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A facile and efficient pretreatment of corncob for bioproduction of butanol
Bioresource Technology 140 (2013) 86–89
Contents lists available at SciVerse ScienceDirect
Bioresource Technology journal homepage: www.elsevier.com/locate/biortech
A facile and efficient pretreatment of corncob for bioproduction of butanol Wei Luo ⇑, Jie Wang, Xb Liu, Hanguang Li, Hepeng Pan, Qiuya Gu, Xiaobin Yu ⇑ The Key Laboratory of Carbohydrate Chemistry & Biotechnology, Ministry of Education and School of Biotechnology, Jiangnan University, Wuxi 214122, PR China.
h i g h l i g h t s An efficient and convenient method was proposed for corncob pretreatment. Simultaneous CBMAS pretreatment and hydrolysis could reduce the processing time. The hydrolysates from CBMAS-pretreated corncob could be utilized without detoxication. Fermentable sugars were completely consumed with a yield of 9.52 g/L butanol.
a r t i c l e
i n f o
Article history: Received 12 January 2013 Received in revised form 15 April 2013 Accepted 16 April 2013 Available online 24 April 2013 Keywords: Corncob Butanol fermentation Pretreatment Combined ball milling-aqueous swelling
a b s t r a c t The present study developed a combined ball milling-aqueous swelling (CBMAS) pretreatment to accelerate the hydrolysis of corncob. The enzymatic hydrolysis of microcrystalline cellulose carried out in the plates and flasks indicated that the response of enzymatic hydrolysis to CBMAS was quite evident. The fermentable reducing sugars of hydrolysates from CBMAS-pretreated corncob was 59.8 g/L, which was 1.3 and 1.7 folds higher than those from diluted acid and alkaline pretreated corncob hydrolysates, respectively. Simultaneous CBMAS pretreatment and enzymatic hydrolysis was also conducted, reducing the processing time from 66 h to 28 h. The enzymatic hydrolysates from CBMAS-pretreated corncob could be directly utilized as the substrate for butanol fermentation without detoxication. Under the optimal conditions, fermentable sugars in the corncob hydrolysate were completely consumed to generate 9.52 g/L butanol. Ó 2013 Elsevier Ltd. All rights reserved.
1. Introduction Corncob is a major waste from corn (maize) production, which contains 30–35% hemicellulose, 40–45% cellulose and 10–20% lignin (Sheng and Marquis, 2006). As a low-cost lignocellulosic biomass, corncob is a reliable and renewable source for the production of various value-added products, such as biofuel. However, the highly ordered structure of lignocellulose constitutes the major obstacle for corncob saccharification to generate fermentable sugars (Demiral et al., 2012). Although various pretreatment methods have been developed to improve the hydrolysis efficiency of lignocellulosic complex, the main challenges still exist in the matter of saccharification performance and environmental pollution, etc. (Kumar et al., 2009). Furthermore, by-products generating during the hydrolysis process are often toxic to the fermenting microorganisms and affects the subsequent fermentation process (Palmqvist and Hahn-Hägerdal, 2000).
⇑ Corresponding authors. Tel.: +86 510 85329272. E-mail addresses: [email protected] (W. Luo), [email protected] (X. Yu). 0960-8524/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.biortech.2013.04.063
To aim at above problems, the present work has developed a facile and efficient method called combined ball milling-aqueous swelling (CBMAS) for corncob pretreatment. The shaker-driving ball milling was of low energy consumption, but notably improved the enzymatic hydrolysis of microcrystalline cellulose combined with the aqueous swelling effect. This method was then adopted for corncob pretreatment, where the processing performance was compared with that of chemical pretreatment. Thereafter, the pretreatment and saccharification of corncob was optimized to shorten the processing time. Finally, the generated corncob hydrolysates were directly used as the substrate for biobutanol production. 2. Methods 2.1. Materials and reagents Corncob was collected from local market and air dried. After chipping, corncob was sieved by a 40 mesh screen and stored for future use. Cellulase (150 FPU/g) and xylanase (1.67 105 U/g) were supplied by Sino Enzymes Co. Ltd. (China).
W. Luo et al. / Bioresource Technology 140 (2013) 86–89
2.2. Pretreatment and saccharification of microcrystalline cellulose and corncob
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3. Results and discussion 3.1. Evaluation of CBMAS pretreatment
The weighed sample (20 g) of microcrystalline cellulose or chipped corncob was prepared in a flask with the supplement of 200 mL water and 50 glass balls (1 mm diameter). The sample was stirred at 200 rpm in a shaker at room temperature for adequate time (24 h). In addition, chipped corncob was pretreated with diluted acid or alkaline, as described by previous reports (Chang et al., 2012; Torre et al., 2008). To evaluate the effectiveness of CBMAS method, 1.0% (w/v) CBMAS- and non-pretreated microcrystalline cellulose was paved in the plates supplemented with 2% agar, respectively. One microlitre cellulase solution of 5 FPU/mL was distributed in the centre of plates and placed in the 50 °C incubator for 24 h. For liquid hydrolysis, samples of 20 g pretreated microcrystalline cellulose or corncob were thrown in the 200 mL solution of 50 mM sodium citrate buffer (pH 5.0) in all experimental cases. Enzymatic hydrolysis was performed in the shake flasks agitated at 200 rpm and 50 °C for 28 h, initiated by an enzyme cocktail constituting 10 FPU cellulase and 2500 U xylanase per gram dry substrate, except only 10 FPU cellulase per gram substrate for microcrystalline cellulose.
2.3. Butanol fermentation Clostridium beijerinckii G-23 was isolated from forest soil and used as the host for butanol fermentation. Tryptone-yeast extract-acetate (TYA) medium was used for the preparation of inoculum (Tanaka et al., 2012). Batch fermentation for butanol production was conducted for 88 h at 37 °C within the P2 medium (Lu et al., 2011) containing corncob hydrolysate as the carbon source. The samples were periodically withdrawn from the broth and centrifuged at 15,000 rpm for 2 min. The remaining supernatants were subjected for subsequent analysis.
A drop of cellulase solution caused the formation of a distinct transparent zone on the plate with CBMAS-treated microcrystalline cellulose, while no change was observed on the plate with non-treated microcrystalline cellulose. This result suggested that the CBMAS pretreatment could effectively disrupt the configuration of microcrystalline cellulose and increase its accessibility to cellulase. Accordingly, the resulted glucose in the hydrolysate of CBMAS-treated microcrystalline cellulose was 112.1 g/L, with a conversion rate of 100.0% (Fig. 1), which were about two folds higher than those of non-treated microcrystalline cellulose. The enhanced performance of enzymatic hydrolysis of CBMAStreated microcrystalline cellulose may derive from the combined effects of both mechanical attrition and aqueous swelling. The reduction of crystallinity and the crystal size caused by mechanical attrition (Abdullah and Wu, 2009) could increase the content of amorphous cellulose, which was considered as the main reason for improved digestibility of milled cellulose (Yu et al., 2010). Compared with other mechanical milling methods, glass ball milling assisted by the shaker rotation in this study was mild and consumed low energy, but giving a much satisfactory processing performance. This could be the contribution of aqueous swelling. It was found that the treatment performance of wet milling was much better than that of dry milling (Taherzadeh and Karimi, 2008). And the enzymatic hydrolysis of microcrystalline cellulose (cellulose I) was greatly accelerated via its conversion to the cellulose II hydrate form (Zhao et al., 2006). Thus, with the assistance of water, the crystalline state of cellulose was easily changed and facilitated the simultaneous mechanical milling process. In spite of literature support, the above-mentioned assumptions should be verified in the subsequent study regarding the mechanism of CBMAS pretreatment.
3.2. Comparison of different pretreatment methods for corncob saccharification 2.4. Analytical methods The concentration of total reducing sugars was analyzed by DNS method (Chang et al., 2012). Monosaccharides in the liquid were determined by Dionex ion chromatography ICS-5000 (Sunnyvale, CA, USA) with a Dionex pulsed amperometric detector equipped with an Au electrode and a Dionex Carbopac PA20 column (150 mm 3 mm). The mobile phase was composed of 250 mM NaOH, 1 M NaAc and water, with a flow rate of 0.5 mL/min. The gradient elution program was indicated in Table 1. The fermentation products were analyzed using a gas chromatograph (Agilent 6820, America) equipped with a flame ionization detector (FID) and a 3000 0.32 mm capillary column. The oven temperature was set at 90 °C, and the injector and detector temperatures were set at 240 °C.
As a lignocellulosic biomass, corncob needs pretreatment for the subsequent saccharification. Although various methods have been tested (Demiral et al., 2012), acid or alkaline hydrolysis are still considered as the effective and cost-efficient alternatives. These two methods thus were adopted to compare with CBMAS
Table 1 Gradient elution program of monosaccharide analysis. Time (min)
Water (%, v/v)
250 mM NaOH (%, v/v)
1 M NaAc (%, v/v)
0 21.0 21.1 30.0 30.1 50.0
98.2 98.2 93.2 78.2 20.0 20.0
1.8 1.8 1.8 1.8 80.0 80.0
0 0 5.0 20.0 0 0
Fig. 1. Effects of CBMAS pretreatment on the enzymatic hydrolysis of microcrystalline cellulose in the flask.
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W. Luo et al. / Bioresource Technology 140 (2013) 86–89
Fig. 2. Effect of different pretreatment methods on the formation of the reducing sugars derived from corncob. For diluted acid hydrolysis, 1% (v/v) sulfuric acid was applied for 60 min at room temperature. Alkaline pretreatment was performed with 3% (w/v) NaOH for 24 h at room temperature, and then for enzymatic hydrolysis.
for corncob pretreatment. The data in Fig. 2 show that CBMAS pretreatment generated 59.8 g/L reducing sugars, which was obviously higher than that from acid or alkaline hydrolysis. As it is well known, the fractions of cellulose and hemicellulose in the corncob can be hydrolyzed to generate fermentable sugars, while lignin cannot be but limits the hydrolysis rate by acting as a physical barrier. Alkaline pretreatment breaks the bonds between lignin and carbohydrates and disrupts the lignin structure, increasing the digestibility of cellulose and hemicellulose (Kim and Holtzapple, 2006). Diluted acid hydrolysis can solubilize the hemicellulosic fraction of the biomass and convert it to fermentable sugars, but is difficult to decompose the fraction of cellulose and lignin (Kumar et al., 2009). In the present study, cellulose is the main component (40–45%) in corncob, which would result in the waste of cellulose and limit the yield of glucose when using diluted acid hydrolysis. The comparison result suggests that CBMAS pretreatment could break the outer protection of lignin and looses the cellulose and hemicellulose and facilitate the subsequent enzymatic hydrolysis. Moreover, the continuous ball milling in the water environment could cause the conversion of crystalline structure. Thus, CBMAS pretreatment presented high efficiency and was environmentally friendly because no chemical reagents were supplemented during the process. 3.3. Optimization of processing time The CBMAS pretreatment of corncob was time-dependent (Fig. 3). The reducing sugars concentration in the hydrolysates increased to the plateau until 16 h-processing, and further action gave no obvious improvement. The pretreated corncob was subsequently hydrolyzed with cellulase and xylanase for the saccharification. At least 50 h were required for the complete saccharification (Fig. 3). A simultaneous pretreatment and saccharification processing was proposed and evaluated, in order to shorten the processing time. With this method, 28 h are adequate for the complete saccharification (Fig. 3), leading to a decrease of enzymatic hydrolysis time by 44% and the whole saccharification period (including pretreatment and enzymatic hydrolysis time) by 58%, respectively. Furcht and Silla have also demonstrated that the saccharification time is greatly reduced by simultaneously milling and hydrolyzing the cellulose, as opposed to first milling and then hydrolyzing (Furcht and Silla, 1990).
Fig. 3. Effect of processing time of pretreatment and saccharification on the recovery of reducing sugars. j represents the effect of CBMAS pretreatment time; the time of enzymatic hydrolysis for all experimental groups was 50 h. d indicates that CBMAS pretreatment was performed for 16 h followed by the addition of hydrolyzing enzymes for different durations. N shows that the CBMAS pretreatment and the addition of hydrolyzing enzymes were conducted at the same time.
3.4. Bioproduction of butanol using corncob hydrolysates Because no chemical was introduced into CBMAS pretreatment, the resulted corncob hydrolysates could be directly used as substrate for butanol production without detoxication. As shown in Table 2, the production of butanol and total solvent was improved with the increasing initial concentration of reducing sugars from 32.6 to 102.5 g/L, while their corresponding yields presented slow reduction, which suggests some possible concentration-dependent inhibition. The initial concentration of 66.3 g/ L reducing sugars gave the highest substrate conversion (90.1%) and its process curve for butanol fermentation was shown in Fig. 4. To further improve the substrate conversion, the initial monosaccharide contents and their utilization were analyzed (Fig. 5). Glucose and xylose are the main components in the corncob hydrolysates, and the conversion of glucose is much better than the conversion of xylose. Culture conditions such as nitrogen source, temperature and pH were optimized to enhance the substrate utilization and butanol production (data not shown). The maximum production and yield of butanol arrived at 9.52 g/L and 0.16 g/g reducing sugars, respectively, with a very good utilization of fermentable sugars. These results prove that CBMAS method is efficient for corncob pretreatment and butanol bioproduction. From an industrial perspective, product yield is an important indicator to determine the efficiency of fermentative production. In the present study, 16.0% and 24.2% of the total reducing sugars were converted to butanol and the total solvents, which are comparable to most previous Clostridium species (Ezeji and Blaschek, 2008; Liu et al., 2010). In a normal batch culture, solvent-producing Clostridium species also produce other compounds such as hydrogen, CO2, acetate, and butyrate. The gases composed of 60% (v/v) CO2 and the balanced hydrogen evolved from butanol fermentation are usually estimated as 1.5 times the mass of solvents produced (Pfromm et al., 2010). The humid hydrogen/CO2 gas mixture issuing from the broth could be separated to recover a small amount of hydrogen but this is not the focus here and likely will not be cost effective.
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W. Luo et al. / Bioresource Technology 140 (2013) 86–89 Table 2 Effect of initial sugar titers of CBMAS-pretreated corncob enzymatic hydrolysates on the butanol production and reducing sugar utilization. Initial reducing sugars titer (g/L)
Butanol titer (g/L)
Butanol yield (g/g)
Total solvents (g/L)
Solvent yield (g/g)
Reducing sugars conversion rate (%)
32.6 44.6 66.3 102.5 133.1
4.68 ± 0.18 6.07 ± 0.16 7.42 ± 0.14 8.68 ± 0.24 6.36 ± 0.72
0.18 0.16 0.12 0.11 0.08
7.42 ± 0.09 8.98 ± 0.36 12.07 ± 0.21 12.08 ± 1.50 9.75 ± 0.48
0.28 0.23 0.20 0.15 0.13
80.3 87.8 90.1 76.3 56.3
Acknowledgements This work was supported by National Natural Science Foundation of China (Grant No. 21176105), the Fundamental Research Funds for the Central Universities (Grant No. JUSRP111A24), Program of the Key Laboratory of Industrial Biotechnology, Ministry of Education, China (Grant No. KLIB-KF201105), the 111 Project (No. 111-2-06) and a Project Funded by the Priority Academic Program Development of Jiangsu Higher Education Institutions. References
Fig. 4. Time courses of butanol fermentation by Clostridium beijerinckii G-23.
Fig. 5. The initial monosaccharide composition in the broth and their conversion rates.
4. Conclusions This investigation concludes that the developed CBMAS pretreatment is a much efficient, convenient, and environmentally friendly method for the generation of fermentable sugar from corncob. Meanwhile, simultaneous CBMAS pretreatment and enzymatic hydrolysis could greatly reduce the processing time. The enzymatic hydrolysates from CBMAS-pretreated corncob were non-toxic to the fermentation host microbe and may be applied for the practical production of biofuels and sugar-derived chemical stocks.
Abdullah, H., Wu, H., 2009. Biochar as a fuel: 1. Properties and grindability of biochars produced from the pyrolysis of mallee wood under slow-heating conditions. Energy Fuels 23 (8), 4174–4181. Chang, Y.H., Chang, K.S., Huang, C.W., Hsu, C.L., Jang, H.D., 2012. Comparison of batch and fed-batch fermentations using corncob hydrolysate for bioethanol production. Fuel 97, 166–173. _ Eryazıcı, A., Sßensöz, S., 2012. Bio-oil production from pyrolysis of Demiral, I., corncob (Zea mays L.). Biomass Bioenergy 36, 43–49. Ezeji, T., Blaschek, H.P., 2008. Fermentation of dried distillers’ grains and solubles (DDGS) hydrolysates to solvents and value-added products by solventogenic clostridia. Bioresour. Technol. 99 (12), 5232–5242. Furcht, P.W., Silla, H., 1990. Comparison of simultaneous wet milling and enzymatic hydrolysis of cellulose in ball mill and attrition mill reactors. Biotechnol. Bioeng. 35 (6), 630–645. Kim, S., Holtzapple, M.T., 2006. Delignification kinetics of corn stover in lime pretreatment. Bioresour. Technol. 97 (5), 778–785. Kumar, P., Barrett, D.M., Delwiche, M.J., Stroeve, P., 2009. Methods for pretreatment of lignocellulosic biomass for efficient hydrolysis and biofuel production. Ind. Eng. Chem. Res. 48 (8), 3713–3729. Liu, Z., Ying, Y., Li, F., Ma, C., Xu, P., 2010. Butanol production by Clostridium beijerinckii ATCC 55025 from wheat bran. J. Ind. Microbiol. Biotechnol. 37 (5), 495–501. Lu, C., Zhao, J., Yang, S.T., Wei, D., 2011. Fed-batch fermentation for n-butanol production from cassava bagasse hydrolysate in a fibrous bed bioreactor with continuous gas stripping. Bioresour. Technol. 104, 380–387. Palmqvist, E., Hahn-Hägerdal, B., 2000. Fermentation of lignocellulosic hydrolysates. II: inhibitors and mechanisms of inhibition. Bioresour. Technol. 74 (1), 25–33. Pfromm, P.H., Amanor-Boadu, V., Nelson, R., Vadlani, P., Madl, R., 2010. Bio-butanol vs. bio-ethanol: a technical and economic assessment for corn and switchgrass fermented by yeast or Clostridium acetobutylicum. Biomass Bioenergy 34 (4), 515–524. Sheng, J., Marquis, R.E., 2006. Enhanced acid resistance of oral streptococci at lethal pH values associated with acid-tolerant catabolism and with ATP synthase activity. FEMS Microbiol. Lett. 262 (1), 93–98. Taherzadeh, M.J., Karimi, K., 2008. Pretreatment of lignocellulosic wastes to improve ethanol and biogas production: a review. Int. J. Mol. Sci. 9 (9), 1621– 1651. Tanaka, S., Tashiro, Y., Kobayashi, G., Ikegami, T., Negishi, H., Sakaki, K., 2012. Membrane-assisted extractive butanol fermentation by Clostridium saccharoperbutylacetonicum N1–4 with 1-dodecanol as the extractant. Bioresour. Technol. 116, 448–452. Torre, P., Aliakbarian, B., Rivas, B., Domínguez, J.M., Converti, A., 2008. Release of ferulic acid from corn cobs by alkaline hydrolysis. Biochem. Eng. J. 40 (3), 500– 506. Yu, J., Li, Z., Ye, Q., Yang, Y., Chen, S., 2010. Development of succinic acid production from corncob hydrolysate by Actinobacillus succinogenes. J. Ind. Microbiol. Biotechnol. 37 (10), 1033–1040. Zhao, H.B., Kwak, J.H., Wang, Y., Franz, J.A., White, J.M., Holladay, J.E., 2006. Effects of crystallinity on dilute acid hydrolysis of cellulose by cellulose ball-milling study. Energy Fuels 20 (2), 807–811.
Contents lists available at SciVerse ScienceDirect
Bioresource Technology journal homepage: www.elsevier.com/locate/biortech
A facile and efficient pretreatment of corncob for bioproduction of butanol Wei Luo ⇑, Jie Wang, Xb Liu, Hanguang Li, Hepeng Pan, Qiuya Gu, Xiaobin Yu ⇑ The Key Laboratory of Carbohydrate Chemistry & Biotechnology, Ministry of Education and School of Biotechnology, Jiangnan University, Wuxi 214122, PR China.
h i g h l i g h t s An efficient and convenient method was proposed for corncob pretreatment. Simultaneous CBMAS pretreatment and hydrolysis could reduce the processing time. The hydrolysates from CBMAS-pretreated corncob could be utilized without detoxication. Fermentable sugars were completely consumed with a yield of 9.52 g/L butanol.
a r t i c l e
i n f o
Article history: Received 12 January 2013 Received in revised form 15 April 2013 Accepted 16 April 2013 Available online 24 April 2013 Keywords: Corncob Butanol fermentation Pretreatment Combined ball milling-aqueous swelling
a b s t r a c t The present study developed a combined ball milling-aqueous swelling (CBMAS) pretreatment to accelerate the hydrolysis of corncob. The enzymatic hydrolysis of microcrystalline cellulose carried out in the plates and flasks indicated that the response of enzymatic hydrolysis to CBMAS was quite evident. The fermentable reducing sugars of hydrolysates from CBMAS-pretreated corncob was 59.8 g/L, which was 1.3 and 1.7 folds higher than those from diluted acid and alkaline pretreated corncob hydrolysates, respectively. Simultaneous CBMAS pretreatment and enzymatic hydrolysis was also conducted, reducing the processing time from 66 h to 28 h. The enzymatic hydrolysates from CBMAS-pretreated corncob could be directly utilized as the substrate for butanol fermentation without detoxication. Under the optimal conditions, fermentable sugars in the corncob hydrolysate were completely consumed to generate 9.52 g/L butanol. Ó 2013 Elsevier Ltd. All rights reserved.
1. Introduction Corncob is a major waste from corn (maize) production, which contains 30–35% hemicellulose, 40–45% cellulose and 10–20% lignin (Sheng and Marquis, 2006). As a low-cost lignocellulosic biomass, corncob is a reliable and renewable source for the production of various value-added products, such as biofuel. However, the highly ordered structure of lignocellulose constitutes the major obstacle for corncob saccharification to generate fermentable sugars (Demiral et al., 2012). Although various pretreatment methods have been developed to improve the hydrolysis efficiency of lignocellulosic complex, the main challenges still exist in the matter of saccharification performance and environmental pollution, etc. (Kumar et al., 2009). Furthermore, by-products generating during the hydrolysis process are often toxic to the fermenting microorganisms and affects the subsequent fermentation process (Palmqvist and Hahn-Hägerdal, 2000).
⇑ Corresponding authors. Tel.: +86 510 85329272. E-mail addresses: [email protected] (W. Luo), [email protected] (X. Yu). 0960-8524/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.biortech.2013.04.063
To aim at above problems, the present work has developed a facile and efficient method called combined ball milling-aqueous swelling (CBMAS) for corncob pretreatment. The shaker-driving ball milling was of low energy consumption, but notably improved the enzymatic hydrolysis of microcrystalline cellulose combined with the aqueous swelling effect. This method was then adopted for corncob pretreatment, where the processing performance was compared with that of chemical pretreatment. Thereafter, the pretreatment and saccharification of corncob was optimized to shorten the processing time. Finally, the generated corncob hydrolysates were directly used as the substrate for biobutanol production. 2. Methods 2.1. Materials and reagents Corncob was collected from local market and air dried. After chipping, corncob was sieved by a 40 mesh screen and stored for future use. Cellulase (150 FPU/g) and xylanase (1.67 105 U/g) were supplied by Sino Enzymes Co. Ltd. (China).
W. Luo et al. / Bioresource Technology 140 (2013) 86–89
2.2. Pretreatment and saccharification of microcrystalline cellulose and corncob
87
3. Results and discussion 3.1. Evaluation of CBMAS pretreatment
The weighed sample (20 g) of microcrystalline cellulose or chipped corncob was prepared in a flask with the supplement of 200 mL water and 50 glass balls (1 mm diameter). The sample was stirred at 200 rpm in a shaker at room temperature for adequate time (24 h). In addition, chipped corncob was pretreated with diluted acid or alkaline, as described by previous reports (Chang et al., 2012; Torre et al., 2008). To evaluate the effectiveness of CBMAS method, 1.0% (w/v) CBMAS- and non-pretreated microcrystalline cellulose was paved in the plates supplemented with 2% agar, respectively. One microlitre cellulase solution of 5 FPU/mL was distributed in the centre of plates and placed in the 50 °C incubator for 24 h. For liquid hydrolysis, samples of 20 g pretreated microcrystalline cellulose or corncob were thrown in the 200 mL solution of 50 mM sodium citrate buffer (pH 5.0) in all experimental cases. Enzymatic hydrolysis was performed in the shake flasks agitated at 200 rpm and 50 °C for 28 h, initiated by an enzyme cocktail constituting 10 FPU cellulase and 2500 U xylanase per gram dry substrate, except only 10 FPU cellulase per gram substrate for microcrystalline cellulose.
2.3. Butanol fermentation Clostridium beijerinckii G-23 was isolated from forest soil and used as the host for butanol fermentation. Tryptone-yeast extract-acetate (TYA) medium was used for the preparation of inoculum (Tanaka et al., 2012). Batch fermentation for butanol production was conducted for 88 h at 37 °C within the P2 medium (Lu et al., 2011) containing corncob hydrolysate as the carbon source. The samples were periodically withdrawn from the broth and centrifuged at 15,000 rpm for 2 min. The remaining supernatants were subjected for subsequent analysis.
A drop of cellulase solution caused the formation of a distinct transparent zone on the plate with CBMAS-treated microcrystalline cellulose, while no change was observed on the plate with non-treated microcrystalline cellulose. This result suggested that the CBMAS pretreatment could effectively disrupt the configuration of microcrystalline cellulose and increase its accessibility to cellulase. Accordingly, the resulted glucose in the hydrolysate of CBMAS-treated microcrystalline cellulose was 112.1 g/L, with a conversion rate of 100.0% (Fig. 1), which were about two folds higher than those of non-treated microcrystalline cellulose. The enhanced performance of enzymatic hydrolysis of CBMAStreated microcrystalline cellulose may derive from the combined effects of both mechanical attrition and aqueous swelling. The reduction of crystallinity and the crystal size caused by mechanical attrition (Abdullah and Wu, 2009) could increase the content of amorphous cellulose, which was considered as the main reason for improved digestibility of milled cellulose (Yu et al., 2010). Compared with other mechanical milling methods, glass ball milling assisted by the shaker rotation in this study was mild and consumed low energy, but giving a much satisfactory processing performance. This could be the contribution of aqueous swelling. It was found that the treatment performance of wet milling was much better than that of dry milling (Taherzadeh and Karimi, 2008). And the enzymatic hydrolysis of microcrystalline cellulose (cellulose I) was greatly accelerated via its conversion to the cellulose II hydrate form (Zhao et al., 2006). Thus, with the assistance of water, the crystalline state of cellulose was easily changed and facilitated the simultaneous mechanical milling process. In spite of literature support, the above-mentioned assumptions should be verified in the subsequent study regarding the mechanism of CBMAS pretreatment.
3.2. Comparison of different pretreatment methods for corncob saccharification 2.4. Analytical methods The concentration of total reducing sugars was analyzed by DNS method (Chang et al., 2012). Monosaccharides in the liquid were determined by Dionex ion chromatography ICS-5000 (Sunnyvale, CA, USA) with a Dionex pulsed amperometric detector equipped with an Au electrode and a Dionex Carbopac PA20 column (150 mm 3 mm). The mobile phase was composed of 250 mM NaOH, 1 M NaAc and water, with a flow rate of 0.5 mL/min. The gradient elution program was indicated in Table 1. The fermentation products were analyzed using a gas chromatograph (Agilent 6820, America) equipped with a flame ionization detector (FID) and a 3000 0.32 mm capillary column. The oven temperature was set at 90 °C, and the injector and detector temperatures were set at 240 °C.
As a lignocellulosic biomass, corncob needs pretreatment for the subsequent saccharification. Although various methods have been tested (Demiral et al., 2012), acid or alkaline hydrolysis are still considered as the effective and cost-efficient alternatives. These two methods thus were adopted to compare with CBMAS
Table 1 Gradient elution program of monosaccharide analysis. Time (min)
Water (%, v/v)
250 mM NaOH (%, v/v)
1 M NaAc (%, v/v)
0 21.0 21.1 30.0 30.1 50.0
98.2 98.2 93.2 78.2 20.0 20.0
1.8 1.8 1.8 1.8 80.0 80.0
0 0 5.0 20.0 0 0
Fig. 1. Effects of CBMAS pretreatment on the enzymatic hydrolysis of microcrystalline cellulose in the flask.
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Fig. 2. Effect of different pretreatment methods on the formation of the reducing sugars derived from corncob. For diluted acid hydrolysis, 1% (v/v) sulfuric acid was applied for 60 min at room temperature. Alkaline pretreatment was performed with 3% (w/v) NaOH for 24 h at room temperature, and then for enzymatic hydrolysis.
for corncob pretreatment. The data in Fig. 2 show that CBMAS pretreatment generated 59.8 g/L reducing sugars, which was obviously higher than that from acid or alkaline hydrolysis. As it is well known, the fractions of cellulose and hemicellulose in the corncob can be hydrolyzed to generate fermentable sugars, while lignin cannot be but limits the hydrolysis rate by acting as a physical barrier. Alkaline pretreatment breaks the bonds between lignin and carbohydrates and disrupts the lignin structure, increasing the digestibility of cellulose and hemicellulose (Kim and Holtzapple, 2006). Diluted acid hydrolysis can solubilize the hemicellulosic fraction of the biomass and convert it to fermentable sugars, but is difficult to decompose the fraction of cellulose and lignin (Kumar et al., 2009). In the present study, cellulose is the main component (40–45%) in corncob, which would result in the waste of cellulose and limit the yield of glucose when using diluted acid hydrolysis. The comparison result suggests that CBMAS pretreatment could break the outer protection of lignin and looses the cellulose and hemicellulose and facilitate the subsequent enzymatic hydrolysis. Moreover, the continuous ball milling in the water environment could cause the conversion of crystalline structure. Thus, CBMAS pretreatment presented high efficiency and was environmentally friendly because no chemical reagents were supplemented during the process. 3.3. Optimization of processing time The CBMAS pretreatment of corncob was time-dependent (Fig. 3). The reducing sugars concentration in the hydrolysates increased to the plateau until 16 h-processing, and further action gave no obvious improvement. The pretreated corncob was subsequently hydrolyzed with cellulase and xylanase for the saccharification. At least 50 h were required for the complete saccharification (Fig. 3). A simultaneous pretreatment and saccharification processing was proposed and evaluated, in order to shorten the processing time. With this method, 28 h are adequate for the complete saccharification (Fig. 3), leading to a decrease of enzymatic hydrolysis time by 44% and the whole saccharification period (including pretreatment and enzymatic hydrolysis time) by 58%, respectively. Furcht and Silla have also demonstrated that the saccharification time is greatly reduced by simultaneously milling and hydrolyzing the cellulose, as opposed to first milling and then hydrolyzing (Furcht and Silla, 1990).
Fig. 3. Effect of processing time of pretreatment and saccharification on the recovery of reducing sugars. j represents the effect of CBMAS pretreatment time; the time of enzymatic hydrolysis for all experimental groups was 50 h. d indicates that CBMAS pretreatment was performed for 16 h followed by the addition of hydrolyzing enzymes for different durations. N shows that the CBMAS pretreatment and the addition of hydrolyzing enzymes were conducted at the same time.
3.4. Bioproduction of butanol using corncob hydrolysates Because no chemical was introduced into CBMAS pretreatment, the resulted corncob hydrolysates could be directly used as substrate for butanol production without detoxication. As shown in Table 2, the production of butanol and total solvent was improved with the increasing initial concentration of reducing sugars from 32.6 to 102.5 g/L, while their corresponding yields presented slow reduction, which suggests some possible concentration-dependent inhibition. The initial concentration of 66.3 g/ L reducing sugars gave the highest substrate conversion (90.1%) and its process curve for butanol fermentation was shown in Fig. 4. To further improve the substrate conversion, the initial monosaccharide contents and their utilization were analyzed (Fig. 5). Glucose and xylose are the main components in the corncob hydrolysates, and the conversion of glucose is much better than the conversion of xylose. Culture conditions such as nitrogen source, temperature and pH were optimized to enhance the substrate utilization and butanol production (data not shown). The maximum production and yield of butanol arrived at 9.52 g/L and 0.16 g/g reducing sugars, respectively, with a very good utilization of fermentable sugars. These results prove that CBMAS method is efficient for corncob pretreatment and butanol bioproduction. From an industrial perspective, product yield is an important indicator to determine the efficiency of fermentative production. In the present study, 16.0% and 24.2% of the total reducing sugars were converted to butanol and the total solvents, which are comparable to most previous Clostridium species (Ezeji and Blaschek, 2008; Liu et al., 2010). In a normal batch culture, solvent-producing Clostridium species also produce other compounds such as hydrogen, CO2, acetate, and butyrate. The gases composed of 60% (v/v) CO2 and the balanced hydrogen evolved from butanol fermentation are usually estimated as 1.5 times the mass of solvents produced (Pfromm et al., 2010). The humid hydrogen/CO2 gas mixture issuing from the broth could be separated to recover a small amount of hydrogen but this is not the focus here and likely will not be cost effective.
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W. Luo et al. / Bioresource Technology 140 (2013) 86–89 Table 2 Effect of initial sugar titers of CBMAS-pretreated corncob enzymatic hydrolysates on the butanol production and reducing sugar utilization. Initial reducing sugars titer (g/L)
Butanol titer (g/L)
Butanol yield (g/g)
Total solvents (g/L)
Solvent yield (g/g)
Reducing sugars conversion rate (%)
32.6 44.6 66.3 102.5 133.1
4.68 ± 0.18 6.07 ± 0.16 7.42 ± 0.14 8.68 ± 0.24 6.36 ± 0.72
0.18 0.16 0.12 0.11 0.08
7.42 ± 0.09 8.98 ± 0.36 12.07 ± 0.21 12.08 ± 1.50 9.75 ± 0.48
0.28 0.23 0.20 0.15 0.13
80.3 87.8 90.1 76.3 56.3
Acknowledgements This work was supported by National Natural Science Foundation of China (Grant No. 21176105), the Fundamental Research Funds for the Central Universities (Grant No. JUSRP111A24), Program of the Key Laboratory of Industrial Biotechnology, Ministry of Education, China (Grant No. KLIB-KF201105), the 111 Project (No. 111-2-06) and a Project Funded by the Priority Academic Program Development of Jiangsu Higher Education Institutions. References
Fig. 4. Time courses of butanol fermentation by Clostridium beijerinckii G-23.
Fig. 5. The initial monosaccharide composition in the broth and their conversion rates.
4. Conclusions This investigation concludes that the developed CBMAS pretreatment is a much efficient, convenient, and environmentally friendly method for the generation of fermentable sugar from corncob. Meanwhile, simultaneous CBMAS pretreatment and enzymatic hydrolysis could greatly reduce the processing time. The enzymatic hydrolysates from CBMAS-pretreated corncob were non-toxic to the fermentation host microbe and may be applied for the practical production of biofuels and sugar-derived chemical stocks.
Abdullah, H., Wu, H., 2009. Biochar as a fuel: 1. Properties and grindability of biochars produced from the pyrolysis of mallee wood under slow-heating conditions. Energy Fuels 23 (8), 4174–4181. Chang, Y.H., Chang, K.S., Huang, C.W., Hsu, C.L., Jang, H.D., 2012. Comparison of batch and fed-batch fermentations using corncob hydrolysate for bioethanol production. Fuel 97, 166–173. _ Eryazıcı, A., Sßensöz, S., 2012. Bio-oil production from pyrolysis of Demiral, I., corncob (Zea mays L.). Biomass Bioenergy 36, 43–49. Ezeji, T., Blaschek, H.P., 2008. Fermentation of dried distillers’ grains and solubles (DDGS) hydrolysates to solvents and value-added products by solventogenic clostridia. Bioresour. Technol. 99 (12), 5232–5242. Furcht, P.W., Silla, H., 1990. Comparison of simultaneous wet milling and enzymatic hydrolysis of cellulose in ball mill and attrition mill reactors. Biotechnol. Bioeng. 35 (6), 630–645. Kim, S., Holtzapple, M.T., 2006. Delignification kinetics of corn stover in lime pretreatment. Bioresour. Technol. 97 (5), 778–785. Kumar, P., Barrett, D.M., Delwiche, M.J., Stroeve, P., 2009. Methods for pretreatment of lignocellulosic biomass for efficient hydrolysis and biofuel production. Ind. Eng. Chem. Res. 48 (8), 3713–3729. Liu, Z., Ying, Y., Li, F., Ma, C., Xu, P., 2010. Butanol production by Clostridium beijerinckii ATCC 55025 from wheat bran. J. Ind. Microbiol. Biotechnol. 37 (5), 495–501. Lu, C., Zhao, J., Yang, S.T., Wei, D., 2011. Fed-batch fermentation for n-butanol production from cassava bagasse hydrolysate in a fibrous bed bioreactor with continuous gas stripping. Bioresour. Technol. 104, 380–387. Palmqvist, E., Hahn-Hägerdal, B., 2000. Fermentation of lignocellulosic hydrolysates. II: inhibitors and mechanisms of inhibition. Bioresour. Technol. 74 (1), 25–33. Pfromm, P.H., Amanor-Boadu, V., Nelson, R., Vadlani, P., Madl, R., 2010. Bio-butanol vs. bio-ethanol: a technical and economic assessment for corn and switchgrass fermented by yeast or Clostridium acetobutylicum. Biomass Bioenergy 34 (4), 515–524. Sheng, J., Marquis, R.E., 2006. Enhanced acid resistance of oral streptococci at lethal pH values associated with acid-tolerant catabolism and with ATP synthase activity. FEMS Microbiol. Lett. 262 (1), 93–98. Taherzadeh, M.J., Karimi, K., 2008. Pretreatment of lignocellulosic wastes to improve ethanol and biogas production: a review. Int. J. Mol. Sci. 9 (9), 1621– 1651. Tanaka, S., Tashiro, Y., Kobayashi, G., Ikegami, T., Negishi, H., Sakaki, K., 2012. Membrane-assisted extractive butanol fermentation by Clostridium saccharoperbutylacetonicum N1–4 with 1-dodecanol as the extractant. Bioresour. Technol. 116, 448–452. Torre, P., Aliakbarian, B., Rivas, B., Domínguez, J.M., Converti, A., 2008. Release of ferulic acid from corn cobs by alkaline hydrolysis. Biochem. Eng. J. 40 (3), 500– 506. Yu, J., Li, Z., Ye, Q., Yang, Y., Chen, S., 2010. Development of succinic acid production from corncob hydrolysate by Actinobacillus succinogenes. J. Ind. Microbiol. Biotechnol. 37 (10), 1033–1040. Zhao, H.B., Kwak, J.H., Wang, Y., Franz, J.A., White, J.M., Holladay, J.E., 2006. Effects of crystallinity on dilute acid hydrolysis of cellulose by cellulose ball-milling study. Energy Fuels 20 (2), 807–811.