A strategy to improve enzymatic saccharification of

Process Biochemistry xxx (xxxx) xxx–xxx

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A strategy to improve enzymatic saccharification of wheat straw by adding water-soluble lignin prepared from alkali pretreatment spent liquor ⁎

Bo Jiang, Jiyao Yu, Xufeng Luo, Yangsu Zhu, Yongcan Jin

Jiangsu Co-Innovation Center of Efficient Processing and Utilization of Forest Resources, Nanjing Forestry University, Nanjing 210037, China

A R T I C LE I N FO

A B S T R A C T

Keywords: Wheat straw Alkali pretreatment Enzymatic saccharification Water-soluble lignin Sugar recovery

In this work, the effect of water-soluble lignin on the enzymatic saccharification of alkali-pretreated wheat straw was investigated using soluble fraction (WAL) and sulfomethylated insoluble fraction (SAL) of alkaline lignin from the pretreatment spent liquor as additives. Results showed the total sugar recovery was improved for pretreated solids but was inhibited for bleached substrate with adding a certain amount of WAL or SAL. The interaction between adsorption domain of cellulase and water-soluble lignin was considered to be the potential reason for reducing the non-productive adsorption of residual lignin to cellulase. SAL was more efficient on enhancing the enzymatic saccharification than WAL, and the effect of WAL and SAL on glucan recovery was rather different from that of xylan recovery. Based on the obtained results, a strategy to improve the enzymatic saccharification of lignocellulosic biomass by adding water-soluble lignin prepared from alkali pretreatment spent liquor was proposed.

1. Introduction Enzymatic hydrolysis is a crucial biorefinery step in bioethanol production from lignocellulosic biomass through sugar platform. This process is influenced by multiple factors, and the presence of lignin in the enzymatic substrate is generally considered to be the most important recalcitrant factor on the bioconversion of lignocelluloses [1,2]. The steric hindrance, non-productive adsorption, lignin-carbohydrate complex as well as deactivating enzymes with soluble lignin fragments are the potential reasons [3–5]. The delignification via chemical pretreatment is an effective and universal pathway to liberate the cellulose and hemicelluloses from the lignin seal, so as to render them accessible for the subsequent enzymatic hydrolysis at low enzyme dosage [6–8]. However, there is always a certain amount of lignin remaining in the pretreated solids regardless of the pretreated methods [9], decreasing the enzymatic saccharification efficiency of lignocelluloses. How the residual lignin impacts the enzymes, what is the relationship between the inhibiting effect and the physicochemical properties of lignin, and how the lignin inhibition could be efficiently countered are still not sufficiently understood [10]. Generally, the interaction between residual lignin and cellulase is considered to be the non-productive adsorption via hydrophobicity, electrostatic binding or hydrogen bond [11,12]. The process of chemical delignification is also accompanied by the loss of carbohydrates (primarily hemicelluloses) if the lignocellulosic

⁎

biomass is subjected to direct pretreated using acid or alkaline agents [13]. How to utilize the carbohydrates and lignin in the spent liquor sufficiently faces challenges of cost, technological breakthroughs and infrastructure needs. Many studies suggest that the adsorption behavior of cellulase onto lignin has a negative effect on enzymatic hydrolysis of cellulose [14,15]. However, recent reports pointed out that a certain amount of lignosulfonate or low molecular weight lignin facilitates the enzymatic saccharification of lignocelluloses [16–19]. Therefore, the water-soluble lignin such as lignosulfonate or low molecular weight lignin may have different effects on enzymatic hydrolysis, which gives a new insight into the application of all cell wall components for the enhancement of enzymatic saccharification. In this work, the watersoluble lignin prepared from the sodium hydroxide pretreatment spent liquor was added to the enzymatic hydrolysis substrates for the investigation of the effect of the water-soluble lignin on the enzymatic sugar recovery. 2. Materials and methods 2.1. Materials Wheat straw (Triticum aestivum L.) was collected from Jiangsu, China. The stems were obtained from wheat straw by removing leaf and sheath, and then were cut to 3–5 cm in length. The air dried stems were stored in sealed plastic bags at 4 °C before use. Cellic CTec2 generously

Corresponding author at: Laboratory of Wood Chemistry, Department of Paper Science and Technology, Nanjing Forestry University, 159 Longpan Rd., Nanjing 210037, China. E-mail address: [email protected] (Y. Jin).

https://doi.org/10.1016/j.procbio.2018.05.007 Received 20 March 2018; Received in revised form 5 May 2018; Accepted 12 May 2018 1359-5113/ © 2018 Elsevier Ltd. All rights reserved.

Please cite this article as: Jiang, B., Process Biochemistry (2018), https://doi.org/10.1016/j.procbio.2018.05.007


B. Jiang et al.

precipitated into the water-soluble alkaline lignin fraction with low molecular weight (WAL, pH 2–5) and the water-insoluble alkaline lignin fraction with high molecular weight (pH > 5) through pH adjustment using 2 mol/L H2SO4, and stirring for 1 h at room temperature, respectively. The WAL and water-insoluble alkaline lignin were washed with deionized water at pH 2.0 and 5.0 (adjusted with 2 mol/L H2SO4) for WAL and water-insoluble alkaline lignin, respectively, and then were freeze-dried to obtain purified lignin preparations. The water-insoluble alkaline lignin was sulfomethylated and purified to prepare sulfomethylated alkaline lignin (SAL). For evaluating the necessary of lignin isolation, a part of spent liquor generated from 8% sodium hydroxide pretreatment was directly sulfomethylated to obtain sulfomethylated spent liquor (SSL). The enzymatic residue was also sulfomethylated (SER) to evaluate the effects of sulfomethylated residual lignin on enzymatic hydrolysis. 2.4. Enzymatic hydrolysis A laboratory KRK refiner (Φ300 mm, 3000 rpm) was used for the defiberization of the pretreated wheat straw samples to produce substrates. The enzymatic hydrolysis of the pretreated and bleached substrates with or without the lignin addition was carried out at a consistency of 2% (w/w) in sodium acetate buffer (pH 5.0) at 50 °C, using a shaking incubator at 180 rpm. The CTec2 loading was 20 FPU/g-cellulose based on cellulase activity. Tetracycline was charged at 40 μg/mLbuffer as an antibiotic to inhibit microbial growth during the enzymatic hydrolysis. The enzymatic hydrolysis residue and hydrolysate was separated by centrifugation (5000 rpm, 20 min). The hydrolysates were sampled for monomeric sugar (glucose, xylose and arabinose) analysis.

Fig. 1. Pretreatment and enzymatic hydrolysis process of pretreated wheat straw by adding water-soluble lignin.

provided by Novozymes (Bagsværd, Denmark) was used for the enzymatic hydrolysis of lignocellulosic materials. All the chemicals were analytical grade and purchased from Nanjing Chemical Reagent Co., Ltd. of China and used as received without further purification. 2.2. Sodium hydroxide pretreatment and bleaching procedure The procedure of pretreatment and enzymatic hydrolysis of wheat straw was illustrated in Fig. 1. A rotary lab-scale cooking system with an electrically heated oil bath was used for the pretreatment. Six 1.25 Lstainless steel bomb reactors with screw cap were contained in the cooking system. The straw samples were directly subjected to pretreatment using sodium hydroxide (4%, 8% and 16%, on the basis of oven dried stems) with the ratio of liquor to straw 10:1 (mL/g). The materials were first impregnated with the pretreatment liquor at 80 °C for 30 min. Then the temperature was raised with the rate of 2 °C/ min–150 °C. The pretreatment was immediately terminated while the designed temperature was reached. At the end of the pretreatment, the pretreated spent liquor was collected, and the solids were washed with hot water to completely remove residual chemicals and dissolved straw compounds. The spent liquor and pretreated solids were stored in a refrigerator at 4 °C before use. The 16% sodium hydroxide pretreated wheat straw was bleached by NaClO2 (0.3 g/g-solid) at pH 4.5 and 75 °C for 2 h to obtain the bleached substrate (BS).

2.5. Analytical methods Cellulase activity in terms of “filter paper unit’’ (FPU) of CTec2 was determined by the filter paper method using Whatman No. 1 filter paper as a standard substrate [20]. The lignin and sugar content of the raw and pretreated materials as well as the sugar content in hydrolysates were analyzed by the methods described by our previous work [21]. Data of glucose, xylose and arabinose content were corrected to glucan, xylan and arabinan respectively for the calculation of sugar recovery. Each data point was the average of duplicate experiments. In this work, sugar recovery is defined as the percentage of sugars (glucan, xylan and total sugar) in enzymatic hydrolysate to that in pretreated substrates. 3. Results and discussion 3.1. Effect of water-soluble lignin on the sugar recovery

2.3. Preparation of water-soluble lignin The yield and chemical composition of the pretreated solids and bleached substrate are given in Table 1. The pretreated solid yield decreased with the alkali charge, and the 8% sodium hydroxide

Lignin in spent liquor generated from 8% sodium hydroxide pretreatment was centrifuged to remove insoluble impurities, and then was Table 1 Chemical composition of wheat straw, alkali pretreated and bleached substrates. Samples

Wheat straw 4% NaOH 8% NaOH 16% NaOH BS a b c

Pretreatment yield (%)

a

100 84.3 60.4 53.3 N.D.b

Lignin (%)c

Carbohydrates (%)

KL

ASL

20.9 ± 0.1 19.5 ± 0.3 13.1 ± 0.3 6.6 ± 0.2 0.2 ± 0.0

2.5 1.5 1.3 1.2 1.6

± ± ± ± ±

0.1 0.0 0.1 0.0 0.0

Total

Glucan

23.3 ± 0.1 20.9 ± 0.3 14.4 ± 0.3 7.6 ± 0.2 1.8 ± 0.0

39.2 47.2 55.7 63.5 64.2

The content of benzene-ethanol extractives is 1.5%. Not detected. KL, Klason lignin; ASL, Acid soluble lignin. 2

± ± ± ± ±

Ash (%)

Xylan 0.3 0.2 0.7 0.3 0.4

19.8 22.8 23.6 23.6 24.4

± ± ± ± ±

0.3 0.2 0.1 0.2 0.3

Arabinan

Total

2.1 4.7 4.8 4.8 4.5

61.1 74.6 84.1 91.9 93.1

± ± ± ± ±

0.2 0.3 0.1 0.1 0.5

± ± ± ± ±

0.8 0.8 0.7 0.4 0.1

6.8 ± 3.8 ± 2.5 ± 1.5 ± N.D.

0.7 0.0 0.0 0.1


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Fig. 2. Effect of water-soluble alkaline lignin (WAL) and sulfomethylated water-insoluble alkaline lignin (SAL) prepared from pretreatment spent liquor on enzymatic sugar recovery.

addition to 78.7% with 0.05 g/g-substrate WAL addition. The xylan recovery of 16% pretreated one was only improved from 80.5% without lignin addition to 87.7% with WAL addition at the same lignin dosage. Therefore, the enzymatic saccharification might be influenced not only by the interaction between lignin and cellulase but also by the interaction between carbohydrates and cellulase. Many studies suggested that the reduction of lignin adsorption to enzyme by adding additives such as BSA [24,25], anionic and non-ionic surfactants [22,23] was because that the hydrophobic domain of the residual lignin was blocked by these additives. However, it is not reasonable to explain the phenomenon that the sugar recovery of green liquor pretreated masson pine increased from 42% without the lignin addition to 75% with 0.3 g/g-substrate sulfomethylated lignin addition [17] as well as the phenomenon observed in this work. The molecular weight of water-soluble lignin is much smaller than that of cellulase, the coupling reaction may occur between water-soluble lignin and the adsorption domain of cellulase forming lignin-cellulase complex under certain conditions [26]. Therefore, the WAL and SAL may block the adsorption domain of the cellulase rather than the hydrophobic domain of the residual lignin. Without the presence of residual lignin, the adsorption domain of cellulase is blocked by water-soluble lignin, reducing the accessibility of cellulase to carbohydrates. Therefore, the enzymatic saccharification of the bleached substrate was slightly inhibited by WAL or SAL. Although the formation of the interaction between water-soluble lignin and cellulase adversely affects the hydrolysis of lignin-free substrates, the cellulase, combined with small molecule of water-soluble lignin, is free in liquid phase. For the case of pretreated substrate with residual lignin, this interaction can effectually prevent from the non-productive adsorption of the residual lignin to cellulase. Therefore, the enzymatic saccharification of pretreated substrates was enhanced with the presence of residual lignin.

pretreatment has higher delignification selectivity (carbohydrates yield/delignification) than the 16% sodium hydroxide pretreatment. After bleaching procedure, the lignin in 16% pretreated substrate was mostly removed and the total sugar content could up to as high as 93%. The enzymatic sugar recovery increased with the pretreated severities, and the BS had the highest saccharification efficiency as shown in Fig. 2. It indicates that the existence of residual lignin in pretreated substrate has an unfavorable effect on enzymatic hydrolysis. The total sugar recovery of the pretreated substrates was improved with the addition of a certain amount WAL. However, that of bleached substrate decreased with WAL addition, indicating the effect of water-soluble lignin on the enzymatic saccharification with or without the presence of residual lignin in pretreated substrates is different. With SAL addition, the enzymatic sugar recovery for the pretreated and bleached substrates showed a trend similar to that with WAL addition. However, the SAL was more efficient on improving the hydrolysis than the WAL. For example, the total sugar recovery of 16% sodium hydroxide pretreated wheat straw increased from 66.8% without the lignin addition to 76.9% with WAL addition, and to 85.2% with SAL addition at a dosage of 0.1 g/g-substrate.. The lignosulfonates are not only water-soluble but also have the characteristics of anionic surfactants. Anionic or non-ionic surfactants were reported to effectively reduce the non-productive adsorption by blocking the adsorption site of lignin to cellulase [22,23]. Therefore, the effect of water-soluble lignin on enzymatic hydrolysis may depend on the existence of residual lignin. Similar results were reported by Wang et al [17] and Zhou et al [19]. The effect of WAL and SAL on enzymatic hydrolysis of cellulose (glucan) was rather different from that of hemicellulose (xylan), as shown in Fig. 2. For pretreated substrates, the hydrolysis yield of xylan was higher than that of glucan without the lignin addition. However, the xylan recovery was lower than glucan recovery for the bleached substrate. Additionally, the increase of glucan recovery with the WAL or SAL addition for pretreated substrates at low concentration level was higher than that of xylan recovery. For example, the glucan recovery of 16% pretreated wheat straw was improved from 64.3% without lignin 3


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adsorption on cellulase during enzymatic hydrolysis. In addition, the molecular weight of lignin present in enzymatic residue could be large, causing the inhibitive effect of SER on enzymatic saccharification. Lou et al pointed out that large molecular weight and low degree of sulfonation have unfavorable effects on cellulose saccharification [28]. Therefore, the separation of water-soluble fraction from SER with low molecular weight and high degree of sulfomethylation is an important step for the enhancement of enzymatic saccharification. The pH is also an important influencing factor of enzymatic hydrolysis. Although an elevated pH (4.5–6.0) increased the lignin surface charge (negative) dramatically, which results in lignin to become more hydrophilic and reduces its coordination affinity to cellulase and, consequently, the nonspecific binding of cellulase for pretreated softwood and hardwood [12]. It may not applicable for gramineous plants such as wheat straw, because the lignin structure in gramineous plants is rather different from that in softwood and hardwood. In addition, Lan et al pointed out that the maximal enzymatic saccharification of lignocellulosic substrates occurred at pH 5.2–6.2 for pure cellulosic substrates [29]. However, the presence of residual lignin in pretreated substrates has different effects on enzymatic hydrolysis comparing with pure cellulosic substrates, as observed in this wotk. Therefore, the enzymatic hydrolysis in this work is still carried out at pH 5.0, which is well-established concept and widely accepted in numerous laboratories throughout the world. What is the optimal range of pH for pretreated gramineous plants needs further investigation based on the physicochemical properties of additives and residual lignin. The obtained results may give a new insight into a strategy for the enhancement of enzymatic saccharification using water-soluble lignin as additives (Fig. 4). The water-soluble lignin from the alkali pretreated spent liquor and the enzymatic residue are added to the enzymatic hydrolysis system of the pretreated substrate, which can not only efficiently utilize all cell wall components, but also increase the sugar recovery and reduce the total cost. Additionally, the remaining lignin isolated in this process can be further used for producing bioenergy and bio-based materials with various biorefinery technologies.

4. Conclusions The enzymatic sugar recovery was improved for pretreated wheat straw solids but was inhibited for bleached one with adding a certain amount of water-soluble lignin, and the promotion degree with SAL addition was higher than that with WAL addition. The interaction between cellulase and water-soluble lignin might be the potential reason for reducing the non-productive adsorption of residual lignin to cellulase. The effect of water-soluble lignin on enzymatic hydrolysis depends

Fig. 3. Effect of sulfomethylated spent liquor (SSL, a) and sulfomethylated enzymatic residue (SER, b) on enzymatic sugar recovery.

3.2. Effect of sulfomethylated spent liquor and enzymatic residue on the sugar recovery The effect of sulfomethylated spent liquor (SSL) and enzymatic residue (SER) addition on the sugar recovery of 8% sodium hydroxide pretreated wheat straw is illustrated in Fig. 3 (the amount of carbohydrates in spent liquor and enzymatic residue was deducted). The improvement of enzymatic sugar recovery was not obvious with a certain amount of SSL addition, and the excessive SSL addition resulted in a drop of enzymatic saccharification dramatically (Fig. 3a). The degradation products in spent liquor may deactivate cellulase causing the decrease of the sugar recovery, as Malgas et al. [27] pointed out that organic acids and furan derivatives derived from the sugar degradation had inhibitory effect on the hydrolytic mannanolytic enzymes. It indicates that lignin isolation from the pretreatment spent liquor is an essential procedure for the subsequent sulfomethylation. The SER exhibited bad effect on enzymatic hydrolysis of the alkalipretreated wheat stem, as shown in Fig. 3b. As only a small part of lignin in enzymatic residue could be dissolved in sodium acetate buffer after sulfomethylation, the addition of SER actually increases the undissolved lignin in substrate due to the low sulfomethylation degree of enzymatic residue, resulting in the increase of the non-productive

Fig. 4. A proposed process to improve the enzymatic saccharification by adding water-soluble lignin derivatives. 4


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on the existence of substrate lignin. The sulfomethylated spent liquor and enzymatic residue exhibited bad effect on the enzymatic hydrolysis of alkali-pretreated wheat stem.

[13] R. Gupta, Y.Y. Lee, Investigation of biomass degradation mechanism in pretreatment of switchgrass by aqueous ammonia and sodium hydroxide, Bioresour. Technol. 101 (2010) 8185–8191. [14] Y. Kim, T. Kreke, J.K. Ko, M.R. Ladisch, Hydrolysis-determining substrate characteristics in liquid hot water pretreated hardwood, Biotechnol. Bioeng. 112 (2015) 677–687. [15] Y.N. Zeng, S. Zhao, S.H. Yang, S.Y. Ding, Lignin plays a negative role in the biochemical process for producing lignocellulosic biofuels, Curr. Opin. Biotechnol. 27 (2014) 38–45. [16] Z.J. Wang, T.Q. Lan, J.Y. Zhu, Lignosulfonate and elevated pH can enhance enzymatic saccharification of lignocelluloses, Biotechnol. Biofuels 6 (2013) 9. [17] W.X. Wang, Y.S. Zhu, J. Du, Y.Q. Yang, Y.C. Jin, Influence of lignin addition on the enzymatic digestibility of pretreated lignocellulosic biomasses, Bioresour. Technol. 189 (2015) 7–12. [18] T. Leskinen, S.S. Kelley, D.S. Argyropoulos, E-beam irradiation & steam explosion as biomass pretreatment, and the complex role of lignin in substrate recalcitrance, Biomass Bioenergy 103 (2017) 21–28. [19] H.F. Zhou, H.M. Lou, D.J. Yang, J.Y. Zhu, X.Q. Qiu, Lignosulfonate to enhance enzymatic saccharification of lignocelluloses: role of molecular weight and substrate lignin, Ind. Eng. Chem. Res. 52 (2013) 8464–8470. [20] T. Ghose, Measurement of cellulase activities, Pure Appl. Chem. 59 (1987) 257–268. [21] Y.C. Jin, T. Huang, W.H. Geng, L.F. Yang, Comparison of sodium carbonate pretreatment for enzymatic hydrolysis of wheat straw stem and leaf to produce fermentable sugars, Bioresour. Technol. 137 (2013) 294–301. [22] T. Eriksson, J. Börjesson, F. Tjerneld, Mechanism of surfactant effect in enzymatic hydrolysis of lignocellulose, Enzyme Microb. Technol. 31 (2002) 353–364. [23] R. Agrawal, A. Satlewal, M. Kapoor, S. Mondal, B. Basu, Investigating the enzymelignin binding with surfactants for improved saccharification of pilot scale pretreated wheat straw, Bioresour. Technol. 224 (2017) 411–418. [24] L. Kumar, V. Arantes, R. Chandra, J. Saddler, The lignin present in steam pretreated softwood binds enzymes and limits cellulose accessibility, Bioresour. Technol. 103 (2012) 201–208. [25] B. Yang, C.E. Wyman, BSA treatment to enhance enzymatic hydrolysis of cellulose in lignin containing substrates, Biotechnol. Bioeng. 94 (2006) 611–617. [26] Z.J. Wang, J.Y. Zhu, Y. Fu, M. Qin, Z. Shao, J. Jiang, F. Yang, Lignosulfonatemediated cellulase adsorption: enhanced enzymatic saccharification of lignocellulose through weakening nonproductive binding to lignin, Biotechnol. Biofuels 6 (2013) 156. [27] S. Malgas, J.S. van Dyk, S. Abboo, B.I. Pletschke, The inhibitory effects of various substrate pre-treatment by-products and wash liquors on mannanolytic enzymes, J. Mol. Catal. B: Enzym. 123 (2016) 132–140. [28] H.M. Lou, H.F. Zhou, X.L. Li, M.X. Wang, J.Y. Zhu, X.Q. Qiu, Understanding the effects of lignosulfonate on enzymatic saccharification of pure cellulose, Cellulose 21 (2014) 1351–1359. [29] T.Q. Lan, H.M. Lou, J.Y. Zhu, Enzymatic saccharification of lignocelluloses should be conducted at elevated pH 5. 2-6. 2, Bioenerg. Res. 6 (2013) 476–485.

Acknowledgment This work was supported by the National Natural Science Foundation of China (grant numbers 31730106, 21704045, 31470593). References [1] X. Li, Y. Zheng, Lignin-enzyme interaction: mechanism, mitigation approach, modeling, and research prospects, Biotechnol. Adv. 35 (2017) 466–489. [2] S.Y. Ding, Y.S. Liu, Y. Zeng, M.E. Himmel, J.O. Baker, E.A. Bayer, How does plant cell wall nanoscale architecture correlate with enzymatic digestibility? Science 338 (2012) 1055–1060. [3] J.V. Vermaas, L. Petridis, X. Qi, R. Schulz, B. Lindner, J.C. Smith, Mechanism of lignin inhibition of enzymatic biomass deconstruction, Biotechnol. Biofuels 8 (2015) 1. [4] Z.Y. Yu, K.S. Gwak, T. Treasure, H. Jameel, H.M. Chang, S. Park, Effect of lignin chemistry on the enzymatic hydrolysis of woody biomass, ChemSusChem 7 (2014) 1942–1950. [5] J.L. Rahikainen, R. Martin-Sampedro, H. Heikkinen, S. Rovio, K. Marjamaa, T. Tamminen, O.J. Rojas, K. Kruus, Inhibitory effect of lignin during cellulose bioconversion: the effect of lignin chemistry on non-productive enzyme adsorption, Bioresour. Technol. 133 (2013) 270–278. [6] K.A. Gray, L. Zhao, M. Emptage, Bioethanol, Curr. Opin. Chem. Biol. 10 (2006) 141–146. [7] C. Zhang, W.J. Xu, P.F. Yan, X.M. Liu, Z.C. Zhang, Overcome the recalcitrance of eucalyptus bark to enzymatic hydrolysis by concerted ionic liquid pretreatment, Process Biochem. 50 (2015) 2208–2214. [8] S. Kumar, S.P. Singh, I.M. Mishra, D.K. Adhikari, Recent advances in production of bioethanol from lignocellulosic biomass, Chem. Eng. Technol. 32 (2009) 517–526. [9] S. Nakagame, R.P. Chandra, J.N. Saddler, The influence of lignin on the enzymatic hydrolysis of pretreated biomass substrates, in: J.Y. Zhu, X. Zhang, X.J. Pan (Eds.), Sustainable Production of Fuels, Chemicals, and Fibers from Forest Biomass, American Chemical Society, Washington, DC, 2011, pp. 145–167. [10] J.K. Saini, A.K. Patel, M. Adsul, R.R. Singhania, Cellulase adsorption on lignin: a roadblock for economic hydrolysis of biomass, Renew. Energy 98 (2016) 29–42. [11] S. Nakagame, R.P. Chandra, J.F. Kadla, J.N. Saddler, The isolation, characterization and effect of lignin isolated from steam pretreated Douglas-fir on the enzymatic hydrolysis of cellulose, Bioresour. Technol. 102 (2011) 4507–4517. [12] H.M. Lou, J.Y. Zhu, T.Q. Lan, H.R. Lai, X.Q. Qiu, pH-induced lignin surface modification to reduce nonspecific cellulase binding and enhance enzymatic saccharification of lignocelluloses, ChemSusChem 6 (2013) 919–927.

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