Cobalt-60 gamma-ray irradiation pretreatment and sludge protein for enhancing enzymatic saccharification of hybrid poplar sawdust

Bioresource Technology 221 (2016) 9–14

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Cobalt-60 gamma-ray irradiation pretreatment and sludge protein for enhancing enzymatic saccharification of hybrid poplar sawdust Yulin Xiang a,⇑, Yuxiu Xiang b, Lipeng Wang a a b

College of Chemistry and Chemical Engineering, Yulin University, Yulin 719000, Shaanxi Province, China Department of Management Engineering, Qiqihar Institute of Engineering, Qiqihar 161005, Heilongjiang Province, China

h i g h l i g h t s
Effects of gamma irradiation pretreatment on HPS were studied.
The sludge proteins could improve enzymatic hydrolysis efficiency of HPS.
Optimal factors were obtained for reducing sugar yield.
The combined method is a promising method for HPS enzymolysis.

a r t i c l e

i n f o

Article history: Received 14 July 2016 Received in revised form 6 September 2016 Accepted 7 September 2016

Keywords: Enzymatic hydrolysis Gamma irradiation pretreatment Hybrid poplar sawdust Sludge protein

a b s t r a c t In order to improve the enzymatic saccharification of hybrid poplar sawdust, gamma irradiation pretreatment and enzymatic hydrolysis in the presence of sludge protein were investigated. The cellulose crystallinity index were significantly decreased after irradiation pretreatment, and adding sludge protein improved enzyme activity and increased the reducing sugar yield. The conditions of irradiation pretreatment and enzymatic hydrolysis in the presence of sludge protein were systematically examined. The maximum reducing sugar yield was 519 mg/g under an irradiation dose of 300 kGy, a sludge protein dosage of 2 mg/mL, an enzymatic hydrolysis temperature of 45 °C, an enzymatic hydrolysis time of 84 h, and a 90 FPU/g enzyme loading. This work indicated that the combined method of gamma irradiation pretreatment and enzymatic hydrolysis in the presence of sludge protein was a promising potential for the saccharification of hybrid poplar sawdust. Ó 2016 Elsevier Ltd. All rights reserved.

1. Introduction With highlighting problems of energy crisis and environmental pollution caused by use of traditional fossil fuels, alternative sustainable biofuels (e.g. biogas, biodiesel, cellulosic ethanol, etc.) have acted as an important research field all over the world (Kaouther et al., 2016). Lignocellulosic biomass is a plentiful natural resources of renewable energy on earth. The biofuel can be obtained from all kinds of lignocellulosic biomass, such as grass, tree, as well as many wastes from agriculture, forestry, municipal solids, and industry (Zhang et al., 2016a). Recently, it has been reported that lignocellulosic biomass have been successfully converted into biogases, bioethanol, and biodiesel (da Silva et al., 2016; Patel et al., 2015). Enzymatic hydrolysis is a key step from lignocellulosic biomass to biofuel (Mateusz et al., 2016). However,

⇑ Corresponding author. E-mail address: [email protected] (Y. Xiang). http://dx.doi.org/10.1016/j.biortech.2016.09.032 0960-8524/Ó 2016 Elsevier Ltd. All rights reserved.

effective enzymatic hydrolysis of unpretreated biomass is not easy because of the compact structure of lignin, cellulose, and hemicellulose in lignocellulosic. Moreover, in terms of the preliminary conversion rate, the preliminary stage of the biorefinery, which is the conversion phase from cellulose to glucose, is a bottleneck (Lynd et al., 2008). To overfulfil this bottleneck, more process time and higher cellulose dosage will be used to improve the conversion rate. Complicated processing technology and high cost restrict wide-ranging production of biofuels. A lot of pretreatment methods, such as hot compressed water pretreatment method (Akihiro et al., 2012), alkali pretreatment (Cai et al., 2016), dilute acid pretreatment (Jeong et al., 2016), organic solvent pretreatment (Zhou et al., 2016), ionic liquids (ILs) pretreatment (Chen et al., 2016), steam explosion pretreatment (Liu and Chen, 2016), and irradiation pretreatment method (Yin and Wang, 2016), have been exploited for strengthening enzymatic hydrolysis efficiency. Among these methods, irradiation pretreatment is considered as a promising pretreatment method because of its high efficiency for enhancing the enzymatic hydrol-

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ysis of biomass (Liu et al., 2015, 2016; Jin et al., 2009). Irradiation pretreatment method can effectively decompose the stubborn lignocellulosic biomass into amorphous conformation and be favorable to the conversion of low molecular carbohydrates from cellulose (Liu et al., 2015; Cheng et al., 2013). Moreover, irradiation pretreatment has several characters as below: short process time, moderate temperature, and scarcely undesirable inhibitory substrates formed during chemical pretreatments (Liu et al., 2016; Chung et al., 2012). But the researches on the effect of c-ray irradiation pretreatment on enzymatic saccharification of lignocellulosic biomass for biofuel production were very little and not systematic. Simultaneously, enzymatic hydrolysis is enhanced when additives, such as surfactants or proteins, are present (Xu et al., 2016; Wang et al., 2015; Brethauer et al., 2011). However, most researches involved the effect of various surfactants, the reports about the effect of proteins are very little. Enhancing enzymatic saccharification efficiency by 60Co c-ray irradiation pretreatment and sludge protein as an additives has not been reported. Therefore, one objective of this study was to investigate the effect of 60Co c-ray irradiation pretreatment on the physicochemical characteristics (including crystallinity and chemical structure) of hybrid poplar sawdust (HPS). A second objective was to study the feasibility and effectiveness of sludge protein as an additive for the improved enzymatic hydrolysis efficiency of HPS for biofuel production, and to determine the optimized operating conditions of enzymatic saccharification. 2. Materials and methods 2.1. Collection and preparation of materials HPS (Aigeiros of Populus) was collected from a wood plant located in Yulin, China. The sawdust was naturally dried at atmospheric condition for 1 week, then crushed and sieved (40 mesh). The obtained sawdust was sealed in plastic bags and stored at room temperature. Sludge protein was fresh-made. Excess sludge from Yuyang zone wastewater treatment plant in Shaanxi (China) was kept in storage tank after diluted with water, and the sample was stirred for 10 min. Experiments were carried out under the action of ultrasonic in a reactor. The effective volume of the reactor was 1 L, working pressure 9.8 MPa. The working power and frequency of ultrasound-assisted extraction were fixed at 108 W and 20.024 kHz, respectively. Extraction time was 40 min, pH was 9, and temperature was 35 °C. After extraction, the flask was immediately cooled to room temperature by using chilled water. Soluble protein in the supernatant was obtained by centrifuging the disintegrated sludge at 3000 r/min for 10 min at ambient temperatures of 21–25 °C. Supernatant was dried at 35 °C for 1 week into protein powder. The powder was mainly consisted of protein, peptides, oligopeptide, amino acid, and a little other material such as polysaccharide (Xiang et al., 2011). Cellulase (derived from and Choi Biotechnology Co., Ltd., Ningxia, China, which was generated by Acremonium cellulolyticus). Activity is 1040 l/g (pH 5.7). 2.2. Irradiation pretreatment 60

Co-source was supplied by Jin-Pengyuan Radiation The Research Center (Tianjin, China). 50 g of the preparative HPS sample was placed in a 100 mL glass bottle, and then the bottle was exposed to the designated doses (0–500 kGy at 50 kGy increments) in a 60Co irradiator (source intensity: 9.99  1015 Bq). In order to achieve uniform target doses, the sample was rotated 360° continuously under the irradiation process. Finally, the sample was cooled to room temperature (about 25 °C) in a water bath before analysis.

2.3. Enzymatic hydrolysis The pretreated HPS of 0.5 g was respectively mixed with 10 mL of 0.075 mol/L (pH 4.8) citric acid-sodium citrate buffer in a 50 mL flask, and then cellulose and a certain amount of sludge protein was blended into the flask. Enzymatic hydrolysis was performed in a shaking incubator at 160 r/min and 50 °C for 48 h. After hydrolysis, the samples were withdrawn at certain time interval for the analysis of reducing sugar by DNS method (Hu et al., 2008). Enzyme activity was measured by IUPAC method (Zou and Guo, 2010), and filter paper size was 1  1 cm2 square piece. 2.4. Analysis methods 2.4.1. SDS-PAGE method SDS-PAGE was performed based on the modified method of Laemmli (Fu and Sapirstein, 1996). Protein solution was centrifuged at 3000g for 10 min. Sludge protein solution (2.5 mg/mL) was mixed 1:1 with Lammeli buffer (25% glycerol, 0.01% bromophenol blue, 2% SDS, 62.5 mM Tris-HCl, pH 6.8) to a final concentration of 1 mg/mL. Electrophoresis buffer contained 0.2 M glycine, 0.1% SDS, and 20 mM Tris-HCl at pH 8.3. SDS-PAGE was run with 4 °C running buffer at room temperature. Protein solutions were loaded onto gels and run at 100 V for 40 min using a Mini-Protean III cell (Bio-Rad) and PowerPac Basic power supply. Gels were stained using a colloidal blue stain kit followed by destaining for 3–5 h. Protein molecular weights were analyzed using the software Gel-Pro analyzer. 2.4.2. Brunauer, Ennett and Teller (BET) analysis The specific surface area of pretreated/untreated HPS was analyzed using the surface area and pore size analyzer (ASAP-2000, MIC, USA), and computed according to BET mothod (Liu et al., 2015). 2.4.3. FT-IR FT-IR spectra of the HPS before and after pretreatment were recorded using a Fourier Transform Spectrometer (IR Prestige21). It is used to investigate the component changes of pretreated and untreated HPS. The wavenumber range of the spectrometer is 4000–500 cm1 using 100 scans at 4 cm1 resolution. The mass ratio of KBr and HPS (10 mg) in was 100:1. 2.4.4. XRD The crystallinity of HPS was determined on a X-ray diffractometer (D/MAX-2400) using Cu ka radiation source carried out at 30 mA and 40 kV over a 2h range from 5° to 40° with a step size of 0.2° (2h) and a Scan speed 2°/s. The crystalline index (CrI) was estimated by Eq. (1) (Segal et al., 1959):

CrI ¼ 100 

I002  Iam I002

ð1Þ

where I002 is maximum intensity of crystalline structure at 2h = 22.6°; Iam is intensity of the amorphous portion at 2h = 18.0°. 2.5. Statistical analysis Enzymatic hydrolysis experiments were performed in triplicate. In order to minimize the systematic error, each experimental measurement was replicated 3 times. The differences were less than 5%, and the results were analyzed using the Origin8.0 and SAS 9.0 software.

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3. Results and discussion 3.1. Effect of c-ray irradiation pretreatment on HPS characterization The specific surface area and crystallinity are foremost factors to measure the merits of a pretreatment method (Liu et al., 2015; Uju et al., 2016). Crystallinity index is the ratio of crystalline cellulose among the lignocellulosic biomass. X-ray measurement of CrI is the best way to investigate the impact of pretreatment on biomass crystallinity (Liu et al., 2015). A higher CrI represents more crystallinity and vice versa. Fig. 1 showed the changes of CrI and SSA of untreated and irradiated HPS. As seen from Fig. 1, the CrI was slowly declined under the doses from 0 to 300 kGy, and then the decline rate of CrI became rapid (from 38.38% to 18.23%) with further strengthening the dose from 300 kGy to 500 kGy. The SSA was dramatically increased with increasing of dose, and reached a maximum (11.20 m2/g) at 300 kGy, which was obviously higher than that of untreated HPS. Therefore, low dose less than 300 kGy pretreatment on HPS presented an excellent effect on SSA of HPS, but slight effect on the material’s CrI value. Otherwise, high dose (P350 kGy) action on HPS showed great influence on its CrI, but little influence on SSA of HPS. The trends are similar to the findings of Liu et al. (2015). XRD image of untreated and irradiated HPS was shown in Fig. S1a–b. The peaks for all curves appeared at 2h = 17.33°, 22.56°, 35.49°, which presented respectively diffraction peaks of 101, 002 and 040 crystalline surfaces for the cellulose of HPS (Lin et al., 2010). Compared to the untreated sample, peak intensities showed different decrease levels at all pretreated samples, especially peak intensity of 002 crystalline surface. The results indicated that the relative content of crystallization region decreased with increasing of dose. The result was in accordance with the previous study which the CrI was gradually declined with increasing of dose. The result is similar to the findings of Lin et al. (2010). As seen from Fig. S1a, no matter untreated or irradiated HPS, their 2h of the most powerful peak remained unchanged. The result indicated that the polymorphic form of cellulose was not changed upon irradiation despite the CrI going down. The FTIR image can effectively reflect the change of functional group in structure of biomass after pretreatment (Lin et al., 2010). FTIR image of untreated and irradiated HPS was shown in Fig. S1b. As well known, in the presence of oxygen, cellulose irradiated by high dose will produce carboxyl and carbonyl groups because of oxidative degradation (Sun et al., 2013). Fig. S1b showed major peaks at 3046, 1739, 1596, 1507, 1242, 1159, 1055 and 897 cm1 in irradiated HPS, which were weak at untreated HPS. The intensity of these peaks became gradually powerful with the increasing of dose.

50

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Enzyme activity(U)

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Sludge protein dosage (mg) Fig. 2. Effect of sludge protein on cellulase activity.

The intensity of the absorption peak at 1739 cm1 ascribed to the carbonyl groups (C@O stretching vibration) increased gradually with the increasing of dose, while the peak was weak in untreated sample. It revealed that the process of 60Co c-ray irradiation on cellulose generated carboxy and carbonyl groups at the sites of bond rupture (Liu et al., 2015). The absorption peaks at 1374, 1159, 1055 and 897 cm1 ascribed respectively to the CAH bending vibration, CAOAC stretching vibration, CAO stretching vibration and anomeric carbon (C1) vibration of cellulose and hemicellulose were strengthened with the increasing of dose. It indicated that the cellulose and hemicellulose of HPS occurred degradation reactions under the irradiation pretreatment condition (Lin et al., 2010), and the content of cellulose and hemicellulose in HPS decreased. This found was consistent with the CrI result of Fig. 1. The increase of the peak intensities at 1595, 1507 and 1241 cm1 ascribed respectively to the carbon skeleton vibration of benzene ring, carbon skeleton vibration of aromatic ring and ArAO stretching vibration of lignin indicated that lignin was gradually degraded with the increasing of dose (Huang et al., 2010).

3.2. Effect of sludge protein on the enzymatic hydrolysis Enzyme activity improved significantly with increasing of sludge protein dosage. The increases of enzyme activity were about 1 time (from 8750 to 15,023 U) with 30 mg sludge protein, 2.5 times (from 8750 to 21,911 U) with 75 mg sludge protein (Fig. 2). Light intensity analysis of sludge protein suggested that the size of protein were between 19.215 kDa and 388.66 kDa and showed the strongest peak of sludge protein at 66.134 kDa (Fig. S2), and the part of the sludge protein had strong hydrophobicity, which was similar to molecular weight and property of bovine serum albumin (66.446 kDa). It was reported that bovine serum albumin with strong hydrophobicity could formed micelles in solution, and the micelles could accelerate relative movement between cellulase and products, and promote the desorption of enzyme and improve the effective adsorption of substrate and enzyme. So the cellulase activity was significantly increased (Jeyachandran et al., 2010; Brethauer et al., 2011). This may be also the reason why sludge protein caused the increase of cellulase activity. The hydrolysis of HPS (300 kGy irradiation pretreatment) using the different enzyme loading with and without sludge protein treatment is shown in Fig. 3a. The presence of sludge protein obviously increased the enzymatic hydrolysis at 45 °C. A gradual increase in the reducing sugar yield was presented when the sludge protein (1.0 mg/mL) was added to the mixture at 45 °C (Fig. 3a). After 12 h, the reducing sugar yields were increased by 38.6% (from 21.1 to 29.2 mg/g) with 5 FPU/g enzyme loading, 37.5% (from 29.1 to 40 mg/g) with 10 FPU/g enzyme loading,

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250

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Fig. 3. Reducing sugar yields and changes in relative enzyme activities at 45 °C (a and c: reducing sugar yields; b and d: changes in the relative enzyme activities).

the 2.0 and 2.5 mg/mL initial sludge protein concentration treatments had no significant difference. This result showed that the influence of the increase in sludge protein on the enzyme activity was inconspicuous when the sludge protein concentration was more than 2.0 mg/mL. Fig. 4 showed sludge protein and cellulase were incubated at 30 and 45 °C. The effect of sludge protein on the remaining enzyme activity at 45 °C was better than 30 °C. It indicated that adding sludge protein could protect the cellulase from thermal deactivation. Similar findings were reported by Wang et al. (2013). Measuring results of relative enzyme activities indicated that sludge protein could increase the remaining cellulase activity by relieving the thermal deactivation. In addition, previous works showed that adding protein could decreased nonspecific adsorp-

100

Relative enzyme activity (%)

24.4% (from 49.9 to 62.1 mg/g) with 15 FPU/g enzyme loading, and 16.7% (from 71.2 to 83.1 mg/g) with 20 FPU/g enzyme loading. After 72 h, the increases were 71.3% (from 40.3 to 69.1 mg/g), 35% (from 71.9 to 97.1 mg/g), 29.9% (from 101 to 131.2 mg/g), and 19.9% (from 141.8 to 170 mg/g), respectively. After 1.0 mg/ mL sludge protein treatment, the reducing sugar yields were increased to 69.1 mg/g with 5 FPU/g enzyme loading, while the reducing sugar yield was 71.9 mg/g without sludge protein treatment when the cellulase loading wad 10 FPU/g. The result revealed that adding sludge protein could save about 50% cellulase loading without reducing the hydrolysis efficiency. Thus, the addition of sludge protein can promote the enzymatic hydrolysis process. This finding can obviously decrease production cost of biofuels from biomass because the cost of enzymes takes up a significant part of the total process cost. The result was similar to the previous study which the addition of bovine serum albumin could improve the enzymatic hydrolysis process and decrease the total production cost of biofuels (Wang et al., 2013; Qin et al., 2013). As shown in Fig. 3c, the reducing sugar yield was increased from 76.13 mg/g (without sludge protein treatment) to 100.1, 127.2, 162.2, 191.9, and 191 mg/g with 15 FPU/g cellulase loading and different sludge protein concentrations, respectively. A total of 2 mg/mL of sludge protein was the best dosage to enhance the reducing sugar yield, followed by 2.5, 1.5, 1.0, and 0.5 mg/mL. Based on a relative enzyme activity of 100% in solution at the initial time, at 45 °C, when sludge protein was added, the enzyme deactivation without the sludge protein treatment was higher than that with the sludge protein treatment (Fig. 3b and d). As shown in Fig. 3d, enzyme activity without the sludge protein treatment disappeared completely after 60 h. However, samples with the sludge protein treatment still had at least 10% at 60 h. The remaining enzyme activities of the 2.0 and 2.5 mg/mL initial sludge protein concentration treatments were higher than 32% at 60 h, and even after 72 h, the remaining enzyme activities still had 22 and 23%, respectively. In addition, the remaining enzyme activity between

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Time (h) Fig. 4. Effect of sludge protein on relative enzyme activities at 30 and 45 °C (2.0 mg/ mL sludge protein, 15 FPU/g cellulase loading).

Y. Xiang et al. / Bioresource Technology 221 (2016) 9–14

Reducing sugar yield (mg/g)

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Time(h) Fig. 5. Reducing sugar yield of HPS under different conditions (enzyme loading of 15 FPU/g, and enzymatic hydrolysis temperature of 45 °C).

tion of cellulase on lignin, saving more cellulase in the solution (Senta et al., 2011; Wang et al., 2013). As for the loss of enzyme activity, it has been pointed by many scholars that cellulase turns into an inactive complex during hydrolysis process, so restricting further hydrolysis (Wallace et al., 2016). 3.3. Mechanism analysis and optimization of enzymatic hydrolysis The enzymatic hydrolysis results of irridiated HPS are shown in Fig. 5. The reducing sugar yield increased as the increase of hydrolysis time, the increase of the control group was slight after 12 h, while the conversion lines of other groups didn’t become flat until 84 h. Compared to the control group, the reducing sugar yield of the samples with irradiation pretreatment and/or sludge protein action significantly improved, and reached 133.2, 115.1 and 216 mg/g for individual sludge protein action, individual irradiation pretreatment and the combined with irradiation pretreatment and sludge protein action, respectively. Clearly, in the system of the combined treatment, the reducing sugar yield was highest of

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all. The lignin and hemicellulose both were main barriers to valid enzymatic hydrolysis, and the hemicellulose was a major part of cellulose-hemicelluloses-lignin network and promoted the stability of the network structure (Jin et al., 2016). But the hemicellulose was not as stable as cellulose. After irradiation pretreatment, the hemicellulose of the HPS was firstly damaged, leading to the structure destroy of cellulose-hemicelluloses-lignin network. Meanwhile, the lignin was another obstacle to effective enzymatic hydrolysis. Delignification was an important method to improve the enzymatic hydrolysis for biomass (Qing et al., 2016). In this work, hemicellulose and lignin could be partly destroyed (as shown in Fig. S1b) and improve accessibility of the enzymes to the inner structure of the HPS (Fig. 6). Thus, the enzymatic hydrolysis of HPS is improved. On the other hand, sludge protein was play a prominent role to increase the accessibility of enzymes onto cellulose. The adding sludge protein decreased nonspecific adsorption of cellulase on lignin or releasing unspecifically bound enzyme, and relieved the thermal deactivation of cellulase, saving more cellulase in the solution. Moreover, the adding sludge protein might be decreased the contact of the enzyme with the air-liquid interface owing to the surface activity of sludge protein (Kim et al., 1982), and this phenomenon needs further research in the future. Based on the above experiments, irradiation pretreatment of 300 kGy, sludge protein dosage of 2 mg/mL, enzymatic hydrolysis temperature of 45 °C, and enzymatic hydrolysis time of 84 h were chosen. Under these conditions, the effect of enzyme loading on the reducing sugar yield of HPS was investigated. The results indicated that increasing enzyme loading significantly improved the reducing sugar yield. The increase trend became flat from 90 to 120 FPU/g after 84 h. It indicated that the cellulose sites were saturated at a cellulase loading of 90 FPU/g and the hydrolysis yield of the cellulose reached the maximum at the time of 84 h. Therefore, irradiation dose of 300 kGy, sludge protein dosage of 2 mg/mL, enzymatic hydrolysis temperature of 45 °C, enzymatic hydrolysis time of 84 h, and enzyme loading of 90 FPU/g were chosen for enhancing enzymatic saccharification of HPS. Under these conditions, the reducing sugar yield reached 519 mg/g, and was higher than previously reported (Jin et al., 2016; Zhang et al., 2016b). Consequently, gamma ray irradiation pretreatment and sludge protein for improving enzymatic saccharification of HPS shows a promising potential for biofuel production.

Fig. 6. Predicted model of irradiation pretreatment and sludge protein enhance enzymatic hydrolysis of HPS.

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4. Conclusions The irradiation pretreatment could effectively improve enzymatic hydrolysis of HPS, and the enzymatic hydrolysis of the irradiated HPS was significantly increased by the presence of sludge protein. The reducing sugar yield achieved 519.03 mg/g under the optimal conditions, irradiation dose of 300 kGy, sludge protein dosage of 2 mg/mL, enzymatic hydrolysis temperature of 45 °C, enzymatic hydrolysis time of 84 h, and enzyme loading of 90 FPU/g. Therefore, the combination of gamma irradiation pretreatment and enzymatic hydrolysis in the presence of sludge protein could enhance enzymatic saccharification of HPS. The combined method was a promising potential for biofuel production. Acknowledgements National Natural Science Foundation of China (21203163). The National Natural Science gold project, Shaanxi Provincial Science and Technology Department foster industrialization projects (15JF035). The authors are grateful for the funding and support provided by the following projects: the Scientific Research Starting Foundation for high-level professionals in Yulin University of China (No. 12GK13). Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.biortech.2016.09. 032. References Akihiro, Hideno, Hiroyuki, Inoue, Takashi, Yanagida, Kenichiro, Tsukahara, Takashi, Endo, Shigeki, Sawayama, 2012. Combination of hot compressed water treatment and wet disk milling for high sugar recovery yield in enzymatic hydrolysis of rice straw. Bioresour. Technol. 104, 743–748. Brethauer, Simone, Studer, Michael H., Yang, Bin, Wyman, Charles E., 2011. The effect of bovine serum albumin on batch and continuous enzymatic cellulose hydrolysis mixed by stirring or shaking. Bioresour. Technol. 102 (10), 6295– 6298. Cai, Ling-Yan, Ma, Yu-Long, Ma, Xiao-Xia, Lv, Jun-Min, 2016. Improvement of enzymatic hydrolysis and ethanol production from corn stalk by alkali and Nmethylmorpholine-N-oxide pretreatments. Bioresour. Technol. 212, 42–46. Chen, Fengli, Zhang, Xinglong, Du, Xinqi, Yang, Lei, Zu, Yuangang, Yang, Fengjian, 2016. A new approach for obtaining trans-resveratrol from tree peony seed oil extracted residues using ionic liquid-based enzymatic hydrolysis in situ extraction. Sep. Purif. Technol. 170, 294–305. Cheng, K., Barber, V.A., Driscoll, M.S., Winter, W.T., Stipanovic, A.J., 2013. Reducing woody biomass recalcitrance by electron beams, biodelignification and hotwater extraction. J. Bioprocess Eng. Biorefinery 2, 143–152. Chung, B.Y., Lee, J.T., Bai, H.-W., Kim, U.-J., Bae, H.-J., Wi, S.G., Cho, J.-Y., 2012. Enhanced enzymatic hydrolysis of poplar bark by combined use of gamma ray and dilute acid for bioethanol production. Radiat. Phys. Chem. 81, 1003–1007. da Silva, André Rodrigues Gurgel, Ortega, Carlo Edgar Torres, Rong, Ben-Guang, 2016. Techno-economic analysis of different pretreatment processes for lignocellulosic-based bioethanol production. Bioresour. Technol. 218, 561–570. Fu, B.X., Sapirstein, H.D., 1996. Procedure for isolating monomeric proteins and polymeric glutenin of wheat flour. J. Cereal Chem. 73, 143–152. Hu, R., Lin, L., Liu, T., Ouyang, P., He, B., Liu, S., 2008. Reducing sugar content in hemicellulose hydrolysate by DNS method: a revisit. J. Biobased Mater. Bioenergy 2, 156–161. Huang, Rongfeng, Lu, Jianxiong, Cao, Yongjian, Zhao, Xiu, Zhao, Youke, Zhou, Yongdong, Wu, Yuzhang, 2010. Impact of heat treatment on chemical composition of Chinese white poplar wood. J. Beijing Forestry Univ. 32 (3), 155–160. Jeong, Han-Seob, Jang, Soo-Kyeong, Kim, Ho-Yong, Yeo, Hwanmyeong, Choi, Joon Weon, Choi, In-Gyu, 2016. Effect of freeze storage on hemicellulose degradation and enzymatic hydrolysis by dilute-acid pretreatment of Mongolian oak. Fuel 165, 145–151. Jeyachandran, Y.L., Mielczarski, J.A., Mielczarski, E., Rai, B., 2010. Efficiency of blocking of non-specific interaction of different proteins by BSA adsorbed on hydrophobic and hydrophilic surfaces. J. Colloid Interface Sci. 341, 136–142. Jin, Seop Bak, Ko, Ja Kyong, Han, Young Hwan, Lee, Byung Cheol, Choi, In-Geol, Kim, Kyoung Heon, 2009. Improved enzymatic hydrolysis yield of rice straw using

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