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Deep eutectic solvents from hemicellulose-derived acids for the cellulosic ethanol refining of Akebia’ herbal residues
Accepted Manuscript Deep eutectic solvents from hemicellulose-derived acids for the cellulosic ethanol refining of Akebia’ herbal residues Qiang Yu, Aiping Zhang, Wen Wang, Long Chen, Ruxue Bai, Xinshu Zhuang, Qiong Wang, Zhongming Wang, Zhenhong Yuan PII: DOI: Reference:
S0960-8524(17)31724-8 https://doi.org/10.1016/j.biortech.2017.09.159 BITE 18987
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Bioresource Technology
Received Date: Revised Date: Accepted Date:
8 August 2017 21 September 2017 22 September 2017
Please cite this article as: Yu, Q., Zhang, A., Wang, W., Chen, L., Bai, R., Zhuang, X., Wang, Q., Wang, Z., Yuan, Z., Deep eutectic solvents from hemicellulose-derived acids for the cellulosic ethanol refining of Akebia’ herbal residues, Bioresource Technology (2017), doi: https://doi.org/10.1016/j.biortech.2017.09.159
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Deep eutectic solvents from hemicellulose-derived acids for the cellulosic ethanol refining of Akebia’ herbal residues Qiang Yu 1, Aiping Zhang2, Wen Wang1, Long Chen1,3, Ruxue Bai1,3, Xinshu Zhuang1,Qiong Wang*,1, Zhongming Wang1, Zhenhong Yuan1 1
Guangzhou Institute of Energy Conversion, Chinese Academy of Sciences,
CAS Key Laboratory of Renewable Energy, Guangdong Provincial Key Laboratory of New and Renewable Energy Research and Development, Guangzhou 510640, China 2
College of Forestry and Landscape Architecture, South China Agricultural University,
Guangzhou 510642, China 3
University of Chinese Academic of Sciences, Beijing 100039, China
Abstract Here, the potential use of herbal residues of Akebia as feedstock for ethanol production is evaluated. Additionally, five deep eutectic solvents from hemicellulose-derived acids were prepared to overcome biomass recalcitrance. Reaction temperatures had more significant influences on solid loss and chemical composition than the molar ratios of choline chloride (ChCl) to derived acids. Glycolic acid resulted in the maximum levels of lignin, xylan and glucan removal, which were 60.0%, 100% and 71.5%, respectively, at 120°C with a 1:6 molar ratio of ChCl-glycolic acid. In contrast, ChCl-formic acid resulted in the greatest level of glucan retention, at 97.8%, with a lignin removal rate of 40.7% under the same pretreatment conditions. Moreover, ChCl loading could significantly enhance the selectivity of carboxylic acid for lignin dissolution. A 98.0% level of subsequent enzymatic saccharification and a 100% ethanol yield were achieved after ChCl-formic acid pretreatments of Akebia’ herbal residues. *
Corresponding author. Tel.: 86-20-37029690 Email address: [email protected] (Q. Wang) 1
Keywords deep eutectic solvents; herb residues; pretreatment; enzymatic digestibility; ethanol
1. Introduction Medicinal herbs, as an important part of traditional Chinese medicine, are widely used in China and are becoming increasingly available in Europe and North America. There are an estimated 1,600 medicinal herb companies in China, and ~30 million metric tonnes of herb residues (solid waste of herbs after decoction) are generated annually. The waste mangement of these herb residues is an ongoing challenge for these companies and the government. For example, Guangzhou Jiaherb Pharmaceutical Company produces~100,000 tons of herb residues per 20,000 tons of extract, and the cost to transport these solid wastes will reach 140,000 US dollars. The traditional disposal methods for disposing of herb residues, like burying and burning, will seriously pollute the environment. Because of their high nutrient contents, however, they can also be used for microbial composting (Wei-qu et al., 2009) and animal feeding (Li et al., 2017b; Su et al., 2016; Xiaoliang et al., 2006; Xiaoming et al., 2007). Recently, biochar (Chen et al., 2017; Shang et al., 2017; Yang & Qiu, 2011) or bioenergy production from herb residues has attracted global attention because of lignocellulose’s characteristics, such as gasification (Dong et al., 2013; Guo et al., 2013; Zeng et al., 2016a; Zeng et al., 2016b) and pyrolysis (Guo et al., 2015; Wang et al., 2010; Zhan et al., 2017). Automobile ownership in Guangdong Province, which has the greatest number of medicinal herb companies and the highest total gross domestic product (GDP) on mainland China, is growing rapidly, with close to 26 million cars on the road in 2016. The implementation of new electric or ethanol-consuming vehicles in Guangdong 2
would reduce its oil dependence and relieve urban air pollution. Here, the potential of herb residues as a source for cellulosic ethanol production is evaluated. The main processes in ethanol production from lignocellulose include pretreatment to reduce biomass recalcitrance, enzymatic hydrolysis to produce fermentable sugars, and microbial fermentation to generate ethanol (Bhatia et al., 2017). Biomass recalcitrance caused by the tight structure of hemicellulose–lignin–cellulose restricts sugar release, which opposes the cost-effective industrial conversion of lignocellulosic biomass to ethanol. The lignin content surrounding hemicelluloses and cellulose in the plant cell walls are negatively correlated not only to enzymatic digestibility but to the pretreatment process (Yu et al., 2016c), and the removal of lignin could be beneficial in reducing the downstream pressure. Compared with low pH-pretreatment methods, such as dilute acid and liquid hot water, alkaline pretreatments with a high pH value, such as sodium hydroxide and aqueous ammonia, perform well in terms of lignin removal (Wang et al., 2017; Yu et al., 2013). However, the formation of a black liquid is not environmentally friendly. Deep eutectic solvents (DESs), sustainable alternatives to ionic liquids derived from natural and renewable components, are a promising solvent for delignification with high yields of 58%–78% (Alvarez-Vasco et al., 2016). Compared with the traditional alkaline methods, DES pretreatments resulted in a greater enhancement in digestibility, lower energy consumption and simpler procedures for lignin purification and solvent recovery (Gunny et al., 2015). DESs based on choline chloride (ChCl) and a polyalcohol, such as ChCl-glycerol and ChCl-ethylene glycol enhanced the delignification and subsequent enzymatic hydrolysis efficiency of corncobs more than DESs from ChCl-carboxylic acids, like ChCl-lactic acid and ChCl-malic acid (Zhang et al., 2016). However, the effectiveness of ChCl-polyalcohol pretreatments was 3
unstable, varying according to the physio-chemical properties of the biomass (Fang et al., 2017), and there was no significant enhancement in the enzymatic digestibility of corn stover after a ChCl-glycerol pretreatment (Xu et al., 2016). Moreover, most of the literature on DES’ applications for biomass refinement neglect to evaluate the effects of DES pretreatments on the subsequent fermentation process. Thus, the applicability of the overall system is uncertain. ChCl-carboxylic pretreatment is in its infant stage, and the research is not yet thorough or extensive. Here, five DES mixtures were prepared using ChCl as the hydrogen-bond acceptors (HBAs) with five hydrogen-bond donors (HBDs) from the hemicellulose derived acids, formic (Fa), acetic (Aa), glucuronic (Glca), glycolic (Glya) and levulinic (La). Then, the effects of the different DESs on the pretreatment of herb residues of Akebia (HRA) were investigated systematically. In addition, the comparative performances of DES and organic acid pretreatments in terms of enzymatic hydrolysis and ethanol fermentation were evaluated. The related results will provide insights into the industrial applications of DES technology and biofuel production from herb residues. 2. Materials and Methods 2.1. Substrate HRA, the solid residues left after the decoction of Akebia, was collected from the Guangdong Second Traditional Chinese Medicine Hospital in Guangzhou City, China. It was first dried at 105°C to a constant weight, then milled and screened to 8−20 mesh. The chemical composition of the raw material (on a dry weight basis) was determined following the standard procedures of NREL (Sluiter et al., 2008), including 42.8% glucan, 24.5% xylan, 24.6% acid-insoluble lignin and 6.8% extractives. Commercial cellulase (151 FPU/g) was purchased from Imperial Jade 4
Biotechnology Co., Ltd. (Ningxia, China). ChCl, Fa, Aa, Glca, Glya, La and other chemicals were analytical grade reagents obtained from Macklin (Shanghai, China). 2.2. DES preparation and pretreatment The DES preparation was carried out by mixing ChCl and five different hemicellulose-derived acids in different molar ratios, followed by stirring at 200 rpm in an oil bath (60°C) to form a homogeneous and transparent liquid. Then, the HRA was added to maintain a solid:liquid ratio of 1:10 (g:g) at 80, 100 and 120°C for 8 h. The slurry was washed with absolute ethanol (~5 times more than the amount of DES) to remove DES and its soluble fraction using a glass crucible. The wet solid fraction was recovered and divided into two portions, one was used for the measurement of weight loss and chemical composition, and the other was evaluated the enzymatic digestibility. The formulas used were as follows: Solid loss % =
initial solid – residual solid × 100, initial solid
Xylan/glucan/ lignin removal % initial xylan/glucan/lignin – residual xylan/glucan/lignin in solid × 100 initial xylan/glucan/lignin and =
Enzymatic digestibility % =
glucose in the liquid fraction × 100 . potential glucose in the substrate
2.3. Enzymatic digestibility and ethanol fermentation tests Enzymatic hydrolysis tests were performed at 50°C for 72 h in 100-mL Erlenmeyer flasks, each containing 40 mL of sodium citrate buffer (pH 4.8) and 5% (w/v) substrate (Yu et al., 2010). The cellulase-loading amounts were 10, 20 and 40 FPU per gram dry solid. The glucose content in the enzymatic hydrolysate was measured using a Waters HPLC. All experiments were performed in duplicate. Details of the ethanol fermentation process using Saccharomyces. cerevisiae Y2034 5
(purchased from the National Center for Agricultural Utilization Research) were described in a previous work (Yu et al., 2016b). Samples were taken and analyzed for their ethanol concentrations and sugar consumption. The yield of ethanol was calculated according to the percentage of theoretical maximum ethanol yield (Chen et al., 2015). 2.4. Analytical methods Sugars, acids, aldehydes and ethanol in the hydrolysate were determined using HPLC with a Shodex SH1011 column coupled with a refractive index-ultraviolet detector. The mobile phase was 0.005 M H2SO4 at a flow rate of 0.5 mL/min, with a column temperature of 50°C. The recovery of xylose, glucose, furfural and 5-HMF in the pretreatment process were calculated based on the percentages of their theoretical maximum yields. 3. Results and discussion 3.1 Solid loss of HRA after DES pretreatment Five DES mixtures were prepared using ChCl as the HBA with five independent HBDs from hemicellulose derived acids, Fa, Aa, Glca, Glya and La. Although a homogeneous and clear liquid was formed by ChCl and each of the five different HBDs at 60°C, solid particles precipitated upon cooling ChCl-Aa at molar ratios of and ChCl-Glca at molar ratios of 2:1, 1:1, 1:2, 1:4 and 1:6. The proton affinities of the HBA and the HBDs for different DES mixtures may contribute to their phase behaviors (Vigier et al., 2015). Therefore, four DESs (ChCl-Fa, ChCl-Aa, ChCl-Glya and ChCl-La) at the molar ratios of 1:2, 1:4 and 1:6 were selected to further evaluate their abilities to solubilize different chemical compositions of HRA. The loss of solids after ChCl-Fa pretreatment increased with the reaction temperature, especially for the low molar ratio of 1:2 (Fig. 1). The solid loss was only 6
7.5% at 80°C but reached 42.5% at 120°C. In contrast, the influence of molar ratio on the solid loss was insignificant. At 10 °C, for example, the losses were 36.6%, 42.8% and 45.1% for the molar ratios of 1:2, 1:4 and 1:6, respectively. Moreover, a similar relationship between reaction conditions and solid losses of HRA were observed for the ChCl-Aa pretreatment, which resulted in a maximum solid loss of 40.1% at 120°C with a molar ratio of 1:2. Glya had properties of both carboxylic acids and alcohols, and a 17.9% solid loss was obtained after ChCl-Glya pretreatment even under of low acid-loading (molar ratio of 1:2) and low reaction-temperature (80°C) conditions. Then, with an increase in temperature, it further increased to 35.3% at 100°C and 55.1% at 120°C. In contrast, at the high molar ratio of 1:6, the loss of solid was not significantly affected by the reaction temperature (from 41.9% at 80°C to 45.0% at 120°C). Moreover, the performance of ChCl-La was completely different compared with the other three DESs, with no solid loss of HRA detected at 80 and 100°C at the molar ratios of 1:2, 1:4 and 1:6. The highest value was only 31.17% at 120°C with a molar ratio of 1:2. Thus, ChCl-Glya performed best in terms of solid loss of HRA (61.1% at a molar ratio of 1:4 at 120°C) among these four DESs. 3.2 Changes in the chemical composition after DES pretreatment DES prepared with ChCl and a polyalcohol can selectively extract lignin from lignocellulosic biomass with high yields of ~70%–87%, and here, the performances of different DESs of hemicellulose-derived acids on the changes in the chemical composition of HRA were further investigated. For the ChCl-Fa pretreatment, the reaction temperature was the limiting factor for lignin removal (Fig. 2a). For example, the lignin removal value was only 0.2% at 80°C with a molar ratio of 1:2; however, it increased to 30.5% and 40.7% at 100 and 120°C respectively. Although the boiling point for Fa solutions under atmospheric pressure occurs at 107°C, a high reaction 7
temperature (120°C) does not appear to negatively influence lignin removal. The maximum of 51.6% was obtained at 120 °C with a molar ratio of 1:4. In contrast, a negative effect of Fa loading on lignin removal was observed at a 1:6 molar ratio, having values that were 1 (80°C), 0.17 (100°C) and 0.65 (120°C) time less than those at a 1:4 molar ratio. Both DESs of ChCl-Fa and Fa itself (Li et al., 2017a) result in lignin dissolution, with the former performing better (Table 1). When Fa loading increased in the ChCl-Fa solution, the excess Fa may cause a decrease in lignin removal, especially at the low temperature of 80°C. In addition, the removal of xylan increased linearly with temperature and Fa loading, and no xylan could be detected in the solid residues after a high acid-loading pretreatment (1:6 molar ratio). ChCl-Fa had a lower selectivity for glucan removal, with a maximum of 29.0% occurring at 120°C, with a molar ratio of 1:6. Compared with ChCl-Fa, the effects of ChCl-Aa pretreatments on lignin removal were less favorable (Fig. 2b). The peak removal ratio of 33.8% occurred at 100°C with a molar ratio of 1:6. Like ChCl-Fa, the reaction temperature was the limiting factor for the chemical compositional changes in the HRA, especially for the lignin and xylan contents. At a 1:2 molar ratio, for example, the lignin, xylan and glucan removal rates were 8.1%, 35.7% and 32.6% at 80°C, and these increased to 30.2%, 48.9% and 35.4%, respectively, at 100°C. When the temperature reached 120°C, the values changed to 26.9%, 78.9% and 37.2%, respectively, which indicated that a reaction temperature higher than the Aa boiling point (117.9 °C) deterred lignin removal. In addition, Aa has a low pKa of 4.8, and the increase in Aa loading slightly enhanced dissolution except at 80°C. In contrast, increases in either the reaction temperature or Glya-loading amount increased lignin, xylan and glucan removal (Fig. 2c). Their maximum removal levels were 60.0%, 100% and 71.5%, respectively, at 8
120°C with a 1:6 molar ratio of ChCl-Glya. ChCl-La removed the lowest amount of lignin, with a maximum level of only 23.5% at 100°C with a molar ratio of 1:6. The peak xylan and glucan removal values of 71.2% and 41.6%, respectively, occurred at 120°C and a 1:2 molar ratio (Fig. 2d). 3.3 The sugars and by-products in the liquid fraction Hemicellulose-derived acids (Fa, Aa, Glya and La) were used independently as catalysts and components of the DES at the same time. In this case, xylan and glucan were decomposed, and the main products recovered are shown in Fig. 3. The xylose recovery with ChCl-Fa and ChCl-Aa first increased with the temperature and acid loading (Fig. 3a, b), and then decreased when the reaction temperature reached 120°C, which is higher than the boiling point of Fa and Aa. For example, with ChCl-Fa, the peak xylose recovery level of 16.6% appeared at a molar ratio of 1:6 at 100°C and was accompanied by an 8.5% furfural recovery. It was estimated that ~74.9% of xylose existed in the form of an oligomer based on the 100% xylan removal rate. For ChCl-Fa, although a similar phenomenon was observed for glucose, it changed little with recovery levels of 1.2%–3.0% and a 5-HMF yield of 0.2%–1.2%. The stable glucose recovery from 0.9%–1.3% even under the mild reaction conditions of a 1:2 molar ratio at 80°C for the four DES pretreatments indicated that the glucose mainly came from starch. And ChCl-Glya resulted in the greatest glucose recovery, reaching 11.9% at a molar ratio 1:6 at 120°C (Fig. 3c). In contrast, glucose was further degraded with increases in Aa/La loading and temperature (Fig. 3b, d). Furthermore, the decreases in xylose recovery levels were insignificant for ChCl-Glya and ChCl-La, which had boiling points that were higher than 120°C. 3.4 Comparisons between DESs and acid pretreatments The main purpose of a biomass pretreatment is to remove lignin- and 9
hemicellulose-related interference, thereby enhancing the accessibility of cellulose to enzyme digestion. Thus, the optimum conditions for different DES pretreatments were determined based on the highest lignin removal and glucan retention rates. Under the optimum conditions for the four DESs, the glucan retention levels were greater than 70% for ChCl-Aa and ChCl-La, but the levels of lignin removal were low (< 35%) (Table 1). ChCl-Glya resulted in a high decomposition rate of not only to lignin but also polymer saccharides, resulting in only 54.5% of glucan being retained despite the high lignin removal level of 58.4%. In contrast, ChCl-Fa resulted in a high glucan retention level of 97.8% and a lignin removal level of 40.7%. Furthermore, control experiments were conducted to evaluate the role of ChCl loading. Pure Fa resulted in a high solid loss of 66.1% because of the high removal ratios of hemicellulose (100%) and glucan (47.6%), but there was limited lignin removal at 9.0%. The ChCl loading could significantly enhance the selectivity of ChCl-Fa for lignin dissolution. Similar phenomena were observed for Aa, Glya and La pretreatments. The Glya pretreatment without ChCl loading resulted in a high lignin removal level of 40.1% because of its carboxylic acid and alcohol characteristics. 3.5 Enzymatic hydrolysis and fermentation Residual solids after independent ChCl-Fa and Fa pretreatments were tested for enzymatic digestibility and ethanol fermentation to further assess the effects of the DES pretreatment. An increase in cellulase loading could enhance the enzymatic digestibility significantly (Fig. 4a). Additionally, there was a 1.6-fold incremental increase in the untreated HRA when the cellulase loading increased from 10 FPU/g to 40 FPU/g, while there were 2.9- and 4.0-fold incremental increases for Fa and ChCl-Fa treated HRAs, respectively. In addition, the ChCl-Fa pretreatment facilitated the enzymatic hydrolysis with a high 72-h digestibility level of 98.0% with 40 FPU/g 10
of enzyme loading. This increased because of the improved accessibility of cellulose to enzymes, which was caused by the removal of lignin and hemicellulose (Yu et al., 2016a). In contrast, the 72-h digestibility level was only 25.3% for Fa-treated samples, which was even lower than the value (43.2%) for untreated HRA. The formylation of cellulose occurs during the formiline process, and an alkaline deformylation process is necessary for cellulase to recognize cellulose substrates (Chen et al., 2015). Moreover, under low enzyme-loading conditions (10 FPU/g), no significant differences in digestibility were observed between ChCl-Fa-treated and untreated samples. The DES remaining in the solid residues may inhibit the activity of cellulase. The ethanol fermentation of enzymatic hydrolysate data in Fig. 4b further support this hypothesis. Ethanol production was delayed in the ChCl-Fa-treated samples, and the peak ethanol yield occurred at 160 h, while it occurred at 12 h in the hot water-pretreated samples (Yu et al., 2016b) and untreated samples. No ethanol was detected in the fermentation broth of Fa-treated samples. Moreover, the conversion ratio of ethanol based on the initial glucose in the enzymatic hydrolysate reached 182.2% at 12 h for the untreated HRA, which resulted from the further saccharification of cellulose. However, this was not the case for the carbon source requirements of yeast. Thus, ethanol was consumed, and no ethanol remained after 48 h of fermentation. Glucose remained in the enzymatic hydrolysate of Fa-treated samples, while ~100% of glucose was fermented into ethanol in the ChCl-Fa-pretreated samples. 4. Conclusion The acid-ionization constants and boiling points of hemicellulose-derived acids contribute to their performances in terms of biomass deconstruction when exposed to different DES mixtures. ChCl-Fa was highly selective for removing lignin and retaining glucan, which were responsible for the significant increase in the enzymatic 11
digestibility of solid residues. Additionally, ~74.9% of xylose existed in the form of an oligomer in the liquid fraction based on the 100% xylan removal rate. Moreover, the results of ethanol fermentation indicated that DESs are biocompatible, although more research is required to improve their efficiency levels. Appendix A. Supplementary data E-supplementary data for this work can be found in the online version of the paper. Acknowledgments This work was supported financially by the Pearl River S&T Nova Program of Guangzhou, China (201610010110), Young Top-notch Talent of Guangdong Province, China (2016TQ03N647), the National Natural Science Foundation of China (31770617, 21506216, 51506207 and 51561145015), the Science and Technology Planning Project of Guangdong Province, China (2014A010106023), the Key Project of the Natural Science Foundation of Guangdong Province (2015A030311022), the Natural Science Foundation for the Research Team of Guangdong Province (2016A030312007) and the Youth Innovation Promotion Association, CAS (2015289). References 1. Alvarez-Vasco, C., Ma, R., Quintero, M., Guo, M., Geleynse, S., Ramasamy, K.K., Wolcott, M., Zhang, X., 2016. Unique low-molecular-weight lignin with high purity extracted from wood by deep eutectic solvents (DES): a source of lignin for valorization. Green Chem. 18(19), 5133-5141. 2. Bhatia, S.K., Kim, S.-H., Yoon, J.-J., Yang, Y.-H., 2017. Current status and strategies for second generation biofuel production using microbial systems. 12
Energ. Convers. Manage. 148, 1142-1156. 3. Chen, H., Zhao, J., Hu, T., Zhao, X., Liu, D., 2015. A comparison of several organosolv pretreatments for improving the enzymatic hydrolysis of wheat straw: Substrate digestibility, fermentability and structural features. Appl. Energ. 150 (S298), 224-232. 4. Chen, S.J., Zhang, J.S., Zhang, H.W., Wang, X.K., 2017. Removal of hexavalent chromium from contaminated water by chinese herb-extraction residues. Water Air Soil Poll. 228(4),145-158. 5. Dong, Y., Zhang, T., Chang, J., Yan, Y., Liang, J., Lu, Z., Yang, S., Fan, P., 2013. An experimental study of gasification of Chinese herb residues in a circulating fluidized bed with a secondary back feeding device. Nat. Gas Ind. 33(10), 127-132. 6. Fang, C., Thomsen, M.H., Frankaer, C.G., Brudecki, G.P., Schmidt, J.E., AlNashef, I.M., 2017. Reviving pretreatment effectiveness of deep eutectic solvents on lignocellulosic date palm residues by prior recalcitrance reduction. Ind. Eng. Chem. Res., 56(12), 3167-3174. 7. Gunny, A.A.N., Arbain, D., Nashef, E.M., Jamal, P., 2015. Applicability evaluation of Deep Eutectic Solvents–Cellulase system for lignocellulose hydrolysis. Bioresour. Technol. 181, 297-302. 8. Guo, F., Dong, Y., Dong, L., Jing, Y., 2013. An innovative example of herb residues recycling by gasification in a fluidized bed. Waste Manage. 33(4), 825-832. 9. Guo, F., Dong, Y., Lv, Z., Fan, P., Yang, S., Dong, L., 2015. Pyrolysis kinetics of 13
biomass (herb residue) under isothermal condition in a micro fluidized bed. Energ. Convers. Manage. 93, 367-376. 10. Li, M.-F., Yu, P., Li, S.-X., Wu, X.-F., Xiao, X., Bian, J., 2017a. Sequential two-step fractionation of lignocellulose with formic acid organosolv followed by alkaline hydrogen peroxide under mild conditions to prepare easily saccharified cellulose and value-added lignin. Energ. Convers. Manage. 148, 1426-1437. 11. Li, Z., Zhu, Q., Ji, Y., Yin, Y., Wang, D., Kong, X., 2017b. Nutrient Content in Six Chinese Herbal Residues. Nat. Prod. Res. Dev. 29(1), 91-95. 12. Shang, J., Zong, M., Yu, Y., Kong, X., Du, Q., Liao, Q., 2017. Removal of chromium (VI) from water using nanoscale zerovalent iron particles supported on herb-residue biochar. J. Environ. Manage. 197, 331-337. 13. Sluiter, A., Hames, B., Ruiz, R., Scarlata, C., Sluiter, J., Templeton, D., Crocker, D., 2008. Laboratory Analytical Procedure. 14. Su, J., Li, H., Li, Z., Yin, Y., Wang, Z., Kong, X., 2016. Effects of fermented Chinese herb residues on growth performance and intestinal mucosal morphology of weaned piglets. Nat. Prod. Res. Dev. 28(9), 1454-1459. 15. Vigier, K.D.O., Chatel, G., Jérôme, F., 2015. Contribution of deep eutectic solvents for biomass processing: opportunities, challenges, and limitations. ChemCatChem, 7(8), 1250-1260. 16. Wang, P., Zhan, S., Yu, H., Xue, X., Hong, N., 2010. The effects of temperature and catalysts on the pyrolysis of industrial wastes (herb residue). Bioresour. 14
Technol. 101(9), 3236-3241. 17. Wang, S., Li, F., Zhang, P., Jin, S., Tao, X., Tang, X., Ye, J., Nabi, M., Wang, H., 2017. Ultrasound assisted alkaline pretreatment to enhance enzymatic saccharification of grass clipping. Energ. Convers. Manage. 149, 409-415. 18. Wei-qu, L., Zhi-bo, Z., Jie-ming, W.E.N., Shi-li, C., Li, Q.-h., 2009. Effect on complex microbial community during the Chinese herb residues composting process. Guangdong Agr. Sci. (5), 150-152. 19. Xiaoliang, L.I., Dongyou, Y.U., Ya, Q., Zhaozheng, Y.I.N., 2006. Effects of ＂ Shiquan Dabu＂ Chinese herb residues on growth, carcass composition and meat quality in finishing pigs. Journal of Zhejiang University. Agr. Life Sci. 32(4), 433-437. 20. Xiaoming, C., Xiaoliang, L.I., Liping, T., Zhaozheng, Y.I.N., Dongyou, Y.U., Weihuan, F., 2007. Effects of Chinese herb residues on growth performance, intestinal microflora, and immune function of weaning piglets. Acta Agr. Zhejiangensis, 19(1), 50-54. 21. Xu, G.-C., Ding, J.-C., Han, R.-Z., Dong, J.-J., Ni, Y.,2016. Enhancing cellulose accessibility of corn stover by deep eutectic solvent pretreatment for butanol fermentation. Bioresour. Technol. 203, 364-369. 22. Yang, J., Qiu, K., 2011. Development of high surface area mesoporous activated carbons from herb residues. Chem. Eng. J. 167(1), 148-154. 23. Yu, Q., Liu, J., Zhuang, X., Yuan, Z., Wang, W., Qi, W., Wang, Q., Tan, X., Kong, X., 2016a. Liquid hot water pretreatment of energy grasses and its influence of 15
physico-chemical changes on enzymatic digestibility. Bioresour.Technol.199, 265-270. 24. Yu, Q., Zhuang, X., Lv, S., He, M., Zhang, Y., Yuan, Z., Qi, W., Wang, Q., Wang, W., Tan, X., 2013. Liquid hot water pretreatment of sugarcane bagasse and its comparison with chemical pretreatment methods for the sugar recovery and structural changes. Bioresour. Technol. 129, 592-598. 25. Yu, Q., Zhuang, X., Wang, W., Qi, W., Wang, Q., Tan, X., Kong, X., Yuan, Z., 2016b. Hemicellulose and lignin removal to improve the enzymatic digestibility and ethanol production. Biomass Bioenerg. 94, 105-109. 26. Yu, Q., Zhuang, X., Yuan, Z., Kong, X., Qi, W., Wang, W., Wang, Q., Tan, X., 2016c. Influence of lignin level on release of hemicellulose-derived sugars in liquid hot water. Int. J. Biol. Macromol. 82, 967-972. 27. Yu, Q., Zhuang, X., Yuan, Z., Wang, Q., Qi, W., Wang, W., Zhang, Y., Xu, J., Xu, H., 2010. Two-step liquid hot water pretreatment of Eucalyptus grandis to enhance sugar recovery and enzymatic digestibility of cellulose. Bioresour. Technol. 101(13), 4895-4899. 28. Zeng, X., Dong, Y.P., Wang, F., Xu, P.J., Shao, R.Y., Dong, P.W., Xu, G.W., Dong, L., 2016a. Fluidized bed two-stage gasification process for clean fuel gas production from herb residue: fundamentals and demonstration. Energ. Fuel. 30(9), 7277-7283. 29. Zeng, X., Shao, R., Wang, F., Dong, P., Yu, J., Xu, G., 2016b. Industrial demonstration plant for the gasification of herb residue by fluidized bed 16
two-stage process. Bioresour. Technol. 206, 93-98. 30. Zhan, H., Yin, X., Huang, Y., Zhang, X., Yuan, H., Xie, J., Wu, C., 2017. Characteristics of NO_x precursors and their formation mechanism during pyrolysis of herb residues. J. Fuel Chem. Tech. 45(3), 279-288. 31. Zhang, C.-W., Xia, S.-Q., Ma, P.-S., 2016. Facile pretreatment of lignocellulosic biomass using deep eutectic solvents. Bioresour. Technol. 219, 1-5.
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Figure Captions Fig. 1 Solid loss of HRA after different DES pretreatments Fig. 2 Changes in the chemical composition of HRA after different DES pretreatments Fig. 3 The sugars and by-products in the liquid fraction after different DES pretreatments Fig. 4 Enzymatic hydrolysis (a) and ethanol fermentation (b) of residual solids after DES pretreatments
18
70
Solid loss (%)
60 50
ChCl:Fa (1:2) ChCl:Fa (1:4) ChCl:Fa (1:6) ChCl:Aa (1:2) ChCl:Aa (1:4) ChCl:Aa (1:6)
ChCl:Glya (1:2) ChCl:Glya (1:4) ChCl:Glya (1:6) ChCl:La (1:2) ChCl:La (1:4) ChCl:La (1:6)
40 30 20 10 0 80
100
120
Temp. ( )
Fig.1 Solid loss of HRA after different DES pretreatment
19
Removal of lignin (%)
50
X-80 X-100 X-120
90 80
40 70 60
30
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L-80 L-100 L-120
50 G-80 G-100 G-120
40 30
10
20
Removal of glucan or xylan (%)
100
a
10 0 1:2
1:4
1:6
Molar ratio of ChCl-Fa
90
30
Removal of lignin (%)
80
20
L-80 L-100 L-120
70 X-80 X-100 X-120
60 50 40
10 30
0
G-80 G-100 G-120 1:2
Removal of glucan or xylan (%)
100
b
20 10 1:4
1:6
Molar ratio of ChCl-Aa 100 90
60
Removal of lignin (%)
80 50
70 X-80 X-100 X-120
40 30 20 10
60 50 40 30
G-80 G-100 G-120
L-80 L-100 L-120
0
20 10 0
1:2
1:4
1:6
Molar ratio of ChCl-Glya 20
Removal of glucan or xylan (%)
c 70
X-80 X-100 X-120
70 60
Removal of lignin (%)
30
G-80 G-100 G-120
50 40
20
30 20
10 L-80 L-100 L-120
10
0
Removal of glucan or xylan (%)
d 40
0 1/2
1/4
1/6
Molar ratio of ChCl-La
Fig.2 The changes of chemical composition of HRA after different DES pretreatment
21
a 30 25
5-HMF Furfural Glucose Xylose
Recovery (%)
20
15
10
5
0 1:2-80 1:4-80 1:6-80 1:2-1001:4-1001:6-1001:2-1201:4-1201:6-120
Molar ratio-Temp. for ChCl-Fa
b 15 5-HMF Furfural Glucose Xylose
Recovery (%)
10
5
0 1:2-80 1:4-80 1:6-80 1:2-1001:4-1001:6-1001:2-1201:4-1201:6-120
Molar ratio-Temp. for ChCl-Aa c 25
Recovery (%)
20
5-HMF Furfural Glucose Xylose
15
10
5
0 1:2-80 1:4-80 1:6-80 1:2-1001:4-1001:6-1001:2-1201:4-1201:6-120
Molar ratio-Temp. for ChCl-Glya
22
Recovery (%)
d 10
5-HMF Furfural Glucose Xylose
5
0 1:2-80 1:4-80 1:6-80 1:2-1001:4-1001:6-1001:2-1201:4-1201:6-120
Molar ratio-Temp. for ChCl-La
Fig.3 The sugars and by-products in the liquid fraction after different DES pretreatment
23
a 100
Untreated-10FPU/g Untreated-20FPU/g Untreated-40FPU/g Fa-10FPU/g Fa-20FPU/g Fa-40FPU/g
90
Enzymatic digestibility (%)
80 70
ChCl-Fa-10FPU/g ChCl-Fa-20FPU/g ChCl-Fa-40FPU/g
60 50 40 30 20 10 0 0
10
20
30
40
50
60
70
80
Time (h)
Glucose from untreated-40FPU/g Glucose from ChCl-Fa-40FPU/g Glucose from Fa-40FPU/g Ethanol from untreated-40FPU/g Ethanol from ChCl-Fa-40FPU/g Ethanol from Fa-40FPU/g
Conc. of glucose (g/l)
12 10 8
100
6 4 2 0 0
20
40
60
80
100
120
140
160
0 180
Time (h)
Fig.4 Enzymatic hydrolysis (a) and ethanol fermentation (b)
24
Yield of ethanol (%)
200
b 14
Table 1 The optimum conditions of different DES pretreatment and comparison with acid method DES or acid
ChCl-Fa
Molar
Temp.
Solid
Lignin
Xylan
Cellulose
ratio
( °C)
loss
removal
removal
remaining
(%)
(%)
(%)
(%)
42.5
40.7
87.0
97.8
66.1
9.0
100
52.4
24.6
33.8
55.4
71.1
8.9
11.3
30.9
93.4
61.1
58.4
89.7
54.5
40.1
40.1
66.1
46.6
22.4
20.2
47.2
82.1
0.1
0
29.2
100
1:2
120
Formic acid ChCl-Aa
1:6
100
Acetic acid ChCl-Glya
1:4
120
Glycolic acid ChCl-La Levulinic acid
1:4
120
25
Highlights Herb residues has the potential to be a feedstock for ethanol production ChCl-Fa has a high selectivity of lignin removal and glucan remaining ChCl loading could enhance the selectivity of lignin dissolution significantly DES has a good biocompatibility with high enzymatic digestibility and ethanol yield
26
S0960-8524(17)31724-8 https://doi.org/10.1016/j.biortech.2017.09.159 BITE 18987
To appear in:
Bioresource Technology
Received Date: Revised Date: Accepted Date:
8 August 2017 21 September 2017 22 September 2017
Please cite this article as: Yu, Q., Zhang, A., Wang, W., Chen, L., Bai, R., Zhuang, X., Wang, Q., Wang, Z., Yuan, Z., Deep eutectic solvents from hemicellulose-derived acids for the cellulosic ethanol refining of Akebia’ herbal residues, Bioresource Technology (2017), doi: https://doi.org/10.1016/j.biortech.2017.09.159
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.
Deep eutectic solvents from hemicellulose-derived acids for the cellulosic ethanol refining of Akebia’ herbal residues Qiang Yu 1, Aiping Zhang2, Wen Wang1, Long Chen1,3, Ruxue Bai1,3, Xinshu Zhuang1,Qiong Wang*,1, Zhongming Wang1, Zhenhong Yuan1 1
Guangzhou Institute of Energy Conversion, Chinese Academy of Sciences,
CAS Key Laboratory of Renewable Energy, Guangdong Provincial Key Laboratory of New and Renewable Energy Research and Development, Guangzhou 510640, China 2
College of Forestry and Landscape Architecture, South China Agricultural University,
Guangzhou 510642, China 3
University of Chinese Academic of Sciences, Beijing 100039, China
Abstract Here, the potential use of herbal residues of Akebia as feedstock for ethanol production is evaluated. Additionally, five deep eutectic solvents from hemicellulose-derived acids were prepared to overcome biomass recalcitrance. Reaction temperatures had more significant influences on solid loss and chemical composition than the molar ratios of choline chloride (ChCl) to derived acids. Glycolic acid resulted in the maximum levels of lignin, xylan and glucan removal, which were 60.0%, 100% and 71.5%, respectively, at 120°C with a 1:6 molar ratio of ChCl-glycolic acid. In contrast, ChCl-formic acid resulted in the greatest level of glucan retention, at 97.8%, with a lignin removal rate of 40.7% under the same pretreatment conditions. Moreover, ChCl loading could significantly enhance the selectivity of carboxylic acid for lignin dissolution. A 98.0% level of subsequent enzymatic saccharification and a 100% ethanol yield were achieved after ChCl-formic acid pretreatments of Akebia’ herbal residues. *
Corresponding author. Tel.: 86-20-37029690 Email address: [email protected] (Q. Wang) 1
Keywords deep eutectic solvents; herb residues; pretreatment; enzymatic digestibility; ethanol
1. Introduction Medicinal herbs, as an important part of traditional Chinese medicine, are widely used in China and are becoming increasingly available in Europe and North America. There are an estimated 1,600 medicinal herb companies in China, and ~30 million metric tonnes of herb residues (solid waste of herbs after decoction) are generated annually. The waste mangement of these herb residues is an ongoing challenge for these companies and the government. For example, Guangzhou Jiaherb Pharmaceutical Company produces~100,000 tons of herb residues per 20,000 tons of extract, and the cost to transport these solid wastes will reach 140,000 US dollars. The traditional disposal methods for disposing of herb residues, like burying and burning, will seriously pollute the environment. Because of their high nutrient contents, however, they can also be used for microbial composting (Wei-qu et al., 2009) and animal feeding (Li et al., 2017b; Su et al., 2016; Xiaoliang et al., 2006; Xiaoming et al., 2007). Recently, biochar (Chen et al., 2017; Shang et al., 2017; Yang & Qiu, 2011) or bioenergy production from herb residues has attracted global attention because of lignocellulose’s characteristics, such as gasification (Dong et al., 2013; Guo et al., 2013; Zeng et al., 2016a; Zeng et al., 2016b) and pyrolysis (Guo et al., 2015; Wang et al., 2010; Zhan et al., 2017). Automobile ownership in Guangdong Province, which has the greatest number of medicinal herb companies and the highest total gross domestic product (GDP) on mainland China, is growing rapidly, with close to 26 million cars on the road in 2016. The implementation of new electric or ethanol-consuming vehicles in Guangdong 2
would reduce its oil dependence and relieve urban air pollution. Here, the potential of herb residues as a source for cellulosic ethanol production is evaluated. The main processes in ethanol production from lignocellulose include pretreatment to reduce biomass recalcitrance, enzymatic hydrolysis to produce fermentable sugars, and microbial fermentation to generate ethanol (Bhatia et al., 2017). Biomass recalcitrance caused by the tight structure of hemicellulose–lignin–cellulose restricts sugar release, which opposes the cost-effective industrial conversion of lignocellulosic biomass to ethanol. The lignin content surrounding hemicelluloses and cellulose in the plant cell walls are negatively correlated not only to enzymatic digestibility but to the pretreatment process (Yu et al., 2016c), and the removal of lignin could be beneficial in reducing the downstream pressure. Compared with low pH-pretreatment methods, such as dilute acid and liquid hot water, alkaline pretreatments with a high pH value, such as sodium hydroxide and aqueous ammonia, perform well in terms of lignin removal (Wang et al., 2017; Yu et al., 2013). However, the formation of a black liquid is not environmentally friendly. Deep eutectic solvents (DESs), sustainable alternatives to ionic liquids derived from natural and renewable components, are a promising solvent for delignification with high yields of 58%–78% (Alvarez-Vasco et al., 2016). Compared with the traditional alkaline methods, DES pretreatments resulted in a greater enhancement in digestibility, lower energy consumption and simpler procedures for lignin purification and solvent recovery (Gunny et al., 2015). DESs based on choline chloride (ChCl) and a polyalcohol, such as ChCl-glycerol and ChCl-ethylene glycol enhanced the delignification and subsequent enzymatic hydrolysis efficiency of corncobs more than DESs from ChCl-carboxylic acids, like ChCl-lactic acid and ChCl-malic acid (Zhang et al., 2016). However, the effectiveness of ChCl-polyalcohol pretreatments was 3
unstable, varying according to the physio-chemical properties of the biomass (Fang et al., 2017), and there was no significant enhancement in the enzymatic digestibility of corn stover after a ChCl-glycerol pretreatment (Xu et al., 2016). Moreover, most of the literature on DES’ applications for biomass refinement neglect to evaluate the effects of DES pretreatments on the subsequent fermentation process. Thus, the applicability of the overall system is uncertain. ChCl-carboxylic pretreatment is in its infant stage, and the research is not yet thorough or extensive. Here, five DES mixtures were prepared using ChCl as the hydrogen-bond acceptors (HBAs) with five hydrogen-bond donors (HBDs) from the hemicellulose derived acids, formic (Fa), acetic (Aa), glucuronic (Glca), glycolic (Glya) and levulinic (La). Then, the effects of the different DESs on the pretreatment of herb residues of Akebia (HRA) were investigated systematically. In addition, the comparative performances of DES and organic acid pretreatments in terms of enzymatic hydrolysis and ethanol fermentation were evaluated. The related results will provide insights into the industrial applications of DES technology and biofuel production from herb residues. 2. Materials and Methods 2.1. Substrate HRA, the solid residues left after the decoction of Akebia, was collected from the Guangdong Second Traditional Chinese Medicine Hospital in Guangzhou City, China. It was first dried at 105°C to a constant weight, then milled and screened to 8−20 mesh. The chemical composition of the raw material (on a dry weight basis) was determined following the standard procedures of NREL (Sluiter et al., 2008), including 42.8% glucan, 24.5% xylan, 24.6% acid-insoluble lignin and 6.8% extractives. Commercial cellulase (151 FPU/g) was purchased from Imperial Jade 4
Biotechnology Co., Ltd. (Ningxia, China). ChCl, Fa, Aa, Glca, Glya, La and other chemicals were analytical grade reagents obtained from Macklin (Shanghai, China). 2.2. DES preparation and pretreatment The DES preparation was carried out by mixing ChCl and five different hemicellulose-derived acids in different molar ratios, followed by stirring at 200 rpm in an oil bath (60°C) to form a homogeneous and transparent liquid. Then, the HRA was added to maintain a solid:liquid ratio of 1:10 (g:g) at 80, 100 and 120°C for 8 h. The slurry was washed with absolute ethanol (~5 times more than the amount of DES) to remove DES and its soluble fraction using a glass crucible. The wet solid fraction was recovered and divided into two portions, one was used for the measurement of weight loss and chemical composition, and the other was evaluated the enzymatic digestibility. The formulas used were as follows: Solid loss % =
initial solid – residual solid × 100, initial solid
Xylan/glucan/ lignin removal % initial xylan/glucan/lignin – residual xylan/glucan/lignin in solid × 100 initial xylan/glucan/lignin and =
Enzymatic digestibility % =
glucose in the liquid fraction × 100 . potential glucose in the substrate
2.3. Enzymatic digestibility and ethanol fermentation tests Enzymatic hydrolysis tests were performed at 50°C for 72 h in 100-mL Erlenmeyer flasks, each containing 40 mL of sodium citrate buffer (pH 4.8) and 5% (w/v) substrate (Yu et al., 2010). The cellulase-loading amounts were 10, 20 and 40 FPU per gram dry solid. The glucose content in the enzymatic hydrolysate was measured using a Waters HPLC. All experiments were performed in duplicate. Details of the ethanol fermentation process using Saccharomyces. cerevisiae Y2034 5
(purchased from the National Center for Agricultural Utilization Research) were described in a previous work (Yu et al., 2016b). Samples were taken and analyzed for their ethanol concentrations and sugar consumption. The yield of ethanol was calculated according to the percentage of theoretical maximum ethanol yield (Chen et al., 2015). 2.4. Analytical methods Sugars, acids, aldehydes and ethanol in the hydrolysate were determined using HPLC with a Shodex SH1011 column coupled with a refractive index-ultraviolet detector. The mobile phase was 0.005 M H2SO4 at a flow rate of 0.5 mL/min, with a column temperature of 50°C. The recovery of xylose, glucose, furfural and 5-HMF in the pretreatment process were calculated based on the percentages of their theoretical maximum yields. 3. Results and discussion 3.1 Solid loss of HRA after DES pretreatment Five DES mixtures were prepared using ChCl as the HBA with five independent HBDs from hemicellulose derived acids, Fa, Aa, Glca, Glya and La. Although a homogeneous and clear liquid was formed by ChCl and each of the five different HBDs at 60°C, solid particles precipitated upon cooling ChCl-Aa at molar ratios of and ChCl-Glca at molar ratios of 2:1, 1:1, 1:2, 1:4 and 1:6. The proton affinities of the HBA and the HBDs for different DES mixtures may contribute to their phase behaviors (Vigier et al., 2015). Therefore, four DESs (ChCl-Fa, ChCl-Aa, ChCl-Glya and ChCl-La) at the molar ratios of 1:2, 1:4 and 1:6 were selected to further evaluate their abilities to solubilize different chemical compositions of HRA. The loss of solids after ChCl-Fa pretreatment increased with the reaction temperature, especially for the low molar ratio of 1:2 (Fig. 1). The solid loss was only 6
7.5% at 80°C but reached 42.5% at 120°C. In contrast, the influence of molar ratio on the solid loss was insignificant. At 10 °C, for example, the losses were 36.6%, 42.8% and 45.1% for the molar ratios of 1:2, 1:4 and 1:6, respectively. Moreover, a similar relationship between reaction conditions and solid losses of HRA were observed for the ChCl-Aa pretreatment, which resulted in a maximum solid loss of 40.1% at 120°C with a molar ratio of 1:2. Glya had properties of both carboxylic acids and alcohols, and a 17.9% solid loss was obtained after ChCl-Glya pretreatment even under of low acid-loading (molar ratio of 1:2) and low reaction-temperature (80°C) conditions. Then, with an increase in temperature, it further increased to 35.3% at 100°C and 55.1% at 120°C. In contrast, at the high molar ratio of 1:6, the loss of solid was not significantly affected by the reaction temperature (from 41.9% at 80°C to 45.0% at 120°C). Moreover, the performance of ChCl-La was completely different compared with the other three DESs, with no solid loss of HRA detected at 80 and 100°C at the molar ratios of 1:2, 1:4 and 1:6. The highest value was only 31.17% at 120°C with a molar ratio of 1:2. Thus, ChCl-Glya performed best in terms of solid loss of HRA (61.1% at a molar ratio of 1:4 at 120°C) among these four DESs. 3.2 Changes in the chemical composition after DES pretreatment DES prepared with ChCl and a polyalcohol can selectively extract lignin from lignocellulosic biomass with high yields of ~70%–87%, and here, the performances of different DESs of hemicellulose-derived acids on the changes in the chemical composition of HRA were further investigated. For the ChCl-Fa pretreatment, the reaction temperature was the limiting factor for lignin removal (Fig. 2a). For example, the lignin removal value was only 0.2% at 80°C with a molar ratio of 1:2; however, it increased to 30.5% and 40.7% at 100 and 120°C respectively. Although the boiling point for Fa solutions under atmospheric pressure occurs at 107°C, a high reaction 7
temperature (120°C) does not appear to negatively influence lignin removal. The maximum of 51.6% was obtained at 120 °C with a molar ratio of 1:4. In contrast, a negative effect of Fa loading on lignin removal was observed at a 1:6 molar ratio, having values that were 1 (80°C), 0.17 (100°C) and 0.65 (120°C) time less than those at a 1:4 molar ratio. Both DESs of ChCl-Fa and Fa itself (Li et al., 2017a) result in lignin dissolution, with the former performing better (Table 1). When Fa loading increased in the ChCl-Fa solution, the excess Fa may cause a decrease in lignin removal, especially at the low temperature of 80°C. In addition, the removal of xylan increased linearly with temperature and Fa loading, and no xylan could be detected in the solid residues after a high acid-loading pretreatment (1:6 molar ratio). ChCl-Fa had a lower selectivity for glucan removal, with a maximum of 29.0% occurring at 120°C, with a molar ratio of 1:6. Compared with ChCl-Fa, the effects of ChCl-Aa pretreatments on lignin removal were less favorable (Fig. 2b). The peak removal ratio of 33.8% occurred at 100°C with a molar ratio of 1:6. Like ChCl-Fa, the reaction temperature was the limiting factor for the chemical compositional changes in the HRA, especially for the lignin and xylan contents. At a 1:2 molar ratio, for example, the lignin, xylan and glucan removal rates were 8.1%, 35.7% and 32.6% at 80°C, and these increased to 30.2%, 48.9% and 35.4%, respectively, at 100°C. When the temperature reached 120°C, the values changed to 26.9%, 78.9% and 37.2%, respectively, which indicated that a reaction temperature higher than the Aa boiling point (117.9 °C) deterred lignin removal. In addition, Aa has a low pKa of 4.8, and the increase in Aa loading slightly enhanced dissolution except at 80°C. In contrast, increases in either the reaction temperature or Glya-loading amount increased lignin, xylan and glucan removal (Fig. 2c). Their maximum removal levels were 60.0%, 100% and 71.5%, respectively, at 8
120°C with a 1:6 molar ratio of ChCl-Glya. ChCl-La removed the lowest amount of lignin, with a maximum level of only 23.5% at 100°C with a molar ratio of 1:6. The peak xylan and glucan removal values of 71.2% and 41.6%, respectively, occurred at 120°C and a 1:2 molar ratio (Fig. 2d). 3.3 The sugars and by-products in the liquid fraction Hemicellulose-derived acids (Fa, Aa, Glya and La) were used independently as catalysts and components of the DES at the same time. In this case, xylan and glucan were decomposed, and the main products recovered are shown in Fig. 3. The xylose recovery with ChCl-Fa and ChCl-Aa first increased with the temperature and acid loading (Fig. 3a, b), and then decreased when the reaction temperature reached 120°C, which is higher than the boiling point of Fa and Aa. For example, with ChCl-Fa, the peak xylose recovery level of 16.6% appeared at a molar ratio of 1:6 at 100°C and was accompanied by an 8.5% furfural recovery. It was estimated that ~74.9% of xylose existed in the form of an oligomer based on the 100% xylan removal rate. For ChCl-Fa, although a similar phenomenon was observed for glucose, it changed little with recovery levels of 1.2%–3.0% and a 5-HMF yield of 0.2%–1.2%. The stable glucose recovery from 0.9%–1.3% even under the mild reaction conditions of a 1:2 molar ratio at 80°C for the four DES pretreatments indicated that the glucose mainly came from starch. And ChCl-Glya resulted in the greatest glucose recovery, reaching 11.9% at a molar ratio 1:6 at 120°C (Fig. 3c). In contrast, glucose was further degraded with increases in Aa/La loading and temperature (Fig. 3b, d). Furthermore, the decreases in xylose recovery levels were insignificant for ChCl-Glya and ChCl-La, which had boiling points that were higher than 120°C. 3.4 Comparisons between DESs and acid pretreatments The main purpose of a biomass pretreatment is to remove lignin- and 9
hemicellulose-related interference, thereby enhancing the accessibility of cellulose to enzyme digestion. Thus, the optimum conditions for different DES pretreatments were determined based on the highest lignin removal and glucan retention rates. Under the optimum conditions for the four DESs, the glucan retention levels were greater than 70% for ChCl-Aa and ChCl-La, but the levels of lignin removal were low (< 35%) (Table 1). ChCl-Glya resulted in a high decomposition rate of not only to lignin but also polymer saccharides, resulting in only 54.5% of glucan being retained despite the high lignin removal level of 58.4%. In contrast, ChCl-Fa resulted in a high glucan retention level of 97.8% and a lignin removal level of 40.7%. Furthermore, control experiments were conducted to evaluate the role of ChCl loading. Pure Fa resulted in a high solid loss of 66.1% because of the high removal ratios of hemicellulose (100%) and glucan (47.6%), but there was limited lignin removal at 9.0%. The ChCl loading could significantly enhance the selectivity of ChCl-Fa for lignin dissolution. Similar phenomena were observed for Aa, Glya and La pretreatments. The Glya pretreatment without ChCl loading resulted in a high lignin removal level of 40.1% because of its carboxylic acid and alcohol characteristics. 3.5 Enzymatic hydrolysis and fermentation Residual solids after independent ChCl-Fa and Fa pretreatments were tested for enzymatic digestibility and ethanol fermentation to further assess the effects of the DES pretreatment. An increase in cellulase loading could enhance the enzymatic digestibility significantly (Fig. 4a). Additionally, there was a 1.6-fold incremental increase in the untreated HRA when the cellulase loading increased from 10 FPU/g to 40 FPU/g, while there were 2.9- and 4.0-fold incremental increases for Fa and ChCl-Fa treated HRAs, respectively. In addition, the ChCl-Fa pretreatment facilitated the enzymatic hydrolysis with a high 72-h digestibility level of 98.0% with 40 FPU/g 10
of enzyme loading. This increased because of the improved accessibility of cellulose to enzymes, which was caused by the removal of lignin and hemicellulose (Yu et al., 2016a). In contrast, the 72-h digestibility level was only 25.3% for Fa-treated samples, which was even lower than the value (43.2%) for untreated HRA. The formylation of cellulose occurs during the formiline process, and an alkaline deformylation process is necessary for cellulase to recognize cellulose substrates (Chen et al., 2015). Moreover, under low enzyme-loading conditions (10 FPU/g), no significant differences in digestibility were observed between ChCl-Fa-treated and untreated samples. The DES remaining in the solid residues may inhibit the activity of cellulase. The ethanol fermentation of enzymatic hydrolysate data in Fig. 4b further support this hypothesis. Ethanol production was delayed in the ChCl-Fa-treated samples, and the peak ethanol yield occurred at 160 h, while it occurred at 12 h in the hot water-pretreated samples (Yu et al., 2016b) and untreated samples. No ethanol was detected in the fermentation broth of Fa-treated samples. Moreover, the conversion ratio of ethanol based on the initial glucose in the enzymatic hydrolysate reached 182.2% at 12 h for the untreated HRA, which resulted from the further saccharification of cellulose. However, this was not the case for the carbon source requirements of yeast. Thus, ethanol was consumed, and no ethanol remained after 48 h of fermentation. Glucose remained in the enzymatic hydrolysate of Fa-treated samples, while ~100% of glucose was fermented into ethanol in the ChCl-Fa-pretreated samples. 4. Conclusion The acid-ionization constants and boiling points of hemicellulose-derived acids contribute to their performances in terms of biomass deconstruction when exposed to different DES mixtures. ChCl-Fa was highly selective for removing lignin and retaining glucan, which were responsible for the significant increase in the enzymatic 11
digestibility of solid residues. Additionally, ~74.9% of xylose existed in the form of an oligomer in the liquid fraction based on the 100% xylan removal rate. Moreover, the results of ethanol fermentation indicated that DESs are biocompatible, although more research is required to improve their efficiency levels. Appendix A. Supplementary data E-supplementary data for this work can be found in the online version of the paper. Acknowledgments This work was supported financially by the Pearl River S&T Nova Program of Guangzhou, China (201610010110), Young Top-notch Talent of Guangdong Province, China (2016TQ03N647), the National Natural Science Foundation of China (31770617, 21506216, 51506207 and 51561145015), the Science and Technology Planning Project of Guangdong Province, China (2014A010106023), the Key Project of the Natural Science Foundation of Guangdong Province (2015A030311022), the Natural Science Foundation for the Research Team of Guangdong Province (2016A030312007) and the Youth Innovation Promotion Association, CAS (2015289). References 1. Alvarez-Vasco, C., Ma, R., Quintero, M., Guo, M., Geleynse, S., Ramasamy, K.K., Wolcott, M., Zhang, X., 2016. Unique low-molecular-weight lignin with high purity extracted from wood by deep eutectic solvents (DES): a source of lignin for valorization. Green Chem. 18(19), 5133-5141. 2. Bhatia, S.K., Kim, S.-H., Yoon, J.-J., Yang, Y.-H., 2017. Current status and strategies for second generation biofuel production using microbial systems. 12
Energ. Convers. Manage. 148, 1142-1156. 3. Chen, H., Zhao, J., Hu, T., Zhao, X., Liu, D., 2015. A comparison of several organosolv pretreatments for improving the enzymatic hydrolysis of wheat straw: Substrate digestibility, fermentability and structural features. Appl. Energ. 150 (S298), 224-232. 4. Chen, S.J., Zhang, J.S., Zhang, H.W., Wang, X.K., 2017. Removal of hexavalent chromium from contaminated water by chinese herb-extraction residues. Water Air Soil Poll. 228(4),145-158. 5. Dong, Y., Zhang, T., Chang, J., Yan, Y., Liang, J., Lu, Z., Yang, S., Fan, P., 2013. An experimental study of gasification of Chinese herb residues in a circulating fluidized bed with a secondary back feeding device. Nat. Gas Ind. 33(10), 127-132. 6. Fang, C., Thomsen, M.H., Frankaer, C.G., Brudecki, G.P., Schmidt, J.E., AlNashef, I.M., 2017. Reviving pretreatment effectiveness of deep eutectic solvents on lignocellulosic date palm residues by prior recalcitrance reduction. Ind. Eng. Chem. Res., 56(12), 3167-3174. 7. Gunny, A.A.N., Arbain, D., Nashef, E.M., Jamal, P., 2015. Applicability evaluation of Deep Eutectic Solvents–Cellulase system for lignocellulose hydrolysis. Bioresour. Technol. 181, 297-302. 8. Guo, F., Dong, Y., Dong, L., Jing, Y., 2013. An innovative example of herb residues recycling by gasification in a fluidized bed. Waste Manage. 33(4), 825-832. 9. Guo, F., Dong, Y., Lv, Z., Fan, P., Yang, S., Dong, L., 2015. Pyrolysis kinetics of 13
biomass (herb residue) under isothermal condition in a micro fluidized bed. Energ. Convers. Manage. 93, 367-376. 10. Li, M.-F., Yu, P., Li, S.-X., Wu, X.-F., Xiao, X., Bian, J., 2017a. Sequential two-step fractionation of lignocellulose with formic acid organosolv followed by alkaline hydrogen peroxide under mild conditions to prepare easily saccharified cellulose and value-added lignin. Energ. Convers. Manage. 148, 1426-1437. 11. Li, Z., Zhu, Q., Ji, Y., Yin, Y., Wang, D., Kong, X., 2017b. Nutrient Content in Six Chinese Herbal Residues. Nat. Prod. Res. Dev. 29(1), 91-95. 12. Shang, J., Zong, M., Yu, Y., Kong, X., Du, Q., Liao, Q., 2017. Removal of chromium (VI) from water using nanoscale zerovalent iron particles supported on herb-residue biochar. J. Environ. Manage. 197, 331-337. 13. Sluiter, A., Hames, B., Ruiz, R., Scarlata, C., Sluiter, J., Templeton, D., Crocker, D., 2008. Laboratory Analytical Procedure. 14. Su, J., Li, H., Li, Z., Yin, Y., Wang, Z., Kong, X., 2016. Effects of fermented Chinese herb residues on growth performance and intestinal mucosal morphology of weaned piglets. Nat. Prod. Res. Dev. 28(9), 1454-1459. 15. Vigier, K.D.O., Chatel, G., Jérôme, F., 2015. Contribution of deep eutectic solvents for biomass processing: opportunities, challenges, and limitations. ChemCatChem, 7(8), 1250-1260. 16. Wang, P., Zhan, S., Yu, H., Xue, X., Hong, N., 2010. The effects of temperature and catalysts on the pyrolysis of industrial wastes (herb residue). Bioresour. 14
Technol. 101(9), 3236-3241. 17. Wang, S., Li, F., Zhang, P., Jin, S., Tao, X., Tang, X., Ye, J., Nabi, M., Wang, H., 2017. Ultrasound assisted alkaline pretreatment to enhance enzymatic saccharification of grass clipping. Energ. Convers. Manage. 149, 409-415. 18. Wei-qu, L., Zhi-bo, Z., Jie-ming, W.E.N., Shi-li, C., Li, Q.-h., 2009. Effect on complex microbial community during the Chinese herb residues composting process. Guangdong Agr. Sci. (5), 150-152. 19. Xiaoliang, L.I., Dongyou, Y.U., Ya, Q., Zhaozheng, Y.I.N., 2006. Effects of ＂ Shiquan Dabu＂ Chinese herb residues on growth, carcass composition and meat quality in finishing pigs. Journal of Zhejiang University. Agr. Life Sci. 32(4), 433-437. 20. Xiaoming, C., Xiaoliang, L.I., Liping, T., Zhaozheng, Y.I.N., Dongyou, Y.U., Weihuan, F., 2007. Effects of Chinese herb residues on growth performance, intestinal microflora, and immune function of weaning piglets. Acta Agr. Zhejiangensis, 19(1), 50-54. 21. Xu, G.-C., Ding, J.-C., Han, R.-Z., Dong, J.-J., Ni, Y.,2016. Enhancing cellulose accessibility of corn stover by deep eutectic solvent pretreatment for butanol fermentation. Bioresour. Technol. 203, 364-369. 22. Yang, J., Qiu, K., 2011. Development of high surface area mesoporous activated carbons from herb residues. Chem. Eng. J. 167(1), 148-154. 23. Yu, Q., Liu, J., Zhuang, X., Yuan, Z., Wang, W., Qi, W., Wang, Q., Tan, X., Kong, X., 2016a. Liquid hot water pretreatment of energy grasses and its influence of 15
physico-chemical changes on enzymatic digestibility. Bioresour.Technol.199, 265-270. 24. Yu, Q., Zhuang, X., Lv, S., He, M., Zhang, Y., Yuan, Z., Qi, W., Wang, Q., Wang, W., Tan, X., 2013. Liquid hot water pretreatment of sugarcane bagasse and its comparison with chemical pretreatment methods for the sugar recovery and structural changes. Bioresour. Technol. 129, 592-598. 25. Yu, Q., Zhuang, X., Wang, W., Qi, W., Wang, Q., Tan, X., Kong, X., Yuan, Z., 2016b. Hemicellulose and lignin removal to improve the enzymatic digestibility and ethanol production. Biomass Bioenerg. 94, 105-109. 26. Yu, Q., Zhuang, X., Yuan, Z., Kong, X., Qi, W., Wang, W., Wang, Q., Tan, X., 2016c. Influence of lignin level on release of hemicellulose-derived sugars in liquid hot water. Int. J. Biol. Macromol. 82, 967-972. 27. Yu, Q., Zhuang, X., Yuan, Z., Wang, Q., Qi, W., Wang, W., Zhang, Y., Xu, J., Xu, H., 2010. Two-step liquid hot water pretreatment of Eucalyptus grandis to enhance sugar recovery and enzymatic digestibility of cellulose. Bioresour. Technol. 101(13), 4895-4899. 28. Zeng, X., Dong, Y.P., Wang, F., Xu, P.J., Shao, R.Y., Dong, P.W., Xu, G.W., Dong, L., 2016a. Fluidized bed two-stage gasification process for clean fuel gas production from herb residue: fundamentals and demonstration. Energ. Fuel. 30(9), 7277-7283. 29. Zeng, X., Shao, R., Wang, F., Dong, P., Yu, J., Xu, G., 2016b. Industrial demonstration plant for the gasification of herb residue by fluidized bed 16
two-stage process. Bioresour. Technol. 206, 93-98. 30. Zhan, H., Yin, X., Huang, Y., Zhang, X., Yuan, H., Xie, J., Wu, C., 2017. Characteristics of NO_x precursors and their formation mechanism during pyrolysis of herb residues. J. Fuel Chem. Tech. 45(3), 279-288. 31. Zhang, C.-W., Xia, S.-Q., Ma, P.-S., 2016. Facile pretreatment of lignocellulosic biomass using deep eutectic solvents. Bioresour. Technol. 219, 1-5.
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Figure Captions Fig. 1 Solid loss of HRA after different DES pretreatments Fig. 2 Changes in the chemical composition of HRA after different DES pretreatments Fig. 3 The sugars and by-products in the liquid fraction after different DES pretreatments Fig. 4 Enzymatic hydrolysis (a) and ethanol fermentation (b) of residual solids after DES pretreatments
18
70
Solid loss (%)
60 50
ChCl:Fa (1:2) ChCl:Fa (1:4) ChCl:Fa (1:6) ChCl:Aa (1:2) ChCl:Aa (1:4) ChCl:Aa (1:6)
ChCl:Glya (1:2) ChCl:Glya (1:4) ChCl:Glya (1:6) ChCl:La (1:2) ChCl:La (1:4) ChCl:La (1:6)
40 30 20 10 0 80
100
120
Temp. ( )
Fig.1 Solid loss of HRA after different DES pretreatment
19
Removal of lignin (%)
50
X-80 X-100 X-120
90 80
40 70 60
30
20
L-80 L-100 L-120
50 G-80 G-100 G-120
40 30
10
20
Removal of glucan or xylan (%)
100
a
10 0 1:2
1:4
1:6
Molar ratio of ChCl-Fa
90
30
Removal of lignin (%)
80
20
L-80 L-100 L-120
70 X-80 X-100 X-120
60 50 40
10 30
0
G-80 G-100 G-120 1:2
Removal of glucan or xylan (%)
100
b
20 10 1:4
1:6
Molar ratio of ChCl-Aa 100 90
60
Removal of lignin (%)
80 50
70 X-80 X-100 X-120
40 30 20 10
60 50 40 30
G-80 G-100 G-120
L-80 L-100 L-120
0
20 10 0
1:2
1:4
1:6
Molar ratio of ChCl-Glya 20
Removal of glucan or xylan (%)
c 70
X-80 X-100 X-120
70 60
Removal of lignin (%)
30
G-80 G-100 G-120
50 40
20
30 20
10 L-80 L-100 L-120
10
0
Removal of glucan or xylan (%)
d 40
0 1/2
1/4
1/6
Molar ratio of ChCl-La
Fig.2 The changes of chemical composition of HRA after different DES pretreatment
21
a 30 25
5-HMF Furfural Glucose Xylose
Recovery (%)
20
15
10
5
0 1:2-80 1:4-80 1:6-80 1:2-1001:4-1001:6-1001:2-1201:4-1201:6-120
Molar ratio-Temp. for ChCl-Fa
b 15 5-HMF Furfural Glucose Xylose
Recovery (%)
10
5
0 1:2-80 1:4-80 1:6-80 1:2-1001:4-1001:6-1001:2-1201:4-1201:6-120
Molar ratio-Temp. for ChCl-Aa c 25
Recovery (%)
20
5-HMF Furfural Glucose Xylose
15
10
5
0 1:2-80 1:4-80 1:6-80 1:2-1001:4-1001:6-1001:2-1201:4-1201:6-120
Molar ratio-Temp. for ChCl-Glya
22
Recovery (%)
d 10
5-HMF Furfural Glucose Xylose
5
0 1:2-80 1:4-80 1:6-80 1:2-1001:4-1001:6-1001:2-1201:4-1201:6-120
Molar ratio-Temp. for ChCl-La
Fig.3 The sugars and by-products in the liquid fraction after different DES pretreatment
23
a 100
Untreated-10FPU/g Untreated-20FPU/g Untreated-40FPU/g Fa-10FPU/g Fa-20FPU/g Fa-40FPU/g
90
Enzymatic digestibility (%)
80 70
ChCl-Fa-10FPU/g ChCl-Fa-20FPU/g ChCl-Fa-40FPU/g
60 50 40 30 20 10 0 0
10
20
30
40
50
60
70
80
Time (h)
Glucose from untreated-40FPU/g Glucose from ChCl-Fa-40FPU/g Glucose from Fa-40FPU/g Ethanol from untreated-40FPU/g Ethanol from ChCl-Fa-40FPU/g Ethanol from Fa-40FPU/g
Conc. of glucose (g/l)
12 10 8
100
6 4 2 0 0
20
40
60
80
100
120
140
160
0 180
Time (h)
Fig.4 Enzymatic hydrolysis (a) and ethanol fermentation (b)
24
Yield of ethanol (%)
200
b 14
Table 1 The optimum conditions of different DES pretreatment and comparison with acid method DES or acid
ChCl-Fa
Molar
Temp.
Solid
Lignin
Xylan
Cellulose
ratio
( °C)
loss
removal
removal
remaining
(%)
(%)
(%)
(%)
42.5
40.7
87.0
97.8
66.1
9.0
100
52.4
24.6
33.8
55.4
71.1
8.9
11.3
30.9
93.4
61.1
58.4
89.7
54.5
40.1
40.1
66.1
46.6
22.4
20.2
47.2
82.1
0.1
0
29.2
100
1:2
120
Formic acid ChCl-Aa
1:6
100
Acetic acid ChCl-Glya
1:4
120
Glycolic acid ChCl-La Levulinic acid
1:4
120
25
Highlights Herb residues has the potential to be a feedstock for ethanol production ChCl-Fa has a high selectivity of lignin removal and glucan remaining ChCl loading could enhance the selectivity of lignin dissolution significantly DES has a good biocompatibility with high enzymatic digestibility and ethanol yield
26