Using highly recyclable sodium caseinate to enhance lignocellulosic hydrolysis and cellulase recovery

Journal Pre-proofs Using highly recyclable sodium caseinate to enhance lignocellulosic hydrolysis and cellulase recovery Cheng Cai, Yu Bao, Feiyun Li, Yuxia Pang, Hongming Lou, Yong Qian, Xueqing Qiu PII: DOI: Reference:

S0960-8524(20)30243-1 https://doi.org/10.1016/j.biortech.2020.122974 BITE 122974

To appear in:

Bioresource Technology

Received Date: Revised Date: Accepted Date:

22 January 2020 4 February 2020 5 February 2020

Please cite this article as: Cai, C., Bao, Y., Li, F., Pang, Y., Lou, H., Qian, Y., Qiu, X., Using highly recyclable sodium caseinate to enhance lignocellulosic hydrolysis and cellulase recovery, Bioresource Technology (2020), doi: https://doi.org/10.1016/j.biortech.2020.122974

This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. 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.

© 2020 Published by Elsevier Ltd.

Using highly recyclable sodium caseinate to enhance lignocellulosic hydrolysis and cellulase recovery Cheng Cai a, Yu Bao a, Feiyun Li a, Yuxia Pang a, Hongming Lou a,b,* Yong Qian a, Xueqing Qiu a,b,c a School

of Chemistry and Chemical Engineering, Guangdong Provincial Engineering Research

Center for Green Fine Chemicals, South China University of Technology, Guangzhou, China b State

Key Laboratory of Pulp and Paper Engineering, South China University of Technology, Guangzhou, China

c School

of Chemical Engineering and Light Industry, Guangdong University of Technology, Guangzhou, China

Corresponding auther: Tel.: 86-20-87114722; Fax: 86-20-87114721; e-mail: [email protected] (H.M. Lou), [email protected] (X.Q. Qiu)

Abstract Most additives that capable of enhancing enzymatic hydrolysis of lignocellulose are petroleum-based, which are not easy to recycle with poor biodegradability. In this work, highly recyclable and biodegradable sodium caseinate (SC) was used to enhance lignocellulosic hydrolysis with improved cellulase recyclability. When the pH decreased from 5.5 to 4.8, more than 96% SC could be precipitated from the solution and recovered. Adding SC increased enzymatic digestibility of dilute acid pretreated eucalyptus (Eu-DA) from 39.5% to 78.2% under Eu-DA loading of 10 wt% and pH = 5.5, and increase cellulase content in 72 h hydrolysate from only 15.2% of the original to 60.0%, which facilitated the recovery of cellulases through readsorption by fresh substrates. With multiple cycles of re-adsorption, application of SC not only increased the sugar yield of Eu-DA by 95.5%, but also reduced cellulase loading by 40%. 1

Keywords：sodium caseinate, recyclable, enzymatic hydrolysis, cellulase recovery 1. Introduction With the increasing awareness of the environment, countries around the world are striving to find and develop renewable energy and chemicals (Cavusoglu et al., 2017; Lam et al., 2011). Plant biomass has received extensive attention for its large reserve, easy access and convertible to renewable materials and chemicals (Chum & Overend, 2001; Dodds & Gross, 2007; Feng et al., 2019; Huang et al., 2019b). The conversion of lignocelluloses into fermentable sugars by enzymatic hydrolysis, and further conversion into liquid fuel or chemicals is one of the research priorities (Chen et al., 2015; Sun et al., 2016b). The enzymatic saccharification of lignocelluloses is not only environmental friendly, but also can be carried out under mild conditions with low capital cost (Sindhu et al., 2016). However, the low hydrolysis efficiency and high cellulase cost hinder the commercialization of enzymatic saccharification of lignocelluloses (Meng et al., 2016; Sun et al., 2016a). The non-productive adsorption of cellulase on lignin is the main reason for the low enzymatic hydrolysis efficiency of lignocelluloses (Cai et al., 2020; Huang et al., 2019a; Huang et al., 2018). Surfactants, especially non-ionic surfactants, can effectively reduce the non-productive adsorption of cellulase on lignin (Eriksson et al., 2002). Therefore, the addition of surfactants is a common method for improving the efficiency of lignocellulosic hydrolysis and saving cellulase (Kristensen et al., 2007; Monschein et al., 2014). In the presence of PEG6000 at 4.35% w/w of dry feedstock, 97% of glucan and 44% of xylan in spent mushroom compost were converted into the corresponding monosaccharides (Kapu et al., 2012). Adding 5 g/L Tween 20 could increase the glucan conversion of H2SO4-pretreated substrates from 40.4 to 74.3% (Chen et al., 2018). However, the quality of these surfactants usually accounts for 5 wt% of the substrate to achieve good results (Cai et al., 2017), and they are difficult to recover, which leads to an increase in overall cost. Besides, they are not easily degraded and can cause potential environmental problems. Cellulase recovery is also a good way to save cellulase (Ding et al., 2016; Jorgensen & Pinelo, 2017). And recovering cellulase by re-adsorption is the easiest to achieve industrial application due to its simple process and no need of special equipment (Li et al., 2010; Wang et al., 2016). Tu et al have found that 82% of free cellulase in hydrolysate could be recovered 2

through fresh substrate re-adsorption at each round of hydrolysis (Tu et al., 2007a). But lignin in substrates would adsorb cellulase, which reduce the efficiency of enzyme recycling (Cai et al., 2019). It has been reported that approximately 76% of the cellulase could be recovered in the liquid phase after a 48 h hydrolysis of Avicel, whereas only 51% of the applied cellulase was recovered from the hydrolysis of an ethanol-pretreated mixed softwood substrate, which contained only 3 wt% of lignin (Tu et al., 2007b). In order to improve cellulase recovery, additives such as tween 80 and PEG are commonly used to increase cellulase concentration in hydrolysate after hydrolysis (Jorgensen & Pinelo, 2017; Tu et al., 2009). Although more cellulase can be saved, the additives are consumed in large quantities in each round of recycling. Recently, some research results provide new ideas for enhancing enzymatic hydrolysis and saving cellulase. Zhu et al have found that by simply increasing the pH of hydrolysate, the electrostatic repulsion of cellulase and lignin in substrates can be increased, cellulase concentration in hydrolysate and hydrolysis efficiency of substrates can be obviously increased (Lou et al., 2013). For example, cellulose saccharification efficiency of a SPORL-pretreated lodgepole pine substrate increased from approximately 43 to 78 % simply by increasing the buffer solution pH from 4.8 to 5.5 at cellulase loading of 11.3 FPU/g glucan (Lan et al., 2013). Some natural compounds can also effectively promote the hydrolysis of lignocelluloses (Lin et al., 2019). Inexpensive soy protein (SP) extracted from defatted soy powder was used to enhance the enzymatic hydrolysis of pretreated lignocellulosic substrates (Brondi et al., 2019; Florencio et al., 2016), near-complete saccharification of glucan could be achieved and the soy protein in hydrolysate could be reused by vacuum distillation of ethanol after fermentation (Luo et al., 2019). However, it is easy to accumulate hydrolysis and fermentation inhibitors (such as monosaccharide and soluble lignin) in the reused SP solution and SP might be consumed by yeast during fermentation. Sodium caseinate (SC, sodium salt of casein) is a safe and harmless thickener and emulsifier with a relative molecular mass of 75,000 ~ 375,000 Da. Because of its good emulsification and thickening effects, it is widely used in almost all food industries (Perrechil & Cunha, 2010). In this work, the biodegradable SC was selected as a hydrolysis additive due to its sensitive pH responsiveness. The isoelectric point of casein is about 4.8. Under pH 4.8, casein will be protonated and precipitated. When the pH increases to 5.5 (which can be set as enzymatic pH), casein will gradually dissolve. Therefore, SC can be easily recovered by adjusting the pH of the 3

hydrolysate before fermentation. The purpose of this study is to clarify the following issues: 1) pH responsiveness of SC; 2) effect of SC on the enzymatic hydrolysis of lignocelluloses; 3) effect of SC on cellulase distribution during enzymatic hydrolysis; 4) evaluate how much cellulase can be saved by fresh substrate re-absorption after adding SC.

2. Materials and methods 2.1. Materials Sodium caseinate (SC, Lot#: C10031090) and acrylamide (30 wt% in solution; Lot#: C10084782) were purchased from Shanghai Macklin Biochemical Co., Ltd. Protein molecular weight marker was purchased from Thermo scientific (made in Lithuania). Sodium hydroxide and hydrochloric acid solutions were used to adjust the pH of the solution in this work. Cellic CTec2 (provided by Novozyme China) had a protein concentration of 73.6 mg / mL with activity 147 filter paper units (FPU)/mL as determined by filter paper enzyme activity (Wood & Bhat, 1988). Eucalyptus was pretreated with 1.1 wt% (based on wood) sulfuric acid at 165 °C for 1 h, with a solid-liquid ratio of 3: 1. The resulting substrate was abbreviated as EuDA. Corncob residue (CCR) was provided by Shengquan Corp. Ltd. (Jinan, China), and its hemicelluloses were almost removed by dilute acid treatment. The composition of the substrates was analyzed by a two-stage acid hydrolysis method (Luo et al., 2010), as shown in Table 1. 2.2. pH responsiveness of SC Add 0.05 g SC to 50 mL acetate buffers (50 mM) with different pH (4.0, 4.3, 4.5, 4.8, 5.0, 5.3, 5.5, 6.0), with shaking at 20 °C and 200 rpm for 2 h in a shaker (DDHZ-300, Jiangsu Taicang equipment factory, China), then separate the solid and liquid phases by filtration. The Zeta potentials of the filtrates were measured with ZetaPALS instrument (Brookhaven Instruments Co., America). The filter cake mass m1 (g) was recorded after freeze drying. The dissolution percentage (DP) of SC was calculated as follows: DP%= m1/0.05×100% 2.3. Enzymatic hydrolysis

4

Enzymatic hydrolysis of lignocelluloses was conducted in 50 mL acetate buffer (pH 5.5, 50 mM) in the shaker (DDHZ-300, China) at 50 °C and 200 rpm. SC was added before adding cellulase. The cellulase loadings for Eu-DA and CCR were both at 10 FPU/g glucan. 1 mL of samples were taken at 72 h for glucose analysis. Glucose concentration was measured with a commercial SBA-40E biosensor (Institute of Biology of the Shandong Academy of Sciences, China). Reported results were the average of three analyses with standard deviations as error bars shown in the figures. Substrate enzymatic digestibility (SED) was used as the enzymatic hydrolysis efficiency of substrates and calculated as follows: SED% = mglucose in hydrolysis/mglucan in substrate×0.9×100% SED@72h represented the SED at 72 h, ΔSED@72h is the difference between SED@72h with and without the addition of SC. 2.4. SDS-PAGE analyses of cellulase during enzymatic hydrolysis 1 mL of the slurry was taken out at different times during enzymatic hydrolysis of Eu-DA to obtain a supernatant by centrifugation. For the sample without SC addition, the supernatant was used directly for SDS-PAGE analyses. For the SC-added sample, the pH of the supernatant was adjusted to 4.8 to precipitate SC, and the supernatant obtained by secondary centrifugation was used for SDS-PAGE analyses. SDS-PAGE was performed in mini-PROTEAN Tetra Electrophoresis System using 12% (w/v) separating gel, power supply was PowerPac Basic. Specific operations referred to our previous work (Cai et al., 2018b). The gel images were analyzed by Image-Pro Plus software. The relative concentrations of cellulase components were obtained by comparing the intensities of corresponding protein bands (Hu et al., 2010). 2.5. Desorption of cellulase on substrates after hydrolysis at different pH After substrate Eu-DA was hydrolyzed for 72 h at 10 wt%, the pH of the slurry was raised from 5.5 to different pH, then put the slurry in the shaker (DDHZ-300, China) at 20 °C for 2 h to improve cellulase desorption from the substrate. After centrifugation, the hydrolysate was used for SDS-PAGE analyses. The specific SDS-PAGE analysis procedure is as described in Method 2.4.

5

2.6. Re-adsorption of cellulase by fresh substrate After Eu-DA (substrate concentration: 10 wt%) hydrolysis for 72 h, the pH of the slurry was adjusted to 7.0, then shook it in the shaker (DDHZ-300, China) at 20 °C for 2 h. After that, the slurry was centrifuged to obtain the hydrolysate, and the pH of the hydrolysate was adjusted to 4.8. For the sample without SC added, fresh substrate was directly added to the hydrolysate for re-adsorbing cellulase; and for the SC-added sample, the precipitated SC (pH 4.8) was centrifuged before adding fresh substrate. Then, the fresh substrate with hydrolysate was placed in the shaker (DDHZ-300, China) and shook at 200 rpm and 20 °C for 2 h, and then centrifuged the slurry to get the cellulase-adsorbed substrate and soaked it in 50 mL acetate buffer (pH 4.8, 20 °C) for 5 min to replace the residual sugar. After that, centrifuged the buffer and added fresh buffer (pH 5.5, 50 mM) to a substrate concentration of 10 wt% (re-added 0.05 g/g substrate SC for SC-added samples), then put the sample in the shaker (DDHZ-300, China) at 50 °C and 200 rpm and hydrolyzed for 72 h, and the next re-adsorption operation was carried out. The readsorption operation was performed three times. The amount of cellulase that can be saved by re-adsorption after adding SC was evaluated by supplementing different amount of cellulase, and the re-adsorption operation was the same as above, except that 40%, 50% or 60% of original cellulase amount was supplemented before each round of hydrolysis.

3. Results and discussion 3.1. pH responsiveness of SC It can be seen from Fig. 1 that when pH = 5.5 or 6.0, SC was completely dissolved in the solution, and when pH = 4.0 ~ 4.8, SC precipitated from the solution. When the pH of the solution gradually decreased from 6.0 to 4.0, the zeta potential of casein solution gradually increased from -19.2 mV to 7.6 mV. When the pH was decreased from 5.3 to 5.0, the dissolution percentage of casein dropped sharply from 100% to 13.0%, and the zeta potential of corresponding solution rose from -15.4 mV to -4.6 mV. This is because the protonation of the carboxyl group in casein reduced the charge repulsion between protein molecules, which caused casein to precipitate out. When pH was 4.5 and 4.8, the dissolution percentage of casein was the smallest, which was 3.6% and 3.8%, respectively. When the pH was further lowered, the 6

dissolution percentage of casein increased, because the ionization of amino groups in casein increased, which increased the electrostatic repulsion between the protein molecules and dissolved the protein in buffer. 3.2. Effect of SC on the hydrolysis of lignocelluloses Effect of SC on the SED@72h of two lignocellulosic substrates at different pH was shown in Fig. 2A. Without SC, the SED@72h of Eu-DA and CCR both increased first and then decreased when pH increased from 4.0 to 6.0, reaching their maximal of 44.6% and 73.4%, respectively, at pH 5.5. This is in agreement with literature (Lan et al., 2013; Wang et al., 2013) due to enhanced electrostatic repulsion between cellulase and lignin in substrates (Lou et al., 2013). Increase pH, lignin will be more negatively charged so does cellulase when pH > the isoelectric point of cellulase (pI). However, too high a pH would reduce cellulase activity (Lin et al., 2016). As a result, an optimal pH for achieving maximal saccharification was at approximately 5.5. After the addition of SC, the SED@72h of Eu-DA and CCR was increased at all the pH tested as shown in Fig. 2. The highest SED were 81.5% and 96.3% achieved at pH 5.5, respectively. Effect of SC on the digestibility of two substrates varied with pH. As shown in Fig. 2B, the ΔSED@72h of both substrates increased first and then decreased with the increase of hydrolysis pH. At pH 4.0 and 4.5, the ΔSED@72h of the two substrates was small, because casein was not soluble at this pH, and it could not be evenly dispersed in the solution to play a role. When pH > 5.0, the ΔSED@72h of both substrates increased significantly, and at pH = 5.5, the ΔSED@72h of them reached the maximum, which were 34.9% and 22.8%, respectively. When the pH was further increased to 6.0, the ΔSED@72h of the two substrates decreased. This is consistent with previous research using polyvinylpyrrolidone as additive (Cai et al., 2016). Effect of SC concentration on the enzymatic hydrolysis of Eu-DA and CCR is shown in Fig. 3A. When SC concentration was less than 0.01 g/g substrate, SC had negligible effect on the hydrolysis. When the concentration was over 0.01 g/g substrate, SC could significantly promote the enzymatic hydrolysis. When SC concentration was 0.05 g/g substrate, the SED@72h of EuDA and CCR reached 81.4% and 97.2%, respectively. As SC concentration continued to increase, the SED of both substrates did not increase further, but instead showed a slight downward trend. This might be due to excessive surfactants preventing cellulase from contacting the substrate (Zhou et al., 2015). 7

Effect of SC on the enzymatic hydrolysis of two lignocelluloses under different substrate concentrations is shown in Fig. 3B. When the substrate concentration was increased from 2 wt% to 5 wt%, the SED@72 of both substrates did not change significantly whether SC was added or not. When the substrate concentration increased from 5 wt% to 10 wt%, the SED@72h of EuDA and CCR after adding SC decreased from 81.0% and 95.5% to 78.2% and 91.2%, respectively. This is because high substrate concentrations can cause mass transfer problems and product inhibition (Kristensen et al., 2009). Although there was a certain decrease in digestibility at 10 wt% substrate concentrations, the promotion effect of SC on substrates hydrolysis remained highly effective. 3.3. Effect of SC on cellulase distribution during the hydrolysis of Eu-DA In order to make the research more representative, Eu-DA (substrate concentration: 10 wt%) was selected for its high lignin content in this study. The change of cellulase components in hydrolysate with time during Eu-DA hydrolysis was measured by SDS-PAGE (Fig. 4A). Standard protein molecular weight was used to distinguish cellulase components. Cellulase component identification was based on previous work (Cai et al., 2018a). In cellulase CTec2, βglucosidase (β-GL) has the largest molecular weight (78.4 kDa) and is at the top of the gel; xylanase (Xyl) has the smallest molecular weight (about 24 kDa) and is at the bottom of the gel (Ko et al., 2015). Since exoglucanase (CBH) I and CBH II are the main components in CTec2, their bands in the gel have the darkest color (Goyal et al., 1991). When SC was not added, different components of cellulase in the hydrolysate gradually decreased with hydrolysis time (Fig. 4B). After 2, 8 and 72 h of hydrolysis, the total cellulase protein in the hydrolysate was 36.4%, 22.0% and 15.2% of the initial amount, respectively. After hydrolysis for 2 h, almost no β-GL and Xyl were contained in the hydrolysate. Because both components have high isoelectric points, they were positively charged during hydrolysis and were easily adsorbed by negatively charged substrates (Yarbrough et al., 2015). At the same time, β-GL has strong hydrophobic interaction with lignin (da Silva et al., 2016), while Xyl has poor thermal stability and is easily deactivated and precipitated (Cai et al., 2018a). With the hydrolysis of Eu-DA, cellulase was not gradually released into the hydrolysate. possibly because the internal hydrophobic amino acids of cellulase were exposed due to high temperature and

8

shearing, resulting in an increase in the hydrophobic interaction between cellulase and lignin in Eu-DA and being adsorbed (Cai et al., 2018a; Lou et al., 2017). After adding SC, the total cellulase protein in the hydrolysate showed a trend of first decreased and then increased (Fig. 4C). The initial decrease was because the substrate continuously adsorbed cellulase in the solution, and the next increase was because cellulose in the substrate was hydrolyzed, and enzyme adsorbed on the cellulose returned to the solution (Tu et al., 2007). After hydrolysis for 2 h, the content of cellulase protein in the hydrolysate was the lowest, which was 18.1% of the initial content. After hydrolysis for 30 h, the content of cellulase protein rose back to 54.0% of the initial, of which β-GL and Xyl content was almost 0, and CBH & EG content was 64.5% the initial. This shows that SC is beneficial for cellulase to return to hydrolysate after cellulose hydrolysis. For β-GL and Xyl, they hardly existed in the hydrolysate after 8 h of hydrolysis. This means that SC had little effect on the distribution of these two components during hydrolysis. In general, the addition of SC increased the content of cellulase protein in hydrolysate from 15.2% to 60.0% after 72 h of hydrolysis. 3.4. Adjust pH to release more cellulase in hydrolysate for recycling Since increasing the pH is beneficial to increase the electrostatic repulsion between cellulase and substrates (Lan et al., 2013), After Eu-DA hydrolysis for 72 h, the pH of the slurry was raised to desorb cellulase adsorbed on substrate. As shown in Fig. 5A, cellulase protein in hydrolysate was severely reduced without SC after 72 h hydrolysis. CBH & EG were only approximately 25% of the original amount (Fig. 5B), and β-GL and Xyl basically disappeared (Fig. 5C). After increasing the pH of slurry to 9.0 and desorbing for 2 h, the protein content of CBH & EG in hydrolysate did not change much, but the content of β-GL increased significantly, from almost 0 to 54.5%. This shows that increasing pH does facilitate the release of β-GL from the substrate. However, Xyl was not found in hydrolysate after desorption. This might be due to the poor thermal stability of Xyl, which lead to its inactivation and precipitation during hydrolysis. After adding SC, the content of cellulase protein in hydrolysate after 72 h hydrolysis increased significantly. The protein content of CBH & EG was approximately 70% of the initial amount, but the content of β-GL and Xyl was close to zero. When pH was raised to 7.0 for cellulase desorption, the protein content of CBH & EG in hydrolysate increased from 69.7% of 9

the initial amount to 76.1%, and the content of β-GL also increased from almost 0 to 20.1% of the initial amount. As the desorption pH continued to increase, the protein content of CBH & EG decreased. At the same time, an obvious band appeared in the small molecular region of the gel, indicating that cellulase protein was hydrolyzed into peptides with smaller molecular weight under alkaline conditions. However, increasing the desorption pH was beneficial for the desorption of β-GL. When the desorption pH was 8.0, 9.0 and 10.0, the protein content of β-GL in the hydrolysate was 26.1%, 54.8% and 50.4% of the initial amount, respectively. 3.5. Using SC to enhance cellulase recovery through re-adsorption Process for enhancing substrate hydrolysis and cellulase recovery by SC is shown in Fig. 6A. When no cellulase was supplemented during the recovery process, the SED@72h of Eu-DA by recovered cellulase by the first round of recovered cellulase through re-adsorption was only 12.0% without SC as shown in Fig. 6B, which was only approximately 30% of the SED in the control run using fresh cellulase. And the second and third round of recovered cellulase had almost no ability to hydrolyze Eu-DA. It meant that cellulase activity was almost completely lost. After adding SC, the SED@72h of Eu-DA by the first round of recovered cellulase through re-adsorption was 48.0%, or 61% of the SED of the control run. And the SED@72h of Eu-DA by adding the second and third round of recovered cellulase was 20% and 5%, respectively. This indicated that the addition of SC could significantly increase the amount of cellulase recovery through re-adsorption. However, as the number of cycles increased, the digestibility of Eu-DA decreased sharply. This might be due to the low recovery of some cellulase components, making the overall hydrolysis ability of cellulase complex poor (Cai et al., 2018b). The amount of cellulase that can be saved through re-adsorption after adding SC was evaluated by supplementing different amounts of cellulase (Fig. 6C). When 40% of the initial cellulase amount was supplemented, the SED@72h of Eu-DA for the three rounds was 73.3%, 67.4% and 57.4%, respectively, showing a significant downward trend. When supplemented 60% cellulase per round, the SED@72h for the three rounds was 80.1%, 83.2% and 82.3%, respectively, which were slightly higher than the initial digestibility. This shown that after adding SC, at least 40% of cellulase could be saved through re-adsorption. Although the price of SC is more expensive than that of petroleum-based nonionic surfactants, the high recyclability of SC can greatly reduce its cost. And SC is safe for the human 10

body and will not leave hidden dangers to the ecological environment. In summary, using highly recyclable sodium caseinate to enhance lignocellulosic hydrolysis and cellulase recovery is a green and sustainable method.

4. Conclusions Due to the special pH-responsive solubility, highly recyclable and biodegradable SC could most effectively enhance lignocellulosic hydrolysis at pH 5.5. Adding 0.05 g/g substrate SC could increase the SED@72h of Eu-DA and CCR from 44.6% and 73.4% to 81.5% and 96.3%, respectively. SDS-PAGE analysis showed that SC could increase cellulase content in the 72 h hydrolysate of Eu-DA from 15.2% to 60.0%. Through multiple rounds of re-adsorption, it was found that the addition of SC could not only double the digestibility of Eu-DA, but also save more than 40% of cellulase.

CRediT authorship contribution statement Cheng Cai: Conceptualization, Methodology, Investigation, Writing - original draft. Yu Bao: Data curation, Investigation. Feiyun Li: Data curation, Investigation. Yuxia Pang: Visualization, Investigation. Hongming Lou: Methodology, Project administration, Funding acquisition. Yong Qian: Visualization, Investigation. Xueqing Qiu: Supervision. Writing Reviewing and Editing.

Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Acknowledgements The authors acknowledge the financial support from the National Natural Science Foundation of China (21676109, 21978106), the Guangdong Special Support Plan (2016TX03Z298). Thanks to Prof. Junyong Zhu in forest service, USA, for helping to modify the manuscript.

11

References 1. Brondi, M.G., Vasconcellos, V.M., Giordano, R.C., Farinas, C.S., 2019. Alternative Low-Cost Additives to Improve the Saccharification of Lignocellulosic Biomass. Appl. Biochem. Biotech. 187(2), 461-473. 2. Cai, C., Bao, Y., Zhan, X.J., Lin, X.L., Lou, H.M., Pang, Y.X., Qian, Y., Qiu, X., 2019. Recovering cellulase and increasing glucose yield during lignocellulosic hydrolysis using ligninMPEG with a sensitive pH response. Green Chem. 21(5), 1141-1151. 3. Cai, C., Hirth, K., Gleisner, R., Lou, H., Qiu, X., Zhu, J., 2020. Maleic acid as a dicarboxylic acid hydrotrope for green fractionation of wood at atmospheric pressure and≤ 100 °C: Mode and utility of lignin esterification. Green Chem. DOI: 10.1039/C9GC04267A. 4. Cai, C., Jin, Y., Pang, Y., Ke, Q., Qiu, W., Qiu, X., Qin, Y., Lou, H., 2018a. Tracing cellulase components in hydrolyzate during the enzymatic hydrolysis of corncob residue and its analysis. Bioresour. Technol. Rep. 4, 137-144. 5. Cai, C., Qiu, X., Zeng, M., Lin, M., Lin, X., Lou, H., Zhan, X., Pang, Y., Huang, J., Xie, L., 2016. Using polyvinylpyrrolidone to enhance the enzymatic hydrolysis of lignocelluloses by reducing the cellulase non-productive adsorption on lignin. Bioresour. Technol. 227, 74-81. 6. Cai, C., Zhan, X., Lou, H., Li, Q., Pang, Y., Qian, Y., Zhou, H., Qiu, X., 2018b. Recycling Cellulase by a pH-Responsive Lignin-Based Carrier through Electrostatic Interaction. ACS Sustain. Chem. Eng. 6(8), 10679-10686. 7. Cai, C., Zhan, X., Zeng, M., Lou, H., Pang, Y., Yang, J., Yang, D., Qiu, X., 2017. Using recyclable pH-responsive lignin amphoteric surfactant to enhance the enzymatic hydrolysis of lignocelluloses. Green Chem. 19(22), 5479-5487. 8. Cavusoglu, A.-H., Chen, X., Gentine, P., Sahin, O., 2017. Potential for natural evaporation as a reliable renewable energy resource. Nat. Commun. 8(1), 617. 9. 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, 224-232. 10. Chen, Y.A., Zhou, Y., Qin, Y.L., Liu, D.H., Zhao, X.B., 2018. Evaluation of the action of Tween 20 non-ionic surfactant during enzymatic hydrolysis of lignocellulose: Pretreatment, hydrolysis conditions and lignin structure. Bioresour. Technol. 269, 329-338.

12

11. Chum, H.L., Overend, R.P., 2001. Biomass and renewable fuels. Fuel Process. Technol. 71(1-3), 187-195. 12. da Silva, V.M., de Souza, A.S., Negrao, D.R., Polikarpov, I., Squina, F.M., de Oliveira Neto, M., Muniz, J.R., Garcia, W., 2016. Non-productive adsorption of bacterial beta-glucosidases on lignins is electrostatically modulated and depends on the presence of fibronection type III-like domain. Enzyme Microb. Technol. 87-88, 1-8. 13. Ding, Z., Zheng, X., Li, S., Cao, X., 2016. Immobilization of cellulase onto a recyclable thermo-responsive polymer as bioconjugate. J. Mol. Catal. B: Enzym. 128, 39-45. 14. Dodds, D.R., Gross, R.A., 2007. Chemicals from biomass. Science 318(5854), 1250-1251. 15. Eriksson, T., Börjesson, J., Tjerneld, F., 2002. Mechanism of surfactant effect in enzymatic hydrolysis of lignocellulose. Enzyme Microb. Technol. 31(3), 353-364. 16. Feng, J., Zhang, L., Jiang, J., Hse, C., Shupe, T.F., Pan, H., 2019. Directional synergistic conversion of lignocellulosic biomass with matching-solvents for added-value chemicals. Green Chem. 21(18), 4951-4957. 17. Florencio, C., Badino, A.C., Farinas, C.S., 2016. Soybean protein as a cost-effective ligninblocking additive for the saccharification of sugarcane bagasse. Bioresour. Technol. 221, 172180. 18. Goyal, A., Ghosh, B., Eveleigh, D., 1991. Characteristics of fungal cellulases. Bioresour. Technol. 36(1), 37-50. 19. Hu, G., Heitmann, J.A., Rojas, O.J., Pawlak, J.J., Argyropoulos, D.S., 2010. Monitoring Cellulase Protein Adsorption and Recovery Using SDS-PAGE. Ind. Eng. Chem. Res. 49(18), 8333-8338. 20. Huang, C., Lin, W., Lai, C., Li, X., Jin, Y., Yong, Q., 2019a. Coupling the post-extraction process to remove residual lignin and alter the recalcitrant structures for improving the enzymatic digestibility of acid-pretreated bamboo residues. Bioresour. Technol. 285, 121355. 21. Huang, C., Ma, J., Liang, C., Li, X., Yong, Q., 2018. Influence of sulfur dioxide-ethanolwater pretreatment on the physicochemical properties and enzymatic digestibility of bamboo residues. Bioresour. Technol. 263, 17-24. 22. Huang, S., Shuyi, S., Gan, H., Linjun, W., Lin, C., Danyuan, X., Zhou, H., Lin, X., Qin, Y., 2019b. Facile fabrication and characterization of highly stretchable lignin-based hydroxyethyl cellulose self-healing hydrogel. Carbohyd. Polym. 223, 115080. 13

23. Jorgensen, H., Pinelo, M., 2017. Enzyme recycling in lignocellulosic biorefineries. Biofuel. Bioprod. Bior. 11(1), 150-167. 24. Kapu, N., Manning, M., Hurley, T., Voigt, J., Cosgrove, D.J., Romaine, C., 2012. Surfactantassisted pretreatment and enzymatic hydrolysis of spent mushroom compost for the production of sugars. Bioresour. Technol. 114, 399-405. 25. Ko, J.K., Ximenes, E., Kim, Y., Ladisch, M.R., 2015. Adsorption of enzyme onto lignins of liquid hot water pretreated hardwoods. Biotechnol. Bioeng. 112(3), 447-456. 26. Kristensen, J.B., Börjesson, J., Bruun, M.H., Tjerneld, F., Jørgensen, H., 2007. Use of surface active additives in enzymatic hydrolysis of wheat straw lignocellulose. Enzyme Microb. Technol. 40(4), 888-895. 27. Kristensen, J.B., Felby, C., Jørgensen, H., 2009. Yield-determining factors in high-solids enzymatic hydrolysis of lignocellulose. Biotechnol. Biofuels 2(1), 11. 28. Lam, H.L., Varbanov, P.S., Klemeš, J.J., 2011. Regional renewable energy and resource planning. Appl. Energ. 88(2), 545-550. 29. Lan, T.Q., Lou, H.M., Zhu, J.Y., 2013. Enzymatic Saccharification of Lignocelluloses Should be Conducted at Elevated pH 5.2-6.2. Bioenerg. Res. 6(2), 476-485. 30. Li, Q., Zhang, M., Su, R., Qi, W., He, Z., 2010. Process optimization of cellulase readsorption for reutilization. Chem. Eng. (China) 38, 62-65. 31. Lin, W., Chen, D., Yong, Q., Huang, C., Huang, S., 2019. Improving enzymatic hydrolysis of acid-pretreated bamboo residues using amphiphilic surfactant derived from dehydroabietic acid. Bioresour. Technol. 293, 122055. 32. Lin, X., Cai, C., Lou, H., Qiu, X., Pang, Y., Yang, D., 2016. Effect of cationic surfactant cetyltrimethylammonium bromide on the enzymatic hydrolysis of cellulose. Cellulose 24(1), 6168. 33. Lou, H., Zeng, M., Hu, Q., Cai, C., Lin, X., Qiu, X., Yang, D., Pang, Y., 2017. Nonionic surfactants enhanced enzymatic hydrolysis of cellulose by reducing cellulase deactivation caused by shear force and air-liquid interface. Bioresour. Technol. 249, 1-8. 34. Lou, H., Zhu, J.Y., Lan, T.Q., Lai, H., Qiu, X., 2013. pH-Induced lignin surface modification to reduce nonspecific cellulase binding and enhance enzymatic saccharification of lignocelluloses. ChemSusChem 6(5), 919-27.

14

35. Luo, X., Gleisner, R., Tian, S., Negron, J., Zhu, W., Horn, E., Pan, X.J., Zhu, J.Y., 2010. Evaluation of Mountain Beetle-Infested Lodgepole Pine for Cellulosic Ethanol Production by Sulfite Pretreatment to Overcome Recalcitrance of Lignocellulose. Ind. Eng. Chem. Res. 49(17), 8258-8266. 36. Luo, X.L., Liu, J., Zheng, P.T., Li, M., Zhou, Y., Huang, L.L., Chen, L.H., Shuai, L., 2019. Promoting enzymatic hydrolysis of lignocellulosic biomass by inexpensive soy protein. Biotechnol. Biofuels 12(1), 51. 37. Meng, X., Sun, Q., Kosa, M., Huang, F., Pu, Y., Ragauskas, A.J., 2016. Physicochemical structural changes of poplar and switchgrass during biomass pretreatment and enzymatic hydrolysis. ACS Sustain. Chem. Eng. 4(9), 4563-4572. 38. Monschein, M., Reisinger, C., Nidetzky, B., 2014. Dissecting the effect of chemical additives on the enzymatic hydrolysis of pretreated wheat straw. Bioresour. Technol. 169, 713-722. 39. Perrechil, F., Cunha, R., 2010. Oil-in-water emulsions stabilized by sodium caseinate: Influence of pH, high-pressure homogenization and locust bean gum addition. J. Food Eng. 97(4), 441-448. 40. Sindhu, R., Binod, P., Pandey, A., 2016. Biological pretreatment of lignocellulosic biomass– An overview. Bioresour. Technol. 199, 76-82. 41. Sun, S., Huang, Y., Sun, R., Tu, M., 2016a. The strong association of condensed phenolic moieties in isolated lignins with their inhibition of enzymatic hydrolysis. Green Chem. 18(15), 4276-4286. 42. Sun, S., Sun, S., Cao, X., Sun, R., 2016b. The role of pretreatment in improving the enzymatic hydrolysis of lignocellulosic materials. Bioresour. Technol. 199, 49-58. 43. Tu, M.B., Chandra, R.P., Saddler, J.N., 2007a. Evaluating the distribution of cellulases and the recycling of free cellulases during the hydrolysis of lignocellulosic substrates. Biotechnol. Prog. 23(2), 398-406. 44. Tu, M.B., Chandra, R.P., Saddler, J.N., 2007b. Recycling cellulases during the hydrolysis of steam exploded and ethanol pretreated lodgepole pine. Biotechnol. Prog. 23(5), 1130-1137. 45. Tu, M.B., Zhang, X., Paice, M., MacFarlane, P., Saddler, J.N., 2009. The potential of enzyme recycling during the hydrolysis of a mixed softwood feedstock. Bioresour. Technol. 100(24), 6407-6415.

15

46. Wang, Q., Liu, S., Yang, G., Chen, J., Ji, X., Ni, Y., 2016. Recycling cellulase towards industrial application of enzyme treatment on hardwood kraft-based dissolving pulp. Bioresour. Technol. 212, 160-163. 47. Wang, Z.J., Zhu, J.Y., Fu, Y.J., Qin, M.H., Shao, Z.Y., Jiang, J.G., Yang, F., 2013. Lignosulfonate-mediated cellulase adsorption: enhanced enzymatic saccharification of lignocellulose through weakening nonproductive binding to lignin. Biotechnol. Biofuels 6(1), 156. 48. Wood, T.M., Bhat, K.M., 1988. Methods for measuring cellulase activities. in: Methods in Enzymology, Vol. Volume 160, Academic Press, pp. 87-112. 49. Yarbrough, J.M., Mittal, A., Mansfield, E., Taylor, L.E., Hobdey, S.E., Sammond, D.W., Bomble, Y.J., Crowley, M.F., Decker, S.R., Himmel, M.E., Vinzant, T.B., 2015. New perspective on glycoside hydrolase binding to lignin from pretreated corn stover. Biotechnol. Biofuels 8(1), 214. 50. Zhou, Y., Chen, H., Qi, F., Zhao, X., Liu, D., 2015. Non-ionic surfactants do not consistently improve the enzymatic hydrolysis of pure cellulose. Bioresour. Technol. 182, 136-143.

16

Table 1 Composition analysis of substrates.

Substrate

Cellulose (wt%)

Acid-

Other

insoluble

Xylan (wt%)

lignin (wt%)

compositions (wt%)

Eucalyptus

43.4 ± 1.1

22.4 ± 0.9

15.0 ± 1.0

19.3 ± 0.8

Eu-DA

60.5 ± 0.5

38.0 ± 0.6

0.52 ± 0.22

0.94 ± 0.15

CCR

87.5 ± 1.7

7.6 ± 0.45

0.33 ± 0.11

3.8 ± 0.4

17

List of Figures Fig. 1. Zeta potential and dissolution percentage of SC at different pH (1 g/L, 50 mM buffer). Fig. 2. A: Enhancing the enzymatic hydrolysis of Eu-DA and CCR with SC at different pH; B: ΔSED@72h of Eu-DA and CCR with and without the addition of SC at different pH. (substrate concentration: 2 wt%, cellulase loading: 10 FPU/g glucan; SC concentration: 0.05 g/g substrate) Fig. 3. A: Effect of SC concentration on the enzymatic hydrolysis of substrates (substrate concentration: 2 wt%; pH 5.5, 10 FPU/g glucan); B: Effect of SC on substrate hydrolysis at different substrate concentrations (SC concentration: 0.05 g/g substrate; pH 5.5, 10 FPU/g glucan). Fig. 4. A: Changes of cellulase components in hydrolysate during the enzymatic hydrolysis of Eu-DA with and without the addition of SC (β-GL: β-glucosidase; CBH: exoglucanase; EG: endoglucanase; Xyl: xylanase); B: Free protein in hydrolysate during the enzymatic hydrolysis of Eu-DA without the addition of SC (CBH & EG: exoglucanase and endoglucanase); C: Free protein in hydrolysate during the enzymatic hydrolysis of Eu-DA with the addition of SC (substrate concentration: 10 wt%; pH 5.5; 10 FPU/g glucan; SC concentration: 0.05 g/g substrate). Fig. 5. A: Adjust the pH of slurry with or without SC after Eu-DA hydrolysis for 72 h to release more cellulase in hydrolysate (desorption for 2 h); B: Free CBH & EG protein in hydrolysate after desorbing cellulase at different pH; C: Free β-GL protein in hydrolysate after desorbing cellulase at different pH. (substrate concentration: 10 wt%; pH 5.5; 10 FPU/g glucan; SC concentration: 0.05 g/g substrate) Fig. 6. A: Using SC to enhance substrate hydrolysis and cellulase recovery; B: Enzymatic digestibilities of fresh substrates by recovered cellulase through re-absorption with or without the addition of SC; C: By supplementing different amount of cellulase to evaluate how much cellulase can be saved by re-adsorption after adding SC (substrate concentration: 10 wt%; pH 5.5; initial cellulase loading: 10 FPU/g glucan; initial SC concentration: 0.05 g/g substrate).

18

Zeta potential(mV)

9

100

6 80

3 0

60

-3 -6

40

-9 -12

20

-15 -18 3.5

Dissolution percentage (%)

Fig. 1.

4.0

4.5

5.0

pH

19

5.5

6.0

0 6.5

A

Eu-DA Eu-DA+SC CCR CCR+SC

100

SED@72h

80 60 40 20 0

4.0

4.5

5.0

Initial pH

5.5

B

40

ΔSED@72h after adding SC

Fig. 2.

35

6.0

20

Eu-DA CCR

30 25 20 15 10 5 0

4

4.5

5

Initial pH

5.5

6

Fig. 3. B 100

90

90

80

80

70 60 Eu-DA CCR

50

SED@72h(%)

100

SED@72h(%)

A

0.001

0.01

70 60 50 40

40 0.0

CCR CCR+SC Eu-DA Eu-DA+SC

30

0.1

Concentration of SC (g/g substrate)

2

5

10

Substrate concentration （wt%)

21

Fig. 4.

C 100

Free protein in hydrolysate (%)

Free protein in hydrolysate (%)

B 100 Total protein CBH & EG β-GI Xyl

80 60 40 20 0 0

10

20

30

40

Time (h)

50

60

70

80

22

Total protein CBH & EG β-GI Xyl

80 60 40 20 0 0

10

20

30

40

Time (h)

50

60

70

80

Fig. 5.

90 80

CBH & EG

Without SC With SC

70 60 50 40 30 20 10 0

5.5

9

5.5

6

7

8

Desorption pH

9

C Free protein in hydrolysate (%)

Free protein in hydrolysate (%)

B

10

23

60

β-GI

Without SC With SC

50 40 30 20 10 0

5.5

9

5.5

6

7

8

Desorption pH

9

10

Fig. 6.

70

SED@72h(%)

C 100

80

60

Without SC With SC

Control

50 40 Control

30

90

SED@72h(%)

B

Supplement 40% cellulase Supplement 50% cellulase Supplement 60% cellulase

Control

80 70 60

20 50

10 0

First

Second

40

Third

Number of cycles

First

Second

Number of cycles

Third

CRediT authorship contribution statement Cheng Cai: Conceptualization, Methodology, Investigation, Writing - original draft. Yu Bao: Data curation, Investigation. Feiyun Li: Data curation, Investigation. Yuxia Pang: Visualization, Investigation. Hongming Lou: Methodology, Project administration, Funding

24

acquisition. Yong Qian: Visualization, Investigation. Xueqing Qiu: Supervision. Writing Reviewing and Editing.

Declaration of interests

☒ The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

☐The authors declare the following financial interests/personal relationships which may be considered as potential competing interests:

Highlights: (1) Highly recyclable SC was used in lignocellulosic enzymatic hydrolysis. (2) Using SC to enhance cellulase recovery through ingenious pH regulation. (3) SC increased the sugar yield of Eu-DA by 95.5% and save at least 40% cellulase.

25

26