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Simultaneous saccharification and fermentation of alkaline-pretreated corn stover to ethanol using a recombinant yeast strain
Fuel Processing Technology 90 (2009) 1193–1197
Contents lists available at ScienceDirect
Fuel Processing Technology j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / f u p r o c
Simultaneous saccharification and fermentation of alkaline-pretreated corn stover to ethanol using a recombinant yeast strain Jing Zhao, Liming Xia ⁎ Department of Chemical and Biochemical Engineering, Zhejiang University, Hangzhou 310027, China
a r t i c l e
i n f o
Article history: Received 13 February 2009 Received in revised form 8 May 2009 Accepted 18 May 2009 Keywords: Simultaneous saccharification and fermentation Corn stover Ethanol Cellulose Hemicellulose Recombinant yeast
a b s t r a c t Bio-ethanol converted from cheap and abundant lignocellulosic materials is a potential renewable resource to replace depleting fossil fuels. Simultaneous saccharification and fermentation (SSF) of alkaline-pretreated corn stover for the production of ethanol was investigated using a recombinant yeast strain Saccharomyces cerevisiae ZU-10. Low cellobiase activity in Trichoderma reesei cellulase resulted in cellobiose accumulation. Supplementing the simultaneous saccharification and fermentation system with cellobiase greatly reduced feedback inhibition caused by cellobiose to the cellulase reaction, thereby increased the ethanol yield. 12 h of enzymatic prehydrolysis at 50 °C prior to simultaneous saccharification and fermentation was found to have a negative effect on the overall ethanol yield. Glucose and xylose produced from alkaline-pretreated corn stover could be co-fermented to ethanol effectively by S. cerevisiae ZU-10. An ethanol concentration of 27.8 g/ L and the corresponding ethanol yield on carbohydrate in substrate of 0.350 g/g were achieved within 72 h at 33 °C with 80 g/L of substrate and enzyme loadings of 20 filter paper activity units (FPU)/g substrate and 10 cellobiase units (CBU)/g substrate. The results are meaningful in co-conversion of cellulose and hemicellulose fraction of lignocellulosic materials to fuel ethanol. © 2009 Elsevier B.V. All rights reserved.
1. Introduction Recently, the use of bio-ethanol as a clean, safe alternative source of fuel has raised considerable interest due to the diminishing fossil fuel reserves and increased air pollution [1,2]. Currently industrial bioethanol is mainly produced from sugarcane or starch materials; however, the limited quantity of food stuff in China and their comparatively high prices greatly restrict large scale production of bio-ethanol. Lignocellulosic materials are cheap, abundant renewable resources and promising raw materials for ethanol production, yet, they are usually disposed or directly burned due to lack of effective utilization, thus often causing serious environmental pollution. Therefore, study on bio-ethanol production from lignocellulosic materials is of far reaching importance in both new energy resources development and environmental protection [3,4]. In the process of ethanol production from lignocellulosic materials, enzymatic hydrolysis and fermentation can be performed separately or simultaneously. Compared with separate hydrolysis and fermentation (SHF), simultaneous saccharification and fermentation (SSF) is more favored because in SSF glucose released by the action of cellulase is converted quickly to ethanol by the fermenting microorganism, thus minimizing end-product inhibition to cellulase caused by glucose and cellobiose accumulation. The improved saccharification rates and higher
⁎ Corresponding author. Tel./fax: +86 571 8795 1840. E-mail address: [email protected] (L. Xia). 0378-3820/$ – see front matter © 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.fuproc.2009.05.018
yield of ethanol are observed in SSF. Furthermore, the presence of ethanol in the culture broth helps to avoid undesired microbial contamination [5–7]. Lignocellulosic materials are the largest sources of hexose and pentose sugars. The conversion of hexose (mainly glucose) to ethanol has been the research focus for the past decades. However, exploitation of lignocellulosics can be enhanced by the efficient utilization of pentose alongside the hexose fraction. Xylose is the most abundant pentose sugar in hemicellulose (20–30% of dry weight of lignocellulosic biomass), and it is second only to glucose in natural abundance of all monosaccharides in lignocellulosic hydrolysate. Saccharomyces cerevisiae, which is one of the most prominent ethanol-producing microorganisms utilizing hexose, has been found unable to utilize xylose due to lack of the key enzymes in the xylose-metabolising pathway [8]. Thus, the efficient utilization of xylose in hemicellulose in addition to glucose in cellulose by a recombinant xylose-fermenting S. cerevisiae strain would offer an opportunity to reduce the production cost of bio-ethanol significantly [9,10]. In China, concentrated agricultural residue corn stover is produced annually. Corn stover contains high contents of cellulose (around 39% of dry weight) and hemicellulose (around 22% of dry weight); therefore, it is an ideal feedstock for the production of fuel ethanol. In this work, the SSF of alkaline-pretreated corn stover for ethanol production was investigated using a recombinant xylose-utilizing S. cerevisiae strain. The main objective of the article was to evaluate the feasibility of co-conversion of cellulose and hemicellulose to ethanol by a recombinant yeast strain using SSF.
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2. Materials and methods
2.6. Analytical methods
2.1. Microorganism
Cellulose was determined by HNO3–ethanol method, lignin by 72% H2SO4 method, and hemicellulose by two-brominating method [16]. Reducing sugars were estimated by the 3, 5-dinitrosalicylic acid (DNS) method [14]. Samples for analysis of glucose, xylose, cellobiose, arabinose and ethanol contents were centrifuged at 10,000 rpm for 10 min. The supernatant was filtered through 0.45 μm membrane filters and then analyzed on a HPLC system (Model 500, Syltech, USA) equipped with an organic acid column (IC Sep ICE-Coregel 87H3, Transgenomic, USA) and a refractive index detector (Model 6040 XR, Spectra-Physics, USA). 5 mM H2SO4 solution was used as the mobile phase at a flow rate of 0.5 mL/min. The column temperature was fixed at 60 °C. The ethanol yield was calculated as concentration of produced ethanol (in g/L) divided by carbohydrate (monosaccharides, i.e. the sum of glucose and xylose) concentration in the substrate (in g/L). The theoretical weight of glucose and xylose released during the hydrolysis is (due to the addition of water when the glycosidic bonds are hydrolysed) 1.11 times the weight of cellulose and 1.14 times the weight of hemicellulose, respectively. Three parallel samples were used in all analytical determinations, and data are presented as the mean of three replicates.
The xylose-utilizing yeast strain S. cerevisiae ZU-10 expresses xylose reductase (XR) and xylitol dehydrogenase (XDH) from the chromosomally integrated Pichia stipitis genes XYL1 and XYL2, respectively, and over-expresses the homologous XKS1 gene encoding xylulokinase (XK). It was maintained on YPX-agar slants containing 10 g/L yeast extract, 20 g/L peptone, 20 g/L xylose and 20 g/L agar [11]. 2.2. Lignocellulosic material and pretreatment Corn stover from local farms was milled to pass a 2.0 mm screen. The milled corn stover was washed thoroughly with tap water to remove sticky clay, then filtered and air-dried, and had the following chemical composition (dry weight basis): cellulose 38.7%, hemicellulose 21.7%, lignin 19.3% and others 20.3%. Corn stover sample was pretreated with 0.5 M NaOH solution at 80 °C for 75 min with a solid-to-liquid ratio of 1:8 (w/v). The solid cellulosic residues were collected and washed to neutral pH, filtered and stored for later use. The chemical composition of corn stover residue was as follows (dry weight basis): cellulose 64.1%, hemicellulose 24.6%, lignin 8.6% and others 2.7%.
3. Results and discussion
2.3. Enzymes
3.1. SSF process by S. cerevisiae ZU-10 with T. reesei ZU-02 cellulase
Cellulase and cellobiase were produced according to Xia and Cen [12] and Shen and Xia [13], respectively. Each gram of dry koji produced by Trichoderma reesei ZU-02 contained 146 filter paper activity units (FPU), 12 cellobiase units (CBU) and 1458 units of xylanase activity. Each gram of dry koji produced by Aspergillus niger ZU-07 contained 376 CBU and no detectable filter paper activity. Filter paper activity (FPA) and cellobiase activity (CBA) were measured according to the method recommended by Ghose [14] and expressed as international units (IU). One unit of filter paper activity (FPU) is the amount of enzyme that forms 1 µmol glucose (reducing sugar as glucose) per minute. One unit of cellobiase activity (CBU) is the amount of enzyme that forms 2 µmol glucose per minute from cellobiose. Xylanase activity was assayed using the Bailey method [15].
SSF process of alkaline-pretreated corn stover was performed with cellulase from T. reesei ZU-02 (20 FPU/g substrate) at 80 g/L of substrate (dry weight basis). As shown in Fig. 1, results indicated that glucose and xylose released from enzymatic hydrolysis can be cofermented to ethanol by recombinant S. cerevisiae ZU-10, yet with different consumption rates. Glucose produced was quickly converted to ethanol with no accumulation, while xylose was utilized slower than glucose. It was found that cellobiose increased gradually to 10.3 g/L within 72 h, indicating relatively low cellobiase activity in T. reesei cellulase. At the T. reesei cellulase dosage of 20 FPU/g substrate, only 1.64 CBU/g substrate of cellobiase was present in the SSF system. Accumulated cellobiose caused feedback inhibitory effect to enzymatic hydrolysis of cellulase, as the enzyme is more susceptible to end-product inhibition
2.4. Inoculum preparation For the inoculum preparation of recombinant S. cerevisiae ZU-10, a loopful of cells were transferred into each 250 mL E-flask with 50 mL of sterile culture medium containing 10 g/L glucose, 10 g/L xylose, 5 g/L peptone and 3 g/L yeast extract. The flasks were sealed with gauze and incubated in a rotary shaker at 30 °C and 180 rpm for 24 h. Cells were harvested by centrifugation (4800 rpm, 5 min), suspended in sterilized water and used as inoculum in the SSF process. 2.5. Simultaneous saccharification and fermentation (SSF) SSF experiments were performed in 250 mL E-flasks in a rotary shaker at 120 rpm and 33 °C. The flasks were sealed with rubber stoppers equipped with needles for CO2 venting. Each 100 mL of SSF reaction mixtures containing alkaline-pretreated corn stover 8 g (dry weight), peptone 0.5 g, yeast extract 0.3 g, CaCl2 0.025 g, MgCl2 0.025 g and KH2PO4 2.5 g was previously autoclaved at 110 °C for 20 min. After cooling down, the initial pH was adjusted to 5.0 with Ca(OH)2. SSF experiments were started by inoculation with 2 mL cell suspension corresponding to 1 g/L dry yeast and addition of cellulase koji (20 FPU/g substrate) and Tween 80 (0.5 g). Exceptions are pointed out in the text.
Fig. 1. Time course of SSF of alkaline-pretreated corn stover by S. cerevisiae ZU-10 with T. reesei cellulase (20 FPU/g substrate; 1.64 CBU/g substrate). (○) glucose; (●) xylose; (□) cellobiose; (◊) arabinose; (▲) ethanol.
J. Zhao, L. Xia / Fuel Processing Technology 90 (2009) 1193–1197
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(2 FPU:1 CBU) could effectively avoid cellobiose accumulation and improve SSF (Table 1). At 72 h, ethanol concentration and yield significantly increased with an increase in the enzyme loadings up to 20 FPU/g substrate, beyond which the increase leveled off. Therefore, the suitable enzyme loading for the SSF process was identified as 20 FPU/g substrate, i.e. an enzyme complex including cellulase of 20 FPU/g substrate and cellobiase of 10 CBU/g substrate. This enzyme loading was therefore chosen in the following experiments. 3.4. Effects of temperature
Fig. 2. Time course of SSF of alkaline-pretreated corn stover by S. cerevisiae ZU-10 with T. reesei cellulase and A. niger cellobiase (20 FPU/g substrate; 10 CBU/g substrate). (○) glucose; (●) xylose; (□) cellobiose; (◊) arabinose; (▲) ethanol.
The optimal temperature for enzymatic hydrolysis by cellulase complex is 45–50 °C; while for fermentation the optimal temperature is generally 30–35 °C. SSF experiments at three different temperatures were performed (Fig. 3). At 33 °C, sugars (mainly glucose and xylose) produced by enzymatic hydrolysis were quickly fermented to ethanol by recombinant S. cerevisiae ZU-10, the reducing sugar concentration was maintained at a low level and ethanol concentration increased steadily during the SSF process. At 36 °C, the vitality of S. cerevisiae ZU-10 was weakened. The production rate of reducing sugars from hydrolysis was higher than its consumption rate, thereby the reducing sugars accumulated continuously and the ethanol concentration
caused by cellobiose than glucose [17,18]. Meanwhile, cellobiose could not be utilized to produce ethanol by recombinant S. cerevisiae ZU-10, thus restricting the final ethanol concentration to 17.9 g/L at 72 h with the ethanol yield of 0.226 g/g. 3.2. Effects of cellobiase supplement To weaken the feedback inhibition caused by cellobiose accumulation, cellobiase produced by A. niger ZU-07 was supplemented to the SSF system to enhance the total cellobiase activity to 10 CBU/g substrate. The time course of SSF with T. reesei cellulase and A. niger cellobiase (20 FPU/g substrate; 10 CBU/g substrate) was shown in Fig. 2. Due to the improvement of cellobiase activity in the SSF system, the concentration of cellobiose was a relatively low level (within 1 g/L) throughout the SSF process without obvious accumulation. Therefore, feedback inhibition by cellobiose to cellulase was greatly reduced, resulting in more effective saccharification of cellulose and a higher ethanol concentration. At 72 h, the ethanol concentration was 27.8 g/L, with the ethanol yield of 0.350 g/g. This was greatly improved compared with SSF without cellobiase supplement. In the following SSF experiments, T. reesei cellulase was supplemented with A. niger cellobiase (2 FPU:1 CBU) to reduce feedback inhibition caused by cellobiose accumulation. 3.3. Effects of enzyme loadings Enzyme loading is considered as one of the most important factors in ethanol production of lignocellulosic materials [19]. Effects of enzyme loadings (presented as FPU/g substrate) on SSF was investigated. Synergetic hydrolysis by a more balanced cellulase complex
Table 1 Effects of enzyme loadings (presented as filter paper activity per gram of substrate, FPU/g substrate) on SSF of alkaline-pretreated corn stover by S. cerevisiae ZU-10. Enzyme loading (FPU/g substrate)
Glucose (g/L)
Xylose (g/L)
Arabinose (g/L)
Ethanol (g/L)
Ethanol yield (g/g)
5 10 15 20 25
ND ND ND ND ND
6.9 4.6 1.8 1.6 1.2
1.1 2.2 2.3 2.7 2.8
14.7 20.5 25.8 27.8 28.1
0.185 0.258 0.325 0.350 0.354
ND — not detected.
Fig. 3. SSF of alkaline-pretreated corn stover by S. cerevisiae ZU-10 at temperatures of 33 °C, 36 °C, and 40 °C, respectively. (A) reducing sugar concentration in SSF; (B) ethanol concentration in SSF. (♦) 33 °C; (●) 36 °C; (▲) 40 °C.
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leveled off from 24 h to 72 h. When the temperature reached 40 °C, the fermentation performance of S. cerevisiae ZU-10 was badly inhibited and ethanol production almost ceased. Only enzymatic hydrolysis of corn stover residue was carried out effectively. The results indicated that S. cerevisiae ZU-10 was not thermo-resistant and the suitable temperature for the SSF is 33 °C. This temperature was therefore chosen in the following experiments. 3.5. Effects of enzymatic prehydrolysis Enzymatic prehydrolysis prior to SSF may help to decrease the viscosity of the slurry for easier stirring and better mass and heat transfer, making the subsequent SSF possible even with high waterinsoluble substances and shortening SSF cycle. In this consideration, 12 h of prehydrolysis was performed at 50 °C with a higher substrate concentration of 120 g/L (dry weight basis) to investigate the effect of enzymatic prehydrolysis on SSF. Yeast cells were added afterwards to initiate SSF process. The concentration profiles of glucose, xylose, arabinose and ethanol in SSF with 12 h prehydrolysis compared with pure SSF were shown in Fig. 4. In the SSF process preceded by 12 h of prehydrolysis, ethanol concentration reached 21.2 g/L and 35.8 g/L at
12 h and 108 h respectively. The corresponding highest ethanol yield was 0.301 g/g. However, in pure SSF process, ethanol concentration was 16.5 g/L and 38.2 g/L at 12 h and 108 h respectively with the highest ethanol yield of 0.321 g/g. Enzymatic prehydrolysis increased the ethanol productivity during the initial 12 h in SSF process, but had a negative effect on the overall ethanol yield. Similar results were also obtained in SSF process at 80 g/L and 200 g/L of substrate content (dry weight basis) (data not shown). The reasons for the lower ethanol yield due to prehydrolysis were not clear now. Maybe sudden changes in the osmotic pressure in the surroundings caused yeast cells to make physiological changes and produced less ethanol in the experiments with prehydrolysis [20]. 4. Conclusions Both the glucose from cellulose and the xylose from hemicellulose in corn stover could be converted to ethanol in SSF process by a recombinant yeast strain. Synergetic enzymatic hydrolysis by cellulase from T. reesei ZU-02 and cellobiase from A. niger ZU-07 (20 FPIU/g substrate; 10 CBIU/g substrate) greatly reduced the feedback inhibition caused by cellobiose accumulation, thereby effectively improve SSF performance. Ethanol concentration reached 27.8 g/L in SSF at 33 °C within 72 h, with the ethanol yield of 0.350 g/g. Enzymatic prehydrolysis prior to SSF increased the ethanol productivity during the initial 12 h in SSF process, but had a negative effect on the overall ethanol yield. Acknowledgments Support from Hi-tech Research and Development Program of China (2007AA05Z401) and Major Project of Natural Science Foundation of Zhejiang Province (Z407010) is gratefully acknowledged. References
Fig. 4. Time course of SSF of alkaline-pretreated corn stover by S. cerevisiae ZU-10 at 120 g/L of substrate concentration. (A) 12 h of enzymatic prehydrolysis prior to SSF; (B) pure SSF. (○) glucose; (●) xylose; (◊) arabinose; (▲) ethanol.
[1] M. Moniruzzaman, B.S. Dien, C.D. Skory, Z.D. Chen, R.B. Hespell, N.W.Y. Ho, B.E. Dale, R.J. Bothast, Fermentation of corn fibre sugars by an engineered xylose utilizing Saccharomyces yeast strain, World J. Microbiol. Biotechnol. 13 (1997) 341–346. [2] S. Ye, Y.C. Jia, Hydrolysis of lignocellulosic materials for ethanol production: a review, Bioresour. Technol. 83 (2002) 1–11. [3] J. Lee, Biological conversion of lignocellulosic biomass to ethanol, J. Biotechnol. 56 (1997) 1–24. [4] N.W.Y. Ho, Z.D. Chen, A. Brainard, Genetically engineered Saccharomyces yeast capable of effective cofermentation of glucose and xylose, Appl. Environ. Microbiol. 64 (1998) 1852–1859. [5] E. Tomás-Pejó, J.M. Oliva, M. Ballesteros, L. Olsson, Comparison of SHF and SSF processes from steam-exploded wheat straw for ethanol production by xylosefermenting and robust glucose-fermenting Saccharomyces cerevisiae strains, Biotechnol. Bioeng. 100 (2008) 1122–1131. [6] K. Öhgren, R. Bura, G. Lesnicki, J. Saddler, G. Zacchi, A comparison between simultaneous saccharification and fermentation and separate hydrolysis and fermentation using steam-pretreated corn stover, Process Biochem. 42 (2007) 834–839. [7] C.E. Wyman, D.D. Spindler, K. Grohmann, Simultaneous saccharification and fermentation of several lignocellulosic feedstocks to fuel ethanol, Biomass Bioenergy 3 (1992) 301–307. [8] N.Q. Meinander, I. Boels, B. Hahn-Hägerdal, Fermentation of xylose/glucose mixtures by metabolically engineered Saccharomyces cerevisiae strains expressing XYL1 and XYL2 from Pichia stipitis with and without overexpression of TALl, Bioresour. Technol. 68 (1999) 79–87. [9] P. Chandrakant, V.S. Bisaria, Simultaneous bioconversion of glucose and xylose to ethanol by Saccharomyces cerevisiae in the presence of xylose isomerase, Appl. Microbiol. Biotechnol. 53 (2000) 301–309. [10] J. Zaldivar, J. Nielsen, L. Olsson, Fuel ethanol production from lignocellulose: a challenge for metabolic engineering and process integration, Appl. Microbiol. Biotechnol. 56 (2001) 17–34. [11] M. Chen, Z.F. Wang, L.M. Xia, Ethanol fermentation on xylose by immobilized recombinant yeast, J. Zhejiang University (Eng. Sci.) 42 (2) (2008) 290–293. [12] L.M. Xia, P.L. Cen, Cellulase production by solid state fermentation on lignocellulosic waste from the xylose industry, Process Biochem. 34 (1999) 909–912. [13] X.L. Shen, L.M. Xia, Production and immobilization of cellobiase from Aspergillus niger ZU-07, Process Biochem. 39 (2004) 1363–1367. [14] T.K. Ghose, Measurement of cellulase activities, Pure Appl. Chem. 59 (1987) 257–268. [15] M.J. Bailey, P. Biely, K. Poutanen, Interlaboratory testing of methods for assay of xylanase activity, J. Biotechnol. 23 (1992) 257–270.
J. Zhao, L. Xia / Fuel Processing Technology 90 (2009) 1193–1197 [16] S.C. Liu, Analysis and Measurement in Papermaking Industry, Chemical Industry Press, China, 2004, pp. 17–30. [17] S.J.B. Duff, W.D. Murray, Bioconversion of forest products industry waste cellulosics to fuel ethanol: a review, Bioresour. Technol. 55 (1996) 1–33. [18] Z.Y. Wen, W. Liao, S.L. Chen, Hydrolysis of animal manure lignocellulosics for reducing sugar production, Bioresour. Technol. 91 (2004) 31–39.
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[19] C.E. Wyman, Ethanol from lignocellulosic biomass: technology, economics, and opportunities, Bioresour. Technol. 50 (1994) 3–15. [20] K. Öhgren, J. Vehmaanperä, M. Siika-Aho, M. Galbe, L. Viikari, G. Zacchi, High temperature enzymatic prehydrolysis prior to simultaneous saccharification and fermentation of steam pretreated corn stover for ethanol production, Enzyme Microb. Technol. 40 (2007) 607–613.
Contents lists available at ScienceDirect
Fuel Processing Technology j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / f u p r o c
Simultaneous saccharification and fermentation of alkaline-pretreated corn stover to ethanol using a recombinant yeast strain Jing Zhao, Liming Xia ⁎ Department of Chemical and Biochemical Engineering, Zhejiang University, Hangzhou 310027, China
a r t i c l e
i n f o
Article history: Received 13 February 2009 Received in revised form 8 May 2009 Accepted 18 May 2009 Keywords: Simultaneous saccharification and fermentation Corn stover Ethanol Cellulose Hemicellulose Recombinant yeast
a b s t r a c t Bio-ethanol converted from cheap and abundant lignocellulosic materials is a potential renewable resource to replace depleting fossil fuels. Simultaneous saccharification and fermentation (SSF) of alkaline-pretreated corn stover for the production of ethanol was investigated using a recombinant yeast strain Saccharomyces cerevisiae ZU-10. Low cellobiase activity in Trichoderma reesei cellulase resulted in cellobiose accumulation. Supplementing the simultaneous saccharification and fermentation system with cellobiase greatly reduced feedback inhibition caused by cellobiose to the cellulase reaction, thereby increased the ethanol yield. 12 h of enzymatic prehydrolysis at 50 °C prior to simultaneous saccharification and fermentation was found to have a negative effect on the overall ethanol yield. Glucose and xylose produced from alkaline-pretreated corn stover could be co-fermented to ethanol effectively by S. cerevisiae ZU-10. An ethanol concentration of 27.8 g/ L and the corresponding ethanol yield on carbohydrate in substrate of 0.350 g/g were achieved within 72 h at 33 °C with 80 g/L of substrate and enzyme loadings of 20 filter paper activity units (FPU)/g substrate and 10 cellobiase units (CBU)/g substrate. The results are meaningful in co-conversion of cellulose and hemicellulose fraction of lignocellulosic materials to fuel ethanol. © 2009 Elsevier B.V. All rights reserved.
1. Introduction Recently, the use of bio-ethanol as a clean, safe alternative source of fuel has raised considerable interest due to the diminishing fossil fuel reserves and increased air pollution [1,2]. Currently industrial bioethanol is mainly produced from sugarcane or starch materials; however, the limited quantity of food stuff in China and their comparatively high prices greatly restrict large scale production of bio-ethanol. Lignocellulosic materials are cheap, abundant renewable resources and promising raw materials for ethanol production, yet, they are usually disposed or directly burned due to lack of effective utilization, thus often causing serious environmental pollution. Therefore, study on bio-ethanol production from lignocellulosic materials is of far reaching importance in both new energy resources development and environmental protection [3,4]. In the process of ethanol production from lignocellulosic materials, enzymatic hydrolysis and fermentation can be performed separately or simultaneously. Compared with separate hydrolysis and fermentation (SHF), simultaneous saccharification and fermentation (SSF) is more favored because in SSF glucose released by the action of cellulase is converted quickly to ethanol by the fermenting microorganism, thus minimizing end-product inhibition to cellulase caused by glucose and cellobiose accumulation. The improved saccharification rates and higher
⁎ Corresponding author. Tel./fax: +86 571 8795 1840. E-mail address: [email protected] (L. Xia). 0378-3820/$ – see front matter © 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.fuproc.2009.05.018
yield of ethanol are observed in SSF. Furthermore, the presence of ethanol in the culture broth helps to avoid undesired microbial contamination [5–7]. Lignocellulosic materials are the largest sources of hexose and pentose sugars. The conversion of hexose (mainly glucose) to ethanol has been the research focus for the past decades. However, exploitation of lignocellulosics can be enhanced by the efficient utilization of pentose alongside the hexose fraction. Xylose is the most abundant pentose sugar in hemicellulose (20–30% of dry weight of lignocellulosic biomass), and it is second only to glucose in natural abundance of all monosaccharides in lignocellulosic hydrolysate. Saccharomyces cerevisiae, which is one of the most prominent ethanol-producing microorganisms utilizing hexose, has been found unable to utilize xylose due to lack of the key enzymes in the xylose-metabolising pathway [8]. Thus, the efficient utilization of xylose in hemicellulose in addition to glucose in cellulose by a recombinant xylose-fermenting S. cerevisiae strain would offer an opportunity to reduce the production cost of bio-ethanol significantly [9,10]. In China, concentrated agricultural residue corn stover is produced annually. Corn stover contains high contents of cellulose (around 39% of dry weight) and hemicellulose (around 22% of dry weight); therefore, it is an ideal feedstock for the production of fuel ethanol. In this work, the SSF of alkaline-pretreated corn stover for ethanol production was investigated using a recombinant xylose-utilizing S. cerevisiae strain. The main objective of the article was to evaluate the feasibility of co-conversion of cellulose and hemicellulose to ethanol by a recombinant yeast strain using SSF.
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2. Materials and methods
2.6. Analytical methods
2.1. Microorganism
Cellulose was determined by HNO3–ethanol method, lignin by 72% H2SO4 method, and hemicellulose by two-brominating method [16]. Reducing sugars were estimated by the 3, 5-dinitrosalicylic acid (DNS) method [14]. Samples for analysis of glucose, xylose, cellobiose, arabinose and ethanol contents were centrifuged at 10,000 rpm for 10 min. The supernatant was filtered through 0.45 μm membrane filters and then analyzed on a HPLC system (Model 500, Syltech, USA) equipped with an organic acid column (IC Sep ICE-Coregel 87H3, Transgenomic, USA) and a refractive index detector (Model 6040 XR, Spectra-Physics, USA). 5 mM H2SO4 solution was used as the mobile phase at a flow rate of 0.5 mL/min. The column temperature was fixed at 60 °C. The ethanol yield was calculated as concentration of produced ethanol (in g/L) divided by carbohydrate (monosaccharides, i.e. the sum of glucose and xylose) concentration in the substrate (in g/L). The theoretical weight of glucose and xylose released during the hydrolysis is (due to the addition of water when the glycosidic bonds are hydrolysed) 1.11 times the weight of cellulose and 1.14 times the weight of hemicellulose, respectively. Three parallel samples were used in all analytical determinations, and data are presented as the mean of three replicates.
The xylose-utilizing yeast strain S. cerevisiae ZU-10 expresses xylose reductase (XR) and xylitol dehydrogenase (XDH) from the chromosomally integrated Pichia stipitis genes XYL1 and XYL2, respectively, and over-expresses the homologous XKS1 gene encoding xylulokinase (XK). It was maintained on YPX-agar slants containing 10 g/L yeast extract, 20 g/L peptone, 20 g/L xylose and 20 g/L agar [11]. 2.2. Lignocellulosic material and pretreatment Corn stover from local farms was milled to pass a 2.0 mm screen. The milled corn stover was washed thoroughly with tap water to remove sticky clay, then filtered and air-dried, and had the following chemical composition (dry weight basis): cellulose 38.7%, hemicellulose 21.7%, lignin 19.3% and others 20.3%. Corn stover sample was pretreated with 0.5 M NaOH solution at 80 °C for 75 min with a solid-to-liquid ratio of 1:8 (w/v). The solid cellulosic residues were collected and washed to neutral pH, filtered and stored for later use. The chemical composition of corn stover residue was as follows (dry weight basis): cellulose 64.1%, hemicellulose 24.6%, lignin 8.6% and others 2.7%.
3. Results and discussion
2.3. Enzymes
3.1. SSF process by S. cerevisiae ZU-10 with T. reesei ZU-02 cellulase
Cellulase and cellobiase were produced according to Xia and Cen [12] and Shen and Xia [13], respectively. Each gram of dry koji produced by Trichoderma reesei ZU-02 contained 146 filter paper activity units (FPU), 12 cellobiase units (CBU) and 1458 units of xylanase activity. Each gram of dry koji produced by Aspergillus niger ZU-07 contained 376 CBU and no detectable filter paper activity. Filter paper activity (FPA) and cellobiase activity (CBA) were measured according to the method recommended by Ghose [14] and expressed as international units (IU). One unit of filter paper activity (FPU) is the amount of enzyme that forms 1 µmol glucose (reducing sugar as glucose) per minute. One unit of cellobiase activity (CBU) is the amount of enzyme that forms 2 µmol glucose per minute from cellobiose. Xylanase activity was assayed using the Bailey method [15].
SSF process of alkaline-pretreated corn stover was performed with cellulase from T. reesei ZU-02 (20 FPU/g substrate) at 80 g/L of substrate (dry weight basis). As shown in Fig. 1, results indicated that glucose and xylose released from enzymatic hydrolysis can be cofermented to ethanol by recombinant S. cerevisiae ZU-10, yet with different consumption rates. Glucose produced was quickly converted to ethanol with no accumulation, while xylose was utilized slower than glucose. It was found that cellobiose increased gradually to 10.3 g/L within 72 h, indicating relatively low cellobiase activity in T. reesei cellulase. At the T. reesei cellulase dosage of 20 FPU/g substrate, only 1.64 CBU/g substrate of cellobiase was present in the SSF system. Accumulated cellobiose caused feedback inhibitory effect to enzymatic hydrolysis of cellulase, as the enzyme is more susceptible to end-product inhibition
2.4. Inoculum preparation For the inoculum preparation of recombinant S. cerevisiae ZU-10, a loopful of cells were transferred into each 250 mL E-flask with 50 mL of sterile culture medium containing 10 g/L glucose, 10 g/L xylose, 5 g/L peptone and 3 g/L yeast extract. The flasks were sealed with gauze and incubated in a rotary shaker at 30 °C and 180 rpm for 24 h. Cells were harvested by centrifugation (4800 rpm, 5 min), suspended in sterilized water and used as inoculum in the SSF process. 2.5. Simultaneous saccharification and fermentation (SSF) SSF experiments were performed in 250 mL E-flasks in a rotary shaker at 120 rpm and 33 °C. The flasks were sealed with rubber stoppers equipped with needles for CO2 venting. Each 100 mL of SSF reaction mixtures containing alkaline-pretreated corn stover 8 g (dry weight), peptone 0.5 g, yeast extract 0.3 g, CaCl2 0.025 g, MgCl2 0.025 g and KH2PO4 2.5 g was previously autoclaved at 110 °C for 20 min. After cooling down, the initial pH was adjusted to 5.0 with Ca(OH)2. SSF experiments were started by inoculation with 2 mL cell suspension corresponding to 1 g/L dry yeast and addition of cellulase koji (20 FPU/g substrate) and Tween 80 (0.5 g). Exceptions are pointed out in the text.
Fig. 1. Time course of SSF of alkaline-pretreated corn stover by S. cerevisiae ZU-10 with T. reesei cellulase (20 FPU/g substrate; 1.64 CBU/g substrate). (○) glucose; (●) xylose; (□) cellobiose; (◊) arabinose; (▲) ethanol.
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(2 FPU:1 CBU) could effectively avoid cellobiose accumulation and improve SSF (Table 1). At 72 h, ethanol concentration and yield significantly increased with an increase in the enzyme loadings up to 20 FPU/g substrate, beyond which the increase leveled off. Therefore, the suitable enzyme loading for the SSF process was identified as 20 FPU/g substrate, i.e. an enzyme complex including cellulase of 20 FPU/g substrate and cellobiase of 10 CBU/g substrate. This enzyme loading was therefore chosen in the following experiments. 3.4. Effects of temperature
Fig. 2. Time course of SSF of alkaline-pretreated corn stover by S. cerevisiae ZU-10 with T. reesei cellulase and A. niger cellobiase (20 FPU/g substrate; 10 CBU/g substrate). (○) glucose; (●) xylose; (□) cellobiose; (◊) arabinose; (▲) ethanol.
The optimal temperature for enzymatic hydrolysis by cellulase complex is 45–50 °C; while for fermentation the optimal temperature is generally 30–35 °C. SSF experiments at three different temperatures were performed (Fig. 3). At 33 °C, sugars (mainly glucose and xylose) produced by enzymatic hydrolysis were quickly fermented to ethanol by recombinant S. cerevisiae ZU-10, the reducing sugar concentration was maintained at a low level and ethanol concentration increased steadily during the SSF process. At 36 °C, the vitality of S. cerevisiae ZU-10 was weakened. The production rate of reducing sugars from hydrolysis was higher than its consumption rate, thereby the reducing sugars accumulated continuously and the ethanol concentration
caused by cellobiose than glucose [17,18]. Meanwhile, cellobiose could not be utilized to produce ethanol by recombinant S. cerevisiae ZU-10, thus restricting the final ethanol concentration to 17.9 g/L at 72 h with the ethanol yield of 0.226 g/g. 3.2. Effects of cellobiase supplement To weaken the feedback inhibition caused by cellobiose accumulation, cellobiase produced by A. niger ZU-07 was supplemented to the SSF system to enhance the total cellobiase activity to 10 CBU/g substrate. The time course of SSF with T. reesei cellulase and A. niger cellobiase (20 FPU/g substrate; 10 CBU/g substrate) was shown in Fig. 2. Due to the improvement of cellobiase activity in the SSF system, the concentration of cellobiose was a relatively low level (within 1 g/L) throughout the SSF process without obvious accumulation. Therefore, feedback inhibition by cellobiose to cellulase was greatly reduced, resulting in more effective saccharification of cellulose and a higher ethanol concentration. At 72 h, the ethanol concentration was 27.8 g/L, with the ethanol yield of 0.350 g/g. This was greatly improved compared with SSF without cellobiase supplement. In the following SSF experiments, T. reesei cellulase was supplemented with A. niger cellobiase (2 FPU:1 CBU) to reduce feedback inhibition caused by cellobiose accumulation. 3.3. Effects of enzyme loadings Enzyme loading is considered as one of the most important factors in ethanol production of lignocellulosic materials [19]. Effects of enzyme loadings (presented as FPU/g substrate) on SSF was investigated. Synergetic hydrolysis by a more balanced cellulase complex
Table 1 Effects of enzyme loadings (presented as filter paper activity per gram of substrate, FPU/g substrate) on SSF of alkaline-pretreated corn stover by S. cerevisiae ZU-10. Enzyme loading (FPU/g substrate)
Glucose (g/L)
Xylose (g/L)
Arabinose (g/L)
Ethanol (g/L)
Ethanol yield (g/g)
5 10 15 20 25
ND ND ND ND ND
6.9 4.6 1.8 1.6 1.2
1.1 2.2 2.3 2.7 2.8
14.7 20.5 25.8 27.8 28.1
0.185 0.258 0.325 0.350 0.354
ND — not detected.
Fig. 3. SSF of alkaline-pretreated corn stover by S. cerevisiae ZU-10 at temperatures of 33 °C, 36 °C, and 40 °C, respectively. (A) reducing sugar concentration in SSF; (B) ethanol concentration in SSF. (♦) 33 °C; (●) 36 °C; (▲) 40 °C.
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leveled off from 24 h to 72 h. When the temperature reached 40 °C, the fermentation performance of S. cerevisiae ZU-10 was badly inhibited and ethanol production almost ceased. Only enzymatic hydrolysis of corn stover residue was carried out effectively. The results indicated that S. cerevisiae ZU-10 was not thermo-resistant and the suitable temperature for the SSF is 33 °C. This temperature was therefore chosen in the following experiments. 3.5. Effects of enzymatic prehydrolysis Enzymatic prehydrolysis prior to SSF may help to decrease the viscosity of the slurry for easier stirring and better mass and heat transfer, making the subsequent SSF possible even with high waterinsoluble substances and shortening SSF cycle. In this consideration, 12 h of prehydrolysis was performed at 50 °C with a higher substrate concentration of 120 g/L (dry weight basis) to investigate the effect of enzymatic prehydrolysis on SSF. Yeast cells were added afterwards to initiate SSF process. The concentration profiles of glucose, xylose, arabinose and ethanol in SSF with 12 h prehydrolysis compared with pure SSF were shown in Fig. 4. In the SSF process preceded by 12 h of prehydrolysis, ethanol concentration reached 21.2 g/L and 35.8 g/L at
12 h and 108 h respectively. The corresponding highest ethanol yield was 0.301 g/g. However, in pure SSF process, ethanol concentration was 16.5 g/L and 38.2 g/L at 12 h and 108 h respectively with the highest ethanol yield of 0.321 g/g. Enzymatic prehydrolysis increased the ethanol productivity during the initial 12 h in SSF process, but had a negative effect on the overall ethanol yield. Similar results were also obtained in SSF process at 80 g/L and 200 g/L of substrate content (dry weight basis) (data not shown). The reasons for the lower ethanol yield due to prehydrolysis were not clear now. Maybe sudden changes in the osmotic pressure in the surroundings caused yeast cells to make physiological changes and produced less ethanol in the experiments with prehydrolysis [20]. 4. Conclusions Both the glucose from cellulose and the xylose from hemicellulose in corn stover could be converted to ethanol in SSF process by a recombinant yeast strain. Synergetic enzymatic hydrolysis by cellulase from T. reesei ZU-02 and cellobiase from A. niger ZU-07 (20 FPIU/g substrate; 10 CBIU/g substrate) greatly reduced the feedback inhibition caused by cellobiose accumulation, thereby effectively improve SSF performance. Ethanol concentration reached 27.8 g/L in SSF at 33 °C within 72 h, with the ethanol yield of 0.350 g/g. Enzymatic prehydrolysis prior to SSF increased the ethanol productivity during the initial 12 h in SSF process, but had a negative effect on the overall ethanol yield. Acknowledgments Support from Hi-tech Research and Development Program of China (2007AA05Z401) and Major Project of Natural Science Foundation of Zhejiang Province (Z407010) is gratefully acknowledged. References
Fig. 4. Time course of SSF of alkaline-pretreated corn stover by S. cerevisiae ZU-10 at 120 g/L of substrate concentration. (A) 12 h of enzymatic prehydrolysis prior to SSF; (B) pure SSF. (○) glucose; (●) xylose; (◊) arabinose; (▲) ethanol.
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