- Email: [email protected]
oxidative pretreatment of corn straw undergoing enzymatic hydrolysis by cellulase
Journal of Bioscience and Bioengineering VOL. 110 No. 6, 660 – 664, 2010 www.elsevier.com/locate/jbiosc
Evaluation of white-rot fungi-assisted alkaline/oxidative pretreatment of corn straw undergoing enzymatic hydrolysis by cellulase Hongbo Yu, Xiaoyu Zhang,⁎ Lili Song, Jing Ke, Chunyan Xu, Wanqing Du, and Ji Zhang College of Life Science and Technology, Huazhong University of Science and Technology, Wuluo Road 1037, Wuhan 430074, China Received 11 February 2010; accepted 3 August 2010 Available online 3 September 2010
In this study, the effects of biological treatment prior to alkaline/oxidative (A/O) pretreatment using three white-rot fungi (Ganoderma lucidum, Trametes versicolor and Echinodontium taxodii) were evaluated for the enzymatic hydrolysis of corn straw. Among these fungi, Echinodontium taxodii significantly enhanced the efficiency of chemical pretreatment. Subsequent to treatment of corn straw with Echinodontium taxodii for 15 days, the straw was subjected to digestion by 0.0016% NaOH and 3% H2O2 at room temperature for 24 h, which increased the reducing sugar yield by 50.7%. The hydrolysis model and kinetic parameters were determined from time course data collected throughout the hydrolysis. The initial hydrolysis rate, V0, of the corn straw increased by 68.5% compared to A/O pretreatment alone, which resulted from an increase in the initial adsorption. The lignin content of the corn straw decreased more significantly after biological and A/O pretreatment than after A/O pretreatment alone. After 72 h of enzymatic hydrolysis, the adsorbed cellulase decreased by 24.8% (from 3.67 to 2.76 mg ml−1) compared to A/O pretreatment alone. These results indicate that biological treatment improves the desorption of cellulase by enhancing delignification during A/O pretreatment. © 2010, The Society for Biotechnology, Japan. All rights reserved. [Key words: Corn straw; Echinodontium taxodii; Biological pretreatment; Alkaline/oxidative pretreatment; Enzymatic hydrolysis; Adsorption]
Lignocellulosic biomass represents a promising, low-cost resource for the large-scale production of biofuels, such as ethanol and methanol (1–4). Enzymatic hydrolysis of lignocellulose is a key step in biofuel production; however, the natural resistance of lignocellulose to hydrolysis prevents enzymatic treatment and release of fermentable sugars. Some studies have indicated that the porosity of lignocellulose and crystallinity of the cellulose fibrils can affect the enzymatic hydrolysis of lignocellulose (5). Furthermore, the network of lignin and hemicellulose surrounding cellulose fibrils prevents the penetration of cellulase (6). Although the activity of cellulase has been improved upon significantly in the last decade, the removal of lignin, reduction in the crystallinity of the cellulose and increase in porosity of lignocellulose by pretreatment technologies remain as challenges in achieving high hydrolysis yields. Several physical, chemical and physicochemical pretreatment methods have been used for the enzymatic hydrolysis of lignocellulose during ethanol production, including mechanical comminution, pyrolysis, steam explosion, ammonia fiber explosion, organosolv extraction, acid hydrolysis, alkaline hydrolysis and alkaline/oxidative delignification (5,7–12). Recently, some studies have indicated that biological pretreatment with white-rot fungi, which are the only known organisms that can efficiently remove lignin from lignocellulose, may reduce the recalcitrance of lignocellulose to enzymatic hydrolysis (13,14). A number of fungal strains have been evaluated for the enzymatic hydrolysis of lignocellulose. For instance, Taniguchi et ⁎ Corresponding author. Tel.: + 86 27 87792108; fax: + 86 27 87792128. E-mail address: [email protected] (X. Zhang).
al. (15) reported that the total sugar yields of rice straw following treatment with Pleurotus ostreatus for 60 days were 33% after enzymatic hydrolysis. In our previous investigation, 34 isolates of white-rot fungi were screened for use in the biological pretreatment of bamboo culms, and sugar yields increased 8.76-fold after 120-day pretreatment with Echinodontium taxodii (16). Besides biological pretreatment, most chemical and physical pretreatment processes require high temperatures and operating pressures, which results in extremely high demand for heat and power. For example, steam explosion is usually carried out at 160– 260 °C and 0.69–4.83 MPa (5). Dilute acid and alkaline pretreatments are often carried out at temperatures above 100 °C and require special equipment for achieving high hydrolysis yields (17). Although biological pretreatment was considered to be a promising technology because of its low energy requirements and mild environmental effects (5), most biological pretreatment processes require a long time (about 3– 5 months) and result in very high feedstock loss during pretreatment. Recently, several studies indicated that a short-term biological pretreatment enhances the efficiency of physicochemical pretreatment. For example, Itoh et al. (18) reported that the yield of ethanol obtained from beech wood chips after pretreatment for 8 weeks was 1.6 times higher than that obtained without the fungal treatments. Some studies on biopulping indicated that 15-day-biotreatment with Ceriporiopsis subvermispora may decrease the amounts of active alkali because depolymerization of lignin during the initial stages of biodegradation enhanced the release of lignin during alkaline cooking (19). These studies suggest that it is not necessary to remove lignin by long-term biological pretreatment during consecutive treatments with lignin-
1389-1723/$ - see front matter © 2010, The Society for Biotechnology, Japan. All rights reserved. doi:10.1016/j.jbiosc.2010.08.002
VOL. 110, 2010
WHITE ROT FUNGI PRETREATMENT
degrading fungi or alkaline cooking. Yet, few studies have evaluated the effects of biological pretreatment of corn straw prior to alkaline pretreatments at room temperature. In this study, we evaluated a new biological-alkaline/oxidative (Bio-A/O) pretreatment process in which lignocellulose was pretreated by white-rot fungi and then by alkaline/oxidative (A/O) treatment at room temperature and pressure with a low concentration of chemicals. The A/O treatment alone at room temperature and pressure can decrease heat and power demands, but enzymatic hydrolysis yields are not high after this treatment step. We attempted to enhance the efficiency of A/O pretreatment by incubating the corn straw in the presence of white-rot fungi prior to A/O pretreatment. The effects of Bio-A/O pretreatments on the enzymatic hydrolysis of corn straw were evaluated with Ganoderma lucidum, Trametes versicolor and Echinodontium taxodii. Alterations in structure, chemistry, enzymatic hydrolysis and adsorption capacity of pretreated corn straw were also investigated. MATERIALS AND METHODS Microorganisms Ganoderma lucidum, Trametes versicolor and Echinodontium taxodii were obtained from our laboratory and used for the Bio-A/O pretreatments. The fungal strains were cultured on potato dextrose agar (PDA) slants at 25 °C. After 7 days, agar pieces were cut from slants and inoculated in 250-ml Erlenmeyer flasks with 100ml of potato dextrose liquid media. The cultures were incubated at 25 °C with agitation (150 rpm) for 7 days and used for the biological pretreatments. Substrate Corn straw from Henan (China) was cut into chips (400–800 μm mesh). The chips were air-dried and stored under dry conditions. Biological pretreatment with white-rot fungi 10-ml of fungal cultures were inoculated into 250-ml Erlenmeyer flasks and sterilized at 121 °C for 30 min with 10-g of corn straw chips and 25-ml of distilled water. The flasks were covered with film to avoid moisture loss and contamination. The cultures were statically incubated at 25 °C for 10, 15 and 30 days. All the cultures were grown in triplicate. Non-inoculated corn straw was used as a control. The treated corn straw was dried at 60 °C for 3 days and weighed to calculate the percentage of weight loss based on the initial weight and final dry weight. The percentage of lignin, cellulose and hemicellulose in the treated and untreated corn straw was determined according to the procedures of AOAC (20). Lignin and cellulose loss was calculated as follows:
Lignin ðor celluloseÞ loss ð%Þ ligninðor celluloseÞ content in treated straw × final weight of straw after pretreament = 100− lignin ðor celluloseÞ content in untreated straw × initial weight of straw
The selectivity value of lignin degradation was calculated as the ratio of lignin loss over cellulose loss. Alkaline/oxidative pretreatment The bio-pretreated and untreated corn straw chips were exposed to an A/O pretreatment. The A/O pretreatment was carried out with 2% corn straw substrate in alkaline liquor with 0.0016% sodium hydroxide (NaOH) and 3% (vol/vol) hydrogen peroxide (H2O2) at room temperature. After 16 h, the pretreated corn straw samples were separated from the liquid by centrifugation and washed with distilled water to bring them to a neutral pH. Enzymatic hydrolysis Cellulase was obtained from Sigma (cellulase from Aspergillus niger; catalogue no. C1184). The total cellulase activity of commercial cellulase −1 was 100 FPU g based on the filter paper assay (FPA). Enzymatic hydrolysis was carried out with 2% substrate in 50 mM sodium acetate buffer (pH 4.8) with 1, 2, 4, 6, 8 or 10 mg ml−1 of cellulase at 50 °C. After 48 h, the reaction mixtures were interrupted by centrifugation at 10,000 rpm for 5 min. The amount of reducing sugar in the reaction supernatants was determined with the 3,5-dinitrosalicylic acid (DNS) reagent (4). Furthermore, the corn straw treated by Bio-A/O pretreatment with E. taxodii for 15 days was hydrolyzed with 10 mg ml−1 cellulase for 0, 1, 3, 6, 10, 24, 48, 72, 96 or 120 h, and the reducing sugar in the supernatant was determined after centrifugation. The yield of reducing sugar in each sample was calculated as follows:
Reducing sugar yield ð%Þ amount of reducing sugar produced after enzymatic hydrolysis × 0:9 × 100 = amount of hemicellulose þ amount of cellulose Enzymatic adsorption during the hydrolysis The corn straw treated by BioA/O pretreatment with E. taxodii for 15 days was used to investigate cellulase adsorption −1 capacity at an enzyme concentration of 10 mg ml . Enzymatic protein was used as an indicator of adsorption capacity. Subsequent to enzymatic hydrolysis for 1, 3, 6, 10, 24, 48, 72, 96 or 120 h, the corn straw liquor was centrifuged at 10,000 rpm for 5 min, and the
661
amount of protein in the supernatant was measured using the Bradford assay (21). The initial adsorption, which is one of the substrate characteristics, was defined as the cellulase adsorption at hour 0 of the hydrolysis. The initial adsorption reaction was performed as described by Mooney et al. (6) in 50 mM sodium acetate buffer (pH 4.8) for 90 min at 4 °C followed by 10 min at 50 °C. The amount of adsorbed cellulase protein was calculated based on the difference between the amount of initial protein and the amount of soluble protein in the supernatant. Scanning electron microscope observation Corn straws that were untreated, treated by A/O pretreatment, treated by biological pretreatment and by Bio-A/O pretreatment with E. taxodii for 15 days were observed using a scanning electron microscope (Quanta 200) in order to observe potential differences in the structural modifications of the corn straw. The dried samples were coated with gold and scanned at an accelerating voltage of 20 kV.
RESULTS AND DISCUSSION Chemical analyses of corn straw pretreated with white-rot fungi The weight loss, cellulose loss, lignin loss and the selectivity value of corn straw treated by the three fungi are listed in Table 1. All fungi were capable of lignin degradation, and the lignin losses after 10-day biological pretreatment were all above 15%. Trametes versicolor, Ganoderma lucidum and Echinodontium taxodii caused 54.6%, 32.7% and 42.2% lignin loss, respectively, after a 30-day pretreatment; however, these fungi displayed very different abilities to break down cellulose. The cellulose loss caused by E. taxodii was significantly lower than that caused by T. versicolor or G.. lucidum. E. taxodii had a very high selectivity value, and lignin loss was 4.5- to 2.6-fold greater than cellulose loss during the pretreatment. These results indicate that E. taxodii preferentially degrades lignin during the pretreatment of corn straw, which was also observed during the biodegradation of bamboo culms; however, the cellulose loss in the corn straw was higher than in the bamboo culms (16). Effect of Bio-A/O pretreatment on enzymatic hydrolysis To evaluate the effect of Bio-A/O pretreatment on enzymatic hydrolysis, we determined the reducing sugar yield of pretreated corn straw after 48-h enzymatic hydrolysis with 1, 2, 4, 6, 8 or 10 mg ml−1 of cellulase. Fig. 1 shows the reducing sugar yields from corn straw pretreated by A/O pretreatment alone or Bio-A/O pretreatment with G. lucidum, T. versicolor and E. taxodii for 10, 15 or 30 days. The reducing sugar yields increased concomitantly with an increase in the cellulase concentration and usually reached a steady state at a cellulase concentration of 8 mg ml−1. As expected, corn straw treated by A/O pretreatment alone had a high resistance to enzymatic hydrolysis, and the maximum sugar yield was only 40.3% at a cellulase concentration of 8 mg ml−1. However, the yield of reducing sugar increased significantly after the corn straw was pretreated with white-rot fungi followed by pretreatment with alkaline/oxidative reagent. The reducing sugar yield of corn straw pretreated with G. lucidum, T. versicolor and E. taxodii reached the highest levels after a 15-day
Table 1. Weight loss, cellulose loss, lignin loss and selectivity value of corn straw pretreated with the three fungi for 10, 15, 30 days. Decay time (day)
Components loss (%) Weight
Selectivity value
Cellulose
Lignin
Trametes versicolor 10 12.2 ± 0.0 15 17.8 ± 0.0 30 27.1 ± 0.1
14.3 ± 0.7 29.7 ± 1.4 47.5 ± 2.2
22.8 ± 1.1 24.1 ± 1.1 54.6 ± 2.6
1.6 0.8 1.2
Ganoderma lucidum 10 10.8 ± 0.0 15 13.5 ± 0.1 30 15.5 ± 0.1
21.7 ± 1.0 26.3 ± 1.2 39.0 ± 1.8
17.5 ± 0.8 20.0 ± 0.9 32.7 ± 1.5
0.8 0.8 0.8
Echinodontium taxodii 10 10.6 ± 0.1 15 14.5 ± 0.1 30 18.5 ± 0.0
5.9 ± 0.8 7.6 ± 0.9 15.8 ± 1.0
27 ± 0.2 29.5 ± 1.4 42.2 ± 1.9
4.5 3.9 2.6
662
YU ET AL.
J. BIOSCI. BIOENG., E. taxodii was the most effective fungus among the three fungi at increasing the yield of reducing sugars. The amount of reducing sugar from corn straw pretreated with E. taxodii and A/O reached 57.52% after enzymatic hydrolysis with 2 mg ml−1 cellulase solution, but the sugar yield of corn straw pretreated by A/O alone only reached 40.22% after enzymatic hydrolysis with an 8 mg ml−1 concentration of cellulase solution. These results indicate that biological pretreatment can significantly enhance the efficiency of A/O pretreatment. A higher sugar yield is obtained at a lower cellulase concentration after a combined pretreatment with E. taxodii and A/O in comparison with the A/O pretreatment alone. This combined pretreatment can reduce the cost of cellulase for enzymatic hydrolysis and ethanol production. Although T. versicolor, a non-selective, lignin-degrading fungus, displayed the greatest ability to degrade lignin among all the fungi tested, the sugar yield of corn straw pretreated with this fungus was lowest because of the very high cellulose loss resulting from pretreatment with this fungus. This result highlights the importance of minimizing cellulose loss during biological pretreatment. Therefore, E. taxodii, which preferentially degraded lignin, was the most promising fungus for bio-A/O pretreatment of the three fungi tested. Microstructure analysis of pretreated corn straw The surface morphology of the untreated and treated corn straws was examined by scanning electron microscopy (SEM). SEM images showed that cells within the untreated corn straw had intact cell walls (Fig. 2A), whereas some irregular holes were found on the straw after the A/O pretreatment or biological pretreatment (Fig. 2B–C), which resulted from the removal of lignin and breaking of lignocellulose networks during the pretreatment (6). The ruptures of the corn straw surface were more profound after Bio-A/O pretreatment than after the chemical pretreatment alone. The surface of the Bio-A/O-treated material was significantly corroded and became heterogeneous and loose; many big holes and small pores were observed, as shown in Fig. 4D. This result indicates that the combined pretreatment caused a further increase in porosity and surface area of the substrate (compared to the A/O pretreatment alone), which may result in an increased susceptibility of corn straw to enzymatic hydrolysis. Enzymatic hydrolysis and adsorption Fig. 3 shows the relationships between the reducing sugar yield and hydrolysis time during cellulase-based hydrolysis of corn straw. A substantial increase in the sugar yield of corn straw was observed after E. taxodii-induced Bio-A/O pretreatment for 15 days compared to pretreatment by A/O alone. The effect of the biological pretreatment was relatively constant during the entire 120 h hydrolysis. The amount of reducing sugar increased by 34% (from 9.3% to 12.5%) after 1 h of hydrolysis and by 40.3% (from 43.2% to 60.6%) after 72-h hydrolysis. The hydrolysis rate of the corn straw decreased as the hydrolysis continued. Therefore, to further investigate the initial hydrolysis rate during the hydrolysis of pretreated corn straw, Ohmine's empirical rate expression (22) was applied to the yield-time data in Fig. 3 using the following equation: S0 X = ðS0 = kÞ*lnð1 + V0 *k*t = S0 Þ
FIG. 1. Reducing sugar yield (%) of A/O-pretreated corn straw (Solid square) and bio-A/Opretreated corn straw with Trametes versicolor (A), Ganoderma lucidum (B), Echinodontium taxodii (C) for 10 (Solid triangle up), 15 (Solid triangle down) or 30 (Solid circle) days. Data represent the means of three experiments and error bars represent standard deviations.
fungal pretreatment. In comparison with A/O pretreatment alone, the reducing sugar yields of corn straw pretreated with G. lucidum, T. versicolor and E. taxodii increased by 26.5%, 29.3% and 50.7%, respectively, at a cellulase concentration of 8 mg ml−1 after a 48-h enzymatic hydrolysis. Yet, the sugar yield decreased when pretreated with these fungi for 30 days.
ð1Þ
Where S0 is the initial concentration of cellulose (mg g−1), X is the reducing sugar yield (%), V0 is the initial hydrolysis rate (mg h−1), t is the hydrolysis time and k is the rate constant. When the concentration of cellulosic substrate is constant and the unit of V0 is % h−1, equation (Eq. 1) can be rewritten in the following form: X = ð1 = kÞ*lnð1 + V0 *k*t Þ
ð2Þ:
The regression curves obtained from Eq. 2 are shown in Fig. 3, and a strong agreement with the experimental results was obtained (R2 N 0.95). The two kinetics parameters, V0 and k, were determined using Eq. 2. The results indicate that the initial hydrolysis rate V0 increased by 68.5% from 75.2 when using A/O pretreatment to 126.7
VOL. 110, 2010
WHITE ROT FUNGI PRETREATMENT
663
FIG. 2. Scanning electron micrographs of corn straw with and without pretreatments. (A) Untreated corn straw; (B) Corn straw after A/O pretreatment alone; (C) Corn straw after biological pretreatment with E. taxodii for 15 days; (D) Corn straw following Bio-A/O pretreatment with E. taxodii and A/O for 15 days. The scale bar represents 50 μm.
when employing Bio-A/O pretreatment, and the rate constant k decreased from 0.151 when using A/O pretreatment alone to 0.114 after biological pretreatment. Fig. 3 shows the adsorption of cellulase during the hydrolysis. The adsorption of cellulase on lignocellulose is the first step in the hydrolysis reaction. Previous studies have reported that low adsorption resulted in the lack of accessibility of the enzymes to the lignocellulose (5,23). The initial adsorption of corn straw that was
pretreated by Bio-A/O (2.52 mg ml−1) was higher than with A/O pretreatment alone (2.32 mg ml−1). The increase in the initial adsorption indicated that the accessibility of the cellulase to the lignocellulose increased after biological pretreatment, which resulted in an increase in the initial hydrolysis rate, V0. The cellulase adsorption increased rapidly during the early stages of hydrolysis and decreased after 3 h of hydrolysis because of the desorption of cellulase during the enzymatic hydrolysis; however,
FIG. 3. Reducing sugar yield (solid symbol) and adsorbed enzyme protein (hollow symbol) during the hydrolysis of corn straw pretreated by A/O (square) or E. taxodii-based Bio-A/O for 15 days (circle) at a cellulase concentration of 10 mg ml−1.
FIG. 4. Composition of corn straw components. (A) Untreated, (B) pretreated by A/O, (C) pretreated with E. taxodii and (D) by E. taxodii Bio-A/O pretreatment for 15 days.
664
YU ET AL.
J. BIOSCI. BIOENG.,
cellulase adsorption on the Bio-A/O-pretreated corn straw was always lower than the A/O-pretreated straw after 16 h of hydrolysis. After 72 h, the cellulase adsorption after Bio-A/O pretreatment decreased by 24.8% (from 3.67 to 2.76 mg ml−1) when compared to A/O pretreatment alone. Some studies have indicated that the unproductive adsorption of cellulase on lignin could impede desorption of cellulase (24). Our results indicate that the biological pretreatment may cause a decrease in unproductive adsorption, which could have resulted in the increased yield of reducing sugar. Fig. 4 shows that biological pretreatment and A/O pretreatment decrease the lignin content of corn straw (from 12.4% to 7.8% and 7%, respectively); however, the lignin content decreased more significantly (6.2%) when the two pretreatment methods were combined. This result indicates that biological pretreatment is able to enhance delignification when combined with A/O pretreatment, which results in a decrease in the unproductive adsorption of cellulase. White-rot fungi-mediated Bio-A/O pretreatment can significantly enhance the efficiency of A/O pretreatment for enzymatic hydrolysis of corn straw. The reducing sugar yield increased by 50.7% after 15day pretreatment with E. taxodii compared to A/O pretreatment alone. The initial hydrolysis rate, V0, and the initial adsorption, increased during enzymatic hydrolysis of corn straw following pretreatment by Bio-A/O. The biological pretreatment enhanced delignification by the A/O reagent and increased the desorption of cellulase. ACKNOWLEDGMENTS This work was supported by funds from the National Basic Research Program (2007CB210200) and the National Natural Science Foundation of China (30901137). The authors thank the Center of Analysis and Testing of Huazhong University of Science and Technology for SEM scanning. References 1. Zhao, Y., Wang, Y., Zhu, J. Y., Ragauskas, A., and Deng, Y.: Enhanced enzymatic hydrolysis of spruce by alkaline pretreatment at low temperature, Biotechnol. Bioeng., 99, 1320–1328 (2008). 2. Saha, B. C. and Cotta, M. A.: Enzymatic hydrolysis and fermentation of lime pretreated wheat straw to ethanol, J. Chem. Technol. Biotechnol., 82, 913–919 (2007). 3. Pu, Y., Zhang, D., Singh, P. M., and Ragauskas, A. J.: The new forestry biofuels sector, Biofuels, Bioprod. Biorefin., 2, 58–73 (2008). 4. Behera, B. K., Arora, M., and Sharma, D. K.: Scanning electorn microscopic (SEM) studies on structural architecture of lignocellulosic materials of calotropis procera during its processing for saccharification, Bioresour. Technol., 58, 241–245 (1996). 5. Sun, Y. and Cheng, J.: Hydrolysis of lignocellulosic materials for ethanol production: a review, Bioresour. Technol., 83, 1–11 (2002).
6. Mooney, C. A., Mansfield, S. D., Touhy, M. G., and Saddler, J. N.: The effect of initial pore volume and lignin content on the enzymatic hydrolysis of softwoods, Bioresour. Technol., 64, 113–119 (1998). 7. Carrillo, F., Lis, M. J., Colom, X., López-Mesas, M., and Valldeperas, J.: Effect of alkali pretreatment on cellulase hydrolysis of wheat straw: Kinetic study, Process Biochem., 40, 3360–3364 (2005). 8. Stenberg, K., Bollok, M., Reczey, K., Galbe, M., and Zacchi, G.: Effect of substrate and cellulase concentration on simultaneous saccharification and fermentation of steam-pretreated softwood for ethanol production, Biotechnol. Bioeng., 68, 204–210 (2000). 9. Teymouri, F., Laureano-Perez, L., Alizadeh, H., and Dale, B. E.: Optimization of the ammonia fiber explosion (AFEX) treatment parameters for enzymatic hydrolysis of corn stover, Bioresour. Technol., 96, 2014–2018 (2005). 10. Pan, X., Arato, C., Gilkes, N., Gregg, D., Mabee, W., Pye, K., Xiao, Z., Zhang, X., and Saddler, J.: Biorefining of softwoods using ethanol organosolv pulping: preliminary evaluation of process streams for manufacture of fuel-grade ethanol and coproducts, Biotechnol. Bioeng., 90, 473–481 (2005). 11. Lloyd, T. A. and Wyman, C. E.: Combined sugar yields for dilute sulfuric acid pretreatment of corn stover followed by enzymatic hydrolysis of the remaining solids, Bioresour. Technol., 96, 1967–1977 (2005). 12. Curreli, N., Fadda, M. B., Rescigno, A., Rinaldi, A. C., Soddu, G., Sollai, F., Vaccargiu, S., Sanjust, E., and Rinaldi, A.: Mild alkaline/oxidative pretreatment of wheat-straw, Process Biochem., 32, 665–670 (1997). 13. Hakala, T. K., Maijala, P., Konnb, J., and Hatakka, A.: Evaluation of novel woodrotting polypores and corticioid fungi for the decay and biopulping of Norway spruce (Picea abies) wood, Enzyme Microb. Technol., 34, 255–263 (2004). 14. Vares, T., Kalsi, M., and Hatakka, A.: Lignin peroxidases, manganese peroxidases, and other ligninolytic enzymes produced by phlebia radiata during solid-state fermentation of wheat straw, Appl. Environ. Microbiol., 61, 3515–3520 (1995). 15. Taniguchi, M., Suzuki, H., Watanabe, D., Sakai, K., Hoshino, K., and Tanaka, T.: Evaluation of pretreatment with Pleurotus ostreatus for enzymatic hydrolysis of rice straw, J. Biosci. Bioeng., 100, 637–643 (2005). 16. Zhang, X., Yu, H., Huang, H., and Liu, Y.: Evaluation of biological pretreatment with white rot fungi for the enzymatic hydrolysis of bamboo culms, Int. Biodeterior. Biodegrad., 60, 159–164 (2007). 17. Yang, B. and Wyman, C. E.: Pretreatment: the key to unlocking low-cost cellulosic ethanol, Biofuels, Bioprod. Biorefin., 2, 26–40 (2008). 18. Itoh, H., Wada, M., Honda, Y., Kuwahara, M., and Watanabe, T.: Bioorganosolve pretreatments for simultaneous saccharification and fermentation of beech wood by ethanolysis and white rot fungi, J. Biotechnol., 103, 273–280 (2003). 19. Mendonca, R., Guerra, A., and Ferraz, A.: Delignification of Pinus taeda wood chips treated with ceriporiopsis subvermispora for preparing high-yield kraft pulps, J. Chem. Technol. Biotechnol., 77, 411–418 (2002). 20. Horwitz, W.: Official methods of analysis of the Association of Official Analytical Chemists, Association of Official Analytical Chemists, Washington D.C. (1980). 21. Mooney, C. A., Mansfield, S. D., Beatson, R. P., and Saddler, J. N.: The effect of fiber characteristics on hydrolysis and cellulase accessibility to softwood substrates, Enzyme Microb. Technol., 25, 644–650 (1999). 22. Ohmine, K., Ooshima, H., and Harano, Y.: Kinetic study on enzymatic hydrolysis of cellulose by collulase from trichoderma viride, Biotechnol. Bioeng., XXV, 2041–2053 (1983). 23. Saddler, J. N.: Adsorption and activity profiles of cellulases during the hydrolysis of two Douglas fir pulps, Enzyme Microb. Technol., 24, 138–143 (1999). 24. Gregg, D. J. and Saddler, J. N.: Factors affecting cellulose hydrolysis and the potential of enzyme recycle to enhance the efficiency of an integrated wood to ethanol process, Biotechnol. Bioeng., 51, 375–383 (1996).
Evaluation of white-rot fungi-assisted alkaline/oxidative pretreatment of corn straw undergoing enzymatic hydrolysis by cellulase Hongbo Yu, Xiaoyu Zhang,⁎ Lili Song, Jing Ke, Chunyan Xu, Wanqing Du, and Ji Zhang College of Life Science and Technology, Huazhong University of Science and Technology, Wuluo Road 1037, Wuhan 430074, China Received 11 February 2010; accepted 3 August 2010 Available online 3 September 2010
In this study, the effects of biological treatment prior to alkaline/oxidative (A/O) pretreatment using three white-rot fungi (Ganoderma lucidum, Trametes versicolor and Echinodontium taxodii) were evaluated for the enzymatic hydrolysis of corn straw. Among these fungi, Echinodontium taxodii significantly enhanced the efficiency of chemical pretreatment. Subsequent to treatment of corn straw with Echinodontium taxodii for 15 days, the straw was subjected to digestion by 0.0016% NaOH and 3% H2O2 at room temperature for 24 h, which increased the reducing sugar yield by 50.7%. The hydrolysis model and kinetic parameters were determined from time course data collected throughout the hydrolysis. The initial hydrolysis rate, V0, of the corn straw increased by 68.5% compared to A/O pretreatment alone, which resulted from an increase in the initial adsorption. The lignin content of the corn straw decreased more significantly after biological and A/O pretreatment than after A/O pretreatment alone. After 72 h of enzymatic hydrolysis, the adsorbed cellulase decreased by 24.8% (from 3.67 to 2.76 mg ml−1) compared to A/O pretreatment alone. These results indicate that biological treatment improves the desorption of cellulase by enhancing delignification during A/O pretreatment. © 2010, The Society for Biotechnology, Japan. All rights reserved. [Key words: Corn straw; Echinodontium taxodii; Biological pretreatment; Alkaline/oxidative pretreatment; Enzymatic hydrolysis; Adsorption]
Lignocellulosic biomass represents a promising, low-cost resource for the large-scale production of biofuels, such as ethanol and methanol (1–4). Enzymatic hydrolysis of lignocellulose is a key step in biofuel production; however, the natural resistance of lignocellulose to hydrolysis prevents enzymatic treatment and release of fermentable sugars. Some studies have indicated that the porosity of lignocellulose and crystallinity of the cellulose fibrils can affect the enzymatic hydrolysis of lignocellulose (5). Furthermore, the network of lignin and hemicellulose surrounding cellulose fibrils prevents the penetration of cellulase (6). Although the activity of cellulase has been improved upon significantly in the last decade, the removal of lignin, reduction in the crystallinity of the cellulose and increase in porosity of lignocellulose by pretreatment technologies remain as challenges in achieving high hydrolysis yields. Several physical, chemical and physicochemical pretreatment methods have been used for the enzymatic hydrolysis of lignocellulose during ethanol production, including mechanical comminution, pyrolysis, steam explosion, ammonia fiber explosion, organosolv extraction, acid hydrolysis, alkaline hydrolysis and alkaline/oxidative delignification (5,7–12). Recently, some studies have indicated that biological pretreatment with white-rot fungi, which are the only known organisms that can efficiently remove lignin from lignocellulose, may reduce the recalcitrance of lignocellulose to enzymatic hydrolysis (13,14). A number of fungal strains have been evaluated for the enzymatic hydrolysis of lignocellulose. For instance, Taniguchi et ⁎ Corresponding author. Tel.: + 86 27 87792108; fax: + 86 27 87792128. E-mail address: [email protected] (X. Zhang).
al. (15) reported that the total sugar yields of rice straw following treatment with Pleurotus ostreatus for 60 days were 33% after enzymatic hydrolysis. In our previous investigation, 34 isolates of white-rot fungi were screened for use in the biological pretreatment of bamboo culms, and sugar yields increased 8.76-fold after 120-day pretreatment with Echinodontium taxodii (16). Besides biological pretreatment, most chemical and physical pretreatment processes require high temperatures and operating pressures, which results in extremely high demand for heat and power. For example, steam explosion is usually carried out at 160– 260 °C and 0.69–4.83 MPa (5). Dilute acid and alkaline pretreatments are often carried out at temperatures above 100 °C and require special equipment for achieving high hydrolysis yields (17). Although biological pretreatment was considered to be a promising technology because of its low energy requirements and mild environmental effects (5), most biological pretreatment processes require a long time (about 3– 5 months) and result in very high feedstock loss during pretreatment. Recently, several studies indicated that a short-term biological pretreatment enhances the efficiency of physicochemical pretreatment. For example, Itoh et al. (18) reported that the yield of ethanol obtained from beech wood chips after pretreatment for 8 weeks was 1.6 times higher than that obtained without the fungal treatments. Some studies on biopulping indicated that 15-day-biotreatment with Ceriporiopsis subvermispora may decrease the amounts of active alkali because depolymerization of lignin during the initial stages of biodegradation enhanced the release of lignin during alkaline cooking (19). These studies suggest that it is not necessary to remove lignin by long-term biological pretreatment during consecutive treatments with lignin-
1389-1723/$ - see front matter © 2010, The Society for Biotechnology, Japan. All rights reserved. doi:10.1016/j.jbiosc.2010.08.002
VOL. 110, 2010
WHITE ROT FUNGI PRETREATMENT
degrading fungi or alkaline cooking. Yet, few studies have evaluated the effects of biological pretreatment of corn straw prior to alkaline pretreatments at room temperature. In this study, we evaluated a new biological-alkaline/oxidative (Bio-A/O) pretreatment process in which lignocellulose was pretreated by white-rot fungi and then by alkaline/oxidative (A/O) treatment at room temperature and pressure with a low concentration of chemicals. The A/O treatment alone at room temperature and pressure can decrease heat and power demands, but enzymatic hydrolysis yields are not high after this treatment step. We attempted to enhance the efficiency of A/O pretreatment by incubating the corn straw in the presence of white-rot fungi prior to A/O pretreatment. The effects of Bio-A/O pretreatments on the enzymatic hydrolysis of corn straw were evaluated with Ganoderma lucidum, Trametes versicolor and Echinodontium taxodii. Alterations in structure, chemistry, enzymatic hydrolysis and adsorption capacity of pretreated corn straw were also investigated. MATERIALS AND METHODS Microorganisms Ganoderma lucidum, Trametes versicolor and Echinodontium taxodii were obtained from our laboratory and used for the Bio-A/O pretreatments. The fungal strains were cultured on potato dextrose agar (PDA) slants at 25 °C. After 7 days, agar pieces were cut from slants and inoculated in 250-ml Erlenmeyer flasks with 100ml of potato dextrose liquid media. The cultures were incubated at 25 °C with agitation (150 rpm) for 7 days and used for the biological pretreatments. Substrate Corn straw from Henan (China) was cut into chips (400–800 μm mesh). The chips were air-dried and stored under dry conditions. Biological pretreatment with white-rot fungi 10-ml of fungal cultures were inoculated into 250-ml Erlenmeyer flasks and sterilized at 121 °C for 30 min with 10-g of corn straw chips and 25-ml of distilled water. The flasks were covered with film to avoid moisture loss and contamination. The cultures were statically incubated at 25 °C for 10, 15 and 30 days. All the cultures were grown in triplicate. Non-inoculated corn straw was used as a control. The treated corn straw was dried at 60 °C for 3 days and weighed to calculate the percentage of weight loss based on the initial weight and final dry weight. The percentage of lignin, cellulose and hemicellulose in the treated and untreated corn straw was determined according to the procedures of AOAC (20). Lignin and cellulose loss was calculated as follows:
Lignin ðor celluloseÞ loss ð%Þ ligninðor celluloseÞ content in treated straw × final weight of straw after pretreament = 100− lignin ðor celluloseÞ content in untreated straw × initial weight of straw
The selectivity value of lignin degradation was calculated as the ratio of lignin loss over cellulose loss. Alkaline/oxidative pretreatment The bio-pretreated and untreated corn straw chips were exposed to an A/O pretreatment. The A/O pretreatment was carried out with 2% corn straw substrate in alkaline liquor with 0.0016% sodium hydroxide (NaOH) and 3% (vol/vol) hydrogen peroxide (H2O2) at room temperature. After 16 h, the pretreated corn straw samples were separated from the liquid by centrifugation and washed with distilled water to bring them to a neutral pH. Enzymatic hydrolysis Cellulase was obtained from Sigma (cellulase from Aspergillus niger; catalogue no. C1184). The total cellulase activity of commercial cellulase −1 was 100 FPU g based on the filter paper assay (FPA). Enzymatic hydrolysis was carried out with 2% substrate in 50 mM sodium acetate buffer (pH 4.8) with 1, 2, 4, 6, 8 or 10 mg ml−1 of cellulase at 50 °C. After 48 h, the reaction mixtures were interrupted by centrifugation at 10,000 rpm for 5 min. The amount of reducing sugar in the reaction supernatants was determined with the 3,5-dinitrosalicylic acid (DNS) reagent (4). Furthermore, the corn straw treated by Bio-A/O pretreatment with E. taxodii for 15 days was hydrolyzed with 10 mg ml−1 cellulase for 0, 1, 3, 6, 10, 24, 48, 72, 96 or 120 h, and the reducing sugar in the supernatant was determined after centrifugation. The yield of reducing sugar in each sample was calculated as follows:
Reducing sugar yield ð%Þ amount of reducing sugar produced after enzymatic hydrolysis × 0:9 × 100 = amount of hemicellulose þ amount of cellulose Enzymatic adsorption during the hydrolysis The corn straw treated by BioA/O pretreatment with E. taxodii for 15 days was used to investigate cellulase adsorption −1 capacity at an enzyme concentration of 10 mg ml . Enzymatic protein was used as an indicator of adsorption capacity. Subsequent to enzymatic hydrolysis for 1, 3, 6, 10, 24, 48, 72, 96 or 120 h, the corn straw liquor was centrifuged at 10,000 rpm for 5 min, and the
661
amount of protein in the supernatant was measured using the Bradford assay (21). The initial adsorption, which is one of the substrate characteristics, was defined as the cellulase adsorption at hour 0 of the hydrolysis. The initial adsorption reaction was performed as described by Mooney et al. (6) in 50 mM sodium acetate buffer (pH 4.8) for 90 min at 4 °C followed by 10 min at 50 °C. The amount of adsorbed cellulase protein was calculated based on the difference between the amount of initial protein and the amount of soluble protein in the supernatant. Scanning electron microscope observation Corn straws that were untreated, treated by A/O pretreatment, treated by biological pretreatment and by Bio-A/O pretreatment with E. taxodii for 15 days were observed using a scanning electron microscope (Quanta 200) in order to observe potential differences in the structural modifications of the corn straw. The dried samples were coated with gold and scanned at an accelerating voltage of 20 kV.
RESULTS AND DISCUSSION Chemical analyses of corn straw pretreated with white-rot fungi The weight loss, cellulose loss, lignin loss and the selectivity value of corn straw treated by the three fungi are listed in Table 1. All fungi were capable of lignin degradation, and the lignin losses after 10-day biological pretreatment were all above 15%. Trametes versicolor, Ganoderma lucidum and Echinodontium taxodii caused 54.6%, 32.7% and 42.2% lignin loss, respectively, after a 30-day pretreatment; however, these fungi displayed very different abilities to break down cellulose. The cellulose loss caused by E. taxodii was significantly lower than that caused by T. versicolor or G.. lucidum. E. taxodii had a very high selectivity value, and lignin loss was 4.5- to 2.6-fold greater than cellulose loss during the pretreatment. These results indicate that E. taxodii preferentially degrades lignin during the pretreatment of corn straw, which was also observed during the biodegradation of bamboo culms; however, the cellulose loss in the corn straw was higher than in the bamboo culms (16). Effect of Bio-A/O pretreatment on enzymatic hydrolysis To evaluate the effect of Bio-A/O pretreatment on enzymatic hydrolysis, we determined the reducing sugar yield of pretreated corn straw after 48-h enzymatic hydrolysis with 1, 2, 4, 6, 8 or 10 mg ml−1 of cellulase. Fig. 1 shows the reducing sugar yields from corn straw pretreated by A/O pretreatment alone or Bio-A/O pretreatment with G. lucidum, T. versicolor and E. taxodii for 10, 15 or 30 days. The reducing sugar yields increased concomitantly with an increase in the cellulase concentration and usually reached a steady state at a cellulase concentration of 8 mg ml−1. As expected, corn straw treated by A/O pretreatment alone had a high resistance to enzymatic hydrolysis, and the maximum sugar yield was only 40.3% at a cellulase concentration of 8 mg ml−1. However, the yield of reducing sugar increased significantly after the corn straw was pretreated with white-rot fungi followed by pretreatment with alkaline/oxidative reagent. The reducing sugar yield of corn straw pretreated with G. lucidum, T. versicolor and E. taxodii reached the highest levels after a 15-day
Table 1. Weight loss, cellulose loss, lignin loss and selectivity value of corn straw pretreated with the three fungi for 10, 15, 30 days. Decay time (day)
Components loss (%) Weight
Selectivity value
Cellulose
Lignin
Trametes versicolor 10 12.2 ± 0.0 15 17.8 ± 0.0 30 27.1 ± 0.1
14.3 ± 0.7 29.7 ± 1.4 47.5 ± 2.2
22.8 ± 1.1 24.1 ± 1.1 54.6 ± 2.6
1.6 0.8 1.2
Ganoderma lucidum 10 10.8 ± 0.0 15 13.5 ± 0.1 30 15.5 ± 0.1
21.7 ± 1.0 26.3 ± 1.2 39.0 ± 1.8
17.5 ± 0.8 20.0 ± 0.9 32.7 ± 1.5
0.8 0.8 0.8
Echinodontium taxodii 10 10.6 ± 0.1 15 14.5 ± 0.1 30 18.5 ± 0.0
5.9 ± 0.8 7.6 ± 0.9 15.8 ± 1.0
27 ± 0.2 29.5 ± 1.4 42.2 ± 1.9
4.5 3.9 2.6
662
YU ET AL.
J. BIOSCI. BIOENG., E. taxodii was the most effective fungus among the three fungi at increasing the yield of reducing sugars. The amount of reducing sugar from corn straw pretreated with E. taxodii and A/O reached 57.52% after enzymatic hydrolysis with 2 mg ml−1 cellulase solution, but the sugar yield of corn straw pretreated by A/O alone only reached 40.22% after enzymatic hydrolysis with an 8 mg ml−1 concentration of cellulase solution. These results indicate that biological pretreatment can significantly enhance the efficiency of A/O pretreatment. A higher sugar yield is obtained at a lower cellulase concentration after a combined pretreatment with E. taxodii and A/O in comparison with the A/O pretreatment alone. This combined pretreatment can reduce the cost of cellulase for enzymatic hydrolysis and ethanol production. Although T. versicolor, a non-selective, lignin-degrading fungus, displayed the greatest ability to degrade lignin among all the fungi tested, the sugar yield of corn straw pretreated with this fungus was lowest because of the very high cellulose loss resulting from pretreatment with this fungus. This result highlights the importance of minimizing cellulose loss during biological pretreatment. Therefore, E. taxodii, which preferentially degraded lignin, was the most promising fungus for bio-A/O pretreatment of the three fungi tested. Microstructure analysis of pretreated corn straw The surface morphology of the untreated and treated corn straws was examined by scanning electron microscopy (SEM). SEM images showed that cells within the untreated corn straw had intact cell walls (Fig. 2A), whereas some irregular holes were found on the straw after the A/O pretreatment or biological pretreatment (Fig. 2B–C), which resulted from the removal of lignin and breaking of lignocellulose networks during the pretreatment (6). The ruptures of the corn straw surface were more profound after Bio-A/O pretreatment than after the chemical pretreatment alone. The surface of the Bio-A/O-treated material was significantly corroded and became heterogeneous and loose; many big holes and small pores were observed, as shown in Fig. 4D. This result indicates that the combined pretreatment caused a further increase in porosity and surface area of the substrate (compared to the A/O pretreatment alone), which may result in an increased susceptibility of corn straw to enzymatic hydrolysis. Enzymatic hydrolysis and adsorption Fig. 3 shows the relationships between the reducing sugar yield and hydrolysis time during cellulase-based hydrolysis of corn straw. A substantial increase in the sugar yield of corn straw was observed after E. taxodii-induced Bio-A/O pretreatment for 15 days compared to pretreatment by A/O alone. The effect of the biological pretreatment was relatively constant during the entire 120 h hydrolysis. The amount of reducing sugar increased by 34% (from 9.3% to 12.5%) after 1 h of hydrolysis and by 40.3% (from 43.2% to 60.6%) after 72-h hydrolysis. The hydrolysis rate of the corn straw decreased as the hydrolysis continued. Therefore, to further investigate the initial hydrolysis rate during the hydrolysis of pretreated corn straw, Ohmine's empirical rate expression (22) was applied to the yield-time data in Fig. 3 using the following equation: S0 X = ðS0 = kÞ*lnð1 + V0 *k*t = S0 Þ
FIG. 1. Reducing sugar yield (%) of A/O-pretreated corn straw (Solid square) and bio-A/Opretreated corn straw with Trametes versicolor (A), Ganoderma lucidum (B), Echinodontium taxodii (C) for 10 (Solid triangle up), 15 (Solid triangle down) or 30 (Solid circle) days. Data represent the means of three experiments and error bars represent standard deviations.
fungal pretreatment. In comparison with A/O pretreatment alone, the reducing sugar yields of corn straw pretreated with G. lucidum, T. versicolor and E. taxodii increased by 26.5%, 29.3% and 50.7%, respectively, at a cellulase concentration of 8 mg ml−1 after a 48-h enzymatic hydrolysis. Yet, the sugar yield decreased when pretreated with these fungi for 30 days.
ð1Þ
Where S0 is the initial concentration of cellulose (mg g−1), X is the reducing sugar yield (%), V0 is the initial hydrolysis rate (mg h−1), t is the hydrolysis time and k is the rate constant. When the concentration of cellulosic substrate is constant and the unit of V0 is % h−1, equation (Eq. 1) can be rewritten in the following form: X = ð1 = kÞ*lnð1 + V0 *k*t Þ
ð2Þ:
The regression curves obtained from Eq. 2 are shown in Fig. 3, and a strong agreement with the experimental results was obtained (R2 N 0.95). The two kinetics parameters, V0 and k, were determined using Eq. 2. The results indicate that the initial hydrolysis rate V0 increased by 68.5% from 75.2 when using A/O pretreatment to 126.7
VOL. 110, 2010
WHITE ROT FUNGI PRETREATMENT
663
FIG. 2. Scanning electron micrographs of corn straw with and without pretreatments. (A) Untreated corn straw; (B) Corn straw after A/O pretreatment alone; (C) Corn straw after biological pretreatment with E. taxodii for 15 days; (D) Corn straw following Bio-A/O pretreatment with E. taxodii and A/O for 15 days. The scale bar represents 50 μm.
when employing Bio-A/O pretreatment, and the rate constant k decreased from 0.151 when using A/O pretreatment alone to 0.114 after biological pretreatment. Fig. 3 shows the adsorption of cellulase during the hydrolysis. The adsorption of cellulase on lignocellulose is the first step in the hydrolysis reaction. Previous studies have reported that low adsorption resulted in the lack of accessibility of the enzymes to the lignocellulose (5,23). The initial adsorption of corn straw that was
pretreated by Bio-A/O (2.52 mg ml−1) was higher than with A/O pretreatment alone (2.32 mg ml−1). The increase in the initial adsorption indicated that the accessibility of the cellulase to the lignocellulose increased after biological pretreatment, which resulted in an increase in the initial hydrolysis rate, V0. The cellulase adsorption increased rapidly during the early stages of hydrolysis and decreased after 3 h of hydrolysis because of the desorption of cellulase during the enzymatic hydrolysis; however,
FIG. 3. Reducing sugar yield (solid symbol) and adsorbed enzyme protein (hollow symbol) during the hydrolysis of corn straw pretreated by A/O (square) or E. taxodii-based Bio-A/O for 15 days (circle) at a cellulase concentration of 10 mg ml−1.
FIG. 4. Composition of corn straw components. (A) Untreated, (B) pretreated by A/O, (C) pretreated with E. taxodii and (D) by E. taxodii Bio-A/O pretreatment for 15 days.
664
YU ET AL.
J. BIOSCI. BIOENG.,
cellulase adsorption on the Bio-A/O-pretreated corn straw was always lower than the A/O-pretreated straw after 16 h of hydrolysis. After 72 h, the cellulase adsorption after Bio-A/O pretreatment decreased by 24.8% (from 3.67 to 2.76 mg ml−1) when compared to A/O pretreatment alone. Some studies have indicated that the unproductive adsorption of cellulase on lignin could impede desorption of cellulase (24). Our results indicate that the biological pretreatment may cause a decrease in unproductive adsorption, which could have resulted in the increased yield of reducing sugar. Fig. 4 shows that biological pretreatment and A/O pretreatment decrease the lignin content of corn straw (from 12.4% to 7.8% and 7%, respectively); however, the lignin content decreased more significantly (6.2%) when the two pretreatment methods were combined. This result indicates that biological pretreatment is able to enhance delignification when combined with A/O pretreatment, which results in a decrease in the unproductive adsorption of cellulase. White-rot fungi-mediated Bio-A/O pretreatment can significantly enhance the efficiency of A/O pretreatment for enzymatic hydrolysis of corn straw. The reducing sugar yield increased by 50.7% after 15day pretreatment with E. taxodii compared to A/O pretreatment alone. The initial hydrolysis rate, V0, and the initial adsorption, increased during enzymatic hydrolysis of corn straw following pretreatment by Bio-A/O. The biological pretreatment enhanced delignification by the A/O reagent and increased the desorption of cellulase. ACKNOWLEDGMENTS This work was supported by funds from the National Basic Research Program (2007CB210200) and the National Natural Science Foundation of China (30901137). The authors thank the Center of Analysis and Testing of Huazhong University of Science and Technology for SEM scanning. References 1. Zhao, Y., Wang, Y., Zhu, J. Y., Ragauskas, A., and Deng, Y.: Enhanced enzymatic hydrolysis of spruce by alkaline pretreatment at low temperature, Biotechnol. Bioeng., 99, 1320–1328 (2008). 2. Saha, B. C. and Cotta, M. A.: Enzymatic hydrolysis and fermentation of lime pretreated wheat straw to ethanol, J. Chem. Technol. Biotechnol., 82, 913–919 (2007). 3. Pu, Y., Zhang, D., Singh, P. M., and Ragauskas, A. J.: The new forestry biofuels sector, Biofuels, Bioprod. Biorefin., 2, 58–73 (2008). 4. Behera, B. K., Arora, M., and Sharma, D. K.: Scanning electorn microscopic (SEM) studies on structural architecture of lignocellulosic materials of calotropis procera during its processing for saccharification, Bioresour. Technol., 58, 241–245 (1996). 5. Sun, Y. and Cheng, J.: Hydrolysis of lignocellulosic materials for ethanol production: a review, Bioresour. Technol., 83, 1–11 (2002).
6. Mooney, C. A., Mansfield, S. D., Touhy, M. G., and Saddler, J. N.: The effect of initial pore volume and lignin content on the enzymatic hydrolysis of softwoods, Bioresour. Technol., 64, 113–119 (1998). 7. Carrillo, F., Lis, M. J., Colom, X., López-Mesas, M., and Valldeperas, J.: Effect of alkali pretreatment on cellulase hydrolysis of wheat straw: Kinetic study, Process Biochem., 40, 3360–3364 (2005). 8. Stenberg, K., Bollok, M., Reczey, K., Galbe, M., and Zacchi, G.: Effect of substrate and cellulase concentration on simultaneous saccharification and fermentation of steam-pretreated softwood for ethanol production, Biotechnol. Bioeng., 68, 204–210 (2000). 9. Teymouri, F., Laureano-Perez, L., Alizadeh, H., and Dale, B. E.: Optimization of the ammonia fiber explosion (AFEX) treatment parameters for enzymatic hydrolysis of corn stover, Bioresour. Technol., 96, 2014–2018 (2005). 10. Pan, X., Arato, C., Gilkes, N., Gregg, D., Mabee, W., Pye, K., Xiao, Z., Zhang, X., and Saddler, J.: Biorefining of softwoods using ethanol organosolv pulping: preliminary evaluation of process streams for manufacture of fuel-grade ethanol and coproducts, Biotechnol. Bioeng., 90, 473–481 (2005). 11. Lloyd, T. A. and Wyman, C. E.: Combined sugar yields for dilute sulfuric acid pretreatment of corn stover followed by enzymatic hydrolysis of the remaining solids, Bioresour. Technol., 96, 1967–1977 (2005). 12. Curreli, N., Fadda, M. B., Rescigno, A., Rinaldi, A. C., Soddu, G., Sollai, F., Vaccargiu, S., Sanjust, E., and Rinaldi, A.: Mild alkaline/oxidative pretreatment of wheat-straw, Process Biochem., 32, 665–670 (1997). 13. Hakala, T. K., Maijala, P., Konnb, J., and Hatakka, A.: Evaluation of novel woodrotting polypores and corticioid fungi for the decay and biopulping of Norway spruce (Picea abies) wood, Enzyme Microb. Technol., 34, 255–263 (2004). 14. Vares, T., Kalsi, M., and Hatakka, A.: Lignin peroxidases, manganese peroxidases, and other ligninolytic enzymes produced by phlebia radiata during solid-state fermentation of wheat straw, Appl. Environ. Microbiol., 61, 3515–3520 (1995). 15. Taniguchi, M., Suzuki, H., Watanabe, D., Sakai, K., Hoshino, K., and Tanaka, T.: Evaluation of pretreatment with Pleurotus ostreatus for enzymatic hydrolysis of rice straw, J. Biosci. Bioeng., 100, 637–643 (2005). 16. Zhang, X., Yu, H., Huang, H., and Liu, Y.: Evaluation of biological pretreatment with white rot fungi for the enzymatic hydrolysis of bamboo culms, Int. Biodeterior. Biodegrad., 60, 159–164 (2007). 17. Yang, B. and Wyman, C. E.: Pretreatment: the key to unlocking low-cost cellulosic ethanol, Biofuels, Bioprod. Biorefin., 2, 26–40 (2008). 18. Itoh, H., Wada, M., Honda, Y., Kuwahara, M., and Watanabe, T.: Bioorganosolve pretreatments for simultaneous saccharification and fermentation of beech wood by ethanolysis and white rot fungi, J. Biotechnol., 103, 273–280 (2003). 19. Mendonca, R., Guerra, A., and Ferraz, A.: Delignification of Pinus taeda wood chips treated with ceriporiopsis subvermispora for preparing high-yield kraft pulps, J. Chem. Technol. Biotechnol., 77, 411–418 (2002). 20. Horwitz, W.: Official methods of analysis of the Association of Official Analytical Chemists, Association of Official Analytical Chemists, Washington D.C. (1980). 21. Mooney, C. A., Mansfield, S. D., Beatson, R. P., and Saddler, J. N.: The effect of fiber characteristics on hydrolysis and cellulase accessibility to softwood substrates, Enzyme Microb. Technol., 25, 644–650 (1999). 22. Ohmine, K., Ooshima, H., and Harano, Y.: Kinetic study on enzymatic hydrolysis of cellulose by collulase from trichoderma viride, Biotechnol. Bioeng., XXV, 2041–2053 (1983). 23. Saddler, J. N.: Adsorption and activity profiles of cellulases during the hydrolysis of two Douglas fir pulps, Enzyme Microb. Technol., 24, 138–143 (1999). 24. Gregg, D. J. and Saddler, J. N.: Factors affecting cellulose hydrolysis and the potential of enzyme recycle to enhance the efficiency of an integrated wood to ethanol process, Biotechnol. Bioeng., 51, 375–383 (1996).