate nanoscopic porous cell wall structure, enhancing the mechanical fibrillation degree and the enzyme accessibility of cellulose (Lee et al., 2010). Fig. 1 shows the effect of the extrusion after HCW treatment and HCW treatment itself on glucose production yield. In HCW treatment alone, the glucose production yield in Doulas fir was less than 5 wt.% in the entire temperature range, whereas glucose yields of 18 and 26 wt.% were obtained at 170 and 180 °C respectively, in Eucalyptus. Generally, HCW treatment is known to be less effective in softwood than in hardwood, which is possibly due to different lignin molecular structure and hemicellulose content between hardwoods and softwoods. For example, degradation of the b-O-4 bond-rich hardwoods is generally known to be more sensitive by hydrothermal treatment than that of softwoods with fewer b-O-4 bonds (Assor et al., 2000). Glucose production yield was improved by extrusion in both woods. It is interesting to note that extrusion drastically improved glucose production yield in Douglas fir. The yields obtained by extrusion were 5 times higher than those by HCW treatment alone across the entire temperature range. This result shows great potential of the extrusion process
Glucose production yield based on original weight (wt%)
40
30
: Douglas fir : Eucalyptus Black: Only HCW White: Extrusion after HCW
20
10
0 120
140
160
180
200
HCW treatment temperature ( °C) Fig. 1. Effect of hot-compressed water (HCW) treatment temperature and HCW + extrusion on glucose production yield. Substrate concentration, 33 mg/mL with 10 FPU of enzymes (Acremonium cellulase, Novozyme 188, Optimash BG); saccharification temperature, 45 °C; time, 72 h.
after HCW treatment in softwood, compensating for the lesser effect of HCW treatment alone on it. In our previous report using batch-type kneader, only mechanical kneading of Douglas fir with water showed limitation in enhancing enzymatic accessibility, but kneading after hot-water treatment improved it (Lee et al., 2009). It was explained by the structural viewpoint of wood cell wall formation. The heterogeneous nature of cellulose microfibril bundles encased by hemicellulose and lignin causes significant recalcitrance against enzyme accessibility. Therefore, loosening this structure by partially removing hemicellulose and lignin by HCW treatment is very effective to enhance enzymatic saccharification and to facilitate fibrillation. Eucalyptus also had a higher glucose production yield upon extrusion than with HCW treatment alone. The total glucose production yield was higher in Eucalyptus than in Douglas fir, which may be due to the higher HCW treatment effect in Eucalyptus. Table 2 summarizes the other monosaccharides obtained by HCW treatment and extrusion. As seen with glucose production yield, extrusion effectively enhanced the hydrolysis of hemicellulose in both woods. The total monosaccharide yield in Douglas fir decreased with increased HCW treatment temperature. Total yields were higher in Eucalyptus than in Douglas fir. For comparison to previous reports (Lee et al., 2008, 2009, 2010), saccharification using the same saccharification conditions and enzyme (Meicelase) was also conducted. Table 3 summarizes the effect of HCW treatment temperature and extrusion on monosaccharide production yield. In a study for extrusion process using additives with cellulose affinity, a 62.40% glucose yield based on cellulose content (29.20 wt.% based on initial wood weight) was achieved from Douglas fir (Lee et al., 2008). Glucose production yield obtained using a batch-type kneader with twinscrew elements was 54.20% (25.40 wt.% based on initial wood weight) in the fibrillated Douglas fir kneaded for 20 min after ball milling for 20 min (Lee et al., 2009). Disk-milling (3 passes) of Eucalyptus treated by HCW at 170 °C had a glucose production yield of 35.11%, which is almost same value as that obtained in this study (Lee et al., 2010). Mannose production yields in Douglas fir and xylose in Eucalyptus were found to be 2.15 and 3.69 wt.%, respectively. The effect of the number of extrusion passes on glucose production yield was also investigated. As shown in Fig. 2, there is no significant difference in yield between numbers of extrusion passes after the drastic improvement seen with 1-pass extrusion. In the context, it can be said that combining the extrusion process with HCW treatment is an effective pretreatment. Fig. 3 shows the effect of rotation speed on discharge amount and glucose production yield of the extruded products. Glucose production yield was slightly decreased with an increase of rota-
Table 2 Monosaccharide production yield from the fibrillated product after hot-compressed water (HCW) treatment. Sample
HCW temperature (°C)
Extrusion
Monosaccharide production yield (wt.% of original wood weight) Xylose
Galactose
Mannose
Total
Douglas fir
140 – 160 – 170 – 180 –
– Extrusion – Extrusion – Extrusion – Extrusion
0.08 1.45 0.11 1.42 0.13 1.07 0.15 0.94
0.48 0.77 0.51 0.68 0.45 0.50 0.49 0.60
0.33 5.06 0.77 3.12 0.60 2.15 0.44 1.08
0.89 7.28 1.39 5.22 1.18 3.72 1.08 2.62
Eucalyptus
170 – 180 –
– Extrusion – Extrusion
3.69 4.45 3.65 3.88
0.83 1.04 0.86 0.99
0.46 0.32 0.41 0.38
4.98 5.81 4.92 5.25
Enzymes: Acremonium cellulase, Novozyme 188, Optimash BG; substrate concentration: 33 mg/mL with 10 FPU enzymes; saccharification at 45 °C for 72 h.
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S.-H. Lee et al. / Bioresource Technology 101 (2010) 9645–9649 Table 3 Effect of extrusion on monosaccharide production yields from fibrillated product after hot-compressed water (HCW) treatment. Sample
Extrusion
Monosaccharide production yield (wt.% of original wood weight) Glucose
Xylose
Galactose
Mannose
Total
Douglas fir
– Extrusion
2.85 27.72
0.13 1.07
0.45 0.50
0.60 2.15
4.03 31.44
Eucalyptus
– Extrusion
24.75 34.32
4.45 3.69
1.04 0.83
0.32 0.46
30.56 39.30
HCW treatment temperature: 170 °C; substrate concentration: 0.118 mg/mL with 10 FPU of enzymes (Meicelase); saccharification temperature, 45 °C; time, 48 h.
Morphology of HCW-treated products at 170 °C and their extruded products was observed. Fibrous morphology with a wide diameter range (approximately 20–50 lm) was found in HCWtreated products, which was mainly due to lignin separation from the middle lamella between the fibers. Our previous study found that cellulose microfibrills can be partially exposed on the surface. In particular, the nanopores between microfibrills were found at a scale of about 20 nm in the cell wall possibly due to partial removal of the hemicellulose and lignin. A detailed discussion of this morphology is discussed in our previous report (Lee et al., 2010). On the other hand, the extruded products showed finer fibrous morphology than HCW-treated ones due to the high shearing force of the extruder passing materials into narrow clearance between the screws and barrel (Blechschmidt et al., 2004). Most fibers were fibrillated into a sub-micron scale (less than approximately 5 lm), and some of them had diameters <100 nm. This fine morphology can increase cellulose surface area and improve enzymatic saccharification.
Glucose production yield based on original weight (wt%)
40
30
20
10
0
0
1
2
3
4
5
6
Extrusion passing number Fig. 2. Effect of number of extrusion passes on glucose production yield. Substrate concentration, 33 mg/mL with 10 FPU of enzymes (Acremonium cellulase, Novozyme 188, Optimash BG); saccharification temperature, 45 °C; time, 72 h.
tion speed, but the discharge amount increased linearly. Dependency of the extruder discharge capacity on electrical power consumption was investigated by using the motor capacity of an EA series extruder (Suehiro EPM Co., Japan). It was found that electric power consumption is linearly decreased on a logarithmic scale as extrusion discharge capacity increases, showing that the bigger the extruder the less the energy consumption. High productivity is one of the benefits of utilizing an extruder to increase economical feasibility of the pretreatment.
An extrusion process was utilized to compensate for the limitations of HCW treatment alone and improve enzymatic saccharification yield. Monosaccharide production yield was improved by extrusion after HCW treatment in the case of both Douglas fir and Eucalyptus wood. In particular, this process is beneficial for softwood, which is less effectively treated by HCW. The obtained results are expected to contribute to technical developments in effective and economical methods of exposure of cellulose surfaces to enzymes. Acknowledgements The authors are deeply grateful to Dr. Minowa for calculating the extruder’s electric power consumption.
40 6
References
30
4 20
2
10
0
0
50
100
Dischare amount (g/min)
Glucose production yield from original weight (wt%)
4. Conclusion
0 150
Rotation speed (rpm) Fig. 3. Effect of rotation speed on discharge amount and glucose production yields of extruded products by enzymatic saccharification.
Ando, H., Sakaki, T., Kokusho, T., Shibata, M., Uemura, Y., Hatate, Y., 2000. Decomposition behavior of plant biomass in hot-compressed water. Industrial & Engineering Chemistry Research 39, 3688–3693. Assor, C., Placet, V., Chabbert, B., Habrant, A., Lapierre, C., Pollet, B., Perre, P., 2000. Concomitant changes in viscoelastic properties and amorphous polymers during the hydrothermal treatment of hardwood and softwood. Journal of Agricultural and Food Chemistry 57, 6830–6837. Biofuels Research Advisory Council, ‘‘Biofuels in the European Union: A Vision for 2030 and Beyond” 2006; available online (http://www.biomatnet.org/ publications/1919rep.pdf). Blechschmidt, J., Engert, P., Stephan, M., 2004. The glass transition of wood from the viewpoint of mechanical pulping. Wood Science and Technology 20, 263–272. Dale, B.E., Weaver, J., Byers, F.M., 1999. Extrusion processing for ammonia fiber explosion (AFEX). Applied Biochemistry and Biotechnology 77, 35–45. Farrel, A.E., Plevin, R.J., Turner, B.T., Jones, A.D., O’Hare, M., Kammen, D.M., 2007. Ethanol can contribute to energy and environmental goals. Science 311, 506– 508. Hideno, A., Inoue, H., Tsukahara, K., Fujimoto, S., Minowa, T., Inoue, S., Endo, T., Sawayama, S., 2009. Wet disk milling pretreatment without sulfuric acid for enzymatic hydrolysis of rice straw. Bioresource Technology 100, 2706–2711.
S.-H. Lee et al. / Bioresource Technology 101 (2010) 9645–9649 Himmel, M.E., Ding, S.Y., Johnson, D.K., Adney, W.S., Nimlos, M.R., Brady, J.W., Thomas, D.F., 2007. Biomass recalcitrance. Engineering plants and enzymes for biofuels production. Science 315, 5813–5815. Inoue, H., Yano, S., Endo, T., Sasaki, T., Sawayama, S., 2008. Combining hotcompressed water and ball milling pretreatments to improve the efficiency of the enzymatic hydrolysis of eucalyptus. Biotechnology for Biofuels 1, 1–9. Lee, S.H., Teramoto, Y., Endo, T., 2008. Enzymatic saccharification of woody biomass micro/nanofibrillated by continuous extrusion process I- Effect of additives with cellulose affinity. Bioresource Technology 100, 275–279. Lee, S.H., Teramoto, Y., Endo, T., 2009. Enhancement of enzymatic accessibility by fibrillation of woody biomass using batch-type kneader with twin-screw elements. Bioresource Technology 101, 769–774. Lee, S.H., Chang F., Inoue S., Endo, T., 2010. Increase in enzyme accessibility by generation of nanospace in cell wall supramolecular structure. Bioresources Technology, doi:10.1016/j.biortech.2010.04.069. Mani, S., Tabil, L.G., Sokhansanj, S., 2004. Grinding performance and physical properties of wheat and barley straws, corn stover and switchgrass. Biomass and Bioenergy 27, 339–352. Office of the Biomass Program (OBP), ‘‘Multi-Year Program Plan, 2007-2012” (OBP, U.S. Department of Energy, Washington, DC, 2005) (http:// www1.eere.energy.gov/biomass/pdfs/mypp.pdf).
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Pilli, T.D., Severini, C., Baiano, A., Derossi, A., Arhaliass, A., Legrand, J., 2005. Effects of operating conditions on oil loss and properties of products obtained by corotating twin-screw extrusion of fatty meal, preliminary study. Journal of Food Engineering 70, 109–116. Sakaki, T., Shibata, M., Miki, T., Hirosue, H., Hayashi, N., 1996a. Decomposition of cellulose in near-critical water and fermentability of the products. Energy Fuels 10, 684–688. Sakaki, T., Shibata, M., Miki, T., Hirosue, H., Hayashi, N., 1996b. Reaction model of cellulose decomposition in near-critical water and fermentation of products. Bioresource Technology 58, 197–202. Thymi, S., Krokida, M.K., Pappa, A., Maroulis, Z.B., 2005. Structural properties of extruded corn starch. Journal of Food Engineering 68, 519–526. Zhu, J.Y., Wang, G.S., Pan, X.J., Gleisner, R., 2009. Specific surface to evaluate the efficiencies of milling and pretreatment of wood for enzymatic saccharification. Chemical Engineering Science 64, 474–485. Zhu, W., Zhu, J.Y., Gleisner, R., Pan, X.J., 2010. On energy consumption for sizereduction and yields from subsequent enzymatic saccharification of pretreated lodgepole pine. Bioresource Technology 101, 2782–2792.
Contents lists available at ScienceDirect
Bioresource Technology journal homepage: www.elsevier.com/locate/biortech
Enzymatic saccharification of woody biomass micro/nanofibrillated by continuous extrusion process II: Effect of hot-compressed water treatment Seung-Hwan Lee a,*, Seiichi Inoue a, Yoshikuni Teramoto b, Takashi Endo a a b
Biomass Technology Research Center, National Institute of Advanced Industrial Science and Technology (AIST), 3-11-32, Kagamiyama, Higashihiroshima, Hiroshima 739 0046, Japan Division of Forest and Biomaterials Science, Graduate School of Agriculture, Kyoto University, Kyoto 606 8502, Japan
a r t i c l e
i n f o
Article history: Received 31 May 2010 Received in revised form 15 July 2010 Accepted 15 July 2010 Available online 27 July 2010 Keywords: Extrusion Bioethanol Enzymatic saccharification Hot-compressed water treatment Fibrillation
a b s t r a c t An extrusion process involving a twin-screw extruder was used for the micro/nanofibrillation of Douglas fir and Eucalyptus treated with hot-compressed water (HCW). Partial removal of hemicellulose and lignin by HCW treatment effectively improved the fibrillation by extrusion. Only HCW treatment produced glucose less than 5 weight percent (wt.%) in Douglas fir in a temperature range of 140–180 °C by enzymatic hydrolysis. Glucose production yields of 18 and 26 wt.% were obtained by HCW treatment at 170 and 180 °C, respectively, in Eucalyptus. Use of extrusion after HCW treatment drastically improved monosaccharide production yield in both woods. In the case of Douglas fir, the obtained values were 5 times higher than those obtained by HCW treatment alone. Total monosaccharide production yields were higher in Eucalyptus than in Douglas fir. The extruded production had a fine fibrous morphology on a sub-micro/nanoscopic scale. This result shows the great potential of the extrusion process after HCW treatment as a cost-effective pretreatment for enzymatic saccharification of woody biomass. Ó 2010 Elsevier Ltd. All rights reserved.
1. Introduction Extensive research and development programs in the area of renewable bioenergy have been initiated in many countries. As part of this initiative, researchers have been encouraged to develop technologies for the production of bioethanol from lignocellulosic biomass. According to the U.S. Department of Energy, Office of the Biomass Program (Office of the Biomass Program, 2010), a reliable biofuels market of at least 7.5 billion gallons will be available by 2010 (Himmel et al., 2007). The European Union has also envisioned that by 2030, one-fourth of the EU’s transportation fuel requirement will be met by biofuels (Biofuels Research Advisory Council, 2006). It is expected that this change will significantly decrease the EU’s dependence on imported fossil fuels. It has been reported that bioethanol from lignocellulosic resources is much less petroleum-intensive than gasoline and can contribute to reducing greenhouse gas emissions (Farrel et al., 2007). However, in order to realize the large-scale use of bioethanol as a liquid fuel, new technology needs to be developed to allow for more efficient utilization of lignocellulose. Mechanical size reduction as a pretreatment for enzymatic saccharification of lignocellulosic biomass is known as one of the most energy-intensive processes (Himmel et al., 2007; Mani et al., 2004). Some research has been conducted to enhance the economical * Corresponding author. Tel./fax: +81 82 420 8278. E-mail addresses: [email protected], [email protected] (S.-H. Lee). 0960-8524/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.biortech.2010.07.068
feasibility of this process along with its effectiveness to increase enzymatic accessibility (Lee et al., 2008, 2009, 2010; Zhu et al., 2009, 2010). Recently, Zhu et al. (2010) reported on the energy consumption of the post-chemical pretreatment size-reduction method; they investigated the effects of chemical treatment and disk-milling conditions on energy consumption for size reduction and the efficiency of enzymatic cellulose saccharification of lodgepole pine wood. They treated lodgepole pine with 3 different methods, namely, hot-water, dilute-acid, and sulfite treatment, and milled the treated product using a disk mill. They found that this combined method decreased size-reduction energy consumption by 20–80%, depending on the conditions. In an earlier study, they also found that chemical pretreatment not only increased cellulose conversion but also reduced mechanical milling energy consumption (Zhu et al., 2009). Thermomechanical disk milling was more energy intensive than high-pressure thermomechanical disk milling, but the former was more effective than the latter in accomplishing enzymatic saccharification. Our research group has reported that mechanical size reduction by ball and disk milling of hot-compressed water (HCW)-treated wood at mild temperatures (below 180 °C) can reduce energy consumption, which then reduces the amount of water required for HCW treatment and enzyme loading for saccharification (Hideno et al., 2009; Inoue et al., 2008; Lee et al., 2009, 2010). HCW treatment can partially remove hemicellulose and lignin from the cell wall supramolecular structure to produce nanoscopic pores between cellulose microfibrils, facilitating mechanical fibrillation.
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This process prevents the formation of fermentation inhibitors such as furaldehydes and hydroxylmethyl furan. In particular, development of new pretreatment technology has also been attempted to achieve high cost feasibility by utilizing a batch-type kneader with combination-available twin-screw elements and a twin-screw extruder (Lee et al., 2008, 2009). A batch-type kneader with combination-available twin-screw elements was used in a pre-experiment for the utilization of a twin-screw extruder. The fibrillated product showed sub-micro/ nanoscopic morphology, which increased the surface area of cellulose. However, use of mechanical kneading alone appears to have some limitations in exposing cellulose for complete enzymatic saccharification. It was found that cooking of the fibrillated products using hot water at 130 °C under 0.25 MPa can be an option to overcome this limitation. Furthermore, a continuous extrusion process using additives with cellulose affinity was employed as a practical and economical method (Lee et al., 2008). As described in this report, this process has great potential for large-scale production with various advantages: highly efficient pulverization by application of high shearing force, high throughputs, and excellent temperature control (Dale et al., 1999; Pilli et al., 2005; Thymi et al., 2005). Based on the experimental results of our previous studies, this study was undertaken in an attempt to improve the enzymatic saccharification yield of Douglas fir and Eucalyptus by using an HCW treatment extrusion process. 2. Experimental 2.1. Materials Douglas fir and Eucalyptus wood chips were purchased from Oji Paper Co., Ltd. (Tokyo, Japan) and cutter-milled under 3 mm using a milling cutter (Masuko Sangyo Co., Ltd., Japan). All chemicals were purchased from commercial sources and used without further purification. Acremonium cellulose (Meiji Seika Co., Ltd., Japan), Novozyme 188 (cellobiase from Aspergillus niger, Sigma– Aldrich Japan, Japan), and Optimash BG (Multifect xylanase, Genencor Kyowa Inc. Japan) were used for the saccharification of the fibrillated product. The Meicelase (Meiji Seika Co., Ltd., Japan) from the fungus Trichoderma viride was also used for cellulose hydrolysis in both woods. 2.2. Chemical composition determination Chemical composition was determined as follows. The holocellulose, a-cellulose, and lignin content were determined as the residue obtained after delignification by NaClO2, the residue insoluble in 17.5% aqueous NaOH solution (as determined by TAPPI-203), and the residue insoluble in 72% aqueous sulfuric acid solution, respectively. The dry-basis content of holocellulose, a-cellulose, and Klason lignin in the starting materials were calculated from their respective weights. The a-cellulose content was quantified as cellulose content. The hemicellulose content was determined by subtracting the a-cellulose content from the holocellulose content. The dry-basis contents of a-cellulose, hemicellulose, and Klason lignin were 46.8, 28.9, and 25.3 weight percent (wt.%) in Douglas fir and 42.1, 34.6, and 28.8 wt.% in Eucalyptus, respectively. 2.3. HCW treatment and extrusion HCW treatment was performed by the same method and the detailed experimental procedure is described in previous report (Lee et al., 2010). Briefly described, the used temperatures ranged from 140 °C to 180 °C and reaction time that excludes the warming-up period was 30 min. The initial pressure and the
water:starting material weight ratio were adjusted to 1 MPa of nitrogen gas and 5:1, respectively. After the treatment, the reactor was immediately soaked in a water bath maintained at room temperature. The treated products were filtrated and washed with water. The obtained water-soluble fraction was pre-condensed and dried at 40 °C in vacuo to calculate its amount. The water-insoluble residue was provided to the twin-screw extruder (2D15 W; Toyo Seiki, Tokyo, Japan). The residual product was extruded through the co-rotating twin screws 1–5 times. The barrel diameter and length/diameter (L/D) of screws were 15.4 and 17 mm respectively. Extrusion was conducted at room temperature with the rotating speed of 45–120 rpm. 2.4. Enzymatic saccharification and morphology Enzymatic hydrolysis of the extruded products was conducted using Acremonium cellulase, Novozyme 188, and Optimash BG. The substrate concentration was set to 33 mg/mL with 10 FPU of enzymes. Saccharification was carried out with 30 mL of acetate buffer (pH 5.0) at 45 °C for 72 h. The samples were stored at 18 °C and then heated at 95 °C for 15 min to deactivate the enzymes. The produced monomeric sugars were analyzed by LC-2000 Plus HPLC system (JASCO Corporation, Tokyo, Japan) equipped with a column (Aminex HPX-87, Bio-Rad Laboratories, K. K., Tokyo, Japan) at 80 °C; the eluent used was distilled water at a flow rate of 1 mL/ min. To compare the data in previous reports, hydrolysis using Meicelase was also carried out using ca. 50 mg of the fibrillated product in 17 mL of 50 mM acetate buffer (pH 5.0) containing 2 mg Meicelase (10 FPU). The morphological characteristics of the HCW-treated and the extruded products were analyzed by scanning electron microscopy (SEM) (S-4800, Hitachi Co. Tokyo, Japan). The samples were freezedried and coated with a 1 nm-thick layer by an osmium plasma coater (NEOC-AN, Meiwa Fosis, Tokyo, Japan). 3. Results and discussion HCW treatment is often called ‘‘autohydrolysis treatment” because the acetyl groups of hemicellulose and the water itself act as acids during treatment, thereby dissolving both the hemicellulose and the lignin of lignocellulosic materials. In particular, this treatment very effectively removes selectively hemicellulose without hydrolyzing cellulose at mild temperatures less than 200 °C (Ando et al., 2000; Sakaki et al., 1996 a,b). In this study, the temperature range of 140–180 °C was used to prevent cellulose degradation and formation of fermentation inhibitors seen at higher temperatures. Further, a water:starting material weight ratio of 5:1 was used to save operating energy required to heat water. Table 1 shows the amount of water-soluble fraction depending on HCW treatment temperature. Of the initial composition of hemicellulose (34.6 and 28.9 wt.% in Eucalyptus and Douglas fir, respectively), about half was removed by HCW at 180 °C. In our previous report, partial removal of hemicellulose and lignin by HCW treatment in the same temperature range was discussed. Experimental
Table 1 Amount of water-soluble fraction (wt.%) obtained according to temperature of hotcompressed water (HCW) treatment. Sample
Douglas fir Eucalyptus a
Lee et al., 2010.
HCW temperature (°C) 140
160
170
180
5.3 2.9a
11.3 14.3a
13.4 14.5
14.7 15.8
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results using Eucalyptus showed that most of the fraction was found to be xylooligosaccharides and only a small amount of lignin (<2.4 wt.%) was removed in a manner that was not dependent on HCW treatment temperature (Lee et al., 2010). It is also reported that removal of the water-soluble fraction can generate nanoscopic porous cell wall structure, enhancing the mechanical fibrillation degree and the enzyme accessibility of cellulose (Lee et al., 2010). Fig. 1 shows the effect of the extrusion after HCW treatment and HCW treatment itself on glucose production yield. In HCW treatment alone, the glucose production yield in Doulas fir was less than 5 wt.% in the entire temperature range, whereas glucose yields of 18 and 26 wt.% were obtained at 170 and 180 °C respectively, in Eucalyptus. Generally, HCW treatment is known to be less effective in softwood than in hardwood, which is possibly due to different lignin molecular structure and hemicellulose content between hardwoods and softwoods. For example, degradation of the b-O-4 bond-rich hardwoods is generally known to be more sensitive by hydrothermal treatment than that of softwoods with fewer b-O-4 bonds (Assor et al., 2000). Glucose production yield was improved by extrusion in both woods. It is interesting to note that extrusion drastically improved glucose production yield in Douglas fir. The yields obtained by extrusion were 5 times higher than those by HCW treatment alone across the entire temperature range. This result shows great potential of the extrusion process
Glucose production yield based on original weight (wt%)
40
30
: Douglas fir : Eucalyptus Black: Only HCW White: Extrusion after HCW
20
10
0 120
140
160
180
200
HCW treatment temperature ( °C) Fig. 1. Effect of hot-compressed water (HCW) treatment temperature and HCW + extrusion on glucose production yield. Substrate concentration, 33 mg/mL with 10 FPU of enzymes (Acremonium cellulase, Novozyme 188, Optimash BG); saccharification temperature, 45 °C; time, 72 h.
after HCW treatment in softwood, compensating for the lesser effect of HCW treatment alone on it. In our previous report using batch-type kneader, only mechanical kneading of Douglas fir with water showed limitation in enhancing enzymatic accessibility, but kneading after hot-water treatment improved it (Lee et al., 2009). It was explained by the structural viewpoint of wood cell wall formation. The heterogeneous nature of cellulose microfibril bundles encased by hemicellulose and lignin causes significant recalcitrance against enzyme accessibility. Therefore, loosening this structure by partially removing hemicellulose and lignin by HCW treatment is very effective to enhance enzymatic saccharification and to facilitate fibrillation. Eucalyptus also had a higher glucose production yield upon extrusion than with HCW treatment alone. The total glucose production yield was higher in Eucalyptus than in Douglas fir, which may be due to the higher HCW treatment effect in Eucalyptus. Table 2 summarizes the other monosaccharides obtained by HCW treatment and extrusion. As seen with glucose production yield, extrusion effectively enhanced the hydrolysis of hemicellulose in both woods. The total monosaccharide yield in Douglas fir decreased with increased HCW treatment temperature. Total yields were higher in Eucalyptus than in Douglas fir. For comparison to previous reports (Lee et al., 2008, 2009, 2010), saccharification using the same saccharification conditions and enzyme (Meicelase) was also conducted. Table 3 summarizes the effect of HCW treatment temperature and extrusion on monosaccharide production yield. In a study for extrusion process using additives with cellulose affinity, a 62.40% glucose yield based on cellulose content (29.20 wt.% based on initial wood weight) was achieved from Douglas fir (Lee et al., 2008). Glucose production yield obtained using a batch-type kneader with twinscrew elements was 54.20% (25.40 wt.% based on initial wood weight) in the fibrillated Douglas fir kneaded for 20 min after ball milling for 20 min (Lee et al., 2009). Disk-milling (3 passes) of Eucalyptus treated by HCW at 170 °C had a glucose production yield of 35.11%, which is almost same value as that obtained in this study (Lee et al., 2010). Mannose production yields in Douglas fir and xylose in Eucalyptus were found to be 2.15 and 3.69 wt.%, respectively. The effect of the number of extrusion passes on glucose production yield was also investigated. As shown in Fig. 2, there is no significant difference in yield between numbers of extrusion passes after the drastic improvement seen with 1-pass extrusion. In the context, it can be said that combining the extrusion process with HCW treatment is an effective pretreatment. Fig. 3 shows the effect of rotation speed on discharge amount and glucose production yield of the extruded products. Glucose production yield was slightly decreased with an increase of rota-
Table 2 Monosaccharide production yield from the fibrillated product after hot-compressed water (HCW) treatment. Sample
HCW temperature (°C)
Extrusion
Monosaccharide production yield (wt.% of original wood weight) Xylose
Galactose
Mannose
Total
Douglas fir
140 – 160 – 170 – 180 –
– Extrusion – Extrusion – Extrusion – Extrusion
0.08 1.45 0.11 1.42 0.13 1.07 0.15 0.94
0.48 0.77 0.51 0.68 0.45 0.50 0.49 0.60
0.33 5.06 0.77 3.12 0.60 2.15 0.44 1.08
0.89 7.28 1.39 5.22 1.18 3.72 1.08 2.62
Eucalyptus
170 – 180 –
– Extrusion – Extrusion
3.69 4.45 3.65 3.88
0.83 1.04 0.86 0.99
0.46 0.32 0.41 0.38
4.98 5.81 4.92 5.25
Enzymes: Acremonium cellulase, Novozyme 188, Optimash BG; substrate concentration: 33 mg/mL with 10 FPU enzymes; saccharification at 45 °C for 72 h.
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Extrusion
Monosaccharide production yield (wt.% of original wood weight) Glucose
Xylose
Galactose
Mannose
Total
Douglas fir
– Extrusion
2.85 27.72
0.13 1.07
0.45 0.50
0.60 2.15
4.03 31.44
Eucalyptus
– Extrusion
24.75 34.32
4.45 3.69
1.04 0.83
0.32 0.46
30.56 39.30
HCW treatment temperature: 170 °C; substrate concentration: 0.118 mg/mL with 10 FPU of enzymes (Meicelase); saccharification temperature, 45 °C; time, 48 h.
Morphology of HCW-treated products at 170 °C and their extruded products was observed. Fibrous morphology with a wide diameter range (approximately 20–50 lm) was found in HCWtreated products, which was mainly due to lignin separation from the middle lamella between the fibers. Our previous study found that cellulose microfibrills can be partially exposed on the surface. In particular, the nanopores between microfibrills were found at a scale of about 20 nm in the cell wall possibly due to partial removal of the hemicellulose and lignin. A detailed discussion of this morphology is discussed in our previous report (Lee et al., 2010). On the other hand, the extruded products showed finer fibrous morphology than HCW-treated ones due to the high shearing force of the extruder passing materials into narrow clearance between the screws and barrel (Blechschmidt et al., 2004). Most fibers were fibrillated into a sub-micron scale (less than approximately 5 lm), and some of them had diameters <100 nm. This fine morphology can increase cellulose surface area and improve enzymatic saccharification.
Glucose production yield based on original weight (wt%)
40
30
20
10
0
0
1
2
3
4
5
6
Extrusion passing number Fig. 2. Effect of number of extrusion passes on glucose production yield. Substrate concentration, 33 mg/mL with 10 FPU of enzymes (Acremonium cellulase, Novozyme 188, Optimash BG); saccharification temperature, 45 °C; time, 72 h.
tion speed, but the discharge amount increased linearly. Dependency of the extruder discharge capacity on electrical power consumption was investigated by using the motor capacity of an EA series extruder (Suehiro EPM Co., Japan). It was found that electric power consumption is linearly decreased on a logarithmic scale as extrusion discharge capacity increases, showing that the bigger the extruder the less the energy consumption. High productivity is one of the benefits of utilizing an extruder to increase economical feasibility of the pretreatment.
An extrusion process was utilized to compensate for the limitations of HCW treatment alone and improve enzymatic saccharification yield. Monosaccharide production yield was improved by extrusion after HCW treatment in the case of both Douglas fir and Eucalyptus wood. In particular, this process is beneficial for softwood, which is less effectively treated by HCW. The obtained results are expected to contribute to technical developments in effective and economical methods of exposure of cellulose surfaces to enzymes. Acknowledgements The authors are deeply grateful to Dr. Minowa for calculating the extruder’s electric power consumption.
40 6
References
30
4 20
2
10
0
0
50
100
Dischare amount (g/min)
Glucose production yield from original weight (wt%)
4. Conclusion
0 150
Rotation speed (rpm) Fig. 3. Effect of rotation speed on discharge amount and glucose production yields of extruded products by enzymatic saccharification.
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