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Biofuels and Biomass
Dual effect of non-ionic surfactants on improving the enzymatic hydrolysis of lignocellulose Wen Wang, Xinshu Zhuang, Xuesong Tan, Qiong Wang, Xiaoyan Chen, Qiang Yu, Wei Qi, Zhongming Wang, and Zhenhong Yuan Energy Fuels, Just Accepted Manuscript • DOI: 10.1021/acs.energyfuels.8b00225 • Publication Date (Web): 16 Apr 2018 Downloaded from http://pubs.acs.org on April 16, 2018
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Dual effect of non-ionic surfactants on improving the enzymatic hydrolysis of lignocellulose Wen Wanga, Xinshu Zhuanga, Xuesong Tana, Qiong Wanga, Xiaoyan Chena, Qiang Yua,*, Wei Qia, Zhongming Wanga, Zhenhong Yuana,b a
Guangzhou Institute of Energy Conversion, Chinese Academy of Sciences; CAS
Key Laboratory of Renewable Energy; Guangdong Provincial Key Laboratory of New and Renewable Energy Research and Development, Guangzhou 510640, P. R. China b
Collaborative Innovation Centre of Biomass Energy, Zhengzhou 450002, P. R.
China
ABSTRACT The mechanism of non-ionic surfactants improving the cellulose conversion has been still controversial. To protect enzyme stability and prevent unproductive adsorption of cellulase to lignin which have been thought as the chief factors for the improvement of non-ionic surfactants on cellulolytic hydrolysis were evaluated in this work. SDS-PAGE detection showed that the enzyme could not aggregate in the enzymatic hydrolysis process whether the polysorbates (Tweens) were present or not. Tweens had different capabilities to retain and even improve enzymes’ activities, but these capabilities had little relation to enhancing the enzymatic hydrolysis of treated sugarcane bagasse (SCB). Tweens could increase the adsorption of cellulase to lignins and SCB samples, which was different from the current viewpoint that non-ionic surfactants could impede the adsorption of cellulase to lignin. After discussion, it 1
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proposed that the non-ionic surfactants initially lubricated the access of cellulase to cellulose, and subsequently combined with the free chemical groups released from lignin to impede the adsorption of cellulase to lignin with the enzymatic hydrolysis proceeding. KEYWORDS: Surfactant; Lignin; Adsorption; Enzymatic hydrolysis; Sugarcane bagasse 1 INTRODUCTION Bioconversion of lignocellulosic biomass to biofuels and high valuable chemicals has gained more and more attentions due to its potential to reduce the net emissions of greenhouse gases, supplement the national energy supply, and transform the economic growth point.1 However, lignocellulosic biomass, mainly composed of cellulose, hemicellulose and lignin, possesses complex structural and chemical features such as epidermal tissue, vascular bundles, sclerenchymatous tissue, lignification degree, structural heterogeneity and complexity of cell-wall constituents to resist the enzymatic attack.2 Pretreatment including biological, physical, chemical and physico-chemical methods is a prerequisite step to reduce the recalcitrance of lignocellulosic feedstock to enzyme, and thus increases the enzymatic saccharification efficiencies of the cellulose and hemicellulose.3 Moreover, the addition of promoters during the enzymolysis process can not only reduce the enzyme usage, but also significantly improve the conversion of plant polysaccharides into monosaccharides. The non-ionic surfactants,4 chemically modified lignin5 and bovine serum albumin6 have exhibited their significant improvement on enzymatic hydrolysis of 2
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lignocellulose. The non-ionic surfactants especially for polysorbate (Tween) surfactants have more and more applications for the enzymatic hydrolysis of pretreated lignocellulose because of their low costs, easy availabilities and little harm to the environment.7 However, the mechanism of non-ionic surfactants on improving the enzymatic hydrolysis of lignocellulose has been inexplicit and controversial. Kaar and Holtzapple8 investigated the benefit of Tween for enzymatic hydrolysis of corn stover, and speculated that Tween enhanced the hydrolysis efficiency of corn stover through enzyme stabilization, lignocellulose disruption, and enzyme activity promotion. The effects of non-ionic surfactants on stabilizing and promoting cellulase were also reported by several other researchers.9,10 But Eriksson et al7 reported the opposite results that the surfactants had only a weak effect on the temperature stability of cellulase, and Tween20 had no significant effect on enzyme activity. They subsequently concluded that the advantage of the non-ionic surfactants for enzymatic hydrolysis of lignocellulose was chiefly ascribed to their prevention on the unproductive adsorption of enzyme to lignin surfaces.7 This conclusion has become the prevalent theory to explain the improvement of non-ionic surfactants on enzymatic hydrolysis of lignocellulose.4,11-13 To influence the stability and activity of cellulase, and to prevent the unproductive adsorption of cellulase to lignin would be the main controversial viewpoints on non-ionic surfactants improving enzymatic hydrolysis of lignocellulose. In this study, the polyoxyethylene (20) sorbitan monolaurate (Tween20), polyoxyethylene (20) sorbitan monostearate (Tween60), polyoxyethylene (4) sorbitan monostearate (Tween61) and polyoxyethylene (20) 3
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sorbitan tristearate (Tween65) which have the differences in the hydrocarbon chain and polymerization degree were selected to investigate their impacts on the enzyme stability and activity. Sugarcane bagasse (SCB) samples containing different lignin contents were prepared, and the whole lignins of the SCB samples were almost isolated by the milled and enzymolytic method. The effects of polysorbate surfactants on the adsorption of cellulase to lignin and SCB samples were investigated. A modified mechanism for the improvement of non-ionic surfactants on the cellulolytic hydrolysis was proposed. 2. MATERIALS AND METHODS 2.1 Materials SCB provided by Fenghao Alcohol Co., Ltd. (China) was sifted out and washed preliminarily as the previous method.14 Cellulase produced from Penicillium sp. was purchased from Imperial Jade Bio-technology Co. Ltd. (China). The FPA of the cellulase was 113.8 FPU/g powder which was assayed by Ghose’s method.15 Xylanase with instructed activity of 100,000 U/g powder was purchased from Shanghai Macklin Biochemical Co., Ltd. (China). Tween20, Tween60, Tween61 and Tween65 were bought from Shanghai Ekear Bio@Tech Co. Ltd. (China). 2.2 Preparation of SCB samples and lignin SCB was respectively treated in 2% (W/V) NaOH solution at 80 ºC for 10, 30, 60 and 120 min with the solid-to-liquid ratio of 1:10. The treated SCB solid residues were washed until their pH values were neutral, and then oven-dried at 60 ºC until their masses were constant. The milled and enzymolytic method which can isolate nearly 4
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all of lignin from lignocellulose and almost preserve the original characteristics of lignin16 was used to extract lignins from the raw and treated SCB samples through the repeating steps of ball milling and enzymatic hydrolysis as the following. The raw and treated SCB were ground for 3 min by the pulverizer, and then transferred to the agate jars. The raw SCB was milled for 48 h, while the treated SCB samples were milled for 24 h in the planetary ball mill (PMQW, Nanjing Chishun Science & Technology Co., Ltd. China). The milled SCB samples of 5% (W/V) solid concentration were hydrolyzed at 50 ºC, pH 4.8, 150 rpm for 96 h with cellulase and xylanase loadings of 40 FPU/g cellulose and 30 U/g hemicellulose, respectively. The hydrolyzed solid residues were collected through centrifugation at 10000 rpm for 5 min. After being milled for 24 h, these residues were hydrolyzed again as the above condition. The addition of cellulase into raw and 10-min NaOH-treated SCB samples was 40 FPU/g initial cellulose, while into other NaOH-treated SCB samples was 20 FPU/g initial cellulose. The loadings of xylanase to all SCB samples were 1000 U/g initial hemicellulose. This ball milling and enzymatic hydrolysis process was repeated for another time. After the final hydrolyzed solid residues were collected, 20 mg/mL proteinase K was loaded into the reaction system to form work concentration of 100 µg/mL to degrade cellulase and xylanase. After being hydrolyzed at 55 ºC for 24 h, the hydrolyzed slurries were incubated in boiling water for 20 min to destruct proteinase K. After being harvested via centrifugation, the solid residues were washed for three times with deionized water, and then naturally dried at the room temperature. The lignin extracted by milled and enzymolytic method was termed as MEL. 5
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2.3 Enzymatic hydrolysis The filter paper and NaOH-treated SCB samples were loaded into 5 mL penicillin bottles containing 2 mL 0.05 M acetate buffer (pH 4.8) to form 10% solid concentration, respectively. Tween20, Tween60, Tween61 and Tween65 at the loadings of 0.5%, 1%, 1.5% and 2% (V/V) were respectively added into the reaction slurries. After being loaded with 20 FPU cellulase/g cellulose, the bottles were sealed with rubber plugs and parafilm to prevent water loss in the hydrolysis process. The enzymatic hydrolysis was conducted at 50 ºC, 180 rpm for 72 h. The filter paper and NaOH-treated SCB samples without Tween addition which were hydrolyzed under the same condition were used as the controls. 2.4 Cellulase adsorption The cellulase was dissolved in 0.05 M acetate buffer (pH 4.8) to form 17.6 mg/mL enzyme solution, and then centrifuged at 10000 rpm for 5 min to remove the insoluble substances. The SCB samples and their MELs of 0.02 g were respectively added into 25 mL conical flasks containing 2 mL enzyme solution. After being loaded with 0.5% (V/V) Tween20, Tween60, Tween61 and Tween65, respectively, the flasks were sealed with the glass stoppers and parafilm, and shaken at 50 ºC, 150 rpm for 90 min. The slurries were respectively transferred to 2 mL EP tubes, and centrifuged at 12000 rpm for 2 min. The protein contents in the supernatants were measured as the Bradford’s protocol.17 The absorbance was read at 595 nm with the microplate spectrophotometer (Eon, BioTek). The slurries of SCB samples and their MELs containing Tweens without cellulase additions were used as the blank controls, and 6
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those containing cellulase without Tween additions were used as the positive controls. In addition, the impact of Tween60 loadings on the cellulase adsorption of untreated SCB MEL was also analysed according to the above method. The adsorption amount of cellulase could be calculated as a ratio of the adsorbed to the initial cellulase amounts.18 2.5 Enzyme activity assay The effects of Tweens on enzyme activities were analysed as the following. The enzyme solution was prepared as the above mention. After being loaded with 0.5%, 1%, 1.5% and 2% (V/V) Tween20, Tween60, Tween61 and Tween65 respectively, the enzyme solutions were placed in a rotary shaker for 72 h at the condition of 50 ºC, 150 rpm. The FPA, endoglucanase and exoglucanase activities of enzyme solutions were measured by DNS method with 1.2×1.2 cm Whatman NO.1 filter paper, 1% (W/V) sodium carboxymethylcellulose and 1% (W/V) microcrystalline cellulose used as the substrates respectively. The total reaction volumes were 0.2 mL which consisted of 0.1 mL acetate buffer/substrate solution and 0.1 mL enzyme solution. After being incubated at 50 ºC for 30 min, the reactions were terminated via the addition of 0.3 mL DNS solutions. These mixtures were boiled for exactly 10 min in a vigorously boiling water. The spectro zeros and glucose standards were boiled together. The spectro zeros were prepared by adding 0.1 mL enzyme solution into 0.1 mL substrate solution mixed with 0.3 mL DNS solution. After boiling, all samples, the spectro zeros and glucose strandards were transferred to a cold water bath immediately, and added with 2 mL distilled water. After being blended well, 300 µL 7
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solutions were transferred into a 96-well plate. The absorbances were measured against the spectro zeros at 540 nm with the microplate spectrophotometer (Eon, BioTek). The FPA, endoglucanase and exoglucanase activities were defined as the release of glucose amount (mg) in 1 min by 1 mL enzyme solution. The β-glucosidase activity was measured by the described pNPG (p-nitrophenyl-β-D-glucopyranoside) protocol.14 The relative enzyme activity was calculated as the ratio of the enzyme activity with being shaken for 72 h to the initial enzyme activity without being shaken. Additionally, the enzyme solutions individually containing Tween20, Tween60, Tween61 and Tween65 were respectively mixed with SCB samples and their MELs, and then incubated in the ice bath for 90 min with being shaken at every 10 min. After incubation and centrifugation, the FPAs of the enzyme in the supernatants were also measured as the above procedure. The relative FPAs was calculated as the ratio of the FPAs of enzyme solutions with Tweens addition after adsorption to those without Tweens addition. All tests in this study were in duplicate. 2.6 Analytic methods The compositional analysis was carried out according to the NREL method.19 The surface morphologies of samples were observed by the field emission SEM (S 4800, Hitachi) at an accelerating voltage of 2.0 kV. The surface tensions of the acetate buffer with and without Tween addition were detected by the surface tension meter (OCA40 Micro, Dataphysics). The SDS-PAGE method was used to detect the aggregation of cellulase. The sugar concentrations of the hydrolysates were quantified at 50 ºC by the HPLC system (Waters 2698, USA) equipped with a sugar column 8
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(SH1011, Shodex). The flow rate of the mobile phase (5mM H2SO4) was 0.5 mL/min. The glycan conversion (GlyC) was defined as the ratio of the practical releases of cellobiose, glucose and xylose from holocellulose to their theoretical yields, while the glucan conversion was calculated as the proportion of the practical releases of cellobiose, glucose from cellulose to their theoretical yields.18 3 RESULTS AND DISCUSSION 3.1 Effect of Tween on enzymatic hydrolysis of the treated SCB The viewpoint that the non-ionic surfactants improve enzymatic hydrolysis of lignocellulose via preventing the adsorption of cellulase to lignin has been accepted by many researchers.4,7,12,20-24 It means that the improvement of non-ionic surfactant on enzymatic hydrolysis has relation to lignin. Therefore, the impacts of non-ionic surfactants on SCB samples containing different lignin contents and filter paper were investigated (Figure 1). In addition, the various effects of surfactants on the enzymatic hydrolysis of lignocellulose not only depended on the features of lignocellulose, but also related to the characteristics of surfactants.4,7 Four Tweens which have similar chemical formula were used to evaluate the influence of their chemical features on the enzymatic hydrolysis of lignocellulose. Tween60 was used as the reference substance to select other three surfactants. Tween20 and Tween65 have the same polymerization degree as Tween60, while respectively show the different length and amount of hydrocarbon chain as Tween60. Conversely, Tween61 has identical hydrocarbon chain as Tween60, while lower polymerization degree than Tween60 (Table 1). Other different chemical features of these four Tweens were also shown in Table 1. 9
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Although the four Tweens have different physico-chemical features, they had similar influence on the enzymatic hydrolysis of filter paper (Figure 1). The glucan conversions of filter paper were weakly improved by the four Tweens at the concentration of 0.5% (V/V), while decreased with the concentrations of Tweens increasing. It had been reported that Tween surfactants could result in the obvious/slight/none increase in glucose yield from the enzymatic saccharification of different pure celluloses such as newspaper, microcrystalline cellulose, amorphous cellulose, filter paper, and so on.7,9,25-27 This study showed that Tween surfactants had little influence on enzymatic hydrolysis of filter paper. The t-test analysis showed that Tween20, Tween61 and Tween65 had insignificant differences with Tween60 in influencing enzymatic hydrolysis efficiencies of filter paper (t< t0.05, p>0.05). It meant that the changes in length of hydrocarbon chain (Tween20 vs Tween60), polymerization degree (Tween61 vs Tween60) and amount of hydrocarbon chain (Tween65 vs Tween60) had little relation to the hydrolysis of pure cellulose. The relationship between lignin content and enhancement of Tweens on cellulolytic hydrolysis was explored. SCB samples containing different lignin amounts were prepared with sodium hydroxide (NaOH) treatment. The compositions of SCB samples were shown in Table 2. As the treated time prolonging, the relative content of cellulose increased, while that of lignin decreased. The percentage of hemicellulose content increased slightly with the treated time prolonging in 60 min, but declined when the treated time was maintained for 120 min. From Figure 1, without Tweens addition, the glycan conversion (GlyC) of SCB samples increased from 40.7% to 10
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68.1%, and the differences in GlyCs of SCB samples were reduced with the lignin content decreasing. With Tweens addition, the GlyC increment of SCB containing the highest lignin content (10-min treated SCB) was more than other three SCB samples. It seemed that the promotion of Tweens for enzymatic hydrolysis of lignocellulose was indeed related to the lignin content. Interestingly, under the improvement of Tweens, SCB having 9.5% lignin (120-min treated SCB) obtained higher GlyC increment than SCB samples containing 11.8% (30-min treated SCB) and 10.3% (60-min treated SCB) lignins which attained the similar maximum GlyC increment at around 4%. These results hinted that the lignin content was not the sole factor to be overcome by Tweens to enhance the enzymatic hydrolysis of lignocellulose. The hemicellulose removal was reported to be more significant to create cellulase accessible pores than lignin removal.28 As far as the components were concerted, the slightly lower xylan amount of 120-min treated SCB might contribute to the higher incremental improvement of the cellulose conversion. Figure 1 showed that all Tweens could improve the enzymatic hydrolysis of SCB samples containing different lignin contents, but the improvements of Tween loadings on GlyCs of SCB samples were various. In the wake of increasing Tween20 addition, the GlyC of 10-min treated SCB increased, while the GlyCs of 30-min, 60-min and 120-min treated SCB samples decreased. With Tween60 loaded from 0.5% to 1.5%, the GlyC of 10-min treated SCB reduced from 55.9% to 53.1%, while the GlyC of 30-min treated SCB rose from 63.7% to 66.2%. When the addition of Tween60 reached to 2%, 10-min treated SCB obtained its maximum GlyC value of 57.3%, 11
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while 30-min treated SCB got the decreased GlyC value of 65.4%. The GlyCs of 60-min and 120-min treated SCB samples tended to go up with Tween60 concentration increasing. The decreases in the GlyCs of the four treated SCB samples could be observed with the incremental loadings of Tween61. The GlyC of 10-min treated SCB was enhanced with Tween65 addition rising. The GlyCs of 30-min and 120-min treated SCB samples went up within 1.0% concentration of Tween65, and then went down with Tween65 concentration exceeding 1.0%. The impacts of Tween65 loadings on improving GlyCs of 60-min treated SCB were fluctuant. After t-test analysis, Tween20, Tween61 and Tween65 had insignificant differences with Tween60 in improving enzymatic hydrolysis of SCB samples treated by 2% NaOH for above 30 min (t0.05). As for the improvement of enzymatic hydrolysis of 10-min treated SCB, Tween20 had insignificant difference with Tween60 (t0.05), while Tween61 and Tween65 had significant differences with Tween60 (t>t0.05, pt0.05, p
