Investigation of the Strengthening Process for Liquid Hot Water

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Investigation of the Strengthening Process for Liquid Hot Water Pretreatments Huisheng Lu, Shuangyan Liu, Minhua Zhang, Fanmei Meng, Xingfang Shi, and Li Yan Energy Fuels, Just Accepted Manuscript • DOI: 10.1021/acs.energyfuels.5b02658 • Publication Date (Web): 20 Jan 2016 Downloaded from http://pubs.acs.org on January 24, 2016

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Investigation of the Strengthening Process for Liquid Hot Water Pretreatments Huisheng Lu, Shuangyan Liu, Minhua Zhang*, Fanmei Meng, Xingfang Shi, and Li Yan Key Laboratory for Green Chemical Technology of Ministry of Education, Tianjin University R&D Center for Petrochemical Technology, Tianjin 300072, China KEYWORDS: liquid hot water pretreatment, strengthening process, acetic acid, spent liquor recycling, surfactant

ABSTRACT: A lignocellulose liquid hot water (LHW) pretreatment process was strengthened by the direct recycling of spent liquor. The use of rich spent liquor and its by-product acetic acid was proposed as a method of strengthening LHW pretreatments, which can reduce energy consumption while producing less waste water. The results showed that the glucose yield increased from 80.82% to 85.44% during enzymatic hydrolysis after the spent liquor had been recycled three times, at which point the acetic acid content was 8.1 g/L. When the spent liquor was reused three times, the furfural and hydroxymethyl furfural (HMF) concentrations in the pretreatment liquid were 1.95 g/L and 0.96 g/L, respectively. These levels are lower than the levels that significantly inhibit the growth of fermenting microorganisms. Furthermore, experiments were conducted with acetic acid contents between 0 and 40 g/L to investigate the difference between the acetic acid-catalyzed LHW pretreatment and the recycled spent liquor

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LHW pretreatment. The optimum concentration of acetic acid was 10 g/L, which confirmed the feasibility of using recycled spent liquor to strengthen the LHW pretreatment. Surfactant was also added during enzymatic hydrolysis to strengthen the LHW pretreatment process. The glucose yield of corn stover pretreated with recycled spent liquor increased to 89.84% with the addition of Tween 80.

1. INTRODUCTION Bioethanol, is considered to be an ideal alternative transportation fuel to fossil fuels, and it can be produced from lignocellulosic materials.1,2 However, lignocellulose is recalcitrant to chemical and biological hydrolysis because of its complex structure, which is formed by cellulose, hemicellulose and lignin.3 Therefore, pretreating the material prior to enzymatic hydrolysis is necessary to alter the structure of the biomass and make it more readily accessible to the enzyme.4-6 In recent decades, numerous physical, chemical and biological pretreatment methods have been investigated with the aim of improving the rate of enzyme hydrolysis and increasing the yield of fermentable sugars.7 Among the various types of pretreatment, liquid hot water (LHW) pretreatment, which is characterized by an environmentally friendly solvent and attractive reaction media for a variety of applications, is recognized as a promising pretreatment technology.8, 9 LHW pretreatment is extensively employed to remove hemicelluloses and break up the intact structure of the lignocellulosic biomass, to increase the accessibility of the biomass for enzymatic hydrolysis. In LHW pretreatments, lignocellulosic materials undergo hydrolysis reactions in the presence of hydronium ions, which are generated by water autoionization and act as catalysts. Garrote et al.10 has proposed that the heterocyclic ether bonds of hemicelluloses are


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the most susceptible bonds to this type of reaction, thus leading to both the generation of oligosaccharides and the splitting of acetyl groups from the hemicellulosic fraction of the raw materials. In further reaction stages, the hydronium ions generated from acetic acid autoionization also act as catalysts in the degradation of polysaccharides. Under the operational conditions usually employed in hydrothermal treatments, hydronium ions generated from acetic acid are more important than those generated from water.11 Compared with other pretreatment methods, the LHW pretreatment is a mild process that leads to a lower glucose yield during enzymatic hydrolysis. LHW pretreatment cannot remove a significant degree of lignin, which may hinder cellulose digestibility.12 Therefore, additional measures to strengthen LHW pretreatments are required. During the LHW pretreatment, acetic acid is formed from the acetyl groups present in the hemicellulose.10, 13 The presence of acetic acid can further loosen the biomass structure by removing additional hemicellulose and then increasing the convertibility of cellulose. Therefore, using the by-product acetic acid, which is produced during pretreatment, as the catalyst to pretreat new biomass may provide another economically feasible method of strengthening LHW pretreatments. Compared with acetic acid-catalyzed pretreatments, the direct recycling of spent liquor presents additional advantages, such as the absence of additional acetic acid, reduction of energy consumption and reduction of waste water. The objective of this study was to investigate strengthening LHW pretreatments by directly recycling spent liquor, which is rich in acetic acid, as an auto-catalytic process. In addition, the influence of surfactants added during enzymatic hydrolysis was studied. Surfactants are hydrophobic and believed to form a coating on the hydrophobic lignin surface, thus reducing irreversible protein adsorption that could lead to


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deactivation.14 Tween 80 was added during enzymatic hydrolysis to improve the yield of glucose with recycled spent liquor.

2. MATERIAL AND METHODS 2.1. Raw Materials Corn stover was provided by the Zhaodong Bio-energy and Bio-chemical Co. It was dried at 105 ºC for 4 h to a constant weight. The corn stover was milled and screened to collect the 20-60 mesh size fraction, which was used in all experiments.

2.2. Pretreatment of Corn Stover The pretreatments were performed in a high pressure reaction vessel (Parr 4843, Parr Instrument Company, USA). Approximately 10 g corn stover and 200 mL of distilled water were packed into a 1000 mL reaction vessel. The pressure of the reactor was maintained with nitrogen gas at 2.0 MPa, and the magnetic agitator was operated at 400 rpm. Pretreatments were performed at 180 ºC for 30 min, and the temperature and the pressure of the reactor were automatically controlled. The experimental apparatus used in this study to pretreat corn stover is shown in Figure 1. After the pretreatments, the slurry was filtered, and both the solid and liquid components were collected. The concentrations of sugars and degradation products in the liquid fraction were analyzed. The solid component was dried in the oven at 105 ºC for 6 h, and the solid samples were stored for enzymatic hydrolysis. During the spent-liquor direct recycling experiments, the liquid component, which is usually treated as spent liquor, was directly recycled to pretreat the fresh corn stover. The amount of


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spent liquor after pretreating the fresh corn stover decreased as the number of recycling processes increased; therefore, the ratio of solid to liquid was fixed at 1:20. The operating conditions and steps are the same as those described above.

2.3. Enzymatic Hydrolysis The cellulase (70 FPU·g-1; NS50013 and NS50010) used during the enzymatic hydrolysis process was provided by Novozymes. A unit of FPU is defined by the manufacturer as the liberation of 1.0 µmol of glucose from cellulose within 1 min at pH 4.8 and 50 ºC. One gram of cellulase (0.95 g of NS50013 and 0.5 g of NS50010) was added to 30 mL buffer solution (0.1 mol·L-1 acetic acid-sodium acetate, pH 4.8), and the mixture was then incubated in a shaker (ZHWY-1102C, Zhiheng, Shanghai), where enzymatic hydrolysis was conducted at 50 ºC and 120 rpm for 72 h. Samples were collected periodically and analyzed for their sugars content. The enzymatic hydrolysis of each pretreated sample was conducted in triplicate. The glucose yield was calculated as a reliable indicator for assessing cellulose digestibility as follows: glucose yield =[(g of total released glucose) /(g of initial cellulose*180/162) ]×100%

2.4. Analysis Methods The raw material was analyzed according to the standard Laboratory Analytical Procedures (LAP) for biomass analysis provided by the National Renewable Energy Laboratory (NREL).15 Monomeric sugars, oligomeric sugars, and degradation products in the hydrolysate were identified based on the NREL LAP. The oligomeric sugars in the liquid fraction were backcalculated into monomerics after a secondary hydrolysis with 4% sulfuric acid. The composition (on a dry weight basis) of raw corn stover and pretreated corn stover is shown in Table 1.


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The monomeric sugars in the hydrolysate were determined by HPLC using a ZorBax-SB-NH2 column coupled with a refractive index detector. The mobile phase was HPLC-grade water at a flow rate of 1.0 mL/min. All of the samples were filtered through a 0.20 µm filter before HPLC analysis. The concentrations of hydroxymethyl furfural (HMF), furfural and acetic acid in the hydrolysate were determined by gas chromatography-mass spectroscopy (GC-MS, Agilent 69805973N, Tianjin, China) in a HP-INNOWAX column under the following conditions: initial temperature maintained at 40 ºC followed by temperature increases of 10 ºC /min to 230 ºC with high purity helium used as the carrier gas at a flow rate of 1.3 mL/min. The native and pretreated biomass microstructures were analyzed by a scanning electron microscope (SEM, Nanosem 430) purchased from FEI Co. (USA). The content of total phenolics in hydrolysate was determined using the Folin–Ciocalteu assay.16, 17

3. RESULTS AND DISCUSSION 3.1. Acetic Acid/Glucose/Xylose Concentration in Pretreatment Liquid Changes in the acetic acid concentration as the number of recycling processes increased are shown in Figure 2. As shown in the figure, the acetic acid in the pretreatment liquid continuously increased from 3.2 g/L to 9.8 g/L as the number of recycling processes increased from 0 to 6. During LHW pretreatment, acetic acid is formed from acetyl groups present in the hemicellulose; therefore, acetic acid in the pretreatment liquid will accumulate with increased recycling times. The accumulating acetic acid strengthened the acidic environment of the pretreatment system, which can improve the effect of the pretreatment. However, the presence of acetic acid will also inhibit the release of acetyl groups from hemicellulose. The concentration of acetic acid


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accumulated at a decreasing rate after recycling 2 times, although after 4-6 recycling times, the rate at which acetic acid accumulated began to increase again. During the LHW pretreatment, cellulose and hemicellulose partially decomposed to glucose and xylose, respectively, as shown in Figure 3. For all of the treatment conditions, only a small part of the glucose was recovered in the pretreatment liquid in concentrations varying from 0.56 g/L to 1.45 g/L. The concentration of glucose increased slightly with increasing recycling processes, but still remained at a low level. Compared with glucose, more xylose was recovered in the pretreatment liquid. For the pretreatment without spent-liquor recycling, the xylose concentration was 8.02 g/L in the liquid, which was much higher than the glucose levels under the same condition; therefore, hemicellulose was more sensitive to decomposition than cellulose in the LHW pretreatment. When the spent liquor was recycled for the third time, the xylose and glucose increased from 8.02 g/L and 0.56 g/L to 21.34 g/L and 1.39 g/L, respectively. Although the recycling times continued to increase, the xylose and glucose concentrations only slightly fluctuated and remained relatively stable. The low yield of glucose indicated that the cellulose fraction did not degrade at high levels under the experimental pretreatment conditions. Based on these results, the conditions under which the massive hemicellulose started to decompose were less severe compared with those of cellulose in the LHW pretreatment.

3.2. Degradation Products in Pretreatment Liquid Xylose and glucose can be further degraded to furfural and HMF with LHW pretreatment. Figure 4 presents the furfural and HMF produced under different numbers of recycling processes. Under the least severe condition without spent liquor recycling, the concentrations of furfural and HMF were 0.35 g/L and 0.22 g/L, respectively, whereas at the most severe condition


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with 6 recycling processes, furfural and HMF increased to 4.16 g/L and 1.11 g/L, respectively. The concentrations of furfural and HMF increased as the number of recycling processes increases. Furfural and HMF are considered to be the most potent inhibitors of cell metabolism.18, 19

The inhibition mechanism may be the destruction of cell permeability in the microorganism

and the reduction of activity in several enzymes in the sugar fermentation degradation pathway caused by furfural and HMF.20 The inhibitory effects of furfural and HMF on yeast fermentation was dose dependent.21 The furfural and HMF concentrations, which significantly inhibited the growth of fermenting microorganisms, are 2 g/L and 5 g/L, respectively.22, 23 Therefore, the spent liquor should not be recycled more than 3 times for the moderate concentrations of furfural and HMF produced in the pretreatment liquid. Lignin is a complex phenolic polymer that is a primary factor limiting the accessibility of hydrolytic enzymes as well as the crystallinity of cellulose. Zhuang et al.24 proposed the solubilization mechanism of lignin in liquid hot water and suggested that lignin first migrates out of the cell wall in the form of molten bodies and then flushes out of the reactor. A small quantity of lignin is further degraded into monomeric products, such as phenolics, which are major inhibitors of cellulase.25 In addition, phenolics are prototype poisons for all individual life. Therefore, the control of phenolics has important significance with regard to sugar utilization. As shown in Figure 5, the concentration of phenolics increased from 480.64 ppm to 600.63 ppm as the number of recycling processes increased from 0 to 1, although with increasing numbers of recycling processes, the concentration was relatively stable. The phenolics remained stable, suggesting that the presence of phenolics inhibited the hydrolysis of lignin; therefore, the concentration of phenolics in the spent liquor was in dynamic equilibrium.


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3.3. Glucose Recovery in Enzymatic Hydrolysis The yield of glucose during enzymatic hydrolysis is an important index for evaluating the effect of the pretreatment and the most direct indicator for evaluating the digestibility of cellulose. The glucose yield for different pretreatment conditions is shown in Figure 6. After the spent liquor was recycled 3 times, the glucose levels improved further to 85.44%, whereas the level was only 80.82% without the recycling of spent liquor. When the number of recycling processes increased further, the yield of glucose began to decrease and dropped to 82.01% for spent liquor was recycled 6 times. When acetic acid is introduced to LHW pretreatment, there would be two effects on the pretreated material, i.e., the positive effect exists in that acetic acid further loosens the biomass structure through the removal of additional hemicellulose and the subsequent increase of cellulose convertibility, but the negative side also comes with more furan compounds produced, which are potential inhibitors to cellulase.26 The competition between the positive and negative effects of acetic acid on the pretreated material caused the different yields of glucose in the present study. In the experiments investigating the direct recycling of spent liquor, the concentrations of inhibitors continued to increase as the number of recycling processes increased. In our study, the concentrations of furfural and HMF continued to increase, whereas the concentration of phenolics was relatively stable. We conclude that the decreased glucose yield was primarily caused by the inhibitory effect of furfural and HMF. To obtain the highest yield of glucose and produce only small levels of furfural and HMF, the optimum number of recycling processes for the spent liquor was 3, which produced an acetic acid concentration of 8.1 g/L. Increasing the scale of the results presented here has the potential to provide an economically feasible method of further improving cellulose digestibility and reducing energy consumption.


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3.4. SEM Characterization A portion of the residual solids was tested by a SEM to visually assess the effects of the direct recycling of spent liquor on corn stover. The SEM micro-images of the native and pretreated corn stover samples are shown in Figure 7. As shown in Figure 7 a, the untreated corn stover exhibits a smooth and ridged surface structure, whereas Figure 7 (b-d) shows that the pretreated corn stover presented cracks and signs of structural breakdown under different conditions, which are attributed to the degradation of hemicellulose and low levels of lignin. More severe pretreatment conditions removed hemicelluloses and lignin more effectively and provided higher accessibility to cellulase, which was consistent with the yield of glucose during enzyme hydrolysis as shown in Figure 6. Table 1 shows the fractions in the solids after different pretreatment conditions. Compared with raw corn stover, the solid hemicellulose content decreased under the different pretreatment conditions and the cellulose and lignin content increased.

3.5. Acetic Acid-catalyzed LHW Pretreatment Experiments were conducted with additional acetic acid at concentrations between 0 and 40 g/L to further investigate the acetic acid-catalyzed LHW pretreatment on corn stover, and the results are shown in Table 2. When the acetic acid content doubled from 10 g/L to 20 g/L, only a slight glucose yield increase of 0.03% was observed. Because the highest concentration of xylose (9.06 g/L) was obtained with the addition of 10 g/L acetic acid to the pretreatment liquid, the optimum concentration of acetic acid for the LHW pretreatment was 10 g/L in the present tests. With 10 g/L acetic acid, the concentrations of furfural and HMF were 1.59 g/L and 1.21 g/L,


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respectively, and these concentrations are lower than the concentrations that significantly inhibit the growth of fermenting microorganisms. A comparison of the strengthening process using spent liquor recycling and acetic acid catalysis showed that the optimum concentrations of acetic acid were 8.1 g/L and 10 g/L, respectively, and large differences in glucose recovery were not observed during enzymatic hydrolysis. This finding confirmed that the direct recycling of spent liquor had a positive effect on strengthening the LHW pretreatment. The recycling of spent liquor shows great advantages such as without additional acetic acid, lower energy consumption and reduction of waste water. Increasing the scale of the results presented here has the potential to provide an economically feasible method of converting biomass to ethanol through the use of the by-product acetic acid, which is produced during the pretreatment, as a catalyst for the pretreatment of additional biomass.

3.6. Adding Tween 80 to the Enzymatic Hydrolysis LHW pretreatments cannot remove lignin to a large extent. Approximately 90% the lignin observed in the biomass pretreated by LHW was residual,22 which may also absorb cellulase, thus leading to the deactivation of cellulase. Adding surfactant to the enzymatic hydrolysis process can reduce residual lignin from the ineffective adsorption of cellulase; therefore, the use of surfactant can be regarded as an effective method of further enhancing the sugar yield in the LHW pretreatment process with spent liquor. The chemical surfactant Tween 80 has been used to enhance cellulase activities and reduce the cost of bio-alcohol production.27 The yield of glucose after adding 300 mg Tween 80 to 1 g of pretreated solids during enzymatic hydrolysis is shown in Table 3. With the addition of Tween


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80, the yield of glucose was further improved to 89.84% when spent liquor was recycled 3 times. Surfactants are hydrophobic and may form a coating on the hydrophobic lignin surface, thus reducing irreversible protein adsorption, which could lead to the deactivation of cellulase.14 Another plausible mechanism is that cellulase entrapment in the reverse micelles formed by the surfactant reduces the detrimental effects of heat and solvents on enzyme activity.28, 29 In addition, we believe that in our experiments, surfactants can reduce certain by-products in the recycled spent liquor, such as furfural and HMF, produced through the ineffective adsorption of cellulase, thus mitigating the inhibition of cellulase by the by-products, which can further improve the yield of glucose. The use of recycled spent liquor is aimed at practical applications, and additional details will be discussed in our continued work.

4. CONCLUSION The direct recycling of spent liquor adds an acetic acid-catalyzed pretreatment to LHW pretreatments. The results indicate that the yield of glucose during enzymatic hydrolysis was improved from 80.82% to 85.44% when the spent liquor was recycled 3 times, which produced a concentration of acetic acid of 8.1 g/L. Therefore, the optimum number of recycling processes for spent liquor to improve enzymatic hydrolysis and simultaneously avoid the inhibitory effects from furfural and HMF was three times. The optimum concentration of acetic acid was 10 g/L in the acetic acid-catalyzed test, which produced a glucose yield of 88.55%. Regardless of the slight difference in glucose yield, pretreatments that include the direct recycling of spent liquor produced additional advantages over the acetic acid-catalyzed pretreatment, such as lower acetic acid dosages, decreased energy consumption and reduced waste water. Moreover, adding Tween 80 to the enzymatic hydrolysis process can further improve the yield of glucose to 89.84% after


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pretreatment with spent liquor. Increasing the scale of the results presented here has the potential to provide an economically feasible method of strengthening LHW pretreatments through the direct recycling of spent liquor combined with the addition of surfactant.

AUTHOR INFORMATION Corresponding Author * Minhua Zhang

Phone/Fax: +86-22-27406119. E-mail: [email protected]

Notes The authors declare no competing financial interest. Present Addresses Key Laboratory for Green Chemical Technology of Ministry of Education, Tianjin University R&D Center for Petrochemical Technology, Tianjin 300072, China. ABBREVIATIONS LHW, liquid hot water; HMF, hydroxymethyl furfural. REFERENCES (1) Bennett, A. S.; Anex, R. P. Bioresour. Technol. 2009, 100, 1595–1607. (2) Matsushita, Y.; Inomata, T.; Hasegawa, T.; Fukushima, K. Bioresour. Technol. 2009, 100, 1024–1026. (3) Himmel, M. E.; Shi-You, D.; Johnson, D. K.; Adney, W. S.; Nimlos, M. R.; Brady, J. W.; Foust, T. D. Science 2007, 315, 804–807. (4) Zabihi, S.; Alinia, R.; Esmaeilzadeh, F.; Kalajahi, J. F. Biosyst. Eng. 2010, 105, 288–297.


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(5) Jasiukaitytea, E.; Kunaver, M.; Crestini, C. Catal. Today 2010, 156, 23–30. (6) Donohoe, B. S.; Decker, S. R.; Tucker, M. P.; Himmel, M. E.; Vinzant, T. B. Biotechnol. Bioeng. 2008, 101, 913–25. (7) Zhu, J. Y.; Pan, X. J. Bioresour. Technol. 2010, 101, 4992–5002. (8) Kumar, S.; Kothari, U.; Kong, L.; Lee, Y. Y.; Gupta, R. B. Biomass Bioenergy 2011, 35, 956–968. (9) Zhuang, X.; Yuan, Z.; Ma, L.; Wu, C.; Xu, M.; Xu, J.; Zhu, S.; Wei, Q. Biotechnol. Adv. 2009, 27, 578–582. (10) Garrote, G.; Domínguez, H.;

Parajó, J. C. Holz als Roh- und Werkstoff 1999, 57, 191-202.

(11) Heitz, M.; Carrasco, F.; Rubio, M.; Chauvette, G.; Chornet, E.; Jaulin, L.; Overend, R. P. Can. J. Chem. Eng. 1986, 64, 647-650. (12) Lv, H.; Yan, L.; Zhang, M.; Geng, Z.; Ren, M.; Sun, Y. Chem. Eng. Technol. 2013, 36, 1899–1906. (13) Jin, F.; Zhou, Z.; Kishita, A.; Enomoto, H. J. Mater. Sci. 2006, 41, 1495–1500. (14) Kumar, R.; Wyman, C. E. Biotechnol. Bioeng. 2009, 102, 1544–1557. (15) Sluiter, A.; Crocker, D.; Hames, B.; Ruiz, R.; Scarlate, C.; Sluiter, J. National Renewable Energy Laboratory. Gloden, CO., 2008(LAP-002). (16) Mojca, S.; Petra, K.; Majda, H.; Andreja, R. H.; Marjana, S.; Zeljko, K. Food Chem. 2005, 89, 191–198. (17) Kim, J. W.; Mazza, G. Food Chem. 2006, 54, 7575–7584. (18) Luo, C.; Brink, D. L.; Blanch, H. W. Biomass Bioenergy 2002, 22, 125–138. (19) Martin, C.; Jonsson, L. J. Enzyme Microb.Technol. 2003, 32, 386–395. (20) Wang, Y. W.; Wang, Y. J.; Zhang, W. J. Liquor-Making Sci. Technol. 2009, 10, 91–94.


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(21) Roberto, I. C.; Lacis, L. S.; Barbosa, M. F. S.; Mancilha, I. M. D. Process Biochem. 1991, 26, 15–21. (22) Hongdan, Z.; Shaohua, X.; Shubin, W. Bioresour. Technol. 2013, 143, 391–396. (23) Gong, C. S.; Cao, N. J.; Du, J.; Tsao, G. T. Adv. Biochem. Eng. Biotechnol. 1999, 65, 207– 41. (24) Zhuang, X.; Qiang, Y.; Wen, W.; Wei, Q.; Wang, Q.; Tan, X.; Yuan, Z. Appl. Biochem. Biotechnol. 2012, 168, 206–218. (25) Ximenes, E.; Kim, Y.; Mosier, N.; Dien, B.; Ladisch, M. Enzyme Microb. Technol. 2010, 46, 170–176. (26) Jian, X.; Thomsen, M. H.; Thomsen, A. B. Appl. Microb. Biotechnol. 2009, 86, 509–16. (27) Feng, Y.; Jiang, J.; Zhu, L.; Wu X. Chem. Ind. For. Prod. 2009, 29, 154–158. (28) Nan, C.; Fan, J. B.; Jin, X.; Jie, C.; Yi, L. Biochim. Biophys. Acta 2006, 1764, 1029–35. (29) Jin, X.; Fan, J. B.; Nan, C.; Jie, C.; Yi, L. Colloids Surf., B 2006, 49, 175–180.


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Table of Contents Graphic and Synopsis

Figure 1. Experimental apparatus for the pretreatment

Figure 2. The concentration of acetic acid in pretreatment liquid with different recycling times of spent liquor

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Concentration of acetic acid (g/L)

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Figure 3. The concentrations of xylose and glucose in pretreatment liquid with different recycling times of spent liquor


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Figure 4. The concentrations of furfural and HMF in pretreatment liquid with different recycling times of spent liquor

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Figure 5. The concentration of phenolics in pretreatment liquid with different recycling times of spent liquor

Figure 6. The yield of glucose in enzymatic hydrolysis with different recycling times of spent liquor


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80 0

1

2

3

4

5

6

Recycle times

Figure 7. SEM images of pretreated corn stover: a, raw corn stover; b, without recycling of spent liquor; c, with the recycling of spent liquor for 3 times; d, with the recycling of spent liquor for 6 times


19

Energy & Fuels

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Page 20 of 21

Table 1. Compositions of Raw Corn Stover and Solid Part (%) with Different Recycling Times Pretreatment condition

cellulose

hemicellulose

lignin

raw corn stover

37.32

20.61

17.54

0

56.61

10.52

26.41

1

55.90

10.37

26.32

2

56.86

10.71

26.44

3

56.79

10.64

26.19

4

56.92

10.98

26.26

5

57.10

11.32

26.03

6

56.73

11.80

25.91

Table 2. Effect of Different Acetic Acid Concentrations on LHW Pretreatment

Acetic acid Xylose g/L g/L

Glucose g/L

Furfural g/L

HMF g/L

Phenolics ppm

Glucose recoveryEH %

0

8.02

0.56

0.35

0.35

480.64

80.82

2

8.13

0.64

0.64

0.43

517.6

81.62

5

8.58

0.88

1.21

0.96

543.46

84.34

10

9.06

1.03

1.59

1.21

594.84

88.55

20

8.43

1.59

2.34

1.64

612.66

88.58

40

6.78

1.56

3.48

2.12

643.19

82.08

Glucose recoveryEH: Glucose recovery in enzymatic hydrolysis Table 3. Glucose Yield with Tween 80 in Enzymatic Hydrolysis


20

Page 21 of 21

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Energy & Fuels

Glucose yield a %

substrate

Glucose yield b %

corn stover pretreated at 180 ºC for 30 min

80.82

83.21

corn stover pretreated at 180 ºC for 30 min with recycled spent liquor for 3 times

85.44

89.84

a: without Tween 80; b: with Tween 80


21

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