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Efficient conversion of crop stalk wastes into succinic acid production by Actinobacillus succinogenes
Bioresource Technology 101 (2010) 3292–3294
Contents lists available at ScienceDirect
Bioresource Technology journal homepage: www.elsevier.com/locate/biortech
Short Communication
Efficient conversion of crop stalk wastes into succinic acid production by Actinobacillus succinogenes Qiang Li a,b, Maohua Yang a,b, Dan Wang a,b, Wangliang Li a, Yong Wu a,b, Yunjian Zhang c, Jianmin Xing a,*, Zhiguo Su a a b c
National Key Laboratory of Biochemical Engineering, Institute of Process Engineering, Chinese Academy of Sciences, P.O. Box 353, Beijing 100190, PR China Graduate School of the Chinese Academy of Sciences, Beijing 100049, PR China Department of Bioengineering, School of Chemical and Environmental Engineering, China University of Mining and Technology (Beijing), Beijing 100083, PR China
a r t i c l e
i n f o
Article history: Received 14 July 2009 Received in revised form 23 November 2009 Accepted 16 December 2009 Available online 12 January 2010 Keywords: Succinic acid Fermentation Hydrolysis Actinobacillus succinogenes
a b s t r a c t Succinic acid is valued as a key platform chemical for use in a variety of synthetic applications. Efficient biosynthesis of succinic acid from renewable biomass resource is reported in this paper. Batch fermentations were carried out to analyze influence of several carbon sources on succinic acid production from feedstock wastes by Actinobacillus succinogenes BE-1. Crop stalk wastes, including corn stalk and cotton stalk, were enzymatically converted into a carbohydrate-rich feedstock, obtaining glucose concentrations approaching 65–80% of the total reducing sugar. For the anaerobic batch cultivation with cotton stalk hydrolysates, the production of succinic acid was 15.8 g l 1 with a high yield of 1.23 g per g glucose. Glucose and xylose were utilized at same time, while cellubiose was not consumed until glucose and xylose were completely consumed. Ó 2009 Elsevier Ltd. All rights reserved.
1. Introduction Succinic acid is a potential platform chemical for the production of various high value-added derivatives. Compared with traditional synthetic methods by fossil fuels, the production of succinic acid from natural derived biomass would alleviate the dependence on oil supply for the production of these platform chemicals in the future (Delhomme et al., 2009). The fermentative production of succinic acid has been most intensively investigated with bacteria capable of producing large amounts of succinic acid, including Actinobacillus succinogenes, Mannheimia succiniciproducens, Anaerobiospirillum succiniciproducens, and recombinant Escherichia coli (Zheng et al., 2009; Kim et al., 2004; Du et al., 2008). Microbial conversion of biomass into succinic acid is attracting increasing interest as an environmentally friendly and energy-saving process. The development of a cost-effective culture medium to obtain maximum sugar utilization yield in the fermentation process is considered to be a fundamental development to the chemical production industry. Much information is available on the feasibility of converting the biomass containing crude cellulose into fermentation products. Many agricultural and industrial wastes or residues, for example, whey, wheat, straw and wood, have been reported as raw carbon materials for the production of succinic
* Corresponding author. Tel./fax: +86 010 62550913. E-mail address: [email protected] (J. Xing). 0960-8524/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved. doi:10.1016/j.biortech.2009.12.064
acid. Kim et al. (2004) used wood hydrolysate-based medium to culture M. succiniciproducens MBEL55E and the final succinic acid concentration of 11.73 g L 1 was obtained in batch fermentation, resulting in a succinic acid yield of 0.56 g g 1. Du et al. (2008) carried out two wheat-based bio-refining strategies converting wheat to succinic acid in which A. succinogene fermentation using only the wheat-derived feedstock resulted in a succinic acid concentration of 16 g L 1 with an overall yield of 0.19 g g 1. In batch fermentation of succinic acid from straw hydrolysate by A. succinogenes, 45.5 g L 1 succinic acid concentration and 0.81 g g 1 yield were attained after 48 h incubation with 58 g L of initial sugar from corn straw hydrolysate (Zheng et al., 2009). Economical succinic acid production from cane molasses can yield 50.6 g L 1 succinic acid in continuous anaerobic fermentation (Liu et al., 2008). In China, the annual biomass wastes exceed 0.7 billion tons, among which the cornstalk, wheat straw and straw wastes are around 220, 110 and 180 million tons per year, respectively (Fan et al., 2006). However, crop stalk residues can be an efficient renewable source for the cost competitive chemical production platform. Within this strategy a synthetic biomass conversion process was developed to convert feedstock wastes into succinic acid by two-step processes, especially with cotton stalks conversion for the first time. In the first place, biomass residue cornstalk or cotton stalk were converted into fermentation feedstock by enzyme hydrolysis. The feedstock was subsequently fermented by A. succinogenes BE-1 to produce succinic acid.
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Q. Li et al. / Bioresource Technology 101 (2010) 3292–3294
2. Methods 100
A. succinogenes BE-1, a strain that produces a high concentration of succinic acid, had been isolated from bovine rumen in our laboratory, and was collected in China General Microbiological Culture Collection Center (No. CGMCC2650). 2.2. Medium and succinic acid fermentation Effect of different carbon sources on the production of succinic acid were evaluated in batch fermentation under anaerobic conditions. The fermentation medium contained per liter: 30 g sugar, 30 g yeast extract, 2 g urea, 2 g MgCl26H2O, 1.5 g CaCl2, 0.07 g MnCl2, 4.4 g Na2HPO4, 3.3 g NaH2PO4, 30 g MgCO3. Glucose and biomass-derived carbohydrates were separately sterilized at 115 °C for 30 min and added to the medium to maintain the initial concentrations of 30 g L 1. The initial pH of the sterilized medium was adjusted to 7.0 by 10 mol L 1 NaOH solution. Seed inoculum was 5% vol 1. 2.3. Hydrolysates preparation from crop stalk wastes The corn stalk and cotton stalk residues were pretreated by steam explosion for 10 min at 1.5 MPa, and separated by a vegetation disintegrator to pass 40-mesh screen. The dried samples were treated by 1 % (w/v) NaOH and 4% (v/v) H2O2 for 24 h at the room temperature at 150 rpm. Samples were washed to neutral, and dried at 50 °C. Cellulase from Trichoderma reessi (Sunson Ltd., China) with the activity of 1.67 filter paper unit (FPU) per mg soluble protein and 0.29 b-glucosidase activity IU per mg soluble protein was used in this research. Cellulase of 20 FPU/g was added to 12% (w/v) pretreated samples with 0.05 M Tris–HCl as the pH buffer. The suspension was placed in a 500-mL flask at 50 °C, 150 rpm for 72 h. The hydrolysate samples were employed as substrate of succinic acid fermentation. 2.4. Analysis methods The different reductive sugar and products (succinate, lactate, formate, acetate and ethanol) formation during fermentation were analyzed by HPLC using an Aminex HPX-87H ion-exchange column (Bio-Rad, USA) and HP1200 chromatography working station system (Agilent Technologies, USA) equipped with UV absorbance detector (Agilent Technologies, G1315D) and refractive index detector (Agilent Technologies, G1362A). The column was eluted isocratically at a rate of 0.6 ml min 1 with 5 mM H2SO4 under 55 °C. The injection volume was 10 ll. 3. Results and discussion 3.1. Carbohydrates conversion from biomass crop stalks In this case, the hydrolysis of corn stalks and cotton stalks, was used to produce the feedstock for the bacterial fermentation. Fig. 1 shows the time course for reducing sugar concentrations obtained in the experiment. Nearly 90 g L 1 reducing sugar was generated at a high rate by action of cellulases preparations. Among the final corn stalk hydrolysate, the concentrations of glucose were about 80% in the total reducing sugar. While for the cotton stalk hydrolysate, the proportion of glucose in the total reducing sugar was about 65%, with 8% xylose and 14% cellubiose. Under the optimized conditions, the total sugar yield could reach 60 g/L in less than 10 h. The proportion of glucose in the total
Reducing sugar (g/L)
2.1. Microorganism 80 60
corn stalk cotton stalk
40 20 0 0
10
20
30
40
50
60
70
80
Time (h) Fig. 1. Time course of total sugar in batch hydrolysates of corn straw.
mixed-sugar was over 50%, which meant the hydrolysis carbohydrates could be used as the replacement of single carbon source in the traditional fermentation of succinic acid. The component on the hydrolysate did not seem to inhibit cell growth and fermentation after sterilization. 3.2. Batch fermentation from different carbon sources Succinic acid fermentation was performed in a simulated medium, containing the salts and nutrients as described earlier except the sorts of the carbohydrates. Crop stalk hydrolysate contained quantities of reducing carbohydrates, among which the concentrations of glucose were the most. In this study, main kinds of carbohydrates, such as glucose, fructose, xylose, maltose and cellobiose were chosen for further batch fermentation tests to compare the fermentability of single sugar and the hydrolysates. Each carbon source was added to give a concentration equivalent to 30 g L 1 glucose. Glucose, fructose, xylose and maltose were consumed completely in 34 h. At the end of the fermentation, about 17.2 g L 1 succinic acid, 8.4 g L 1 acetic acid, and 5.2 g L 1 lactic acid were produced. Fructose was consumed completely by 10 h, but the productivity of succinic acid was not very high. The titers of succinic acid, acetic acid and lactic acid were about 14.2 g L 1, 8.9 g L 1, and 6.0 g L 1, respectively. For the fermentation using xylose as the carbon source, the succinic acid concentration, productivity and yield were relatively very low. The concentrations of succinic acid, acetic acid and lactic acid were only 7.0 g L 1, 5.4 g L 1, and 5.3 g L 1, respectively. Compared to the fermentation using the glucose, the fermentation using maltose as the carbon source resulted in a similar final succinic acid concentration of about 16.9 g L 1 and a similar acetic, lactic acid concentration of about 9.7 g L 1, 4.9 g L 1, respectively. The utilization yield of cellobiose was relatively slow. Nearly half of the initial cellobiose was not consumed. This batch test led to 0.97 g g 1 yield with 14.7 g L 1 succinic acid, 8.2 g L 1 acetic acid, and 5.8 g L 1 lactic acid, confirming that the carbon sources are crucial components in succinic acid fermentations. 3.3. Fermentation of hydrolysates Enzymatic hydrolysis was a more effective method for treating the crop stalks in order that the enzymatic hydrolysate could be used as an inexpensive carbon source for succinic acid production. Accordingly, the initial glucose concentrations were maintained
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Q. Li et al. / Bioresource Technology 101 (2010) 3292–3294
Table 1 Comparison of succinic acid production from various biomass-derived sources.
a
Carbon source
Concentration (g L
Glucose Fructose Xylose Maltose Cellubiose Corn stalk Cotton stalk
17.2 ± 0.1 14.2 ± 0.2 6.9 ± 0.3 16.8 ± 0.1 14.7 ± 0.2 17.8 ± 0.2 15.8 ± 0.1
1
)
Productivity (g L
1
h
1
)
0.51 ± 0.02 0.42 ± 0.03 0.21 ± 0.05 0.50 ± 0.03 0.43 ± 0.02 0.56 ± 0.01 0.62 ± 0.01
Yielda (g g
1
)
0.70 ± 0.02 0.48 ± 0.02 0.28 ± 0.01 0.65 ± 0.04 0.97 ± 0.02 0.66 ± 0.02 1.23 ± 0.02
Reference Supplementary Supplementary Supplementary Supplementary Supplementary Supplementary Supplementary
Fig. Fig. Fig. Fig. Fig. Fig. Fig.
S1A S1B S1C S1D S1E S1F S1G
The unit is g succinic acid per g carbohydrate consumed.
30 g L 1 in the fermentation broth. At the end of fermentation, the medium contained up to 17.8 g L 1 and 15.8 g L 1 succinic acid, respectively for corn stalks and cotton stalks as the carbon source for the succinic acid fermentation process. It can be assumed that that the biomass-based generic feedstock contained sufficient essential nutrients for the succinic acid fermentation by A. succinogenes. Table 1 compared succinic acid production on the semi-defined medium and on the crop stalk-derived media. Compared with the fermentation on the semi-defined medium, the fermentation with crop stalk hydrolysate resulted in a similar succinic acid concentration and yield but with a higher productivity. Moreover, for the cotton stalk hydrolysate fermentation, the glucose to succinic acid production yield was 1.23 g g 1, which was the highest yield of the batch fermentations. This was probably due to the nutrient richness of the crop stalk hydrolysate, such as nitrogen source, minerals and vitamins (Wang et al., 2002; Gullon et al., 2008). A relatively concentrated hydrolysate contained sufficient quantity of essential nutrition for A. succinogenes growth and succinic acid formation, suggesting that the usage of stalk hydrolysate would improve process economics. Moreover, Liu et al. (2008) and Zheng et al. (2009) reported that A. succinogenes could use a wide range of carbohydrates as the carbon sources, and ferment straw hydrolysate or cane molasses onto succinic acid. But the utilization order of each sugar in the hydrolysates was different. As shown in Supplementary Fig. S2, glucose and xylose were utilized at the beginning of the fermentation, while cellubiose was not consumed until glucose and xylose completely consumed. A. succinogenes prefers to ferment monosaccharide rather than disaccharide. Thus, much attention can be drawn to increase the proportions of monosaccharide in the enzymatic hydrolysis process of crop stalks. 4. Conclusions The potential of crop stalks wastes for obtaining sugars suitable as carbon sources for succinic acid fermentation was evaluated synthetically. The hydrolysis processes of feedstock from crop stalks on the formation of reducing sugars were assessed at a high hydrolysis rate by action of enzymatic pretreatment. In batch cul-
tivation of A. succinogenes, the conversion yield of succinic acid from the biomass was relatively high. The possibility of developing a crop stalk bio-refinery for the production of succinic acid would result in improvement of process economics by using hydrolysate medium in comparison to semi-defined medium. Acknowledgement This work was supported by the Knowledge Innovation Program of the Chinese Academy of Sciences, Grant No. KSCX2-YWG-021. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.biortech.2009.12.064. References Delhomme, C., Weuster-Botz, D., Kühn, F.E., 2009. Succinic acid from renewable resources as a C4 building-block chemical – a review of the catalytic possibilities in aqueous media. Green Chem. 11, 13–26. Du, C.Y., Carol Lin, S.K., Koutinas, A., Wang, R.H., Dorado, P., Webb, C., 2008. A wheat biorefining strategy based on solid-state fermentation for fermentative production of succinic acid. Bioresour. Technol. 99, 8310–8315. Fan, Y.T., Zhang, Y.H., Zhang, S.F., Hou, H.W., Ren, B.Z., 2006. Efficient conversion of wheat straw wastes into biohydrogen gas by cow dung compost. Bioresour. Technol. 97, 500–505. Gullon, B., Yñez, R., Alonso, J.L., Parajó, J.C., 2008. L-lactic acid production from apple pomace by sequential hydrolysis and fermentation. Bioresour. Technol. 99, 308–319. Kim, D.Y., Yim, S.C., Lee, P.C., Lee, W.G., Lee, S.Y., Chang, H.N., 2004. Batch and continuous fermentation of succinic acid from wood hydrolysate by Mannheimia succiniciproducens MBEL55E. Enzyme Microbiol. Technol. 35, 648–653. Liu, Y.P., Zheng, P., Sun, Z.H., Ni, Y., Dong, J.J., Zhu, L.L., 2008. Economical succinic acid production from cane molasses by Actinobacillus succinogenes. Bioresour. Technol. 99, 1736–1742. Wang, R., Dominguez-Espinosa, R.M., Leonard, K., Koutinas, A., Webb, C., 2002. The application of a generic feedstock from wheat for microbial fermentations. Biotechnol. Prog. 18, 1033–1038. Zheng, P., Dong, J.J., Sun, Z.H., Ni, Y., Fang, L., 2009. Fermentative production of succinic acid from straw hydrolysate by Actinobacillus succinogenes. Bioresour. Technol. 100, 2425–2429.
Contents lists available at ScienceDirect
Bioresource Technology journal homepage: www.elsevier.com/locate/biortech
Short Communication
Efficient conversion of crop stalk wastes into succinic acid production by Actinobacillus succinogenes Qiang Li a,b, Maohua Yang a,b, Dan Wang a,b, Wangliang Li a, Yong Wu a,b, Yunjian Zhang c, Jianmin Xing a,*, Zhiguo Su a a b c
National Key Laboratory of Biochemical Engineering, Institute of Process Engineering, Chinese Academy of Sciences, P.O. Box 353, Beijing 100190, PR China Graduate School of the Chinese Academy of Sciences, Beijing 100049, PR China Department of Bioengineering, School of Chemical and Environmental Engineering, China University of Mining and Technology (Beijing), Beijing 100083, PR China
a r t i c l e
i n f o
Article history: Received 14 July 2009 Received in revised form 23 November 2009 Accepted 16 December 2009 Available online 12 January 2010 Keywords: Succinic acid Fermentation Hydrolysis Actinobacillus succinogenes
a b s t r a c t Succinic acid is valued as a key platform chemical for use in a variety of synthetic applications. Efficient biosynthesis of succinic acid from renewable biomass resource is reported in this paper. Batch fermentations were carried out to analyze influence of several carbon sources on succinic acid production from feedstock wastes by Actinobacillus succinogenes BE-1. Crop stalk wastes, including corn stalk and cotton stalk, were enzymatically converted into a carbohydrate-rich feedstock, obtaining glucose concentrations approaching 65–80% of the total reducing sugar. For the anaerobic batch cultivation with cotton stalk hydrolysates, the production of succinic acid was 15.8 g l 1 with a high yield of 1.23 g per g glucose. Glucose and xylose were utilized at same time, while cellubiose was not consumed until glucose and xylose were completely consumed. Ó 2009 Elsevier Ltd. All rights reserved.
1. Introduction Succinic acid is a potential platform chemical for the production of various high value-added derivatives. Compared with traditional synthetic methods by fossil fuels, the production of succinic acid from natural derived biomass would alleviate the dependence on oil supply for the production of these platform chemicals in the future (Delhomme et al., 2009). The fermentative production of succinic acid has been most intensively investigated with bacteria capable of producing large amounts of succinic acid, including Actinobacillus succinogenes, Mannheimia succiniciproducens, Anaerobiospirillum succiniciproducens, and recombinant Escherichia coli (Zheng et al., 2009; Kim et al., 2004; Du et al., 2008). Microbial conversion of biomass into succinic acid is attracting increasing interest as an environmentally friendly and energy-saving process. The development of a cost-effective culture medium to obtain maximum sugar utilization yield in the fermentation process is considered to be a fundamental development to the chemical production industry. Much information is available on the feasibility of converting the biomass containing crude cellulose into fermentation products. Many agricultural and industrial wastes or residues, for example, whey, wheat, straw and wood, have been reported as raw carbon materials for the production of succinic
* Corresponding author. Tel./fax: +86 010 62550913. E-mail address: [email protected] (J. Xing). 0960-8524/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved. doi:10.1016/j.biortech.2009.12.064
acid. Kim et al. (2004) used wood hydrolysate-based medium to culture M. succiniciproducens MBEL55E and the final succinic acid concentration of 11.73 g L 1 was obtained in batch fermentation, resulting in a succinic acid yield of 0.56 g g 1. Du et al. (2008) carried out two wheat-based bio-refining strategies converting wheat to succinic acid in which A. succinogene fermentation using only the wheat-derived feedstock resulted in a succinic acid concentration of 16 g L 1 with an overall yield of 0.19 g g 1. In batch fermentation of succinic acid from straw hydrolysate by A. succinogenes, 45.5 g L 1 succinic acid concentration and 0.81 g g 1 yield were attained after 48 h incubation with 58 g L of initial sugar from corn straw hydrolysate (Zheng et al., 2009). Economical succinic acid production from cane molasses can yield 50.6 g L 1 succinic acid in continuous anaerobic fermentation (Liu et al., 2008). In China, the annual biomass wastes exceed 0.7 billion tons, among which the cornstalk, wheat straw and straw wastes are around 220, 110 and 180 million tons per year, respectively (Fan et al., 2006). However, crop stalk residues can be an efficient renewable source for the cost competitive chemical production platform. Within this strategy a synthetic biomass conversion process was developed to convert feedstock wastes into succinic acid by two-step processes, especially with cotton stalks conversion for the first time. In the first place, biomass residue cornstalk or cotton stalk were converted into fermentation feedstock by enzyme hydrolysis. The feedstock was subsequently fermented by A. succinogenes BE-1 to produce succinic acid.
3293
Q. Li et al. / Bioresource Technology 101 (2010) 3292–3294
2. Methods 100
A. succinogenes BE-1, a strain that produces a high concentration of succinic acid, had been isolated from bovine rumen in our laboratory, and was collected in China General Microbiological Culture Collection Center (No. CGMCC2650). 2.2. Medium and succinic acid fermentation Effect of different carbon sources on the production of succinic acid were evaluated in batch fermentation under anaerobic conditions. The fermentation medium contained per liter: 30 g sugar, 30 g yeast extract, 2 g urea, 2 g MgCl26H2O, 1.5 g CaCl2, 0.07 g MnCl2, 4.4 g Na2HPO4, 3.3 g NaH2PO4, 30 g MgCO3. Glucose and biomass-derived carbohydrates were separately sterilized at 115 °C for 30 min and added to the medium to maintain the initial concentrations of 30 g L 1. The initial pH of the sterilized medium was adjusted to 7.0 by 10 mol L 1 NaOH solution. Seed inoculum was 5% vol 1. 2.3. Hydrolysates preparation from crop stalk wastes The corn stalk and cotton stalk residues were pretreated by steam explosion for 10 min at 1.5 MPa, and separated by a vegetation disintegrator to pass 40-mesh screen. The dried samples were treated by 1 % (w/v) NaOH and 4% (v/v) H2O2 for 24 h at the room temperature at 150 rpm. Samples were washed to neutral, and dried at 50 °C. Cellulase from Trichoderma reessi (Sunson Ltd., China) with the activity of 1.67 filter paper unit (FPU) per mg soluble protein and 0.29 b-glucosidase activity IU per mg soluble protein was used in this research. Cellulase of 20 FPU/g was added to 12% (w/v) pretreated samples with 0.05 M Tris–HCl as the pH buffer. The suspension was placed in a 500-mL flask at 50 °C, 150 rpm for 72 h. The hydrolysate samples were employed as substrate of succinic acid fermentation. 2.4. Analysis methods The different reductive sugar and products (succinate, lactate, formate, acetate and ethanol) formation during fermentation were analyzed by HPLC using an Aminex HPX-87H ion-exchange column (Bio-Rad, USA) and HP1200 chromatography working station system (Agilent Technologies, USA) equipped with UV absorbance detector (Agilent Technologies, G1315D) and refractive index detector (Agilent Technologies, G1362A). The column was eluted isocratically at a rate of 0.6 ml min 1 with 5 mM H2SO4 under 55 °C. The injection volume was 10 ll. 3. Results and discussion 3.1. Carbohydrates conversion from biomass crop stalks In this case, the hydrolysis of corn stalks and cotton stalks, was used to produce the feedstock for the bacterial fermentation. Fig. 1 shows the time course for reducing sugar concentrations obtained in the experiment. Nearly 90 g L 1 reducing sugar was generated at a high rate by action of cellulases preparations. Among the final corn stalk hydrolysate, the concentrations of glucose were about 80% in the total reducing sugar. While for the cotton stalk hydrolysate, the proportion of glucose in the total reducing sugar was about 65%, with 8% xylose and 14% cellubiose. Under the optimized conditions, the total sugar yield could reach 60 g/L in less than 10 h. The proportion of glucose in the total
Reducing sugar (g/L)
2.1. Microorganism 80 60
corn stalk cotton stalk
40 20 0 0
10
20
30
40
50
60
70
80
Time (h) Fig. 1. Time course of total sugar in batch hydrolysates of corn straw.
mixed-sugar was over 50%, which meant the hydrolysis carbohydrates could be used as the replacement of single carbon source in the traditional fermentation of succinic acid. The component on the hydrolysate did not seem to inhibit cell growth and fermentation after sterilization. 3.2. Batch fermentation from different carbon sources Succinic acid fermentation was performed in a simulated medium, containing the salts and nutrients as described earlier except the sorts of the carbohydrates. Crop stalk hydrolysate contained quantities of reducing carbohydrates, among which the concentrations of glucose were the most. In this study, main kinds of carbohydrates, such as glucose, fructose, xylose, maltose and cellobiose were chosen for further batch fermentation tests to compare the fermentability of single sugar and the hydrolysates. Each carbon source was added to give a concentration equivalent to 30 g L 1 glucose. Glucose, fructose, xylose and maltose were consumed completely in 34 h. At the end of the fermentation, about 17.2 g L 1 succinic acid, 8.4 g L 1 acetic acid, and 5.2 g L 1 lactic acid were produced. Fructose was consumed completely by 10 h, but the productivity of succinic acid was not very high. The titers of succinic acid, acetic acid and lactic acid were about 14.2 g L 1, 8.9 g L 1, and 6.0 g L 1, respectively. For the fermentation using xylose as the carbon source, the succinic acid concentration, productivity and yield were relatively very low. The concentrations of succinic acid, acetic acid and lactic acid were only 7.0 g L 1, 5.4 g L 1, and 5.3 g L 1, respectively. Compared to the fermentation using the glucose, the fermentation using maltose as the carbon source resulted in a similar final succinic acid concentration of about 16.9 g L 1 and a similar acetic, lactic acid concentration of about 9.7 g L 1, 4.9 g L 1, respectively. The utilization yield of cellobiose was relatively slow. Nearly half of the initial cellobiose was not consumed. This batch test led to 0.97 g g 1 yield with 14.7 g L 1 succinic acid, 8.2 g L 1 acetic acid, and 5.8 g L 1 lactic acid, confirming that the carbon sources are crucial components in succinic acid fermentations. 3.3. Fermentation of hydrolysates Enzymatic hydrolysis was a more effective method for treating the crop stalks in order that the enzymatic hydrolysate could be used as an inexpensive carbon source for succinic acid production. Accordingly, the initial glucose concentrations were maintained
3294
Q. Li et al. / Bioresource Technology 101 (2010) 3292–3294
Table 1 Comparison of succinic acid production from various biomass-derived sources.
a
Carbon source
Concentration (g L
Glucose Fructose Xylose Maltose Cellubiose Corn stalk Cotton stalk
17.2 ± 0.1 14.2 ± 0.2 6.9 ± 0.3 16.8 ± 0.1 14.7 ± 0.2 17.8 ± 0.2 15.8 ± 0.1
1
)
Productivity (g L
1
h
1
)
0.51 ± 0.02 0.42 ± 0.03 0.21 ± 0.05 0.50 ± 0.03 0.43 ± 0.02 0.56 ± 0.01 0.62 ± 0.01
Yielda (g g
1
)
0.70 ± 0.02 0.48 ± 0.02 0.28 ± 0.01 0.65 ± 0.04 0.97 ± 0.02 0.66 ± 0.02 1.23 ± 0.02
Reference Supplementary Supplementary Supplementary Supplementary Supplementary Supplementary Supplementary
Fig. Fig. Fig. Fig. Fig. Fig. Fig.
S1A S1B S1C S1D S1E S1F S1G
The unit is g succinic acid per g carbohydrate consumed.
30 g L 1 in the fermentation broth. At the end of fermentation, the medium contained up to 17.8 g L 1 and 15.8 g L 1 succinic acid, respectively for corn stalks and cotton stalks as the carbon source for the succinic acid fermentation process. It can be assumed that that the biomass-based generic feedstock contained sufficient essential nutrients for the succinic acid fermentation by A. succinogenes. Table 1 compared succinic acid production on the semi-defined medium and on the crop stalk-derived media. Compared with the fermentation on the semi-defined medium, the fermentation with crop stalk hydrolysate resulted in a similar succinic acid concentration and yield but with a higher productivity. Moreover, for the cotton stalk hydrolysate fermentation, the glucose to succinic acid production yield was 1.23 g g 1, which was the highest yield of the batch fermentations. This was probably due to the nutrient richness of the crop stalk hydrolysate, such as nitrogen source, minerals and vitamins (Wang et al., 2002; Gullon et al., 2008). A relatively concentrated hydrolysate contained sufficient quantity of essential nutrition for A. succinogenes growth and succinic acid formation, suggesting that the usage of stalk hydrolysate would improve process economics. Moreover, Liu et al. (2008) and Zheng et al. (2009) reported that A. succinogenes could use a wide range of carbohydrates as the carbon sources, and ferment straw hydrolysate or cane molasses onto succinic acid. But the utilization order of each sugar in the hydrolysates was different. As shown in Supplementary Fig. S2, glucose and xylose were utilized at the beginning of the fermentation, while cellubiose was not consumed until glucose and xylose completely consumed. A. succinogenes prefers to ferment monosaccharide rather than disaccharide. Thus, much attention can be drawn to increase the proportions of monosaccharide in the enzymatic hydrolysis process of crop stalks. 4. Conclusions The potential of crop stalks wastes for obtaining sugars suitable as carbon sources for succinic acid fermentation was evaluated synthetically. The hydrolysis processes of feedstock from crop stalks on the formation of reducing sugars were assessed at a high hydrolysis rate by action of enzymatic pretreatment. In batch cul-
tivation of A. succinogenes, the conversion yield of succinic acid from the biomass was relatively high. The possibility of developing a crop stalk bio-refinery for the production of succinic acid would result in improvement of process economics by using hydrolysate medium in comparison to semi-defined medium. Acknowledgement This work was supported by the Knowledge Innovation Program of the Chinese Academy of Sciences, Grant No. KSCX2-YWG-021. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.biortech.2009.12.064. References Delhomme, C., Weuster-Botz, D., Kühn, F.E., 2009. Succinic acid from renewable resources as a C4 building-block chemical – a review of the catalytic possibilities in aqueous media. Green Chem. 11, 13–26. Du, C.Y., Carol Lin, S.K., Koutinas, A., Wang, R.H., Dorado, P., Webb, C., 2008. A wheat biorefining strategy based on solid-state fermentation for fermentative production of succinic acid. Bioresour. Technol. 99, 8310–8315. Fan, Y.T., Zhang, Y.H., Zhang, S.F., Hou, H.W., Ren, B.Z., 2006. Efficient conversion of wheat straw wastes into biohydrogen gas by cow dung compost. Bioresour. Technol. 97, 500–505. Gullon, B., Yñez, R., Alonso, J.L., Parajó, J.C., 2008. L-lactic acid production from apple pomace by sequential hydrolysis and fermentation. Bioresour. Technol. 99, 308–319. Kim, D.Y., Yim, S.C., Lee, P.C., Lee, W.G., Lee, S.Y., Chang, H.N., 2004. Batch and continuous fermentation of succinic acid from wood hydrolysate by Mannheimia succiniciproducens MBEL55E. Enzyme Microbiol. Technol. 35, 648–653. Liu, Y.P., Zheng, P., Sun, Z.H., Ni, Y., Dong, J.J., Zhu, L.L., 2008. Economical succinic acid production from cane molasses by Actinobacillus succinogenes. Bioresour. Technol. 99, 1736–1742. Wang, R., Dominguez-Espinosa, R.M., Leonard, K., Koutinas, A., Webb, C., 2002. The application of a generic feedstock from wheat for microbial fermentations. Biotechnol. Prog. 18, 1033–1038. Zheng, P., Dong, J.J., Sun, Z.H., Ni, Y., Fang, L., 2009. Fermentative production of succinic acid from straw hydrolysate by Actinobacillus succinogenes. Bioresour. Technol. 100, 2425–2429.