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Optimization of ethanol production from hot-water extracts of sugar maple chips
Renewable Energy 34 (2009) 2353–2356
Contents lists available at ScienceDirect
Renewable Energy journal homepage: www.elsevier.com/locate/renene
Optimization of ethanol production from hot-water extracts of sugar maple chips Jian Xu*, Shijie Liu Department of Paper and Bioprocess Engineering, College of Environmental Science and Forestry, State University of New York, 1 Forestry Drive, Syracuse, NY 13210, USA
a r t i c l e i n f o
a b s t r a c t
Article history: Received 11 June 2008 Accepted 18 March 2009 Available online 19 April 2009
Hot-water extracts from sugar maple chips prior to papermaking was employed in this study to produce ethanol by Pichia stipitis 58784. The effects of several factors, seed culture age, fermentation time, inoculum quantity, agitation rate, percent extract, concentration of inorganic nitrogen source (NH4)2SO4 and pH value, on ethanol production were investigated by orthogonal experiments. Orthogonal analysis shows that the optimal fermentation was obtained in the condition of 48-h seed culture, 120-h fermentation, 16% inoculum, 180 rpm, containing 30% extracts, 8% ammonium sulphate supplement and pH 5. This optimal condition was verified at 800-mL level in a 1.3 L fermentor. The ethanol yield reached 82.27% of the theoretical (20.57 g/L) after 120 h. Ó 2009 Elsevier Ltd. All rights reserved.
Keywords: Hot-water extracts Sugar maple chips Ethanol Pichia stipitis 58784 Orthogonal analysis
1. Introduction Compared with the finite supply of fossil fuels, bioethanol can play an important role in achieving sustainable development by reducing greenhouse gas emissions and providing new market opportunities for farmers [1]. Agricultural residuals (corn stover and wheat straw), grass and economical crops (willow) for bioethanol production have been intensively developed and made a rapid progress over the years [2–5]. However, most researches focus on cellulose utilization, ignoring the hemicellulose fraction, especially the residual hemicellulose in the papermaking process was generally underestimated. It was estimated that the US pulp and paper industry collects and processes approximately 108 million tons of wood every year, among which underutilized hemicellulose accounted for 10–20% (14 million tons) [6]. If utilized, this part of hemicellulose can contribute about 7 million tons of ethanol annually to bioethanol industry. The main composition of hemicellulose is xylose. Although most of the natural microorganisms could not metabolize xylose, there are several other naturally occurring and recombinant ones that can convert xylose to ethanol. Olsson and Hahn-Ha¨gerdal [7,8] presented a list of bacteria, yeasts and filamentous fungi that can produce ethanol from xylose. Among the xylose-fermenting microbes, Pichia stipitis can ferment xylose and xylan rapidly with a high ethanol yield. Moreover, P. stipitis has no absolute vitamin requirement for xylose fermentation [9,10].
* Corresponding author. Tel.: þ1 859 333 7425. E-mail address: [email protected] (J. Xu). 0960-1481/$ – see front matter Ó 2009 Elsevier Ltd. All rights reserved. doi:10.1016/j.renene.2009.03.025
In the present study, P. stipitis 58784 was used to ferment hotwater extracts, in which the main component is xylose, of sugar maple chips. The toxic tolerance of the strain to the extracts was evaluated first. Then, an orthogonal experiment was designed to optimize the ethanol fermentation condition, considering seed culture age, fermentation time, inoculum quantity, agitation rate, percent extract, concentration of inorganic nitrogen source, i.e., (NH4)2SO4 and pH value. 2. Materials and methods 2.1. Hot-water extract, and yeast and inoculum preparation Sugar maple wood chips was pretreated in a 65 ft3 pilot-scale batch reactor at a solid concentration of 1/4 (w/v), 165 C and approximate residence time of 2 h. The liquid fraction after pretreatment, hot-water extracts, was concentrated by member separation. Through analysis (see Section 2.4), the hot-water extract contained 24.84 g/L xylose and 4.40 g/L glucose. P. stipitis 58784 was grown at 28 C and maintained at 4 C in a medium containing 3 g/L yeast extract, 3 g/L malt extract, 5 g/L peptone, 10 g/L xylose and 20 g/L agar. Inoculum medium contained (g/L): KH2PO4, 10; (NH4)2SO4, 5; xylose, 50; yeast extract, 5; peptone, 3. The pH was adjusted to 5.0. 2.2. Toxic tolerance test of P. stipitis 58784 P. stipitis 58784 cells were grown in media with sequentially increasing concentrations (10, 20, 30, 40, 50, and 60%) of crude extract supplemented with extra xylose to bring the final
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J. Xu, S. Liu / Renewable Energy 34 (2009) 2353–2356
2.5
concentration to the same level of 50 g/L, with pH 5.0. At 0, 4, 8, 12, 24, 36, 48, and 72 h, samples with triplicate repetitions were measured in terms of optical density at 600 nm.
10% extract 20% extract 30% extract
2.0
40% extract
2.3. Orthogonal experiment of fermentations
50% extract
OD600
We designed the orthogonal experiment (Table 1) to involve seven factors: A (seed culture time, h), B (fermentation time, h), C (inoculum, %), D (agitation, rpm), E (sugar maple extract added into medium, %), F ((NH4)2SO4, g/L) and G (pH buffer, %), following symbol L18(37). Each factor was set at three levels. There were 18 experiments in total and all of them were performed in triplicate. They were performed in screw-capped Pyrex bottles. Samples were harvested at different times for ethanol and xylose determination. Optimal fermentation condition obtained from orthogonal experiments was further carried out in a 1.3 L BioFlo 110 fermentor containing 800 mL medium.
1.5
60% extract
1.0
0.5
0.0
0
20
40
60
80
Culture time (h) Fig. 1. Cell growth in medium containing different proportion of sugar maple extract.
2.4. Analysis Optical density of the yeast culture was measured at 600 nm before inoculating, using a spectrophotometer. The concentrations of xylose, glucose and ethanol were determined by nuclear magnetic resonance (NMR) spectroscopy methods described by Kiemle et al. [11]. 3. Results and discussion 3.1. Toxic tolerance of P. stipitis 58784 to hot-water extracts In the medium with 10–20% of hot-water extract, OD value, representing P. stipitis 58784 growth, sharply increased and reached about 2.0 at 36 h of cultivation and then tended to remain constant from 36 to 72 h (Fig. 1). Compared to cell growth in the medium containing 10% and 20% extract, there was a lag phase of about 8 h found in cell growth in the medium containing 30% extract. Accordingly, the time to get the highest OD value for cells growing in the medium containing 30% extract was postponed for about 12 h and the highest value decreased by 0.4 OD units. When the extract in the medium increased to 40%, the cells proliferation was still observed but became slow (OD ¼ 0.5). No cell growth was observed in the medium containing 50% and 60% extract. The parallel results were found when P. stipitis 58784 was transferred to slant medium containing 10–40% of hot-water extracts. P. stipitis
58784 grew well on slant containing 10–30% extract, while it did not grow on the slant containing 40% extract. The more the sugar maple extract was employed in the medium, the more inhibitors were introduced, and thus, the cell growth would be restrained to a larger extent. Although the best cell growth was obtained in medium containing 10% and 20% extract, sugar content will be too low to meet the industrial needs. P. stipitis 58784 could grow in the medium containing 40% extract, but the growth was restrained to a larger extent. Therefore, medium containing 30% extract was chosen in the subsequent studies, since it slightly decrease only 0.4 OD compared with medium containing 10–20% hot-water extracts, while 30% hot-water extracts might offer desirable sugar concentration.
3.2. Impacts of multi-factors on ethanol yield The ethanol yield, percentage of the actual yield to the theoretical ethanol yield, varied with combined factor treatments (Fig. 2). Fig. 2 shows the orthogonal experimental results of ethanol yield. The range analysis was applied to clarify the importance sequence of seed culture time (factor A), fermentation time
Table 1 Orthogonal experimental design L18(37) of influence of multi-factors on ethanol production from P. stipitis 58784. Experiment no.
A (seed culture time, h)
B (fermentation time, h)
C (inoculum, %)
D (agitation, rpm)
E (sugar maple extract, %)
F (NH4)2SO4, g/L)
G (pH buffer, %)
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18
24 24 24 36 36 36 48 48 48 24 24 24 36 36 36 48 48 48
72 96 120 72 96 120 72 96 120 72 96 120 72 96 120 72 96 120
8 12 16 8 12 16 12 16 8 16 8 12 12 16 8 16 8 12
0 150 180 150 180 0 0 150 180 180 0 150 180 0 150 150 180 0
0 15 30 15 30 0 30 0 15 15 30 0 0 15 30 30 0 15
3 5 8 8 3 5 5 8 3 5 8 3 8 3 5 3 5 8
0 6 10 10 0 6 10 0 6 0 6 10 10 10 0 6 10 0
J. Xu, S. Liu / Renewable Energy 34 (2009) 2353–2356
Ethanol production (% of theoretical)
60 50 40 30 20 10
100
60 ethanol sugar
80
40
60
30 40 20 20
10
0
0 0
0
1
2
3
4
5
6
7
8
20
40
60
80
100
120
140
Fermentation time (h)
9 10 11 12 13 14 15 16 17 18
Orthogonal experimental No.
50
Sugar concentration (g/L)
Ethanol yield (% of theoretical)
70
2355
Fig. 3. Ethanol production of P. stipitis 58784 under optimal fermentation condition.
Fig. 2. Ethanol production of different orthogonal experimental condition.
(factor B), inoculum (factor C), agitation rate (factor D), sugar maple extract ratio (factor E), (NH4)2SO4 (factor F) and pH control (factor G)in the orthogonal experiments (Table 2). The highest range (R) value of 9.29 was found on factor E and the lowest of 2.50 was on factor G. The bigger R value of a factor represents greater effect on the final ethanol yield. The highest ethanol of 15.91 g/L was found from experiment no. 3 with 30% hot-water extract added. Among all the experiments with 15% extract added, experiment no. 9 gave the highest ethanol concentration of 6.32 g/L. For the experiments without hot-water extract, the highest ethanol concentration of 7.29 g/L was obtained in experiment no. 8. According to the range, the order of influence is sugar maple extracts in medium > inoculum concentration > seed supplementaculture time > agitation rate > (NH4)2SO4 tion > fermentation time > pH value. The optimal condition for ethanol yield was determined as A3B3C3D3E3F3G3, i.e., extracts proportion in medium was 30%, inoculum of 16% (V/V), seed culture time of 48 h, agitation rate at 180 rpm, (NH4)2SO4 supplementation with 8 g/L, fermentation time of 120 h, 10% pH buffer added into the medium. Furthermore, the optimal condition A3B3C3D3E3F3G3, 48-h seed culture, 120-h fermentation, 16% inoculum, 180 rpm, containing 30% extracts, 8% ammonium sulphate supplement and pH 5, was carried out in a bigger-scale term, 1.3 L Bioflo 110 fermentor, to detect ethanol yield. The initial total sugar concentration was 50 g/L. As shown in Fig. 3, final ethanol yield and the residual sugar concentration were 82.27% (20.57 g/L) and 1.41 g/L at the end of fermentation (120 h), respectively. In this study, the fermentation medium contained 30% wood extracts, which contributed 15 g/L to the total sugar concentration. Compared with pure xylose fermentation (data not shown), the final ethanol yield had no
Table 2 The range analysis of L18(37) orthogonal experiments of ethanol yield. Factors
A
B
C
D
E
F
G
T1 T2 T3 x1 x2 x3 R Optimal
49.89 42.09 88.04 8.32 7.02 14.67 7.66 A3
52.50 49.29 78.24 8.75 8.22 13.04 4.82 B3
49.23 41.01 89.78 8.21 6.84 14.96 8.13 C3
39.69 68.51 71.82 6.62 11.42 11.97 5.35 D3
40.70 42.91 96.41 6.78 7.15 16.07 9.29 E3
61.46 44.23 74.33 10.24 7.37 12.39 5.02 F3
52.58 59.87 67.57 8.76 9.98 11.26 2.50 G3
distinct difference. But the fermentation time of medium containing wood extracts was shortened by 14 h, which was probably caused by more nutrition introduced by the extract. 4. Conclusions Bioethanol production from biomass xylose is promising as an alternative fuel. In order to obtain a higher yield of ethanol and fermentation rate in ethanol fermentation, the main parameters of ethanol fermentation by P. stipitis 58784 were investigated. Based on the analysis of orthogonal experiments and verification experiments, the optimal fermentation condition was determined as follows: extracts proportion 30%, inoculum 16%, seed culture time 48 h, agitation rate 180 rpm, (NH4)2SO4 supplementation 8 g/L, fermentation time 120 h, pH buffer 10%. This study showed preliminary information on ethanol production from hot-water extract of sugar maple chips. Trials will be made to optimize the hot-water extraction technology which produces less toxic compounds. It would be interesting to determine the trace element content in the extracts and to investigate what could accelerate the fermentation speed. Acknowledgements The authors are indebted to US Department of Energy for financial support. Special thanks are given for the generation and concentration of the wood extract by C.D. Wood and the NMR analysis by D. Kiemle. References [1] Demirbas AH, Demirbas I. Importance of rural bioenergy for developing countries. Energ Convers Manage 2007;48:2386–98. ¨ hgren K, Vehmaanpera¨ J, Siika-Aho M, Galbe M, Viikari L, Zacchi G. High [2] O temperature enzymatic prehydrolysis prior to simultaneous saccharification and fermentation of steam pretreated corn stover for ethanol production. Enzyme Microb Technol 2007;40:607–13. [3] Linde M, Jakobsson EL, Galbe M, Zacchi G. Steam pretreatment of dilute H2SO4-impregnated wheat straw and SSF with low yeast and enzyme loadings for bioethanol production. Biomass Bioenerg 2008;32:326–32. [4] Hurduc N, Teaci D, Serb anescu E, Hartia S. Potential for fuel production from crops. Energ Agric 1986;5:151–9. [5] Sassner P, Mårtensson CG, Galbe M, Zacchi G. Steam pretreatment of H2SO4impregnated salix for the production of bioethanol. Bioresource Technol 2008;99:137–45. [6] Liu S, Amidon TE, Francis RC, Ramarao BV, Lai YZ, Scott GM. From forest biomass to chemicals and energy: biorefinery initiative in New York. Ind Biotechnol 2006;2:113–20.
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J. Xu, S. Liu / Renewable Energy 34 (2009) 2353–2356
[7] Olsson L, Hahn-Ha¨gerdal B. Fermentative performance of bacteria and yeasts in lignocellulose hydrolysates. Process Biochem 1993;28:249–57. [8] Olsson L, Hahn-Ha¨gerdal B. Fermentation of lignocellulosic hydrolysates for ethanol production. Enzyme Microb Technol 1996;18:312–31. [9] Thomas WJ. Engineering yeasts for xylose metabolism. Curr Opin Biotechnol 2006;17:320–6.
[10] Du Preez JC, Bosch M, Prior BA. Xylose fermentation by Candida shehatae and Pichia stipitis: effects of pH, temperature and substrate concentration. Enzyme Microb Technol 1986;8:360–4. [11] Kiemle DJ, Stipanovic AJ, Mayo KE. Proton NMR methods in the compositional characterization of polysaccharides. ACS Symp Ser 2004;864: 122–39.
Contents lists available at ScienceDirect
Renewable Energy journal homepage: www.elsevier.com/locate/renene
Optimization of ethanol production from hot-water extracts of sugar maple chips Jian Xu*, Shijie Liu Department of Paper and Bioprocess Engineering, College of Environmental Science and Forestry, State University of New York, 1 Forestry Drive, Syracuse, NY 13210, USA
a r t i c l e i n f o
a b s t r a c t
Article history: Received 11 June 2008 Accepted 18 March 2009 Available online 19 April 2009
Hot-water extracts from sugar maple chips prior to papermaking was employed in this study to produce ethanol by Pichia stipitis 58784. The effects of several factors, seed culture age, fermentation time, inoculum quantity, agitation rate, percent extract, concentration of inorganic nitrogen source (NH4)2SO4 and pH value, on ethanol production were investigated by orthogonal experiments. Orthogonal analysis shows that the optimal fermentation was obtained in the condition of 48-h seed culture, 120-h fermentation, 16% inoculum, 180 rpm, containing 30% extracts, 8% ammonium sulphate supplement and pH 5. This optimal condition was verified at 800-mL level in a 1.3 L fermentor. The ethanol yield reached 82.27% of the theoretical (20.57 g/L) after 120 h. Ó 2009 Elsevier Ltd. All rights reserved.
Keywords: Hot-water extracts Sugar maple chips Ethanol Pichia stipitis 58784 Orthogonal analysis
1. Introduction Compared with the finite supply of fossil fuels, bioethanol can play an important role in achieving sustainable development by reducing greenhouse gas emissions and providing new market opportunities for farmers [1]. Agricultural residuals (corn stover and wheat straw), grass and economical crops (willow) for bioethanol production have been intensively developed and made a rapid progress over the years [2–5]. However, most researches focus on cellulose utilization, ignoring the hemicellulose fraction, especially the residual hemicellulose in the papermaking process was generally underestimated. It was estimated that the US pulp and paper industry collects and processes approximately 108 million tons of wood every year, among which underutilized hemicellulose accounted for 10–20% (14 million tons) [6]. If utilized, this part of hemicellulose can contribute about 7 million tons of ethanol annually to bioethanol industry. The main composition of hemicellulose is xylose. Although most of the natural microorganisms could not metabolize xylose, there are several other naturally occurring and recombinant ones that can convert xylose to ethanol. Olsson and Hahn-Ha¨gerdal [7,8] presented a list of bacteria, yeasts and filamentous fungi that can produce ethanol from xylose. Among the xylose-fermenting microbes, Pichia stipitis can ferment xylose and xylan rapidly with a high ethanol yield. Moreover, P. stipitis has no absolute vitamin requirement for xylose fermentation [9,10].
* Corresponding author. Tel.: þ1 859 333 7425. E-mail address: [email protected] (J. Xu). 0960-1481/$ – see front matter Ó 2009 Elsevier Ltd. All rights reserved. doi:10.1016/j.renene.2009.03.025
In the present study, P. stipitis 58784 was used to ferment hotwater extracts, in which the main component is xylose, of sugar maple chips. The toxic tolerance of the strain to the extracts was evaluated first. Then, an orthogonal experiment was designed to optimize the ethanol fermentation condition, considering seed culture age, fermentation time, inoculum quantity, agitation rate, percent extract, concentration of inorganic nitrogen source, i.e., (NH4)2SO4 and pH value. 2. Materials and methods 2.1. Hot-water extract, and yeast and inoculum preparation Sugar maple wood chips was pretreated in a 65 ft3 pilot-scale batch reactor at a solid concentration of 1/4 (w/v), 165 C and approximate residence time of 2 h. The liquid fraction after pretreatment, hot-water extracts, was concentrated by member separation. Through analysis (see Section 2.4), the hot-water extract contained 24.84 g/L xylose and 4.40 g/L glucose. P. stipitis 58784 was grown at 28 C and maintained at 4 C in a medium containing 3 g/L yeast extract, 3 g/L malt extract, 5 g/L peptone, 10 g/L xylose and 20 g/L agar. Inoculum medium contained (g/L): KH2PO4, 10; (NH4)2SO4, 5; xylose, 50; yeast extract, 5; peptone, 3. The pH was adjusted to 5.0. 2.2. Toxic tolerance test of P. stipitis 58784 P. stipitis 58784 cells were grown in media with sequentially increasing concentrations (10, 20, 30, 40, 50, and 60%) of crude extract supplemented with extra xylose to bring the final
2354
J. Xu, S. Liu / Renewable Energy 34 (2009) 2353–2356
2.5
concentration to the same level of 50 g/L, with pH 5.0. At 0, 4, 8, 12, 24, 36, 48, and 72 h, samples with triplicate repetitions were measured in terms of optical density at 600 nm.
10% extract 20% extract 30% extract
2.0
40% extract
2.3. Orthogonal experiment of fermentations
50% extract
OD600
We designed the orthogonal experiment (Table 1) to involve seven factors: A (seed culture time, h), B (fermentation time, h), C (inoculum, %), D (agitation, rpm), E (sugar maple extract added into medium, %), F ((NH4)2SO4, g/L) and G (pH buffer, %), following symbol L18(37). Each factor was set at three levels. There were 18 experiments in total and all of them were performed in triplicate. They were performed in screw-capped Pyrex bottles. Samples were harvested at different times for ethanol and xylose determination. Optimal fermentation condition obtained from orthogonal experiments was further carried out in a 1.3 L BioFlo 110 fermentor containing 800 mL medium.
1.5
60% extract
1.0
0.5
0.0
0
20
40
60
80
Culture time (h) Fig. 1. Cell growth in medium containing different proportion of sugar maple extract.
2.4. Analysis Optical density of the yeast culture was measured at 600 nm before inoculating, using a spectrophotometer. The concentrations of xylose, glucose and ethanol were determined by nuclear magnetic resonance (NMR) spectroscopy methods described by Kiemle et al. [11]. 3. Results and discussion 3.1. Toxic tolerance of P. stipitis 58784 to hot-water extracts In the medium with 10–20% of hot-water extract, OD value, representing P. stipitis 58784 growth, sharply increased and reached about 2.0 at 36 h of cultivation and then tended to remain constant from 36 to 72 h (Fig. 1). Compared to cell growth in the medium containing 10% and 20% extract, there was a lag phase of about 8 h found in cell growth in the medium containing 30% extract. Accordingly, the time to get the highest OD value for cells growing in the medium containing 30% extract was postponed for about 12 h and the highest value decreased by 0.4 OD units. When the extract in the medium increased to 40%, the cells proliferation was still observed but became slow (OD ¼ 0.5). No cell growth was observed in the medium containing 50% and 60% extract. The parallel results were found when P. stipitis 58784 was transferred to slant medium containing 10–40% of hot-water extracts. P. stipitis
58784 grew well on slant containing 10–30% extract, while it did not grow on the slant containing 40% extract. The more the sugar maple extract was employed in the medium, the more inhibitors were introduced, and thus, the cell growth would be restrained to a larger extent. Although the best cell growth was obtained in medium containing 10% and 20% extract, sugar content will be too low to meet the industrial needs. P. stipitis 58784 could grow in the medium containing 40% extract, but the growth was restrained to a larger extent. Therefore, medium containing 30% extract was chosen in the subsequent studies, since it slightly decrease only 0.4 OD compared with medium containing 10–20% hot-water extracts, while 30% hot-water extracts might offer desirable sugar concentration.
3.2. Impacts of multi-factors on ethanol yield The ethanol yield, percentage of the actual yield to the theoretical ethanol yield, varied with combined factor treatments (Fig. 2). Fig. 2 shows the orthogonal experimental results of ethanol yield. The range analysis was applied to clarify the importance sequence of seed culture time (factor A), fermentation time
Table 1 Orthogonal experimental design L18(37) of influence of multi-factors on ethanol production from P. stipitis 58784. Experiment no.
A (seed culture time, h)
B (fermentation time, h)
C (inoculum, %)
D (agitation, rpm)
E (sugar maple extract, %)
F (NH4)2SO4, g/L)
G (pH buffer, %)
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18
24 24 24 36 36 36 48 48 48 24 24 24 36 36 36 48 48 48
72 96 120 72 96 120 72 96 120 72 96 120 72 96 120 72 96 120
8 12 16 8 12 16 12 16 8 16 8 12 12 16 8 16 8 12
0 150 180 150 180 0 0 150 180 180 0 150 180 0 150 150 180 0
0 15 30 15 30 0 30 0 15 15 30 0 0 15 30 30 0 15
3 5 8 8 3 5 5 8 3 5 8 3 8 3 5 3 5 8
0 6 10 10 0 6 10 0 6 0 6 10 10 10 0 6 10 0
J. Xu, S. Liu / Renewable Energy 34 (2009) 2353–2356
Ethanol production (% of theoretical)
60 50 40 30 20 10
100
60 ethanol sugar
80
40
60
30 40 20 20
10
0
0 0
0
1
2
3
4
5
6
7
8
20
40
60
80
100
120
140
Fermentation time (h)
9 10 11 12 13 14 15 16 17 18
Orthogonal experimental No.
50
Sugar concentration (g/L)
Ethanol yield (% of theoretical)
70
2355
Fig. 3. Ethanol production of P. stipitis 58784 under optimal fermentation condition.
Fig. 2. Ethanol production of different orthogonal experimental condition.
(factor B), inoculum (factor C), agitation rate (factor D), sugar maple extract ratio (factor E), (NH4)2SO4 (factor F) and pH control (factor G)in the orthogonal experiments (Table 2). The highest range (R) value of 9.29 was found on factor E and the lowest of 2.50 was on factor G. The bigger R value of a factor represents greater effect on the final ethanol yield. The highest ethanol of 15.91 g/L was found from experiment no. 3 with 30% hot-water extract added. Among all the experiments with 15% extract added, experiment no. 9 gave the highest ethanol concentration of 6.32 g/L. For the experiments without hot-water extract, the highest ethanol concentration of 7.29 g/L was obtained in experiment no. 8. According to the range, the order of influence is sugar maple extracts in medium > inoculum concentration > seed supplementaculture time > agitation rate > (NH4)2SO4 tion > fermentation time > pH value. The optimal condition for ethanol yield was determined as A3B3C3D3E3F3G3, i.e., extracts proportion in medium was 30%, inoculum of 16% (V/V), seed culture time of 48 h, agitation rate at 180 rpm, (NH4)2SO4 supplementation with 8 g/L, fermentation time of 120 h, 10% pH buffer added into the medium. Furthermore, the optimal condition A3B3C3D3E3F3G3, 48-h seed culture, 120-h fermentation, 16% inoculum, 180 rpm, containing 30% extracts, 8% ammonium sulphate supplement and pH 5, was carried out in a bigger-scale term, 1.3 L Bioflo 110 fermentor, to detect ethanol yield. The initial total sugar concentration was 50 g/L. As shown in Fig. 3, final ethanol yield and the residual sugar concentration were 82.27% (20.57 g/L) and 1.41 g/L at the end of fermentation (120 h), respectively. In this study, the fermentation medium contained 30% wood extracts, which contributed 15 g/L to the total sugar concentration. Compared with pure xylose fermentation (data not shown), the final ethanol yield had no
Table 2 The range analysis of L18(37) orthogonal experiments of ethanol yield. Factors
A
B
C
D
E
F
G
T1 T2 T3 x1 x2 x3 R Optimal
49.89 42.09 88.04 8.32 7.02 14.67 7.66 A3
52.50 49.29 78.24 8.75 8.22 13.04 4.82 B3
49.23 41.01 89.78 8.21 6.84 14.96 8.13 C3
39.69 68.51 71.82 6.62 11.42 11.97 5.35 D3
40.70 42.91 96.41 6.78 7.15 16.07 9.29 E3
61.46 44.23 74.33 10.24 7.37 12.39 5.02 F3
52.58 59.87 67.57 8.76 9.98 11.26 2.50 G3
distinct difference. But the fermentation time of medium containing wood extracts was shortened by 14 h, which was probably caused by more nutrition introduced by the extract. 4. Conclusions Bioethanol production from biomass xylose is promising as an alternative fuel. In order to obtain a higher yield of ethanol and fermentation rate in ethanol fermentation, the main parameters of ethanol fermentation by P. stipitis 58784 were investigated. Based on the analysis of orthogonal experiments and verification experiments, the optimal fermentation condition was determined as follows: extracts proportion 30%, inoculum 16%, seed culture time 48 h, agitation rate 180 rpm, (NH4)2SO4 supplementation 8 g/L, fermentation time 120 h, pH buffer 10%. This study showed preliminary information on ethanol production from hot-water extract of sugar maple chips. Trials will be made to optimize the hot-water extraction technology which produces less toxic compounds. It would be interesting to determine the trace element content in the extracts and to investigate what could accelerate the fermentation speed. Acknowledgements The authors are indebted to US Department of Energy for financial support. Special thanks are given for the generation and concentration of the wood extract by C.D. Wood and the NMR analysis by D. Kiemle. References [1] Demirbas AH, Demirbas I. Importance of rural bioenergy for developing countries. Energ Convers Manage 2007;48:2386–98. ¨ hgren K, Vehmaanpera¨ J, Siika-Aho M, Galbe M, Viikari L, Zacchi G. High [2] O temperature enzymatic prehydrolysis prior to simultaneous saccharification and fermentation of steam pretreated corn stover for ethanol production. Enzyme Microb Technol 2007;40:607–13. [3] Linde M, Jakobsson EL, Galbe M, Zacchi G. Steam pretreatment of dilute H2SO4-impregnated wheat straw and SSF with low yeast and enzyme loadings for bioethanol production. Biomass Bioenerg 2008;32:326–32. [4] Hurduc N, Teaci D, Serb anescu E, Hartia S. Potential for fuel production from crops. Energ Agric 1986;5:151–9. [5] Sassner P, Mårtensson CG, Galbe M, Zacchi G. Steam pretreatment of H2SO4impregnated salix for the production of bioethanol. Bioresource Technol 2008;99:137–45. [6] Liu S, Amidon TE, Francis RC, Ramarao BV, Lai YZ, Scott GM. From forest biomass to chemicals and energy: biorefinery initiative in New York. Ind Biotechnol 2006;2:113–20.
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J. Xu, S. Liu / Renewable Energy 34 (2009) 2353–2356
[7] Olsson L, Hahn-Ha¨gerdal B. Fermentative performance of bacteria and yeasts in lignocellulose hydrolysates. Process Biochem 1993;28:249–57. [8] Olsson L, Hahn-Ha¨gerdal B. Fermentation of lignocellulosic hydrolysates for ethanol production. Enzyme Microb Technol 1996;18:312–31. [9] Thomas WJ. Engineering yeasts for xylose metabolism. Curr Opin Biotechnol 2006;17:320–6.
[10] Du Preez JC, Bosch M, Prior BA. Xylose fermentation by Candida shehatae and Pichia stipitis: effects of pH, temperature and substrate concentration. Enzyme Microb Technol 1986;8:360–4. [11] Kiemle DJ, Stipanovic AJ, Mayo KE. Proton NMR methods in the compositional characterization of polysaccharides. ACS Symp Ser 2004;864: 122–39.