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Optimization of fed-batch enzymatic hydrolysis from alkali-pretreated sugarcane bagasse for high-concentration sugar production
Accepted Manuscript Optimization of fed-batch enzymatic hydrolysis from alkali-pretreated sugarcane bagasse for high-concentration sugar production Yueshu Gao, Jingliang Xu, Zhenhong Yuan, Yu Zhang, Yunyun Liu, Cuiyi Liang PII: DOI: Reference:
S0960-8524(14)00693-2 http://dx.doi.org/10.1016/j.biortech.2014.05.034 BITE 13442
To appear in:
Bioresource Technology
Received Date: Revised Date: Accepted Date:
9 March 2014 10 May 2014 12 May 2014
Please cite this article as: Gao, Y., Xu, J., Yuan, Z., Zhang, Y., Liu, Y., Liang, C., Optimization of fed-batch enzymatic hydrolysis from alkali-pretreated sugarcane bagasse for high-concentration sugar production, Bioresource Technology (2014), doi: http://dx.doi.org/10.1016/j.biortech.2014.05.034
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Optimization of fed-batch enzymatic hydrolysis from alkali-pretreated sugarcane bagasse for high-concentration sugar production Yueshu Gao 1,2, Jingliang Xu 1, Zhenhong Yuan 1*1,Yu Zhang 1, Yunyun Liu1，Cuiyi Liang1 1. Key Laboratory of Renewable Energy, Guangzhou Institute of Energy Conversion, Chinese Academy of Sciences, Guangzhou 510640, P.R. China 2. University of Chinese Academy of Sciences, Beijing 100049, P.R. China Abstract: Fed-batch enzymatic hydrolysis process from alkali-pretreated sugarcane
bagasse was investigated to increase solids loading, produce high-concentration fermentable sugar and finally to reduce the cost of the production process. The optimal initial solids loading, feeding time and quantities were examined. The hydrolysis system was initiated with 12% (w/v) solids loading in flasks, where 7% fresh solids were fed consecutively at 6 h, 12h, 24h to get a final solids loading of 33%. All the requested cellulase loading (10 FPU/g substrate) was added completely at the beginning of hydrolysis reaction. After 120 h of hydrolysis, the maximal concentrations of cellobiose, glucose and xylose obtained were 9.376g/L，129.50g/L， 56.03g/L, respectively. The final total glucan conversion rate attained to 60% from this fed-batch process. Keywords ： Sugarcane bagasse, Fed-batch process, Enzymatic hydrolysis, High-concentration sugars 1. Introduction Over the last few years, several studies have begun to investigate the effects of *
Corresponding author: Zhenhong Yuan. Tel.: +86 20 87057735; fax: +86 20
87057737. E-mail address: [email protected] (Z. H. Yuan).
high-solid loading (>15% solids, w/w) on different unit operations within the process stream (Hodge, et al., 2008; Jorgense et al., 2007; Kristensen et al., 2009; Lu et al., 2010; Zhang et al., 2010) as a means of improving the process economics of the lignocellulose to ethanol conversion. It has been suggested that using high-solids loading, especially the combination of a high-solids pretreatment followed by high-solids hydrolysis has great potential at improving the process efficiency (Modenbach, A. A. and Nokes, 2012). The conversion process is more environmentally friendly, as less water is consumed and the production cost is due to reduced size of equipment (tanks and distillation column etc.) and reduced energy utilization for distillation (Mohagheghi and Schell, 2010; Stenberg et al.,1998). However, a high content of lignocellulosic substrate might cause poor mass transfer and high viscosity, which would make the mixing difficult, the heat transfer efficiency lower and raise the power consumption in the stirred tank reactors (Fan et al., 2003; Jørgensen et al., 2007). To decrease these negative effects and maximize the end product concentration, fed-batch hydrolysis has been proposed as a feasible method (Laopaiboon et al., 2007; Rudolf et al., 2005; Varga et al., 2004).Yang et al.(Yang et al., 2010) reported that 30% solids could be hydrolyzed with sugar concentration reaching to nearly 220g/L. However, the pretreatment conditions were complex
and
time-consuming
since
both
steam
explosion
and
alkaline
hydrogen-peroxide pretreatments were applied, which inevitably increased the energy consumption and capital cost. A fed-batch process was used to increase the solids loading to 30% by Wang et al (Wang et al., 2012), however, only about 100g/L of reducing sugar and 50% glucan rate were obtained with a cellulase loading of 20FPU/g
substrate.
Therefore,
choosing
a
substrate
pretreated
by
a
low-energy-consuming method and optimization of fed-batch process were necessary for high concentration sugar and subsequent bio-ethanol production. The high-solids alkali pretreatment has been clarified to be a more efficient, and water-saving method than liquid hot water and HCl pretreatment in our precious report (Yu Zhang, 2013). In this study, for the purpose of cheap and efficient processes for the bio-ethanol production, the raw sugarcane bagasse (SCB) was
pretreated with diluted NaOH at a S/L ratio of 1:6. Taking the alkali-pretreated SCB as substrate, the optimal condition of fed-batch hydrolysis was investigated, aiming to improve the overall solids and sugar concentrations under low enzyme loading of 10 FPU/g substrate. 2. Methods 2.1 Materials Sugarcane bagasse (SCB) was provided by Guangxi Fenghao Group Co. Ltd. (Pingxiang, China). It was premilled and screened, with the fraction between 20 and 80 meshes used for these experiments. A cellulase mixture namely Cellic CTec2, was provided from Novozymes A/S (Bagsævrd, Denmark). The cellulase activity was 310 FPU/ml (FPU is the activity unit of cellulase when filter paper is used as the enzymatic substrate), assayed by the description of IUPAC (Ghose, 1987). The βglucosidase activitie was 704U /mL, which was determined by the method of Sylwia, W (Wołosowska and Synowiecki, 2004). 2.2. Alkali-pretreatment Details of the NaOH pretreatment are described elsewhere (Yu zhang, 2013). 1 kg of sugarcane bagasse was mixed with 1.8%(w/v) NaOH solution at a S/L ratio of 1:6. The slurry was incubated in the rotating cooker at 110℃ for 60 min. At the end of reaction, the hydrolyzate was separated by filtering and washed with tap water until neutral pH. The solids residue was dried in a forced-air oven at 50 ℃, and stored in a desiccator for the subsequent chemical analysis and enzymatic digestibility assay. 2.3 Batch enzymatic hydrolysis in shake flasks A series of batch enzymatic hydrolysis experiments were conducted with solids
loading of 8%, 10%, 12%, and 14% dry mass (DM) (w/v) 50℃ and 150 rpm in 250 mL Erlenmeyer flasks, each containing 100 mL of 0.05 M sodium citrate buffer (pH 5.0) sealed with rubber stoppers. The enzyme loading was 10 FPU/g substrate. Samples were collected at 3, 6, 12, 24, 48, 72 h and measured by high performance liquid chromatography (HPLC). All experiments were carried out in duplicate. 2.4. Fed-batch enzymatic hydrolysis in shake flasks The optimum solids loading obtained from the batch enzymatic hydrolysis was 12%, which was set as the concentration for the fed-batch process. Firstly, to identify the mode of adding enzyme, fed-batch processes， “a” and “b” were carried out. 7% of fresh substrates was fed after 6 h for a final solids loading of 19%, with an enzyme loading of 10 FPU/ (g substrate) for both tests. For “a”, all cellulase was completely added into the solution at the beginning of hydrolysis according to 19% DM. In contrast, for experiment “b”, cellulase was added along with the corresponding amount of substrate. Secondly, to further increase the concentration of reducing sugar, more fresh solids were fed and the final solids loading reached 33%. 21% solids were separated into different amounts and fed to the enzymatic hydrolysis systems at different times. The first feeding time was set at 6 hours. And other feeding times and feeding amounts of DM were determined. The other enzymatic hydrolysis conditions were similar to the batch hydrolysis. The detailed operating conditions and time of samples taken at were described in the context. 2.5. Analysis methods The components of sugarcane bagasse before and after pretreatment were
determined according to the standardized methods of the National Renewable Energy Laboratory (NREL, Golden, CO, USA) (Sluiter et al., 2004). Samples for sugar analysis were taken at different time (specified in the text) and centrifuged at 12,000 rpm for 2 min. Sugar concentrations were measured by high performance liquid chromatography (HPLC, Waters 2695), using a Shodex sugar SH-1011 column coupled with a refractive index detector RI 2414. The mobile phase was 5mmol/L H2SO4 at a flow rate of 0.5 mL/min. The analysis was performed with a column temperature of 50℃. The yield of sugars in the hydrolyzate was calculated on the basis of the amount of sugar polymers in the treated solids. 3. Results and discussion 3.1 Compositional changes before and after alkali pretreatment In order to benefit lignocellulosic enzymes accessing the recalcitrant structure of cellulose for maximum recovery of sugars, a pretreatment process should be adapted to remove lignin or hemicellulose. This would decrease the crystallinity of cellulose, and increase the biomass surface area (Balat et al., 2008; Jorgensen et al., 2007). In this work, the sugarcane bagasse was pretreated with sodium hydroxide at a high-solids loading of 16.67%. The chemical composition of raw SCB was 41.95% of glucan, 21.7% of xylan, 23.6% of lignin. After alkali pretreatment, the percentage concentration of glucan and xylan increased to 63.19% and 26.63%. Simultaneously, the lignin content decreased sharply to 8.95%, which could be attributed to the dilute NaOH treatment of lignocellulosic biomass causing swelling. This leads to an increase in the internal surface area, separation of structural linkages between lignin
and carbohydrates, and disruption of the lignin structure (Fan et al. 1987). After the NaOH pretreatment, the solid recovery was 62.4%. Based on the initial content of three compositions in the raw SCB, the recovery of glucan and xylan were 94.0% and 76.58%,respectively and the lignin removal was 76.44%. It can be concluded that dilute NaOH pretreatment was an effective method to remove lignin and retain most of the cellulose and hemicellulose, thus enhancing the reactivity of the remaining
carbohydrates.
Still
it
would
facilitate
subsequent
enzymatic
saccharification operating at high cellulose content to obtain high sugar concentration (Gao et al., 2013). 3.2 Optimal initial substrate loading The rate and extent of enzymatic hydrolysis of lignocellulose were highly dependent on solids loading, enzyme loading, hydrolysis time and structural features of substrates (Zhu et al., 2008). Firstly, the optimal initial solids loading was determined, in which the concentration of glucose and xylose and time of liquefaction were chosen as the criterion. Fig.1 showed the effects of solids loading on the release of glucose from the hydrolysis of SCB at 10 FPU/g substrate and 50℃. It could be found that the hydrolysis rate was high in the first 12 h, which might be explained by the reduction of crystallinity and the increase of exposed available catalytic sites. With time prolonging, the decrease of enzyme accessible sites and the products inhibitive effects caused the hydrolysis rate to decrease in the final hydrolysis stage (Zhu et al., 2008). In order to shorten the total hydrolysis time, especially in the fed-batch process, fresh solids were supposed to be fed early.(Yang et al., 2010)
Therefore, more attention was paid to the first 12 hours in the hydrolysis system and the hydrolysis rate and yield should be kept at a high level during the initial hydrolysis stage. When the solids loading were below 12%, all the hydrolysis systems could be liquefied within 3 h, and the glucose concentration had an approximate linear relationship with solids loading. While when the solids loading was 14%, time of liquefaction retarded over to 6 h and the hydrolysis speed was very slow. During this period, the glucose and xylose concentration were least compared with that of three other solids loadings. Therefore, it could be concluded that 12% was the optimal initial solids loading based on the change of glucose concentration and time of liquefication. 3.3 Fed-batch enzymatic hydrolysis The effect of the enzyme added mode on the release of glucose and xylose was presented in Fig.2. In the first 6 h. the “a” and “b” modes had the same substrate loading of 12%, while for “a”, all of the requested enzymes were added at 0 h, where much more cellulase existed than that of “b”. Therefore, much more glucose and xylose were released in “a” than “b”. After 6 h, though the “a” and “b” hydrolysis system had the same substrate and enzyme loading, the former always produced more sugars than the latter until the final stage of 72 h. It may be due to that, in the first 6 h for “a”, comparatively more cellulase accelerated the hydrolysis process and decreased the content of insoluble substrate which would result in the viscosity decreasing much more rapidly and facilitate to the heat and mass transfer of the whole process. Gradually, with hydrolysis proceeding, the distinction between the two
systems was shrunk because of the more lost of enzyme activity for the “a” process since the cellulase was added earlier which was subjected to high temperature and shear force(Ganesh et al., 2000). Under the conditions for test “a”, the final glucose and xylose concentration of 19% solids loading reached 80.87 g/L and 38.61 g/L. Given the result above, test “a” is more benefiticial to the fed-batch process, and the procedures could be simplified. Therefore, in the subsequent experiments, all the requested cellulase was added at 0 h. Fermentable sugars should be as high as possible in the practical bio-ethanol fermentation system to reduce the cost (Puri et al., 2013). To further increase the concentration of fermentable sugars, it is necessary to carry out some experiments to feed a higher quantity of fresh solids and determine the optimal feeding time and feeding amount. In our studies, the effect of feeding points on the release of sugars from the hydrolysis of SCB was firstly investigated. The fed-batch process was started with 12% of solids loading. 7% fresh solids were fed into the system thirdly to obtain a final solids loading of 33%. The first feeding point was set at 6 h. Four feeding conditions were designed to determine the second and third feeding points. The detailed operating conditions were shown in Table 1. Fig.3 and Fig.4 showed the comparison of sugars concentrations from fed-batch enzymatic hydrolysis with 33% final solids loading under four feeding point conditions. It can be found that feeding points had a specific effect on the release of reducing sugar from the hydrolysis of SCB. There are many factors that lead to a decrease in substrate conversion for hydrolysis at high total solids concentrations,
including high viscosity (Roche et al., 2009), product inhibition as a result of increasing sugar concentration(Xiao et al., 2004), decreasing water availability (Roberts et al., 2011; Selig et al., 2012), irreversible binding of adsorbed enzyme to the substrate (Kristensen et al., 2009), inhibition of enzyme adsorption (Kristensen et al., 2009) and enzyme denaturation (Yu et al., 2012). As for the comparison of four conditions, distinction of feeding points resulted in difference of system viscosity which was the most significant factor. During the enzymatic hydrolysis process, enzyme activity would be gradually decreased since it was subjected to high temperature and shear force. With the time for feeding process needed was prolonged, the decrease of viscosity benefited the hydrolysis efficiency but the remaining enzyme activity was low which caused the last feeding solid was difficulty to be hydrolysised. For condition A, it took only 18 hours to reach to 33% of the solids concentration and the intervals were shortest. Undoubtedly, insufficient mixing might be the biggest problem, while enough enzymatic accessibility sites could lead the hydrolysis of fresh substrates and decrease the viscosity quickly. For condition D, the intervals were longest, the fresh substrates adding could make relatively sufficient liquefaction but enzyme inactivity became the biggest problem. Finally, the concentrations of glucose, xylose and the total sugars obtained were lowest. When the intervals extended to 6 h and 12 h, as condition B, the concentrations of glucose, xylose and the total sugars obtained were highest. Except that the feeding points were determined, the feeding quantities were analysed as well and the detailed scheme was presented in Table 2. According to
Figure 5, glucan and xylan conversion rates with three conditions were shown. For condition 1, the most solids were added at 6 h, thereby the ratio of enzyme to substrate was lowest, which led a lowest conversion at 12h. With more solids added, the cellulose conversion gradually decreased and then increased since the hydrolysis time was extended. Totally, with condition 2, it would obtain the highest glucose and xylose concentrations. Drawn from the above results, it is supposed that the interplay between feeding points and amounts commonly would affect the whole hydrolysis process. Finally, initiated with 12% (w/v) solids loading in flasks, 7% fresh solids were fed consecutively at 6 h, 12h, 24h to get a final solids loading of 33%. The highest concentrations of cellobiose, glucose and xylose could attain to 9.376g/L，129.50g/L， 56.03g/L, respectively, and the total sugar concentration was more than 200g/L. Though the solids concentration had reached 33%, no high viscosity was observed after 48 h. It might be feasible to adding fresh substrate to get much higher solids concentration. Through the fed-batch process, the solids loading increased from 12% to 19% and 33%, and the glucose concentration increased from 58.84 g/L to 80.87 g/L and 109.20 g/L after 72 h enzymatic hydrolysis. Though the glucan conversion rate decreased from 74.16 % to 61.93% and 51.37%, it still demonstrated that fed-batch process was an excellent way to produce high-concentration fermentable sugars, which was consistent with the report from M. Yang (Yang et al., 2010). For the solids loading of 33%, when hydrolysis time was extended to 120 h, the glucan conversion rate could reach 59.88%.
4. Conclusions Fed-batch enzymatic hydrolysis process from alkali-pretreated sugarcane bagasse was investigated to increase solids loading, produce high-concentration fermentable sugar and finally to reduce the cost of the production process. The hydrolysis system was initiated with 12% (w/v) in flasks, and finally reached 33% (w/v) solids loading. After the enzymatic hydrolysis for 120 hours, the maximum concentrations of cellobiose, glucose and xylose obtained were 9.376g/L ， 129.50g/L ，56.03g/L, respectively, and the total sugar concentration was more than 200g/L. From the results, it was found that high-solids NaOH pretreatment and fed-batch process were efficient for high-concentration sugars production. Acknowlegements This work was funded by the National High-tech R&D Program (2013AA065803), National Key Technology R&D Program (2011BAD22B01), Program of National Natural Science Foundation of China (21176237, 21211140237), and Cooperation Project between Chinese Academy of Sciences and Guangxi Academy of Sciences, and Science & Technology Project of Guangzhou（2013J4300026）. References [1] Fan, L., Gharpuray, M., Lee, Y., 1987. Cellulose hydrolysis. Biotechnology monographs. Volume 3. [2] Fan, Z., South, C., Lyford, K., Munsie, J., van Walsum, P., Lynd, L.R., 2003. Conversion of paper sludge to ethanol in a semicontinuous solids-fed reactor. Bioprocess and Biosystems Engineering 26, 93-101.
[3] Ganesh, K., Joshi, J., Sawant, S., 2000. Cellulase deactivation in a stirred reactor. Biochem Eng J 4, 137-141. [4] Gao, Y., Xu, J., Zhang, Y., Yu, Q., Yuan, Z., Liu, Y., 2013. Effects of different pretreatment methods on chemical composition of sugarcane bagasse and enzymatic hydrolysis. Bioresource Technol. 144, 396-400. [5] Ghose, T., 1987. Measurement of cellulase activities. Pure Appl Chem 59, 257-268. [6] Hodge, D.B., Karim, M.N., Schell, D.J., McMillan, J.D., 2008. Soluble and insoluble solids contributions to high-solids enzymatic hydrolysis of lignocellulose. Bioresource Technol 99, 8940-8948. [7] Jørgensen, H., Vibe‐Pedersen, J., Larsen, J., Felby, C., 2007. Liquefaction of lignocellulose at high‐solids concentrations. Biotechnol Bioeng 96, 862-870. [8] Kristensen, J.B., Felby, C., Jørgensen, H., 2009. Yield-determining factors in high-solids enzymatic hydrolysis of lignocellulose. Biotechnol Biofuels 2, 11. [9] Laopaiboon, L., Thanonkeo, P., Jaisil, P., Laopaiboon, P., 2007. Ethanol production from sweet sorghum juice in batch and fed-batch fermentations by Saccharomyces cerevisiae. World Journal of Microbiology and Biotechnology 23, 1497-1501. [10] Lu YF, Wang YH, Xu GQ, Chu J, Zhuang YP, Zhang SL. 2010. Influence of high solid
concentration
steam-exploded 160(2):360–369.
corn
on
enzymatic stover
hydrolysis
biomass.
Appl
and
fermentation
Biochem
of
Biotechnol
[11] Modenbach, A.A., Nokes, S.E., 2012. The use of high‐solids loadings in biomass pretreatment—a review. Biotechnol Bioeng 109, 1430-1442. [12] Mohagheghi A, Schell DJ. 2010. Impact of recycling stillage on conversion of dilute sulfuric acid pretreated corn stover to ethanol. Biotechnol Bioeng 105(5):992–996. [13] Puri, D.J., Heaven, S., Banks, C.J., 2013. Improving the performance of enzymes in hydrolysis of high solids paper pulp derived from MSW. Biotechnol Biofuels 6, 107. [14] Roberts, K.M., Lavenson, D.M., Tozzi, E.J., McCarthy, M.J., Jeoh, T., 2011. The effects of water interactions in cellulose suspensions on mass transfer and saccharification efficiency at high solids loadings. Cellulose 18, 759-773. [15] Roche, C., Dibble, C., Stickel, J., 2009. Laboratory-scale method for enzymatic saccharification of lignocellulosic biomass at high-solids loadings. Biotechnol Biofuels 2, 28. [16] Rudolf, A., Alkasrawi, M., Zacchi, G., Liden, G., 2005. A comparison between batch and fed-batch simultaneous saccharification and fermentation of steam pretreated spruce. Enzyme Microb Tech 37, 195-204. [17] Selig, M.J., Hsieh, C.W.C., Thygesen, L.G., Himmel, M.E., Felby, C., Decker, S.R., 2012. Considering water availability and the effect of solute concentration on high solids saccharification of lignocellulosic biomass. Biotechnol Progr 28, 1478-1490. [18] Sluiter, A., Hames, B., Ruiz, R., Scarlata, C., Sluiter, J., Templeton, D., Crocker,
D., 2004. Determination of structural carbohydrates and lignin in biomass. NREL, Golden, Co. [19] Stenberg K, Tengborg C, Galbe M, Zacchi G, Palmqvist E, Hahn-Hagerdal B. 1998. Recycling of process streams in ethanol production from softwoods based on enzymatic hydrolysis. Appl Biochem Biotechnol 70–72:697–708. [20] Varga, E., Klinke, H.B., Réczey, K., Thomsen, A.B., 2004. High solid simultaneous saccharification and fermentation of wet oxidized corn stover to ethanol. Biotechnol Bioeng 88, 567-574. [21] Wang, W., Zhuang, X., Yuan, Z., Yu, Q., Qi, W., Wang, Q., Tan, X., 2012. High consistency enzymatic saccharification of sweet sorghum bagasse pretreated with liquid hot water. Bioresource Technol 108, 252-257. [22] Wołosowska, S., Synowiecki, J., 2004. Thermostable β-glucosidase with a broad substrate specifity suitable for processing of lactose-containing products. Food chemistry 85, 181-187. [23] Xiao, Z., Zhang, X., Gregg, D.J., Saddler, J.N., 2004. Effects of sugar inhibition on cellulases and β-glucosidase during enzymatic hydrolysis of softwood substrates Proceedings of the Twenty-Fifth Symposium on Biotechnology for Fuels and Chemicals Held May 4–7, 2003, in Breckenridge, CO. Springer, pp. 1115-1126. [24] Yang, M., Li, W., Liu, B., Li, Q., Xing, J., 2010. High-concentration sugars production from corn stover based on combined pretreatments and fed-batch process. Bioresource Technol 101, 4884-4888.
[25] Yu, Z., Jameel, H., Chang, H.m., Philips, R., Park, S., 2012. Evaluation of the factors affecting avicel reactivity using multi‐stage enzymatic hydrolysis. Biotechnol Bioeng 109, 1131-1139. [26] Zhang J, Chu DQ, Huang J, Yu ZC, Dai GC, Bao J. 2010.Simultaneous saccharification and ethanol fermentation at high corn stover solids loading in a helical stirring bioreactor. Biotechnol Bioeng 105(4):718–728. [27] Zhang, Y., Xu, J.-L., Yuan, Z.-H., Liu, Y.-Y., He, M.-C., 2013. Comparisonof different pretreatment methods on sugarcane bagasse and fractal-like kinetics of enzymatic hydrolysis. Advantage in new and renewable energy 1 (2): 166-169 166-169. [28] Zhu, L., O’Dwyer, J.P., Chang, V.S., Granda, C.B., Holtzapple, M.T., 2008. Structural features affecting biomass enzymatic digestibility. Bioresource Technol 99, 3817-3828.
Figure and table Legends Figure1 Concentration of glucose and xylose for sugarcane bagasse with different substrate loadings Figure2 Effect of enzyme added mode on the release of glucose and xylose. “a”
means all of the requested enzymes were added completely at the beginning of hydrolysis, “b” means the enzyme was separately added corresponding with the substrate. Figure3 The curve of glucose and xylose concentrations under four operating conditions of different feeding points for 120 h hydrolysis Figure4 The comparison of sugars concentration among four operating condition of different feeding points after 120 h hydrolysis Figure5 The time course of glucan and xylan conversion rates under three feeding conditions for 120 h hydrolysis
The concentration of glucose: g/l
72
48
8% 10% 12% 14%
24
0 0
24
48
72
Time: h
The concentration of xylose: g/l
30
20
8% 10% 12% 14%
10
0
0
24
48
72
Time: h
Fig.1 Concentration of glucose and xylose for sugarcane bagasse with different substrate loadings
The concentration of sugar (g/L)
Glucose-a Glucose-b Xylose-a Xylose-b
80
60
40
20
0
6
12
24
48
72
Time (h)
Fig.2 Effect of enzyme added mode on the release of glucose and xylose. “a” means all of the requested enzymes were added completely at the beginning of hydrolysis, “b” means the enzyme was separately added corresponding with the substrate.
A-glucose C-glucose A-xylose C-xylose
The concentrations of sugar (g/L)
120
B-glucose D-glucose B-xylose D-xylose
90
60
30
0 0
24
48
72
96
120
Time (h)
Fig.3 The curve of glucose and xylose concentrations under four operating conditions of different feeding points for 120 h hydrolysis
Arabinase Cellubiose Xylose Glucose
The concentration of sugar (g/L)
200
160
120
80
40
0 Condition A
Condition B Condition C
Condition D
Fig.4 The comparison of sugars concentration among four operating conditions of different feeding points after 120 h hydrolysis
Condition 1 Condition 2 Condition 3
Glucan conversion rate (%)
60
45
30
15
0 0
12
36
48 Time (h)
72
96
120
36
48 Time (h)
72
96
120
Condition 1 Condition 2 Condition 3
60 Xylan conversion rate (%)
24
45
30
15
0 0
12
24
Fig. 5 The time course of glucan and xylan conversion rates under three feeding conditions for 120 h hydrolysis
Table1 Hydrolysis conditions and enzymatic conversion rate by fed-batch mode Run No.
Feeding point First time
Second time
Third time
A
6h
12 h
18 h
B
6h
12 h
24 h
C
6h
18 h
30 h
D
6h
18h
42h
Table 2 Hydrolysis conditions and enzymatic conversion rate by fed-batch mode
No. 1 2 3
6h 9 7 5
Feeding amount (%) 12h 7 7 7
24h 5 7 9
Table 3 sugar concentrations produced from high solids loading based fed-batch hydrolysis of different agricultural residues
Substrate
Pretreatment method
Enzyme loading (FPU/g solid)
Sugarcane bagasse
NaOH
10
33
120
129.50(glucose), 56.03 (xylose)
59.88
Sugarcane bagasse
NaOH
9.6
30
144
125.97(glucose), 8.66 (xylose)
50.85
(Zhang et al., 2011)
Wheat straw
NaOH
9.6
30
144
81.99(glucose), 20.30 (xylose)
34.77
(Zhang et al., 2011)
Sweet sorghum bagasse
Hot liquid water
120
88.95(glucose), 9.8 (xylose)
Corn stover
Steam explosion + NaOH-H2O2
144
175 (glucose), 20 (xylose)
19
20
Substrate loading (w/v)
Hydrolytic time (h)
Produced sugar concentration (g/L)
30
30
Glucan conversion rate (%)
49.84
65.57
References
This study
(Wang et al., 2012) (Yang et al., 2010)
Highlights A high-solids alkali-pretreatment followed by high-solids enzymatic hydrolysis with fed-batch process was applied > Fed-batch hydrolysis process was optimized to get a final solids loading of 33%> the maximal total sugar concentration was more than 200 g/L > The final total cellulose conversion attained to 60% from fed-batch process.
S0960-8524(14)00693-2 http://dx.doi.org/10.1016/j.biortech.2014.05.034 BITE 13442
To appear in:
Bioresource Technology
Received Date: Revised Date: Accepted Date:
9 March 2014 10 May 2014 12 May 2014
Please cite this article as: Gao, Y., Xu, J., Yuan, Z., Zhang, Y., Liu, Y., Liang, C., Optimization of fed-batch enzymatic hydrolysis from alkali-pretreated sugarcane bagasse for high-concentration sugar production, Bioresource Technology (2014), doi: http://dx.doi.org/10.1016/j.biortech.2014.05.034
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Optimization of fed-batch enzymatic hydrolysis from alkali-pretreated sugarcane bagasse for high-concentration sugar production Yueshu Gao 1,2, Jingliang Xu 1, Zhenhong Yuan 1*1,Yu Zhang 1, Yunyun Liu1，Cuiyi Liang1 1. Key Laboratory of Renewable Energy, Guangzhou Institute of Energy Conversion, Chinese Academy of Sciences, Guangzhou 510640, P.R. China 2. University of Chinese Academy of Sciences, Beijing 100049, P.R. China Abstract: Fed-batch enzymatic hydrolysis process from alkali-pretreated sugarcane
bagasse was investigated to increase solids loading, produce high-concentration fermentable sugar and finally to reduce the cost of the production process. The optimal initial solids loading, feeding time and quantities were examined. The hydrolysis system was initiated with 12% (w/v) solids loading in flasks, where 7% fresh solids were fed consecutively at 6 h, 12h, 24h to get a final solids loading of 33%. All the requested cellulase loading (10 FPU/g substrate) was added completely at the beginning of hydrolysis reaction. After 120 h of hydrolysis, the maximal concentrations of cellobiose, glucose and xylose obtained were 9.376g/L，129.50g/L， 56.03g/L, respectively. The final total glucan conversion rate attained to 60% from this fed-batch process. Keywords ： Sugarcane bagasse, Fed-batch process, Enzymatic hydrolysis, High-concentration sugars 1. Introduction Over the last few years, several studies have begun to investigate the effects of *
Corresponding author: Zhenhong Yuan. Tel.: +86 20 87057735; fax: +86 20
87057737. E-mail address: [email protected] (Z. H. Yuan).
high-solid loading (>15% solids, w/w) on different unit operations within the process stream (Hodge, et al., 2008; Jorgense et al., 2007; Kristensen et al., 2009; Lu et al., 2010; Zhang et al., 2010) as a means of improving the process economics of the lignocellulose to ethanol conversion. It has been suggested that using high-solids loading, especially the combination of a high-solids pretreatment followed by high-solids hydrolysis has great potential at improving the process efficiency (Modenbach, A. A. and Nokes, 2012). The conversion process is more environmentally friendly, as less water is consumed and the production cost is due to reduced size of equipment (tanks and distillation column etc.) and reduced energy utilization for distillation (Mohagheghi and Schell, 2010; Stenberg et al.,1998). However, a high content of lignocellulosic substrate might cause poor mass transfer and high viscosity, which would make the mixing difficult, the heat transfer efficiency lower and raise the power consumption in the stirred tank reactors (Fan et al., 2003; Jørgensen et al., 2007). To decrease these negative effects and maximize the end product concentration, fed-batch hydrolysis has been proposed as a feasible method (Laopaiboon et al., 2007; Rudolf et al., 2005; Varga et al., 2004).Yang et al.(Yang et al., 2010) reported that 30% solids could be hydrolyzed with sugar concentration reaching to nearly 220g/L. However, the pretreatment conditions were complex
and
time-consuming
since
both
steam
explosion
and
alkaline
hydrogen-peroxide pretreatments were applied, which inevitably increased the energy consumption and capital cost. A fed-batch process was used to increase the solids loading to 30% by Wang et al (Wang et al., 2012), however, only about 100g/L of reducing sugar and 50% glucan rate were obtained with a cellulase loading of 20FPU/g
substrate.
Therefore,
choosing
a
substrate
pretreated
by
a
low-energy-consuming method and optimization of fed-batch process were necessary for high concentration sugar and subsequent bio-ethanol production. The high-solids alkali pretreatment has been clarified to be a more efficient, and water-saving method than liquid hot water and HCl pretreatment in our precious report (Yu Zhang, 2013). In this study, for the purpose of cheap and efficient processes for the bio-ethanol production, the raw sugarcane bagasse (SCB) was
pretreated with diluted NaOH at a S/L ratio of 1:6. Taking the alkali-pretreated SCB as substrate, the optimal condition of fed-batch hydrolysis was investigated, aiming to improve the overall solids and sugar concentrations under low enzyme loading of 10 FPU/g substrate. 2. Methods 2.1 Materials Sugarcane bagasse (SCB) was provided by Guangxi Fenghao Group Co. Ltd. (Pingxiang, China). It was premilled and screened, with the fraction between 20 and 80 meshes used for these experiments. A cellulase mixture namely Cellic CTec2, was provided from Novozymes A/S (Bagsævrd, Denmark). The cellulase activity was 310 FPU/ml (FPU is the activity unit of cellulase when filter paper is used as the enzymatic substrate), assayed by the description of IUPAC (Ghose, 1987). The βglucosidase activitie was 704U /mL, which was determined by the method of Sylwia, W (Wołosowska and Synowiecki, 2004). 2.2. Alkali-pretreatment Details of the NaOH pretreatment are described elsewhere (Yu zhang, 2013). 1 kg of sugarcane bagasse was mixed with 1.8%(w/v) NaOH solution at a S/L ratio of 1:6. The slurry was incubated in the rotating cooker at 110℃ for 60 min. At the end of reaction, the hydrolyzate was separated by filtering and washed with tap water until neutral pH. The solids residue was dried in a forced-air oven at 50 ℃, and stored in a desiccator for the subsequent chemical analysis and enzymatic digestibility assay. 2.3 Batch enzymatic hydrolysis in shake flasks A series of batch enzymatic hydrolysis experiments were conducted with solids
loading of 8%, 10%, 12%, and 14% dry mass (DM) (w/v) 50℃ and 150 rpm in 250 mL Erlenmeyer flasks, each containing 100 mL of 0.05 M sodium citrate buffer (pH 5.0) sealed with rubber stoppers. The enzyme loading was 10 FPU/g substrate. Samples were collected at 3, 6, 12, 24, 48, 72 h and measured by high performance liquid chromatography (HPLC). All experiments were carried out in duplicate. 2.4. Fed-batch enzymatic hydrolysis in shake flasks The optimum solids loading obtained from the batch enzymatic hydrolysis was 12%, which was set as the concentration for the fed-batch process. Firstly, to identify the mode of adding enzyme, fed-batch processes， “a” and “b” were carried out. 7% of fresh substrates was fed after 6 h for a final solids loading of 19%, with an enzyme loading of 10 FPU/ (g substrate) for both tests. For “a”, all cellulase was completely added into the solution at the beginning of hydrolysis according to 19% DM. In contrast, for experiment “b”, cellulase was added along with the corresponding amount of substrate. Secondly, to further increase the concentration of reducing sugar, more fresh solids were fed and the final solids loading reached 33%. 21% solids were separated into different amounts and fed to the enzymatic hydrolysis systems at different times. The first feeding time was set at 6 hours. And other feeding times and feeding amounts of DM were determined. The other enzymatic hydrolysis conditions were similar to the batch hydrolysis. The detailed operating conditions and time of samples taken at were described in the context. 2.5. Analysis methods The components of sugarcane bagasse before and after pretreatment were
determined according to the standardized methods of the National Renewable Energy Laboratory (NREL, Golden, CO, USA) (Sluiter et al., 2004). Samples for sugar analysis were taken at different time (specified in the text) and centrifuged at 12,000 rpm for 2 min. Sugar concentrations were measured by high performance liquid chromatography (HPLC, Waters 2695), using a Shodex sugar SH-1011 column coupled with a refractive index detector RI 2414. The mobile phase was 5mmol/L H2SO4 at a flow rate of 0.5 mL/min. The analysis was performed with a column temperature of 50℃. The yield of sugars in the hydrolyzate was calculated on the basis of the amount of sugar polymers in the treated solids. 3. Results and discussion 3.1 Compositional changes before and after alkali pretreatment In order to benefit lignocellulosic enzymes accessing the recalcitrant structure of cellulose for maximum recovery of sugars, a pretreatment process should be adapted to remove lignin or hemicellulose. This would decrease the crystallinity of cellulose, and increase the biomass surface area (Balat et al., 2008; Jorgensen et al., 2007). In this work, the sugarcane bagasse was pretreated with sodium hydroxide at a high-solids loading of 16.67%. The chemical composition of raw SCB was 41.95% of glucan, 21.7% of xylan, 23.6% of lignin. After alkali pretreatment, the percentage concentration of glucan and xylan increased to 63.19% and 26.63%. Simultaneously, the lignin content decreased sharply to 8.95%, which could be attributed to the dilute NaOH treatment of lignocellulosic biomass causing swelling. This leads to an increase in the internal surface area, separation of structural linkages between lignin
and carbohydrates, and disruption of the lignin structure (Fan et al. 1987). After the NaOH pretreatment, the solid recovery was 62.4%. Based on the initial content of three compositions in the raw SCB, the recovery of glucan and xylan were 94.0% and 76.58%,respectively and the lignin removal was 76.44%. It can be concluded that dilute NaOH pretreatment was an effective method to remove lignin and retain most of the cellulose and hemicellulose, thus enhancing the reactivity of the remaining
carbohydrates.
Still
it
would
facilitate
subsequent
enzymatic
saccharification operating at high cellulose content to obtain high sugar concentration (Gao et al., 2013). 3.2 Optimal initial substrate loading The rate and extent of enzymatic hydrolysis of lignocellulose were highly dependent on solids loading, enzyme loading, hydrolysis time and structural features of substrates (Zhu et al., 2008). Firstly, the optimal initial solids loading was determined, in which the concentration of glucose and xylose and time of liquefaction were chosen as the criterion. Fig.1 showed the effects of solids loading on the release of glucose from the hydrolysis of SCB at 10 FPU/g substrate and 50℃. It could be found that the hydrolysis rate was high in the first 12 h, which might be explained by the reduction of crystallinity and the increase of exposed available catalytic sites. With time prolonging, the decrease of enzyme accessible sites and the products inhibitive effects caused the hydrolysis rate to decrease in the final hydrolysis stage (Zhu et al., 2008). In order to shorten the total hydrolysis time, especially in the fed-batch process, fresh solids were supposed to be fed early.(Yang et al., 2010)
Therefore, more attention was paid to the first 12 hours in the hydrolysis system and the hydrolysis rate and yield should be kept at a high level during the initial hydrolysis stage. When the solids loading were below 12%, all the hydrolysis systems could be liquefied within 3 h, and the glucose concentration had an approximate linear relationship with solids loading. While when the solids loading was 14%, time of liquefaction retarded over to 6 h and the hydrolysis speed was very slow. During this period, the glucose and xylose concentration were least compared with that of three other solids loadings. Therefore, it could be concluded that 12% was the optimal initial solids loading based on the change of glucose concentration and time of liquefication. 3.3 Fed-batch enzymatic hydrolysis The effect of the enzyme added mode on the release of glucose and xylose was presented in Fig.2. In the first 6 h. the “a” and “b” modes had the same substrate loading of 12%, while for “a”, all of the requested enzymes were added at 0 h, where much more cellulase existed than that of “b”. Therefore, much more glucose and xylose were released in “a” than “b”. After 6 h, though the “a” and “b” hydrolysis system had the same substrate and enzyme loading, the former always produced more sugars than the latter until the final stage of 72 h. It may be due to that, in the first 6 h for “a”, comparatively more cellulase accelerated the hydrolysis process and decreased the content of insoluble substrate which would result in the viscosity decreasing much more rapidly and facilitate to the heat and mass transfer of the whole process. Gradually, with hydrolysis proceeding, the distinction between the two
systems was shrunk because of the more lost of enzyme activity for the “a” process since the cellulase was added earlier which was subjected to high temperature and shear force(Ganesh et al., 2000). Under the conditions for test “a”, the final glucose and xylose concentration of 19% solids loading reached 80.87 g/L and 38.61 g/L. Given the result above, test “a” is more benefiticial to the fed-batch process, and the procedures could be simplified. Therefore, in the subsequent experiments, all the requested cellulase was added at 0 h. Fermentable sugars should be as high as possible in the practical bio-ethanol fermentation system to reduce the cost (Puri et al., 2013). To further increase the concentration of fermentable sugars, it is necessary to carry out some experiments to feed a higher quantity of fresh solids and determine the optimal feeding time and feeding amount. In our studies, the effect of feeding points on the release of sugars from the hydrolysis of SCB was firstly investigated. The fed-batch process was started with 12% of solids loading. 7% fresh solids were fed into the system thirdly to obtain a final solids loading of 33%. The first feeding point was set at 6 h. Four feeding conditions were designed to determine the second and third feeding points. The detailed operating conditions were shown in Table 1. Fig.3 and Fig.4 showed the comparison of sugars concentrations from fed-batch enzymatic hydrolysis with 33% final solids loading under four feeding point conditions. It can be found that feeding points had a specific effect on the release of reducing sugar from the hydrolysis of SCB. There are many factors that lead to a decrease in substrate conversion for hydrolysis at high total solids concentrations,
including high viscosity (Roche et al., 2009), product inhibition as a result of increasing sugar concentration(Xiao et al., 2004), decreasing water availability (Roberts et al., 2011; Selig et al., 2012), irreversible binding of adsorbed enzyme to the substrate (Kristensen et al., 2009), inhibition of enzyme adsorption (Kristensen et al., 2009) and enzyme denaturation (Yu et al., 2012). As for the comparison of four conditions, distinction of feeding points resulted in difference of system viscosity which was the most significant factor. During the enzymatic hydrolysis process, enzyme activity would be gradually decreased since it was subjected to high temperature and shear force. With the time for feeding process needed was prolonged, the decrease of viscosity benefited the hydrolysis efficiency but the remaining enzyme activity was low which caused the last feeding solid was difficulty to be hydrolysised. For condition A, it took only 18 hours to reach to 33% of the solids concentration and the intervals were shortest. Undoubtedly, insufficient mixing might be the biggest problem, while enough enzymatic accessibility sites could lead the hydrolysis of fresh substrates and decrease the viscosity quickly. For condition D, the intervals were longest, the fresh substrates adding could make relatively sufficient liquefaction but enzyme inactivity became the biggest problem. Finally, the concentrations of glucose, xylose and the total sugars obtained were lowest. When the intervals extended to 6 h and 12 h, as condition B, the concentrations of glucose, xylose and the total sugars obtained were highest. Except that the feeding points were determined, the feeding quantities were analysed as well and the detailed scheme was presented in Table 2. According to
Figure 5, glucan and xylan conversion rates with three conditions were shown. For condition 1, the most solids were added at 6 h, thereby the ratio of enzyme to substrate was lowest, which led a lowest conversion at 12h. With more solids added, the cellulose conversion gradually decreased and then increased since the hydrolysis time was extended. Totally, with condition 2, it would obtain the highest glucose and xylose concentrations. Drawn from the above results, it is supposed that the interplay between feeding points and amounts commonly would affect the whole hydrolysis process. Finally, initiated with 12% (w/v) solids loading in flasks, 7% fresh solids were fed consecutively at 6 h, 12h, 24h to get a final solids loading of 33%. The highest concentrations of cellobiose, glucose and xylose could attain to 9.376g/L，129.50g/L， 56.03g/L, respectively, and the total sugar concentration was more than 200g/L. Though the solids concentration had reached 33%, no high viscosity was observed after 48 h. It might be feasible to adding fresh substrate to get much higher solids concentration. Through the fed-batch process, the solids loading increased from 12% to 19% and 33%, and the glucose concentration increased from 58.84 g/L to 80.87 g/L and 109.20 g/L after 72 h enzymatic hydrolysis. Though the glucan conversion rate decreased from 74.16 % to 61.93% and 51.37%, it still demonstrated that fed-batch process was an excellent way to produce high-concentration fermentable sugars, which was consistent with the report from M. Yang (Yang et al., 2010). For the solids loading of 33%, when hydrolysis time was extended to 120 h, the glucan conversion rate could reach 59.88%.
4. Conclusions Fed-batch enzymatic hydrolysis process from alkali-pretreated sugarcane bagasse was investigated to increase solids loading, produce high-concentration fermentable sugar and finally to reduce the cost of the production process. The hydrolysis system was initiated with 12% (w/v) in flasks, and finally reached 33% (w/v) solids loading. After the enzymatic hydrolysis for 120 hours, the maximum concentrations of cellobiose, glucose and xylose obtained were 9.376g/L ， 129.50g/L ，56.03g/L, respectively, and the total sugar concentration was more than 200g/L. From the results, it was found that high-solids NaOH pretreatment and fed-batch process were efficient for high-concentration sugars production. Acknowlegements This work was funded by the National High-tech R&D Program (2013AA065803), National Key Technology R&D Program (2011BAD22B01), Program of National Natural Science Foundation of China (21176237, 21211140237), and Cooperation Project between Chinese Academy of Sciences and Guangxi Academy of Sciences, and Science & Technology Project of Guangzhou（2013J4300026）. References [1] Fan, L., Gharpuray, M., Lee, Y., 1987. Cellulose hydrolysis. Biotechnology monographs. Volume 3. [2] Fan, Z., South, C., Lyford, K., Munsie, J., van Walsum, P., Lynd, L.R., 2003. Conversion of paper sludge to ethanol in a semicontinuous solids-fed reactor. Bioprocess and Biosystems Engineering 26, 93-101.
[3] Ganesh, K., Joshi, J., Sawant, S., 2000. Cellulase deactivation in a stirred reactor. Biochem Eng J 4, 137-141. [4] Gao, Y., Xu, J., Zhang, Y., Yu, Q., Yuan, Z., Liu, Y., 2013. Effects of different pretreatment methods on chemical composition of sugarcane bagasse and enzymatic hydrolysis. Bioresource Technol. 144, 396-400. [5] Ghose, T., 1987. Measurement of cellulase activities. Pure Appl Chem 59, 257-268. [6] Hodge, D.B., Karim, M.N., Schell, D.J., McMillan, J.D., 2008. Soluble and insoluble solids contributions to high-solids enzymatic hydrolysis of lignocellulose. Bioresource Technol 99, 8940-8948. [7] Jørgensen, H., Vibe‐Pedersen, J., Larsen, J., Felby, C., 2007. Liquefaction of lignocellulose at high‐solids concentrations. Biotechnol Bioeng 96, 862-870. [8] Kristensen, J.B., Felby, C., Jørgensen, H., 2009. Yield-determining factors in high-solids enzymatic hydrolysis of lignocellulose. Biotechnol Biofuels 2, 11. [9] Laopaiboon, L., Thanonkeo, P., Jaisil, P., Laopaiboon, P., 2007. Ethanol production from sweet sorghum juice in batch and fed-batch fermentations by Saccharomyces cerevisiae. World Journal of Microbiology and Biotechnology 23, 1497-1501. [10] Lu YF, Wang YH, Xu GQ, Chu J, Zhuang YP, Zhang SL. 2010. Influence of high solid
concentration
steam-exploded 160(2):360–369.
corn
on
enzymatic stover
hydrolysis
biomass.
Appl
and
fermentation
Biochem
of
Biotechnol
[11] Modenbach, A.A., Nokes, S.E., 2012. The use of high‐solids loadings in biomass pretreatment—a review. Biotechnol Bioeng 109, 1430-1442. [12] Mohagheghi A, Schell DJ. 2010. Impact of recycling stillage on conversion of dilute sulfuric acid pretreated corn stover to ethanol. Biotechnol Bioeng 105(5):992–996. [13] Puri, D.J., Heaven, S., Banks, C.J., 2013. Improving the performance of enzymes in hydrolysis of high solids paper pulp derived from MSW. Biotechnol Biofuels 6, 107. [14] Roberts, K.M., Lavenson, D.M., Tozzi, E.J., McCarthy, M.J., Jeoh, T., 2011. The effects of water interactions in cellulose suspensions on mass transfer and saccharification efficiency at high solids loadings. Cellulose 18, 759-773. [15] Roche, C., Dibble, C., Stickel, J., 2009. Laboratory-scale method for enzymatic saccharification of lignocellulosic biomass at high-solids loadings. Biotechnol Biofuels 2, 28. [16] Rudolf, A., Alkasrawi, M., Zacchi, G., Liden, G., 2005. A comparison between batch and fed-batch simultaneous saccharification and fermentation of steam pretreated spruce. Enzyme Microb Tech 37, 195-204. [17] Selig, M.J., Hsieh, C.W.C., Thygesen, L.G., Himmel, M.E., Felby, C., Decker, S.R., 2012. Considering water availability and the effect of solute concentration on high solids saccharification of lignocellulosic biomass. Biotechnol Progr 28, 1478-1490. [18] Sluiter, A., Hames, B., Ruiz, R., Scarlata, C., Sluiter, J., Templeton, D., Crocker,
D., 2004. Determination of structural carbohydrates and lignin in biomass. NREL, Golden, Co. [19] Stenberg K, Tengborg C, Galbe M, Zacchi G, Palmqvist E, Hahn-Hagerdal B. 1998. Recycling of process streams in ethanol production from softwoods based on enzymatic hydrolysis. Appl Biochem Biotechnol 70–72:697–708. [20] Varga, E., Klinke, H.B., Réczey, K., Thomsen, A.B., 2004. High solid simultaneous saccharification and fermentation of wet oxidized corn stover to ethanol. Biotechnol Bioeng 88, 567-574. [21] Wang, W., Zhuang, X., Yuan, Z., Yu, Q., Qi, W., Wang, Q., Tan, X., 2012. High consistency enzymatic saccharification of sweet sorghum bagasse pretreated with liquid hot water. Bioresource Technol 108, 252-257. [22] Wołosowska, S., Synowiecki, J., 2004. Thermostable β-glucosidase with a broad substrate specifity suitable for processing of lactose-containing products. Food chemistry 85, 181-187. [23] Xiao, Z., Zhang, X., Gregg, D.J., Saddler, J.N., 2004. Effects of sugar inhibition on cellulases and β-glucosidase during enzymatic hydrolysis of softwood substrates Proceedings of the Twenty-Fifth Symposium on Biotechnology for Fuels and Chemicals Held May 4–7, 2003, in Breckenridge, CO. Springer, pp. 1115-1126. [24] Yang, M., Li, W., Liu, B., Li, Q., Xing, J., 2010. High-concentration sugars production from corn stover based on combined pretreatments and fed-batch process. Bioresource Technol 101, 4884-4888.
[25] Yu, Z., Jameel, H., Chang, H.m., Philips, R., Park, S., 2012. Evaluation of the factors affecting avicel reactivity using multi‐stage enzymatic hydrolysis. Biotechnol Bioeng 109, 1131-1139. [26] Zhang J, Chu DQ, Huang J, Yu ZC, Dai GC, Bao J. 2010.Simultaneous saccharification and ethanol fermentation at high corn stover solids loading in a helical stirring bioreactor. Biotechnol Bioeng 105(4):718–728. [27] Zhang, Y., Xu, J.-L., Yuan, Z.-H., Liu, Y.-Y., He, M.-C., 2013. Comparisonof different pretreatment methods on sugarcane bagasse and fractal-like kinetics of enzymatic hydrolysis. Advantage in new and renewable energy 1 (2): 166-169 166-169. [28] Zhu, L., O’Dwyer, J.P., Chang, V.S., Granda, C.B., Holtzapple, M.T., 2008. Structural features affecting biomass enzymatic digestibility. Bioresource Technol 99, 3817-3828.
Figure and table Legends Figure1 Concentration of glucose and xylose for sugarcane bagasse with different substrate loadings Figure2 Effect of enzyme added mode on the release of glucose and xylose. “a”
means all of the requested enzymes were added completely at the beginning of hydrolysis, “b” means the enzyme was separately added corresponding with the substrate. Figure3 The curve of glucose and xylose concentrations under four operating conditions of different feeding points for 120 h hydrolysis Figure4 The comparison of sugars concentration among four operating condition of different feeding points after 120 h hydrolysis Figure5 The time course of glucan and xylan conversion rates under three feeding conditions for 120 h hydrolysis
The concentration of glucose: g/l
72
48
8% 10% 12% 14%
24
0 0
24
48
72
Time: h
The concentration of xylose: g/l
30
20
8% 10% 12% 14%
10
0
0
24
48
72
Time: h
Fig.1 Concentration of glucose and xylose for sugarcane bagasse with different substrate loadings
The concentration of sugar (g/L)
Glucose-a Glucose-b Xylose-a Xylose-b
80
60
40
20
0
6
12
24
48
72
Time (h)
Fig.2 Effect of enzyme added mode on the release of glucose and xylose. “a” means all of the requested enzymes were added completely at the beginning of hydrolysis, “b” means the enzyme was separately added corresponding with the substrate.
A-glucose C-glucose A-xylose C-xylose
The concentrations of sugar (g/L)
120
B-glucose D-glucose B-xylose D-xylose
90
60
30
0 0
24
48
72
96
120
Time (h)
Fig.3 The curve of glucose and xylose concentrations under four operating conditions of different feeding points for 120 h hydrolysis
Arabinase Cellubiose Xylose Glucose
The concentration of sugar (g/L)
200
160
120
80
40
0 Condition A
Condition B Condition C
Condition D
Fig.4 The comparison of sugars concentration among four operating conditions of different feeding points after 120 h hydrolysis
Condition 1 Condition 2 Condition 3
Glucan conversion rate (%)
60
45
30
15
0 0
12
36
48 Time (h)
72
96
120
36
48 Time (h)
72
96
120
Condition 1 Condition 2 Condition 3
60 Xylan conversion rate (%)
24
45
30
15
0 0
12
24
Fig. 5 The time course of glucan and xylan conversion rates under three feeding conditions for 120 h hydrolysis
Table1 Hydrolysis conditions and enzymatic conversion rate by fed-batch mode Run No.
Feeding point First time
Second time
Third time
A
6h
12 h
18 h
B
6h
12 h
24 h
C
6h
18 h
30 h
D
6h
18h
42h
Table 2 Hydrolysis conditions and enzymatic conversion rate by fed-batch mode
No. 1 2 3
6h 9 7 5
Feeding amount (%) 12h 7 7 7
24h 5 7 9
Table 3 sugar concentrations produced from high solids loading based fed-batch hydrolysis of different agricultural residues
Substrate
Pretreatment method
Enzyme loading (FPU/g solid)
Sugarcane bagasse
NaOH
10
33
120
129.50(glucose), 56.03 (xylose)
59.88
Sugarcane bagasse
NaOH
9.6
30
144
125.97(glucose), 8.66 (xylose)
50.85
(Zhang et al., 2011)
Wheat straw
NaOH
9.6
30
144
81.99(glucose), 20.30 (xylose)
34.77
(Zhang et al., 2011)
Sweet sorghum bagasse
Hot liquid water
120
88.95(glucose), 9.8 (xylose)
Corn stover
Steam explosion + NaOH-H2O2
144
175 (glucose), 20 (xylose)
19
20
Substrate loading (w/v)
Hydrolytic time (h)
Produced sugar concentration (g/L)
30
30
Glucan conversion rate (%)
49.84
65.57
References
This study
(Wang et al., 2012) (Yang et al., 2010)
Highlights A high-solids alkali-pretreatment followed by high-solids enzymatic hydrolysis with fed-batch process was applied > Fed-batch hydrolysis process was optimized to get a final solids loading of 33%> the maximal total sugar concentration was more than 200 g/L > The final total cellulose conversion attained to 60% from fed-batch process.