Rapid fractionation of lignocellulosic biomass by p-TsOH pretreatment

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Biofuels and Biomass

Rapid fractionation of lignocellulosic biomass by p-TsOH pretreatment Mingyan Yang, Xiaofeng Gao, Meng Lan, Yan Dou, and Xiao Zhang Energy Fuels, Just Accepted Manuscript • DOI: 10.1021/acs.energyfuels.8b03770 • Publication Date (Web): 17 Feb 2019 Downloaded from http://pubs.acs.org on February 19, 2019

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Rapid fractionation of lignocellulosic biomass by p-TsOH pretreatment Mingyan Yang†, ‡*, Xiaofeng Gao†, Meng Lan†, Yan Dou†, ‡, and Xiao Zhang† † School

of Environmental Science and Engineering, Chang’an University, Xi’an, 710054, China;

‡

Shaanxi Key Laboratory of Exploration and Comprehensive Utilization of Mineral Resources, 710054, Xi’an, China Corresponding auther: Tel.: 86-29-82339052; Fax: 86-29-82339052; e-mail: [email protected] ORCID: 0000-0002-4553-8600

ACS Paragon Plus Environment

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Abstract: An innovative pretreatment process using a recyclable aromatic acid, p-toluenesulfonic acid (p-TsOH), was developed for lignocellulose fractionation and improvement in cellulose digestibility as well as lignin extraction in this study. Three different feedstocks including corncobs, wheat straw and Miscanthus were pretreated by p-TsOH at a 70% (wt) concentration at 80C for 10 min for comparison. The results showed that p-TsOH pretreatment was highly effective in removing lignin and xylan, and it resulted in cellulose-rich water-insoluble solids (WIS) and spent liquor, primarily containing xylose and lignin. The produced WIS from corncobs, wheat straw and Miscanthus had rather high conversion rates of 100%, 93% and 87%, respectively, at a cellulase loading of 15 FPU/g glucan at 72 h. The high lignin yield of 80.76% was obtained by diluting the spent acid liquor to below the minimal hydrotrope concentration (MHC). This process promotes an innovative approach for biomass fractionation and full utilization of the components. Keywords: lignocellulosic biomass; p-TsOH pretreatment; enzymatic hydrolysis; ethanol fermentation; lignin recovery 1. Introduction Lignocellulosic biomass is a promising alternative source for producing various high value biobased products including biofuels, such as bioethanol and biodiesel, due to its abundance, renewability, and low cost.1 Lignocellulosic biomass is mainly composed of cellulose, hemicellulose, and lignin, which intertwine with one another tightly by chemical linkages to form a recalcitrant structure that is difficult to deconstruct without any pretreatment.2 Therefore, various fractionation and


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pretreatment technologies including chemical, physical, biological, or a combination of these strategies have been developed with the aim of destroying the complex cross-linked structure of the lignin-cellulose-hemicellulose, removing the lignin and hemicellulose as much as possible,3 increasing the accessibility of cellulase4 and improving the utility of lignocellulose.5,6 However, the existing pretreatment technologies attain results by exerting high pressure, high temperature or a combination of both, and they result in high cost and intensive energy consumption.3 For example, the conventional chemical dilute acid/alkaline pretreatments are considered to be one of the most common pretreatment technologies; however, the enzymatic efficiency is positively correlated with the severity of the pretreatment conditions. Thus, the high demands with regards to the temperature, pressure and reaction time leads to a lower efficiency and high cost.7 Pretreatment with organic solvents can provide suitable cellulose for enzymatic digestion by simultaneously solubilizing lignin and hemicelluloses in mild conditions; however, the removal and recycling of organic solvents from the system can increase the cost of the process.8,9 Furthermore, in recent years, in addition to producing highly digestible cellulose for enzymatic saccharification, more research has been focused on the other remaining fractions in the lignocellulosic biomass including the hemicellulose and lignin utilization.10 After separation and purification, hemicellulose and lignin can be economically converted to value-added products such as furfural, health additives, polymeric materials, aromatic chemicals and carbon fiber based on their differing physical and chemical properties. Thus, the simple recovery of chemicals except for


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cellulose is another key issue for pretreatment technologies. In this sense, the ideal pretreatment strategies include a low energy cost, high substrate loading and simple recovery of chemicals for multipurpose lignocellulose-based biorefining.11-13 In recent years, a novel pretreatment strategy with a hydrotrope aromatic acid, p-toluenesulfonic acid (p-TsOH), has captured much attention due to its overwhelming advantages, including the rapid and near-complete dissolution of wood lignin at low temperature, the simple recovery of dissolved lignin and xylose, and the recyclability of the acid.14 p-TsOH pretreatment has been used for fast wood fractionation14 as well as the production of wood-based nanomaterials,15,16 furfural,17 and lignin-based materials.18 However, the feedstock–dependent properties of the pretreatment methods lead to different effects among various biomass species,19,20 and the suitability of p-TsOH on a wide spectrum of lignocellulosic biomass with high substrate loading and sequential ethanol fermentation, which is an important parameter to evaluate the economic feasibility, was not investigated in previous studies. Thus, it is worth comparing the effects of p-TsOH pretreatment on different biomass species to better understand how different lignocellulosic biomass responds to p-TsOH pretreatment. China is one of the largest producers of crops and by-products in the world. Corn and wheat are of fundamental importance among the main crops. There’re millions of tons of lignocellulosic residues in the form of corncob and wheat straw, indicating a great potential for the use of chemical components of the biomass. Miscanthus has been considered as one of the most potential non-food energy plant due to its high biomass,


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cellulose rich content, perennial characteristics, and remarkable adaptability to different environments. In this study, p-TsOH was employed for pretreating three different kinds of biomass, including corncobs, wheat straw and Miscanthus. The component and structural changes of the biomass after p-TsOH pretreatment and their correlations with enzymatic digestibility were investigated. The pretreated biomass was further subjected to enzymatic hydrolysis and ethanol fermentation with a high substrate loading of 15% to test the potential convertibility for developing biofuel. The recovery of the dissolved lignin and the reusability of p-TsOH were investigated as well.

2. Materials and methods

2.1 Materials The corncobs and wheat straw were collected from Chang’an county in Shaanxi Province, China. The Miscanthus was purchase from Qingdao, Shandong Province, China. The three raw feedstocks were milled and screened to obtain fractions with a size of 20 mesh. The main compositions of the three materials are listed in Table 1. A commercial complex cellulase enzyme, Cellic® CTec 2 (abbreviated CTec 2), was provided by Novozyme China (Shanghai, China). The cellulase activity was 147 FPU/mL (filter paper activity unit), as calibrated by a method in the literature.21 A fermentation yeast strain isolated from Angel yeast (Angel Yeast Co., Yichang, Hubei, China) was activated at 30C for 24 h on YPD agar plates containing 10 g/L yeast extract, 20 g/L peptone, 20 g/L glucose, and 20 g/L agar. A colony from the


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plate was transferred to liquid YPD medium in a flask and cultured at 150 rpm at 30C on a shaking bed incubator. The cell concentration was monitored by the optical density at 600 nm (OD600). After the OD600 reached approximately10 (the samples were diluted to appropriate concentration to remain the OD range 0.1 – 1), the yeast pellet was obtained by centrifugation at 2348 g for 20 min and mixed with deionized (DI) water to the original volume for use as the fermentation inoculum.2,22 The chemicals used in this study were of analytical reagent grade. 2.2 Methods 2.2.1 Experimental plan The experimental process flow is shown in Fig.1. p-TsOH was initially used to fractionate the three feedstocks and produce cellulose-rich water-insoluble solids (WIS) as well as spent liquor primarily containing xylose and lignin. The WIS were hydrolyzed by enzymatic hydrolysis, and the resultant sugar hydrolysate was fermented to produce ethanol. The dissolved lignin in the spent liquor obtained from wheat straw was recovered for further application by dilution. The xylose in the spent liquor can be converted to furfural products. 2.2.2 Fractionation process Five grams (dry weight) of the feedstock were mixed with a 70% (wt%) p-TsOH solution (pre-heated to 80°C), and kept at 80°C in a water bath for 10 min at 150 rpm on a shaker. At the end of each reaction, DI water was added to dilute the solution to the final acid concentration of 30% to terminate the reaction. The hydrolysate was


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then separated by vacuum filtration. The solid phase (WIS) was washed with DI water to obtain a until neutral pH and collected for chemical composition analysis as well as sugar and ethanol production. The filtrate was collected for the recovery of the acid and dissolved lignin. 2.2.3 Enzymatic hydrolysis One gram (dry weight) of the WIS samples was used to conducted enzymatic hydrolysis at cellulase enzyme loadings of 5, 10 and 15 FPU/g glucan (CTec 2) at a total solid loading of 2%, respectively. Enzymatic hydrolysis was conducted in a shaker (Model 20P-250, Shanghai Jinghong Experimental Equipment Co., Ltd., China) at 50°C and 150 rpm for 72 h.14,18 Hydrolysates were sampled at 4, 8, 12, 24, 48, 72 h for sugar analysis. The substrate enzymatic digestibility (SED) is described as follows: 𝐶𝐺𝑙𝑢 ∗ 𝑉 ∗ 0.9

SED =

𝑀∗𝐶

∗ 100%

where CGlu (g/L) and V (L) are the concentration of the glucose and volume of the hydrolysate; M is the dry weight of the sample (g); C is the glucan content of the sample (g/g); and 0.9 is the parameter that converts glucose to an equivalent glucan. 2.2.4 Simultaneous saccharification and fermentation (SSF) Simultaneous saccharification and fermentation (SSF) and quasi-simultaneous saccharification and fermentation (Q-SSF) with a 15% (w/w) total solid loading were carried out at 32C with the addition of 15 FPU/g glucan CTec 2 and 8% (v/v) yeast loading.23 In the Q-SSF process, before fermentation, the solid substrate was first


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enzymatically hydrolyzed for approximately 6 h at 50C and 180 rpm. The whole biomass, except the cellulase and inoculum, was autoclaved for sterilization at 121C for 20 min prior to fermentation. Samples were withdrawn at 4, 8, 12, 24, 36, 72, 96 h and centrifuged at 13000 g for 5 min. The supernatants were used for sugar and ethanol analyses. The reported ethanol yield (as the theoretical yield percentage) was calculated according to the following equation: 𝑦 𝐸𝑡𝑂𝐻 𝑇 h𝑒𝑜𝑟𝑒𝑡𝑖𝑐𝑎𝑙(%) =

𝐶𝐸𝑡𝑂𝐻 × 𝑉𝐵𝑟𝑜𝑡h 0.511 ×

𝐶𝐺𝑙𝑢

×

𝑌𝑃𝑟𝑒𝑡𝑟𝑒𝑎𝑡𝑒𝑑 𝑇𝑜𝑡𝑎𝑙 𝑆𝑜𝑙𝑖𝑑𝑠 𝑀𝑃𝑟𝑒𝑡𝑟𝑒𝑎𝑡𝑒𝑑 𝑇𝑜𝑡𝑎𝑙 𝑆𝑜𝑙𝑖𝑑𝑠

× 100

0.9

where 𝐶𝐸𝑡𝑂𝐻 and VBroth are the final ethanol concentration (g/L) and the volume (L) of the fermentation broth, respectively; 𝐶𝐺𝑙𝑢 is the glucan content of the raw material (g/g); 𝑌𝑃𝑟𝑒𝑡𝑟𝑒𝑎𝑡𝑒𝑑 𝑇𝑜𝑡𝑎𝑙 𝑆𝑜𝑙𝑖𝑑𝑠 is the total yield of residue from the raw material (g/g); 𝑀𝑃𝑟𝑒𝑡𝑟𝑒𝑎𝑡𝑒𝑑 𝑇𝑜𝑡𝑎𝑙 𝑆𝑜𝑙𝑖𝑑𝑠 is the weight of the sample used in the fermentation (g); and 0.511 is the theoretical yield of ethanol fermented by glucose.

2.3 Analytical methods

2.3.1 Composition analysis of raw and pretreated biomass

The chemical composition of the raw material and fractionated WIS was determined by two-step acid hydrolysis established by the National Renewable Energy Laboratory (NREL).24 Untreated and pretreated samples were hydrolyzed using 72% (v/v) sulfuric acid at 30°C for 1 h and 3.6% (v/v) at 120°C for 1 h. The residue and the filtrate were collected separately. Glucan and xylan of the acid hydrolysates were


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analyzed by HPLC with pulsed amperometric detection (Ultimate 3000, Thermo Scientific, USA). Klason lignin was quantified gravimetrically. For quick analysis, the glucose in the enzymatic hydrolysates was measured using a commercial glucose analyzer (SBA-40E biosensor, Institute of Biology of the Shandong Academy of Sciences, China). All experimental runs were performed in triplicate, and the results were reported as mean values ± standard deviation.

2.3.2 Ethanol concentration

The concentration of ethanol in the fermentation supernatant was determined by gas chromatography (Agilent 7890A, Agilent Corporation).25

2.3.3 Scanning electron microscopy (SEM)

Scanning electron microscopy (SEM) was used to analyze the microstructural changes and surface characteristics of the pretreated biomass using a SEM instrument (Hitachi S-3700, Hitachi, Tokyo, Japan). Prior to observation by SEM, untreated and p-TsOH-pretreated samples were sputter-coated with a thin layer of gold.2

2.3.4 FTIR analysis

Five milligrams of dried untreated and treated biomass species was mixed with 500 mg of KBr in an agate mortar and pressed in a HY-12 tablet press (Tianjin, China). The tableting pressure was 26 MPa and the tableting duration was 6 s. The samples were analyzed by a Thermo Nicolet FTIR spectrometer (U.S.A.) in the range


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from 400 to 4000 cm-1 with a spectral resolution of 4 cm-1. An infrared spectrum was obtained and analyzed using OPUS software (Bruker, Germany) to investigate changes in chemical structure during pretreatment.2 2.4 Separation of the dissolved lignin The dissolved lignin in the spent p-TsOH liquor can be separated simply through precipitation after diluting the spent acid liquor to below the minimal hydrotrope concentration (MHC) of 11.5 wt%.14 The collected spent liquor from wheat straw at a p-TsOH concentration of 30% was diluted to p-TsOH concentrations of 15%, 10%, and 4%, respectively, and then centrifuged at 13000 g for 10 min to separate the raw lignin. The pellet was washed by DI water three times and then dried at 105°C to calculate the recovery of the lignin. The lignin purity was analyzed following the above-mentioned NREL protocol.10,22 The lignin yield was calculated as a percentage of the recovered pure lignin based on the lignin content of the raw material using the following equation: Lignin yield (%) 

M M 1*C1  M 2 * C2

where M is the weight of lignin obtained from spent liquor (g); M1 is the weight of raw material (g); C1 is the lignin content of the raw material (g/g); M2 is the weight of WIS (g); and C2 is the lignin content of WIS (g/g). 2.5 Reusability of p-TsOH The spent liquor obtained from wheat straw at a concentration of 30% was used to


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evaluate the reusability of p-TsOH. Fresh p-TsOH was added to the collected spent liquor to reach the target concentration of 70% with the assumption that p-TsOH consumption by the reaction was negligible, and then, it was used to treat the fresh wheat straw at 80C for 10 min. The experiment was conducted by recycling the p-TsOH five times. The chemical composition and SED of the WIS of the five reuse cycles were used to compare the reusability of p-TsOH.

3. Results and discussion

3.1 Compositional changes of different biomass species after p-TsOH fractionation As seen from Table 2, the solid residues of the three pretreated biomass species were less than 60% likely due to the loss of the solubilized hemicellulose and lignin in the biomass. Approximately 80% of xylan and 70% lignin from corncobs as well as 72% of xylan and 74% lignin from wheat straw were solubilized by the p-TsOH solution. In the case of Miscanthus, under the same conditions, there was a total mass loss of 57% and approximately 60% of xylan and 47% of lignin removal. Among the three biomass species, corncobs were the most labile to p-TsOH pretreatment, while Miscanthus appeared to be most recalcitrant, which requires harsh treatment conditions to deconstruct the crosslinking bonds between lignin and carbohydrates. The ratio of feedstock recalcitrance appeared to be negatively correlated with its lignin content.26 Meanwhile, cellulose losses were minimal at 10% or less from the three different biomass species, and this resulted in cellulose enrichment of over 60%


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glucose in the three pretreated biomass samples, indicating that p-TsOH pretreatment was highly effective in removing xylan and lignin while retaining cellulose as much as possible in the pretreated biomass. Table 3 shows the effects of different pretreatments on chemical composition changes of corncobs, wheat straw and Miscanthus.27-29 Compared with a prior study, p-TsOH pretreatment presents overwhelming advantages in providing mild pretreatment conditions while achieving an equivalent or higher degree of solubilization of hemicellulose, delignification and cellulose recovery. 3.2 Substrate enzymatic digestibility (SED) of various biomass species after p-TsOH fractionation Different enzyme loadings were used to compare the sugar production of the three pretreated biomass species. The SED values for corncobs, wheat straw, and Miscanthus increased sharply within 24 h and then increased slowly at different cellulase dosages, reaching 100%, 93% and 87% at a cellulase loading of 15 FPU/g glucan at 72 h, respectively. Among the three feedstocks, pretreated corncobs presented a higher hydrolysis rate and could be completely hydrolyzed at 12 h with an enzyme loading of 15 FPU, at 24 h with 10 FPU, and at 72 h with 5 FPU. This may have resulted from the lower lignin content of 10.98% in pretreated corncobs compared to the other two species. Miscanthus also appeared to be more recalcitrant than corncobs and wheat straw due to its high lignin content. Similar reports have been reported for microwave-assisted DES pretreatment of corn stove, switchgrass, and Miscanthus.10 The differences in digestibility among the three feedstocks


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indicated that the removal of hemicellulose and lignin had a positive effect on the enzymatic hydrolysis of lignocelluloses.2,30 The improvement in the hydrolysis of different biomass types indicated the effectiveness of p-TsOH for sugar production. 3.3 Ethanol fermentation SSF and Quasi-SSF of the three pretreated biomass species are shown in Fig. 3. From Fig.3, the glucose consumption and ethanol yield were rapid within the first 24 h and reached the highest ethanol production at approximately 48 h. Compared with SSF, Q-SSF exhibited higher ethanol production during the entire fermentation period in three different biomass species due to the favorable glucose concentration and reduced viscosity in the Q-SSF slurry, which had been hydrolyzed and liquefied in advance. The ethanol concentration of corncobs was higher than that of wheat straw and Miscanthus, reaching values of 55 g/L, 37.5 g/L and 34.5 g/L at 72 h, respectively, which is equivalent to theoretical ethanol yields of 87.86%, 61.71% and 69.42% based on the untreated biomass glucan content. 3.4 Structural characterization of raw and pretreated biomasses

3.4.1 SEM analysis

To gain insight into the relationship between SED and physical structural features, SEM was used to observe structural changes of the pretreated biomasses. The raw materials (Figure 4 (A-C)) had an intact lamellar structure in which cellulose is closely wrapped by hemicellulose and lignin, while the lamellar structures of the


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p-TsOH-pretreated biomass types (Figure 4 (a-c)) were disrupted and had more irregular cracks and large pores. These morphological changes resulted from the release of xylan and removal of lignin by the p-TsOH pretreatment, which led to an increased specific surface area and pore volume of the biomass as well as the marked improvement in the enzymatic hydrolysis.

3.4.2 FTIR analysis

The structural modifications of three different biomass species after p-TsOH pretreatment were investigated by Fourier transform infrared (FTIR) spectra. The peaks were assigned according to previous references. As shown in Fig. 5, the absorption bands at 1162 cm-1 (C-O-C stretching in cellulose) and 897 cm-1 (presence of β-glycosides in cellulose)31 in the three pretreated biomass species increased significantly, indicating the removal of lignin and hemicellulose after pretreatment. The ether band at 1254 cm−1,31 and the ester band at 1736 cm−1,32 had almost disappeared in the three pretreated biomass species indicating that the contents of ether and ester linkages between lignin and carbohydrates of the pretreated biomasses were low due to their low lignin and hemicelluloses contents. Furthermore, the decreased or disappearing bands at 1514 cm−1 and 1601 cm−1 assigned to the aromatic skeletal stretching of lignin in the spectra33,34 are directly related to delignification of the three pretreated biomass species. All spectral changes were consistent with the chemical component analysis.


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3.5 Lignin recovery After centrifugation at 13000 g for 10 min, the precipitated lignins at the bottom can be observed at a p-TsOH concentration of 15% or lower. With further dilution to an acid concentration of 4 wt%, the precipitates increased substantially and the supernatant became clear and colorless. The precipitates obtained from different p-TsOH concentrations were dried, and the lignin purity was analyzed to calculate the lignin yield. The lignin yield increased with the decreasing concentrations of p-TsOH, and reached 59.06%, 77.17%, and 80.76% at p-TsOH concentration of 15%, 10%, and 4% based on the untreated biomass lignin content, respectively. The recovered lignin had a relatively high purity of 94.74%, which may be exploited as a potential feedstock for the downstream production of aromatic chemicals. The lignin recovery and purity were much higher than previous studies reported by Chen,10 Su,27 and Yuan,28 indicating that the precipitated lignin is favorable for further processing to produce high-value lignin-derivatives. The FTIR spectra of the recovered lignin samples are illustrated in Figure 6. The typical signals of aromatic skeletal vibrations in the lignin were intensified at approximately 1601 cm−1, 1515 cm−1, 1460 cm−1and 1424 cm−1.35-37 The peaks at approximately 1035 cm−1 and 1025 cm−1 showed C-H in plane deformation in the aromatic groups.38 The lignin obtained from the three feedstocks presented analogous FTIR spectra, indicating that the core structures of the obtained lignin samples were similar. 3.6 Reusability of p-TsOH p-TsOH could dissolve a substantial amount of wood lignin below 80°C within a 10


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min period, indicating that it is a promising wood fractionation approach. As a strong organic acid, its recyclability and reusability were important for its further application in biomass fractionation. As an example, the spent liquor collected from wheat straw was used for pretreating wheat straw for five runs. The chemical composition and SED of the solid residue shown in Table 4 indicated that the differences between the runs were within measurement error, suggesting the good reusability of p-TsOH. 3.7 Conclusions The p-toluenesulfonic acid (p-TsOH) pretreatment can fractionate lignocelluloses with high selectivity for cellulose over hemicelluloses and lignin at 80C in 10 min and produce cellulose-rich water-insoluble solids (WIS) as well as spent liquor primarily containing xylose and lignin. The higher cellulose digestibility, easy recovery of dissolved lignin oligomers and reusability of the acid demonstrate that it is a promising strategy for biomass fractionation, lignocellulosic biomass conversion and the production of other chemical compounds. Acknowledgements The authors acknowledge the financial support of Science and Technology Development Project of Shaanxi (2015SF263), and Natural Science Foundation of Shaanxi (2018JQ4042).


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10. Chen, Z.; Wan, C. Ultrafast fractionation of lignocellulosic biomass by microwave-assisted deep eutectic solvent pretreatment. Bioresour. Technol. 2018, 250, 532-537. 11. Alvira, P.; Tomás-Pejó, E.; Ballesteros, M. J.; Negro, M. J. Pretreatment technologies for an efficient bioethanol production process based on enzymatic hydrolysis: a review. Bioresour. Technol. 2010, 101(13), 4851-4861. 12. Galbe, M.; Zacchi, G. Pretreatment: the key to efficient utilization of lignocellulosic materials. Biomass Bioenergy 2012, 46, 70-78. 13. Sindhu, R.; Binod, P.; Pandey, A. Biological pretreatment of lignocellulosic biomass–An overview. Bioresour. Technol. 2016, 199, 76-82. 14. Chen, L.; Dou, J.; Ma, Q.; Li, N.; Wu, R.; Bian, H.; Zhu, J. J. Rapid and near-complete dissolution of wood lignin at ≤ 80°C by a recyclable acid hydrotrope. Sci. Adv. 2017, 3(9), e1701735. 15. Bian, H.; Chen, L.; Gleisner, R.; Dai, H.; Zhu, J. Y. Producing wood-based nanomaterials by rapid fractionation of wood at 80°C using a recyclable acid hydrotrope. Green Chem. 2017, 19(14), 3370-3379. 16. Bian, H.; Gao, Y.; Yang, Y.; Fang, G.; Dai, H. Improving cellulose nanofibrillation of waste wheat straw using the combined methods of prewashing, p-toluenesulfonic

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endoglucanase

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17. Ji, H.; Chen, L.; Zhu, J. Y.; Gleisner, R.; Zhang, X. Reaction kinetics based optimization of furfural production from corncob using a fully recyclable solid acid. Ind. Eng. Chem. Res. 2016, 55(43), 11253-11259. 18. Ji, H.; Song, Y.; Zhang, X.; Tan, T. Using a combined hydrolysis factor to balance enzymatic saccharification and the structural characteristics of lignin during pretreatment of Hybrid poplar with a fully recyclable solid acid. Bioresour. Technol. 2017, 238, 575-581. 19. Xu, G. C.; Ding, J. C.; Han, R. Z.; Dong, J. J.; Ni, Y. Enhancing cellulose accessibility of corn stover by deep eutectic solvent pretreatment for butanol fermentation. Bioresour. Technol. 2016, 203, 364-369. 20. Yu, C.; Wang, F.; Zhang, C.; Fu, S.; Lucia, L. A. The synthesis and absorption dynamics

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dye-contaminated effluent. React. Funct. Polym. 2016, 106, 137-142. 21. Wood, T. M.; Bhat, K. M. Methods for measuring cellulase activities. Method. Enzymol. 1988, 160(1), 87-112. 22. Zhou, H.; Lan, T.; Dien, B. S.; Hector,R. E.,Zhu, J. Y. Comparisons of Five Saccharomyces cerevisiae Strains for Ethanol Production from SPORL-Pretreated Lodgepole Pine. Biotechnol. Prog. 2014, 30( 5), 1076-1083. 23. Jang, J.; Cho, Y.; Jeong, G.; Kim, S. Optimization of saccharification and ethanol production by simultaneous saccharification and fermentation (SSF) from seaweed, Saccharina japonica. Bioprocess Biosyst. Eng. 2012, 35, 11–18.


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31. Lü, J.; Zhou, P. Optimization of microwave-assisted FeCl3 pretreatment conditions of rice straw and utilization of Trichodermaviride and Bacillus pumilus for production of reducing sugars. Bioresour. Technol. 2011, 102(13), 6966-6971. 32. Li, M. F.; Yu, P.; Li, S. X.; Wu, X. F.; Xiao, X.; Bian, J. 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. 2017, 148, 1426-1437. 33. Li, J.; Lu, M.; Guo, X.; Zhang, H.; Li, Y.; Han, L. Insights into the improvement of alkaline hydrogen peroxide (AHP) pretreatment on the enzymatic hydrolysis of corn stover: chemical and microstructural analyses. Bioresour. Technol. 2018, 265, 1-7. 34. Hsu, T. C.; Guo, G. L.; Chen, W. H.; Hwang, W. S. Effect of dilute acid pretreatment

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enzymatic

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Page 23 of 34 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Energy & Fuels

List of tables Table 1 Composition of the three biomass species Table 2 Chemical composition and component recoveries of different biomass species after p-TsOH fractionation Table 3 The effects of different pretreatments on chemical composition changes of the three feedstocks Table 4 Chemical composition of reusable p-TsOH fractionated lignocellulosic biomass and the SED of WIS


Energy & Fuels 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

List of figures Figure 1 Flow chart of p-TsOH pretreatment for the fractionation of various biomass species Figure 2 Effect of the enzyme loading on the time-dependent substrate enzymatic digestibility of different pretreated biomass species (A, corncobs; B, wheat straw; C, Miscanthus) Figure 3 Comparison of SSF and Q-SSF for p-TsOH pretreated biomass (A, corncobs; B, wheat straw; C, Miscanthus) Figure 4 SEM images of corncobs (A and a), wheat straw (B and b), and Miscanthus (C and c) before and after pretreatment Figure 5 FTIR spectra of various biomasses prior to and after pretreatment Figure 6 FTIR spectra of lignin obtained from p-TsOH pretreatment


Page 24 of 34

Page 25 of 34 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Energy & Fuels

Table 1 Sample

Glucan (%)

Xylan (%)

Lignin (%)

Corncobs

34.10±0.24

31.97±0.41

16.53±0.13

Wheat straw

34.10±0.28

24.96±0.14

23.04±0.21

Miscanthus

33.23±0.40

20.25±0.16

25.66±0.29

*All sugar and lignin contents are based on a dry biomass basis


Energy & Fuels 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46

Page 26 of 34

Table 2 WISs sample

66.12±0.45

Recovery of glucan (%) (%) 89.51±1.72

47.55±1.41

64.38±0.18

57.09±1.82

60.77±0.09

Solid yield (%)

Glucan (%)

Corncob

46.16±1.24

Wheat straw Miscanthus

Spent liquor

14.28±0.62

Removal of xylan (%) (%) 79.38±1.41

89.77 89.77±1.86

14.86±0.44

104.4 99.4±1.91

13.95±0.28

Xylan (%)

Glucose (g/L)

Xylose (g/L)

10.98±0.07

Removal of Lignin (%) (%) 69.34±1.34

4.35±0.23

29.56±0.25

71.69±1.72

12.87±0.46

73.44±1.09

1.17±0.17

21.61±0.24

60.67±1.51

24.09±0.43

46.41±1.56

0.38±0.26

14.03±0.42

Lignin (%)

*All sugar and lignin contents are based on dry biomass basis


Page 27 of 34 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Energy & Fuels

Table 3 Biomass

Corncobs

Wheat straw

Miscanthus

Pretreatment condition Alkaline hydrogen peroxide (AHP): pH=11.5, H2O22%, 50°C,6 h Ethanol Pre-extraction (50% v/v ethanol, 160°C, 50 min)+ Alkaline pretreatment (11% w/w Na2CO3, 75°C, 50 min) Alkaline hydrogen peroxide (AHP): pH=11.8, H2O2 2%, 35°C, 24 h

Solid yield (%)

Removal of xylan (%)

Removal of lignin (%)

Recovery of glucan (%)

53.6

38.7

75.4

81.3

50.1

74.8

69.3

87.3

58.2

17.0

70.7

99.8


Energy & Fuels 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 28 of 34

Table 4 Solid yield

Glycan

Xylan

Lignin

SED(%)

SED(%)

(%)

%

%

%

(48 h)

(72 h)

1

54.77±1.42

61.77±0.46

14.57±0.06

13.97±0.14

91.35±0.23

97.99±0.41

2

57.83±1.82

66.12±0.40

14.29±0.21

13.67±0.60

86.67±0.18

90.17±0.24

3

53.96±1.26

64.82±0.22

14.38±0.63

13.07±0.25

90.25±0.16

91.99±0.15

4

58.39±1.50

60.47±0.62

14.25±0.42

13.77±0.41

85.22±0.20

93.40±0.10

5

59.62±1.62

59.60±0.32

14.12±0.25

12.90±0.42

85.96±0.44

95.43±0.23

Runs


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Energy & Fuels

Figure 1


Energy & Fuels

Figure 2

100 80 60 5 FPU 10 FPU 15 FPU

40 20

A

0 80

SED (%)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

60 40

5 FPU 10 FPU 15 FPU

20

B

0 80 60 40

5 FPU 10 FPU 15 FPU

20

C

0 0

20

40 60 Time (h)

80


Page 30 of 34

Page 31 of 34

Figure 3

60 50 40 30 20 10 0

Concentration (g/L)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Energy & Fuels

40

A Ethanol SSF Q-SSF

Glucose

B

30 Ethanol Glucose SSF Q-SSF

20 10 0 40 C

30 20

Ethanol Glucose SSF Q-SSF

10 0 0

20 40 60 80 100 Time (h)


Energy & Fuels 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 32 of 34

Figure 4

A

a

50 50m m

50 m

B

b

50 m

C

5050m m

c

50 m


50 50m m

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Energy & Fuels

Figure 5 1736 1601 1514

1254

pretreated corncobs raw material

897 1162

pretreated wheat straw raw mateial

pretreated Miscanthus raw material

2000

1800

1600

1400

1200

1000 -1

Wavenumber (cm )


800

600

400

Energy & Fuels 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 34 of 34

Figure 6

1460 1515 1601

Corncobs

1125 1424

1035

Wheat straw

Miscanthus

2000

1800

1600

1400

1200

1000 -1

Wave length (cm )


800

600

400

Rapid fractionation of lignocellulosic biomass by p-TsOH pretreatment

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