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Combination of biological pretreatment with mild acid pretreatment for enzymatic hydrolysis and ethanol production from water hyacinth
Bioresource Technology 101 (2010) 9600–9604
Contents lists available at ScienceDirect
Bioresource Technology journal homepage: www.elsevier.com/locate/biortech
Combination of biological pretreatment with mild acid pretreatment for enzymatic hydrolysis and ethanol production from water hyacinth Fuying Ma, Na Yang, Chunyan Xu, Hongbo Yu, Jianguo Wu, Xiaoyu Zhang * Key Laboratory of Molecular Biophysics of MOE, College of Life Science and Technology, Huazhong University of Science and Technology, Wuhan 430074, PR China
a r t i c l e
i n f o
Article history: Received 4 May 2010 Received in revised form 16 July 2010 Accepted 20 July 2010 Available online 24 July 2010 Keywords: Water hyacinth Biological pretreatment Mild acid pretreatment Saccharification Ethanol fermentation
a b s t r a c t The mild acid pretreatment and the combination of biological pretreatment by a white rot fungus Echinodontium taxodii or a brown rot fungus Antrodia sp. 5898 with mild acid pretreatment were evaluated under different pretreatment conditions for enzymatic hydrolysis and ethanol production from water hyacinth. The combined pretreatment with E. taxodii (10 days) and 0.25% H2SO4 was proved to be more effective than the sole acid pretreatment. The reducing sugar yield from enzymatic hydrolysis of co-treated water hyacinth increased 1.13–2.11 fold than that of acid-treated water hyacinth at the same conditions. The following study on separate hydrolysis and fermentation with Saccharomyces cerevisiae indicated that the ethanol yield from co-treated water hyacinth achieved 0.192 g/g of dry matter, which increased 1.34-fold than that from acid-treated water hyacinth (0.146 g/g of dry matter). This suggested that the combination of biological and mild acid pretreatment is a promising method to improve enzymatic hydrolysis and ethanol production from water hyacinth with low lignin content. Ó 2010 Elsevier Ltd. All rights reserved.
1. Introduction Water hyacinth (Eichhornia crassipes) was first introduced from South America into China in 1901 and widely planted as pig feedstuff in the 1950s (Xie et al., 2001; Chu et al., 2006). Currently, it is regarded as a nuisance which has seriously impaired the biological diversity and ecological environment because of its extraordinary adaptive ability and remarkable growth rate (Center et al., 1999; Lu et al., 2007; Malik, 2007). However, some studies indicated that water hyacinth is a promising plant for production of fuel ethanol, biogas and other valuable products because it grows quickly and contains high amounts of hemicellulose and cellulose (Nigam, 2002; Isarankura-Na-Ayudhya et al., 2007; Carina and Cecilia, 2007; Malik, 2007; Hronich et al., 2008; Kumar et al., 2009a; Almoustapha et al., 2009; Bhattacharya and Kumar, 2010; Klass and Ghosh, 1981). Fuel ethanol is convenient to be used as a gasoline additive to increase octane and improve vehicle emissions. Fuel ethanol production from biomass involves enzymatic hydrolysis and fermentation. Pretreatment is an effective way to increase the hydrolysis efficiency, because it can break down the complex structure of biomass and increase the accessibility of cellulase protein to cellulose substrate (Mosier et al., 2005; Kumar et al., 2009b).
* Corresponding author. Tel./fax: +86 27 87792128. E-mail address: [email protected] (X. Zhang). 0960-8524/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.biortech.2010.07.084
The acid pretreatments are effective methods used for water hyacinth to ethanol for dissolving hemicellulose and retaining most of the cellulose (Nigam, 2002; Kumar et al., 2009a). Though the conventional acid pretreatments are energy-intensive and environment-unfriendly, the concentrated acid or the dilute acid at high temperature and pressure is usually applied (Sun and Cheng, 2002; Mishima et al., 2006, 2008; Masami et al., 2008). In addition, trials done on water hyacinth acid or enzymatic hydrolysis indicated that the reducing sugar yield was higher with saccharification (Abraham and Kurup, 1996). However, most investigators neglected the residue of acid hydrolysis. Biological pretreatment with fungus has attracted much attention for disrupting the lignin-hemicellulose sheath, requiring relatively low energy and mild environmental conditions (Zhang et al., 2007a,b; Yu et al., 2009a). However, the economic feasibility of fungal pretreatment is poor, because the loss in polysaccharide components during fungal growth and the long cultivation period reduce overall productivity. In order to overcome these disadvantages, the combinations of biological and mild chemical pretreatment were used to enhance the saccharification ratio of rice hull and cornstalks (Yu et al., 2009b, 2010). The white rot fungus Echinodontium taxodii can selectively degrade lignin of woody and non-woody lignocellulose to enhance the enzymatic hydrolysis (Zhang et al., 2007b; Yu et al., 2009a). But, there is no report about biological pretreatment of water hyacinth with low lignin content. Thus, the influences of the sole acid pretreatment and the combination of fungal pretreatment (white
F. Ma et al. / Bioresource Technology 101 (2010) 9600–9604
rot fungus E. taxodii and brown rot fungus Antrodia sp. 5898) with mild acid pretreatment on reducing sugar yield and ethanol production using Saccharomyces cerevisiae from water hyacinth were investigated in this study. 2. Methods 2.1. Microorganisms and cultivation The white rot fungus E. taxodii and brown rot fungus Antrodia sp. 5898 were isolated from Shennongjia Nature Reserve (Hubei, China). The organisms were maintained on potato dextrose agar (PDA) slants at 4 °C. A plug of the two fungi activated for 5–6 days on PDA slants at 28 °C was inoculated onto the center of 9 cm PDA plates and incubated for one week at 28 °C. Thermal-tolerant alcohol active dry yeast S. cerevisiae, provided by Angel Yeast Co. Ltd., was firstly rehydrated in a starter solution (5% dry yeast added to 2% glucose solution) before ‘‘pitching”. To ensure the quality of the culture and to get a vigorous start, the yeast was activated at 35–42 °C for 30 min and then again at 34 °C for 1.5–2 h to get small bubbles. 2.2. Raw material Fresh water hyacinth (E. crassipes) was collected and washed to remove adhering dirt, chopped into small pieces (about 1 cm in length) and air-dried. The samples were ground, and the particles between 0.45 mm and 0.9 mm were prepared for the following pretreatment with basidiomycetes and chemicals. 2.3. Biological pretreatment of water hyacinth The biological pretreatments with E. taxodii and Antrodia sp. 5898 were carried out in a 250-ml Erlenmeyer flask with 5 g of air-dried water hyacinth and 12 ml of distilled water. The samples were sterilized in the autoclave for 30 min at 121 °C and inoculated with five plugs cut from the fungal mat on the PDA plates. Biological pretreatments were conducted statically at 28 °C for 10 days, and then the treated samples were dried at 60 °C for 3 days. The non-inoculated sample was used as the control. All experiments were performed in triplicate. 2.4. Dilute acid pretreatment of water hyacinth The raw and bio-pretreated water hyacinth with E. taxodii or Antrodia sp. 5898 were further treated with 0.25% sulfuric acid at varied temperature (25, 80, and 100 °C) for 15–60 min, and then filtrated. The reducing sugar in filtrate was measured after neutralization with CaCl2. The residues were washed to neutralize with distilled water, and then dried at 60 °C for 3 days and ground to be used for enzymatic hydrolysis and saccharification. 2.5. Separate hydrolysis and fermentation (SHF) Commercial cellulase preparation, produced by Trichoderma reesei, was provided by Henan Tianguan Enterprise Group Co., Ltd. Enzymatic hydrolysis was carried out at 2% substrate concentration in 50 mM sodium acetate buffer (pH 4.8) with cellulase (30 FPU/g of substrate) at 50 °C for 48 h. After filtration, the enzymatic hydrolysate was used for reducing sugar measurement and ethanol fermentation. To make fermentation medium, the enzymatic hydrolysate concentrated to 2% (w/v) of reducing sugar, was supplemented with nutrients (g/L): peptone, 5; yeast extract, 5; KH2PO4, 1; MgSO47H2O, 0.3; NH4Cl, 2. Ethanol fermentation was conducted
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in a 250-ml Erlenmeyer flask with 100 ml fermentation medium (pH 4). Activated yeast (0.3% (v/v)) was inoculated into the fermentation medium. The flasks were incubated at 40 °C for 8 h shaking at 100 rpm and then again at 40 °C for 72 h statically. All experiments were performed in triplicate. 2.6. Analytical methods 2.6.1. Chemical components analysis The content of neutral detergent fiber (NDF), acid detergent fiber (ADF), acid detergent lignin (ADL) and ash were determined according to the procedures of Goering and Van Soest (1971). Hemicellulose content was estimated as the difference between NDF and ADF, and cellulose content was calculated based on the difference between ADF and ADL. 2.6.2. Reducing sugar estimation Total reducing sugar was measured with the DNS (3,5-dinitrosalicylic acid) method (Behera et al., 1996). The amount of reducing sugar was calculated as follows: Reducing sugar yield from enzymatic hydrolysisðmg=g dry water hyacinthÞ ¼
amount of reducing sugar produced after enzymatic hydrolysis amount of dry water hyacinth
2.6.3. Determination of ethanol concentration The ethanol concentration was determined by a high performance liquid chromatography (HPLC) system (Agilent 1200, China) using sugar-pak-1 column (Waters, China) and the refractive index (RI) detector (Agilent G1362A, China). The mobile phase was 50 mg/L CaNa-EDTA solution at a flow rate of 0.6 ml/min with the column temperature of 75 °C. 3. Results and discussion 3.1. Chemical components of water hyacinth The average lignocellulosic composition of water hyacinth is (as total percentage of solids): cellulose: 18.2 ± 0.6, hemicellulose: 29.3 ± 0.5, lignin: 2.8 ± 0.2, ash: 1.2 ± 0.2. The cellulose content is in accordance with the data reported by other investigators, but the hemicellulose and lignin content are lower than their results (Klass and Ghosh, 1981; Nigam, 2002; Kumar et al., 2009a). These differences might originate from different sources (Carina and Cecilia, 2007) and different growth state of water hyacinth. 3.2. Enzymatic hydrolysis of water hyacinth The H2SO4 pretreatment effectively improved the enzymatic hydrolysis of water hyacinth. The reducing sugar yield was slightly increased by increasing the H2SO4 concentration from 0.25% to 1% (data not shown). Thus, 0.25% H2SO4 treatment was used in this study. The reducing sugar yield from enzymatic hydrolysis of acid-treated sample decreased as pretreatment time increased at room temperature (25 °C). However, at high temperature (80 and 100 °C), the reducing sugar yield increased with the increase in pretreatment time (Fig. 1A). After 10 days of biological pretreatment with E. taxodii or Antrodia sp. 5898, the bio-pretreated sample and raw material were further treated with 0.25% H2SO4 at 25, 80, 100 °C for 15–60 min, respectively. The residue of acid hydrolysis was subjected to enzymatic hydrolysis and the reducing sugar yield might be seen in Fig. 1. The reducing sugar yield of water hyacinth co-treated with E. taxodii and mild acid (from 208.8 to 366.0 mg/g dry matter) were higher than that of acid-treated (from 112.9 to 322.3 mg/g dry
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digestibility after the combination pretreatment can be attributed to the synergistic effect of biological and acid treatment. Especially, at room temperature (25 °C), the reducing sugar yield was enhanced 1.79-, 1.90- and 2.11-fold for 60-, 15- and 30-min treatment, respectively. The result suggested that the synergy of combination pretreatment was greater at moderate condition. The reason might be the white rot fungus E. taxodii degraded and modified lignin in water hyacinth, which made the accessibility of cellulase to cellulose easier. But the reducing sugar yield of water hyacinth pretreated with Antrodia sp. 5898 (Fig. 1A and C) was lower than that of control sample. Antrodia sp. 5898, as a brown rot fungus, metabolized the carbohydrates including cellulose and hemicellulose of biomass without removing lignin, producing sugar-free brown residue (Eriksson et al., 1990). On the other hand, the polysaccharide losses caused by Antrodia sp. 5898 might be significantly more than that caused by the white rot fungus E. taxodii. Furthermore, the concentrated lignin brought about more difficulties for cellulase to access cellulose due to carbohydrates degradation. Therefore, the reducing sugar yield from the combined pretreatment with Antrodia sp. 5898 was less than that from the acid or combination pretreatment with E. taxodii. These results suggested that lignin was a major hindrance to enzymatic hydrolysis of water hyacinth.
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The dilute acid pretreatment is primarily a hydrolytic process which causes solubilization of hemicellulosic sugars (Torget et al., 1990). In this work, the reducing sugar of acid hydrolysate was called the reducing sugar loss. At room temperature (25 °C), the reducing sugar loss of control sample had little change with the increasing of acid pretreatment time. At high temperature (80 and 100 °C), the reducing sugar loss increased as acid pretreatment time increased (Fig. 2A). The amount of reducing sugar of acid hydrolyate at mild conditions (72.8–114.7 mg/g dry matter) was relatively low, compared with high acid concentration or high reaction temperature (Nigam, 2002; Isarankura-Na-Ayudhya et al., 2007; Kumar et al., 2009a). These results might be caused by low hemicellulose content of raw material or incomplete hydrolysis of hemicellulose under mild reaction conditions. In fact, high temperature (121–190 °C) and high pressure were usually employed in dilute acid pretreatment to get sufficient hydrolysis of hemicellulose (Isarankura-Na-Ayudhya et al., 2007; Masami et al., 2008; Sun and Cheng, 2002). The reducing sugar loss by acid hydrolysis of the bio-pretreated sample was lower than that of the control except for the combination of mild acid pretreatment with biological pretreatment by Antrodia sp. 5898 at 100 °C for 30–60 min (Fig. 2). This result might result from the hemicellulose and cellulose degradation by Antrodia sp. 5898 and the excess of degradation products during the biological pretreatment. 3.4. Ethanol production
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sulfuric acid treatment time (min) Fig. 1. The reducing sugar yield from enzymatic hydrolysis of water hyacinth treated by 0.25% H2SO4 solution at varied temperature and different time after 10 days of biological pretreatment with E. taxodii or Antrodia sp. 5898. (A) The sole acid pretreatment (control); (B) The combined pretreatment with E. taxodii and acid; (C), the combined pretreatment with Antrodia sp. 5898 and acid.
matter), which increased 1.13–2.11 fold at the same conditions (Fig. 1A and B). The maximum yield of reducing sugar was 322.3 and 366.0 mg/g dry matter for the acid and combined pretreatment at 100 °C for 60 min, respectively. The increase of enzymatic
In order to verify the influence of pretreatment on ethanol production, enzymatic hydrolysate of the maximum reducing sugar yield from the acid treated or the co-treated at 100 °C for 60 min was used to ferment. The result showed that the ethanol yield achieved 0.192 g/g of dry material, which was 1.34-fold higher than that of the sole acid-treated sample (0.146 g/g of dry material). Compared the results of enzymatic hydrolysis and ethanol fermentation from the co-treated sample with the acid-treated sample, it can be seen that the enhancement of ethanol yield (1.34-fold) was significant. This result indicated that the enzymatic hydrolysate of the co-treated sample was easier to be fermented to ethanol by S. cerevisiae than that of the acid-treated sample. The
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The ethanol yield in this study was comparable to those reported from water hyacinth by different chemical pretreatment or enzymatic hydrolysis. Abraham and Kurup (1996) used peracetic acid pretreated water hyacinth to acid or enzymatic hydrolysis and fermentation with S. cerevisiae. The maximum yields of reducing sugar and ethanol were 430 and 163 mg/g pretreated water hyacinth, respectively, which were higher than those achieved by 10.0% H2SO4 acid hydrolysis (375 and 116 mg/g pretreated water hyacinth). Although the reducing sugar yield was higher than that studied here, the ethanol yield was lower. This suggested the combination pretreatment with E. taxodii and mild acid might decrease the fermentation inhibitors. Mishima et al. (2008) used alkaline/oxidative pretreated water hyacinth to produce ethanol by separated hydrolysis and fermentation (SHF) and simultaneous saccharification and fermentation (SSF) with S. cerevisiae NBRC 2346 or recombinant Escherichia coli KO11. The results showed that ethanol yields in SSF achieved 140, or 170 mg/g pretreated biomass. Other studies focused on fermentation of acid hydrolysate of water hyacinth (Nigam, 2002; Isarankura-Na-Ayudhya et al., 2007; Masami et al., 2008; Kumar et al., 2009a), the maximum ethanol yield was 190–425 mg/g sugar utilized and converted into 25–70 mg/g dry water hyacinth. Although ethanol fermentation was preliminary experiment in this study, the combination of mild acid pretreatment and biological pretreatment with the white rot fungus E. taxodii provided a promising method for bio-ethanol production from water hyacinth.
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4. Conclusion 30
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In conclusion, the combined pretreatment with white rot fungus and mild acid was an effective method to improve enzymatic hydrolysis and ethanol production from water hyacinth. The reducing sugar yield from enzymatic hydrolysis of co-treated sample increased 1.13–2.11-fold than that of acid-treated sample at the same conditions, and the synergistic effect of combination of pretreatment with E. taxodii and acid was greater at moderate conditions. Ethanol fermentation showed that the ethanol yield from combined pretreatment increased 1.34-fold than that from sole acid pretreatment. The increase of the ethanol yield was more obvious than the increase of the reducing sugar yield (1.13-fold) at the same acid pretreatment conditions. Acknowledgements
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This work was supported by a grant from the Major State Basic Research Development Program of China (2007CB210200) and the National Natural Science Foundation of China (30901137). The authors thank the Centre of Analysis and Test of Huazhong University of Science and Technology for ethanol analysis.
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sulfuric acid treatment time (min) Fig. 2. The reducing sugar loss of water hyacinth treated by 0.25% H2SO4 solution at varied temperature and different time after 10 days of biological pretreatment with E. taxodii or Antrodia sp. 5898. (A) The sole acid pretreatment (control); (B) The combined pretreatment with E. taxodii and acid; (C) The combined pretreatment with Antrodia sp. 5898 and acid.
combined pretreatment could stimulate ethanol fermentation by increasing glucose concentration, decreasing the fermentation inhibitors or producing fermentation accelerants (Keller et al., 2003; Lloyd and Wyman, 2005; Carmona et al., 2009). Further study on the mechanism of ethanol production enhancement by combined pretreatment is under investigation.
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Masami, G.O.O., Usui, I.Y., Urano, N., 2008. Ethanol production from the water hyacinth Eichhornia crassipes by yeast isolated from various hydrospheres. African Journal of Microbiology Research 2, 110–113. Mishima, D., Tateda, M., Ike, M., Fujita, M., 2006. Comparative study on chemical pretreatments to accelerate enzymatic hydrolysis of aquatic macrophyte biomass used in water purification processes. Bioresource Technology 97, 2166–2172. Mishima, D., Kuniki, M., Sei, K., Soda, S., Ike, M., Fujita, M., 2008. Ethanol production from candidate energy crops: water hyacinth (Eichhornia crassipes) and water lettuce (Pistia stratiotes L.). Bioresource Technology 99, 2495–2500. Mosier, N., Wyman, C., Dale, B., Elander, R., Lee, Y.Y., Holtzapple, M., Ladisch, M., 2005. Features of promising technologies for pretreatment of lignocellulosic biomass. Bioresource Technology 96, 673–686. Nigam, J.N., 2002. Bioconversion of water-hyacinth (Eichhornia crassipes) hemicellulose acid hydrolysate to motor fuel ethanol by xylose-fermenting yeast. Journal of Biotechnology 97, 107–116. Sun, Y., Cheng, J.Y., 2002. Hydrolysis of lignocellulosic materials for ethanol production: a review. Bioresource Technology 83, 1–11. Torget, R., Walter, P., Himmel, M., Grohmann, K., 1990. Dilute acid pretreatment of short rotation woody and herbaceous crops. Applied Biochemistry and Biotechnology 24 (25), 115–126. Xie, Y., Li, Z.Y., William, P.G., Li, D.M., 2001. Invasive species in China – an overview. Biodiversity and Conservation 10, 1317–1341. Yu, H.B., Guo, G.N., Zhang, X.Y., Yan, K.L., Xu, C.Y., 2009a. The effect of biological pretreatment with the selective white-rot fungus Echinodontium taxodii on enzymatic hydrolysis of softwoods and hardwoods. Bioresource Technology 100, 5170–5175. Yu, H.B., Du, W.Q., Zhang, J., Ma, F.Y., Zhang, X.Y., Zhong, W.X., 2010. Fungal treatment of cornstalks enhances the delignification and xylan loss during mild alkaline pretreatment and enzymatic digestibility of glucan. Bioresource Technology 101, 6728–6734. Yu, J., Zhang, J.B., He, J., Liu, Z.D., Yu, Z.N., 2009b. Combinations of mild physical or chemical pretreatment with biological pretreatment for enzymatic hydrolysis of rice hull. Bioresource Technology 100, 903–908. Zhang, X.Y., Xu, C.Y., Wang, H.X., 2007a. Pretreatment of bamboo residues with Coriolus versicolor for enzymatic hydrolysis. Journal of Bioscience and Bioengineering 104, 149–151. Zhang, X.Y., Yu, H.B., Huang, H.Y., Liu, Y.X., 2007b. Evaluation of biological pretreatment with white rot fungi for the enzymatic hydrolysis of bamboo culms. International Biodeterioration & Biodegradation 60, 159–164.
Contents lists available at ScienceDirect
Bioresource Technology journal homepage: www.elsevier.com/locate/biortech
Combination of biological pretreatment with mild acid pretreatment for enzymatic hydrolysis and ethanol production from water hyacinth Fuying Ma, Na Yang, Chunyan Xu, Hongbo Yu, Jianguo Wu, Xiaoyu Zhang * Key Laboratory of Molecular Biophysics of MOE, College of Life Science and Technology, Huazhong University of Science and Technology, Wuhan 430074, PR China
a r t i c l e
i n f o
Article history: Received 4 May 2010 Received in revised form 16 July 2010 Accepted 20 July 2010 Available online 24 July 2010 Keywords: Water hyacinth Biological pretreatment Mild acid pretreatment Saccharification Ethanol fermentation
a b s t r a c t The mild acid pretreatment and the combination of biological pretreatment by a white rot fungus Echinodontium taxodii or a brown rot fungus Antrodia sp. 5898 with mild acid pretreatment were evaluated under different pretreatment conditions for enzymatic hydrolysis and ethanol production from water hyacinth. The combined pretreatment with E. taxodii (10 days) and 0.25% H2SO4 was proved to be more effective than the sole acid pretreatment. The reducing sugar yield from enzymatic hydrolysis of co-treated water hyacinth increased 1.13–2.11 fold than that of acid-treated water hyacinth at the same conditions. The following study on separate hydrolysis and fermentation with Saccharomyces cerevisiae indicated that the ethanol yield from co-treated water hyacinth achieved 0.192 g/g of dry matter, which increased 1.34-fold than that from acid-treated water hyacinth (0.146 g/g of dry matter). This suggested that the combination of biological and mild acid pretreatment is a promising method to improve enzymatic hydrolysis and ethanol production from water hyacinth with low lignin content. Ó 2010 Elsevier Ltd. All rights reserved.
1. Introduction Water hyacinth (Eichhornia crassipes) was first introduced from South America into China in 1901 and widely planted as pig feedstuff in the 1950s (Xie et al., 2001; Chu et al., 2006). Currently, it is regarded as a nuisance which has seriously impaired the biological diversity and ecological environment because of its extraordinary adaptive ability and remarkable growth rate (Center et al., 1999; Lu et al., 2007; Malik, 2007). However, some studies indicated that water hyacinth is a promising plant for production of fuel ethanol, biogas and other valuable products because it grows quickly and contains high amounts of hemicellulose and cellulose (Nigam, 2002; Isarankura-Na-Ayudhya et al., 2007; Carina and Cecilia, 2007; Malik, 2007; Hronich et al., 2008; Kumar et al., 2009a; Almoustapha et al., 2009; Bhattacharya and Kumar, 2010; Klass and Ghosh, 1981). Fuel ethanol is convenient to be used as a gasoline additive to increase octane and improve vehicle emissions. Fuel ethanol production from biomass involves enzymatic hydrolysis and fermentation. Pretreatment is an effective way to increase the hydrolysis efficiency, because it can break down the complex structure of biomass and increase the accessibility of cellulase protein to cellulose substrate (Mosier et al., 2005; Kumar et al., 2009b).
* Corresponding author. Tel./fax: +86 27 87792128. E-mail address: [email protected] (X. Zhang). 0960-8524/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.biortech.2010.07.084
The acid pretreatments are effective methods used for water hyacinth to ethanol for dissolving hemicellulose and retaining most of the cellulose (Nigam, 2002; Kumar et al., 2009a). Though the conventional acid pretreatments are energy-intensive and environment-unfriendly, the concentrated acid or the dilute acid at high temperature and pressure is usually applied (Sun and Cheng, 2002; Mishima et al., 2006, 2008; Masami et al., 2008). In addition, trials done on water hyacinth acid or enzymatic hydrolysis indicated that the reducing sugar yield was higher with saccharification (Abraham and Kurup, 1996). However, most investigators neglected the residue of acid hydrolysis. Biological pretreatment with fungus has attracted much attention for disrupting the lignin-hemicellulose sheath, requiring relatively low energy and mild environmental conditions (Zhang et al., 2007a,b; Yu et al., 2009a). However, the economic feasibility of fungal pretreatment is poor, because the loss in polysaccharide components during fungal growth and the long cultivation period reduce overall productivity. In order to overcome these disadvantages, the combinations of biological and mild chemical pretreatment were used to enhance the saccharification ratio of rice hull and cornstalks (Yu et al., 2009b, 2010). The white rot fungus Echinodontium taxodii can selectively degrade lignin of woody and non-woody lignocellulose to enhance the enzymatic hydrolysis (Zhang et al., 2007b; Yu et al., 2009a). But, there is no report about biological pretreatment of water hyacinth with low lignin content. Thus, the influences of the sole acid pretreatment and the combination of fungal pretreatment (white
F. Ma et al. / Bioresource Technology 101 (2010) 9600–9604
rot fungus E. taxodii and brown rot fungus Antrodia sp. 5898) with mild acid pretreatment on reducing sugar yield and ethanol production using Saccharomyces cerevisiae from water hyacinth were investigated in this study. 2. Methods 2.1. Microorganisms and cultivation The white rot fungus E. taxodii and brown rot fungus Antrodia sp. 5898 were isolated from Shennongjia Nature Reserve (Hubei, China). The organisms were maintained on potato dextrose agar (PDA) slants at 4 °C. A plug of the two fungi activated for 5–6 days on PDA slants at 28 °C was inoculated onto the center of 9 cm PDA plates and incubated for one week at 28 °C. Thermal-tolerant alcohol active dry yeast S. cerevisiae, provided by Angel Yeast Co. Ltd., was firstly rehydrated in a starter solution (5% dry yeast added to 2% glucose solution) before ‘‘pitching”. To ensure the quality of the culture and to get a vigorous start, the yeast was activated at 35–42 °C for 30 min and then again at 34 °C for 1.5–2 h to get small bubbles. 2.2. Raw material Fresh water hyacinth (E. crassipes) was collected and washed to remove adhering dirt, chopped into small pieces (about 1 cm in length) and air-dried. The samples were ground, and the particles between 0.45 mm and 0.9 mm were prepared for the following pretreatment with basidiomycetes and chemicals. 2.3. Biological pretreatment of water hyacinth The biological pretreatments with E. taxodii and Antrodia sp. 5898 were carried out in a 250-ml Erlenmeyer flask with 5 g of air-dried water hyacinth and 12 ml of distilled water. The samples were sterilized in the autoclave for 30 min at 121 °C and inoculated with five plugs cut from the fungal mat on the PDA plates. Biological pretreatments were conducted statically at 28 °C for 10 days, and then the treated samples were dried at 60 °C for 3 days. The non-inoculated sample was used as the control. All experiments were performed in triplicate. 2.4. Dilute acid pretreatment of water hyacinth The raw and bio-pretreated water hyacinth with E. taxodii or Antrodia sp. 5898 were further treated with 0.25% sulfuric acid at varied temperature (25, 80, and 100 °C) for 15–60 min, and then filtrated. The reducing sugar in filtrate was measured after neutralization with CaCl2. The residues were washed to neutralize with distilled water, and then dried at 60 °C for 3 days and ground to be used for enzymatic hydrolysis and saccharification. 2.5. Separate hydrolysis and fermentation (SHF) Commercial cellulase preparation, produced by Trichoderma reesei, was provided by Henan Tianguan Enterprise Group Co., Ltd. Enzymatic hydrolysis was carried out at 2% substrate concentration in 50 mM sodium acetate buffer (pH 4.8) with cellulase (30 FPU/g of substrate) at 50 °C for 48 h. After filtration, the enzymatic hydrolysate was used for reducing sugar measurement and ethanol fermentation. To make fermentation medium, the enzymatic hydrolysate concentrated to 2% (w/v) of reducing sugar, was supplemented with nutrients (g/L): peptone, 5; yeast extract, 5; KH2PO4, 1; MgSO47H2O, 0.3; NH4Cl, 2. Ethanol fermentation was conducted
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in a 250-ml Erlenmeyer flask with 100 ml fermentation medium (pH 4). Activated yeast (0.3% (v/v)) was inoculated into the fermentation medium. The flasks were incubated at 40 °C for 8 h shaking at 100 rpm and then again at 40 °C for 72 h statically. All experiments were performed in triplicate. 2.6. Analytical methods 2.6.1. Chemical components analysis The content of neutral detergent fiber (NDF), acid detergent fiber (ADF), acid detergent lignin (ADL) and ash were determined according to the procedures of Goering and Van Soest (1971). Hemicellulose content was estimated as the difference between NDF and ADF, and cellulose content was calculated based on the difference between ADF and ADL. 2.6.2. Reducing sugar estimation Total reducing sugar was measured with the DNS (3,5-dinitrosalicylic acid) method (Behera et al., 1996). The amount of reducing sugar was calculated as follows: Reducing sugar yield from enzymatic hydrolysisðmg=g dry water hyacinthÞ ¼
amount of reducing sugar produced after enzymatic hydrolysis amount of dry water hyacinth
2.6.3. Determination of ethanol concentration The ethanol concentration was determined by a high performance liquid chromatography (HPLC) system (Agilent 1200, China) using sugar-pak-1 column (Waters, China) and the refractive index (RI) detector (Agilent G1362A, China). The mobile phase was 50 mg/L CaNa-EDTA solution at a flow rate of 0.6 ml/min with the column temperature of 75 °C. 3. Results and discussion 3.1. Chemical components of water hyacinth The average lignocellulosic composition of water hyacinth is (as total percentage of solids): cellulose: 18.2 ± 0.6, hemicellulose: 29.3 ± 0.5, lignin: 2.8 ± 0.2, ash: 1.2 ± 0.2. The cellulose content is in accordance with the data reported by other investigators, but the hemicellulose and lignin content are lower than their results (Klass and Ghosh, 1981; Nigam, 2002; Kumar et al., 2009a). These differences might originate from different sources (Carina and Cecilia, 2007) and different growth state of water hyacinth. 3.2. Enzymatic hydrolysis of water hyacinth The H2SO4 pretreatment effectively improved the enzymatic hydrolysis of water hyacinth. The reducing sugar yield was slightly increased by increasing the H2SO4 concentration from 0.25% to 1% (data not shown). Thus, 0.25% H2SO4 treatment was used in this study. The reducing sugar yield from enzymatic hydrolysis of acid-treated sample decreased as pretreatment time increased at room temperature (25 °C). However, at high temperature (80 and 100 °C), the reducing sugar yield increased with the increase in pretreatment time (Fig. 1A). After 10 days of biological pretreatment with E. taxodii or Antrodia sp. 5898, the bio-pretreated sample and raw material were further treated with 0.25% H2SO4 at 25, 80, 100 °C for 15–60 min, respectively. The residue of acid hydrolysis was subjected to enzymatic hydrolysis and the reducing sugar yield might be seen in Fig. 1. The reducing sugar yield of water hyacinth co-treated with E. taxodii and mild acid (from 208.8 to 366.0 mg/g dry matter) were higher than that of acid-treated (from 112.9 to 322.3 mg/g dry
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digestibility after the combination pretreatment can be attributed to the synergistic effect of biological and acid treatment. Especially, at room temperature (25 °C), the reducing sugar yield was enhanced 1.79-, 1.90- and 2.11-fold for 60-, 15- and 30-min treatment, respectively. The result suggested that the synergy of combination pretreatment was greater at moderate condition. The reason might be the white rot fungus E. taxodii degraded and modified lignin in water hyacinth, which made the accessibility of cellulase to cellulose easier. But the reducing sugar yield of water hyacinth pretreated with Antrodia sp. 5898 (Fig. 1A and C) was lower than that of control sample. Antrodia sp. 5898, as a brown rot fungus, metabolized the carbohydrates including cellulose and hemicellulose of biomass without removing lignin, producing sugar-free brown residue (Eriksson et al., 1990). On the other hand, the polysaccharide losses caused by Antrodia sp. 5898 might be significantly more than that caused by the white rot fungus E. taxodii. Furthermore, the concentrated lignin brought about more difficulties for cellulase to access cellulose due to carbohydrates degradation. Therefore, the reducing sugar yield from the combined pretreatment with Antrodia sp. 5898 was less than that from the acid or combination pretreatment with E. taxodii. These results suggested that lignin was a major hindrance to enzymatic hydrolysis of water hyacinth.
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The dilute acid pretreatment is primarily a hydrolytic process which causes solubilization of hemicellulosic sugars (Torget et al., 1990). In this work, the reducing sugar of acid hydrolysate was called the reducing sugar loss. At room temperature (25 °C), the reducing sugar loss of control sample had little change with the increasing of acid pretreatment time. At high temperature (80 and 100 °C), the reducing sugar loss increased as acid pretreatment time increased (Fig. 2A). The amount of reducing sugar of acid hydrolyate at mild conditions (72.8–114.7 mg/g dry matter) was relatively low, compared with high acid concentration or high reaction temperature (Nigam, 2002; Isarankura-Na-Ayudhya et al., 2007; Kumar et al., 2009a). These results might be caused by low hemicellulose content of raw material or incomplete hydrolysis of hemicellulose under mild reaction conditions. In fact, high temperature (121–190 °C) and high pressure were usually employed in dilute acid pretreatment to get sufficient hydrolysis of hemicellulose (Isarankura-Na-Ayudhya et al., 2007; Masami et al., 2008; Sun and Cheng, 2002). The reducing sugar loss by acid hydrolysis of the bio-pretreated sample was lower than that of the control except for the combination of mild acid pretreatment with biological pretreatment by Antrodia sp. 5898 at 100 °C for 30–60 min (Fig. 2). This result might result from the hemicellulose and cellulose degradation by Antrodia sp. 5898 and the excess of degradation products during the biological pretreatment. 3.4. Ethanol production
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sulfuric acid treatment time (min) Fig. 1. The reducing sugar yield from enzymatic hydrolysis of water hyacinth treated by 0.25% H2SO4 solution at varied temperature and different time after 10 days of biological pretreatment with E. taxodii or Antrodia sp. 5898. (A) The sole acid pretreatment (control); (B) The combined pretreatment with E. taxodii and acid; (C), the combined pretreatment with Antrodia sp. 5898 and acid.
matter), which increased 1.13–2.11 fold at the same conditions (Fig. 1A and B). The maximum yield of reducing sugar was 322.3 and 366.0 mg/g dry matter for the acid and combined pretreatment at 100 °C for 60 min, respectively. The increase of enzymatic
In order to verify the influence of pretreatment on ethanol production, enzymatic hydrolysate of the maximum reducing sugar yield from the acid treated or the co-treated at 100 °C for 60 min was used to ferment. The result showed that the ethanol yield achieved 0.192 g/g of dry material, which was 1.34-fold higher than that of the sole acid-treated sample (0.146 g/g of dry material). Compared the results of enzymatic hydrolysis and ethanol fermentation from the co-treated sample with the acid-treated sample, it can be seen that the enhancement of ethanol yield (1.34-fold) was significant. This result indicated that the enzymatic hydrolysate of the co-treated sample was easier to be fermented to ethanol by S. cerevisiae than that of the acid-treated sample. The
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The ethanol yield in this study was comparable to those reported from water hyacinth by different chemical pretreatment or enzymatic hydrolysis. Abraham and Kurup (1996) used peracetic acid pretreated water hyacinth to acid or enzymatic hydrolysis and fermentation with S. cerevisiae. The maximum yields of reducing sugar and ethanol were 430 and 163 mg/g pretreated water hyacinth, respectively, which were higher than those achieved by 10.0% H2SO4 acid hydrolysis (375 and 116 mg/g pretreated water hyacinth). Although the reducing sugar yield was higher than that studied here, the ethanol yield was lower. This suggested the combination pretreatment with E. taxodii and mild acid might decrease the fermentation inhibitors. Mishima et al. (2008) used alkaline/oxidative pretreated water hyacinth to produce ethanol by separated hydrolysis and fermentation (SHF) and simultaneous saccharification and fermentation (SSF) with S. cerevisiae NBRC 2346 or recombinant Escherichia coli KO11. The results showed that ethanol yields in SSF achieved 140, or 170 mg/g pretreated biomass. Other studies focused on fermentation of acid hydrolysate of water hyacinth (Nigam, 2002; Isarankura-Na-Ayudhya et al., 2007; Masami et al., 2008; Kumar et al., 2009a), the maximum ethanol yield was 190–425 mg/g sugar utilized and converted into 25–70 mg/g dry water hyacinth. Although ethanol fermentation was preliminary experiment in this study, the combination of mild acid pretreatment and biological pretreatment with the white rot fungus E. taxodii provided a promising method for bio-ethanol production from water hyacinth.
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4. Conclusion 30
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In conclusion, the combined pretreatment with white rot fungus and mild acid was an effective method to improve enzymatic hydrolysis and ethanol production from water hyacinth. The reducing sugar yield from enzymatic hydrolysis of co-treated sample increased 1.13–2.11-fold than that of acid-treated sample at the same conditions, and the synergistic effect of combination of pretreatment with E. taxodii and acid was greater at moderate conditions. Ethanol fermentation showed that the ethanol yield from combined pretreatment increased 1.34-fold than that from sole acid pretreatment. The increase of the ethanol yield was more obvious than the increase of the reducing sugar yield (1.13-fold) at the same acid pretreatment conditions. Acknowledgements
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This work was supported by a grant from the Major State Basic Research Development Program of China (2007CB210200) and the National Natural Science Foundation of China (30901137). The authors thank the Centre of Analysis and Test of Huazhong University of Science and Technology for ethanol analysis.
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sulfuric acid treatment time (min) Fig. 2. The reducing sugar loss of water hyacinth treated by 0.25% H2SO4 solution at varied temperature and different time after 10 days of biological pretreatment with E. taxodii or Antrodia sp. 5898. (A) The sole acid pretreatment (control); (B) The combined pretreatment with E. taxodii and acid; (C) The combined pretreatment with Antrodia sp. 5898 and acid.
combined pretreatment could stimulate ethanol fermentation by increasing glucose concentration, decreasing the fermentation inhibitors or producing fermentation accelerants (Keller et al., 2003; Lloyd and Wyman, 2005; Carmona et al., 2009). Further study on the mechanism of ethanol production enhancement by combined pretreatment is under investigation.
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