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Feb 19, 2016 - Xylanase addition could increase the laccase incubation rate. Bonds between lignin−carbohydrate complexes (LCCs) were disrupted with ...
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Research Article pubs.acs.org/journal/ascecg

Laccase and Xylanase Incubation Enhanced the Sulfomethylation Reactivity of Alkali Lignin Haifeng Zhou,†,‡ Xueqing Qiu,*,§,∥ Dongjie Yang,§ and Shaoqu Xie§ †

Key Laboratory of Low Carbon Energy and Chemical Engineering, Shandong University of Science and Technology, 579 Qianwangang Road, Qingdao, China ‡ College of Chemical and Environmental Engineering, Shandong University of Science and Technology, 579 Qianwangang Road, Qingdao, China § School of Chemistry and Chemical Engineering, South China University of Technology, 381 Wushan Road, Guangzhou, China ∥ State Key Laboratory of Pulp and Paper Engineering, South China University of Technology, 381 Wushan Road, Guangzhou, China S Supporting Information *

ABSTRACT: Alkali lignin (AL), from poplar alkali pulping process, was activated by laccase and laccase/xylanase system (LXS). When incubated with 10 U/g laccase for 24 h, the sulfomethylation reactivity of AL could increase by 33%. The functional group content and 1H NMR analysis showed that the cleavage of various ether linkages and demethylation, resulting in the increase of phenolic groups and decrease of steric hindrance, contributed to the improvement of sulfomethylation reactivity. Xylanase addition could increase the laccase incubation rate. Bonds between lignin−carbohydrate complexes (LCCs) were disrupted with the addition of xylanase. Hence, AL was more accessible to laccase, leading to a higher incubation rate. Due to the increased reactivity, the dispersion performance of sulfomethylated lignin on TiO2 slurry was obviously improved. KEYWORDS: Alkali lignin, Laccase, Xylanase, Sulfomethylation reactivity, Dispersion performance

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INTRODUCTION Alkali lignin (AL), a byproduct from alkali pulping process, accounts for nearly 85% of total technical lignin in the world. However, most of AL is used as fuel or discarded as wastewater. Only no more than 10% of AL is used as industrial products. The utilization of AL has both economic and environmental benefits.1 The alkali pulping makes the lignin highly fragmented and insoluble in water at neutral pH.2 Therefore, AL must be modified before industrial application. Chemical modifications, such as sulfomethylation,3 oxidation,4 and copolymerization,5 are the main methods to improve the properties during industrial application. Among all chemical modifications, sulfomethylation is one of the most widely used methods. The introduction of sulfonic groups enhances the water solubility of AL. Therefore, sulfomethylated alkali lignin could be used as cement additive,6 dyestuff and coal−water slurry dispersant,7 and phenol substitute in phenol formaldehyde resins.8 The functional groups content, molecular weight, and structure characterization significantly affect the end-use of the modified AL. However, because of the cleavage of various linkages and condensation of polyphenyl propene units during the violent condition of the alkali pulping process, the reactivity of AL is extremely decreased.3,9 The low reactivity of AL results in the seriously affected modification, which is the bottleneck of © 2016 American Chemical Society

the high-efficiency utilization of AL. Thereby, AL must be activated before modification. So far, the activation methods of AL are classified as physical, chemical, and biological ones. The chemical activation has been used in the industry, including hydroxymethylation,10 phenolation,11 demethylation,12 oxidation,13 and so on. Compared with the chemical method, the biological activation is only at the research stage. Nevertheless, the biological method has the advantages of lower energy consumption, fewer chemical reagents, and a simple technique, which is a promising technique in harmless treatment. Laccase is one of the most widely used ligninolytic enzymes. Laccase possesses both polymerization and depolymerization. It could attack the phenolic groups of lignin to form phenoxyl radicals, leading to the couplings of radicals. It also could result in the cleavages of various linkages, decarboxylation, demethylation, and so on.14−16 Xylanase is a hydrolytic enzyme that catalyzes xylan degradation. Most of the published studies on laccase and xylanase have focused on biopulping, biobleaching, and wastewater treatment.17 The combination of xylanase and laccase may prove to be a promising strategy for achieving higher tensile strength of wet and dry paper and a Received: October 13, 2015 Revised: February 17, 2016 Published: February 19, 2016 1248

DOI: 10.1021/acssuschemeng.5b01291 ACS Sustainable Chem. Eng. 2016, 4, 1248−1254

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Figure 1. (a) Diagram of enzymatic incubation and sulfomethylation of AL. (b) Diagram of sulfomethylation of lignin model compound.

higher degree of pulp bleaching.18,19 The reports on influence of laccase and xylanase incubation on chemical reactivity of lignin are limited. We have previously demonstrated that laccase could improve the sulfonation reactivity of wheat straw AL under the condition of atmosphere pressure.9 However, the influence of laccase/xylanase system (LXS) on chemical reactivity of lignin is scarce. Furthermore, the chemical structure and content of the lignin from woody biomass are very different from those from herbaceous biomass, as are the types and structures of lignin−carbohydrate complexes (LCCs).20 To further investigate the influence of laccase and LXS incubation on chemical reactivity of lignin, we used a poplar wood alkali lignin (AL) as raw material in this work. AL was first incubated by laccase or LXS to obtain the incubated AL. Subsequently, the incubated AL was modified by sulfomethylation in order to investigate the sulfomethylation reactivity of AL. The structural characterization was analyzed before and after laccase and LXS incubation to understand the mechanism of enzymatic incubation. In addition, the dispersion properties of the products were also reported.

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The size analysis of TiO2 was determined by an EyeTech Laser particle size analyzer (Ankersmid Co. Ltd., Holland). The average particle diameter of TiO2 was 5.43 μm. Incubation of AL by Laccase and Laccase/Xylanase System (LXS). A 5 g portion of AL was dissolved in 50 mL of acetate buffer at pH 5.0 in a 250 mL reaction vessel, which was agitated at 480 rpm and 40 °C prior to addition of different dosages of laccase and xylanase. After the incubation, 10% (w/w) sodium hydroxide solution was used to adjust the pH 12.0 of enzymatic incubated samples. Then, all samples were heated in the boiling water bath for 10 min to stop the reaction. The samples were then filtered, and the filtrate was freezingly desiccated. The samples after laccase and LXS incubation were named as LAL and LXAL, respectively. Sulfomethylation of Lignin. The 5 g portions of lignin samples were dissolved in 10% (w/w) sodium hydroxide solution in a reaction vessel and heated to 75 °C. Then, 1.0 g of formaldehyde aqueous solution of 37% (w/v) concentration was added and stirred for 90 min at 480 rpm. Subsequently, the reaction temperature was increased to 90 °C, and 0.5 g of Na2SO3 was added. The reaction was kept for another 2 h. Gel Permeation Chromatography (GPC). The molecular weight distribution of lignin was determined by aqueous GPC, which was conducted using Ultrahydragel 500, Ultrahydragel 250, and Ultrahydragel 120 columns in series and measured with a Waters UV detector 2487 at 280 nm (Waters Co.). A 0.10 M NaNO3 solution at pH 10.0 was used as the eluent at 0.5 mL/min. Polystyrenesulfonates in the range from 1000 to 10 000 g/mol were used for calibration. All samples were prepared by double distilled water and filtrated by a 0.22 μm filter. The injection volume was 50 μL. Functional Group Content Measurements. The sulfonic group content was determined by automatic potentiometric titrator (905 Titrando, Metrohm Corp., Switzerland) as reported previously.3 The methoxyl group content was measured by the headspace gas chromatography (HS-GC) as Li et al. reported.23 All measurements were conducted on a DK-3001A headspace sampler (Beijing Zhongxing Huili Science & Technology Co., Ltd., Beijing, China) and model GC9800 capillary gas chromatography (Kechuang Co., Shanghai, China). The phenolic group content was determined as described by de Sousa et al.24 The absorbance at 760 nm was measured by a UV−vis spectrophotometer (UV-2450, Shimadzu Co., Kyoto, Japan), and vanillin was used as the standard. 1 H NMR Analysis. The 1H NMR spectra were recorded with 30 mg of lignin dissolved in 0.5 mL of DMSO-d6 using a Bruker DRX-400

MATERIALS AND METHODS

Materials. Commercial alkali lignin (AL) of polar wood (Populustomentosa Carr) was a byproduct of alkali pulping process from Shixian Papermaking Co. Ltd. (Jilin, China). 2,2′-Azino-bis(3thylbenzothiazoline-6-sulfonate) (ABTS), Folin Ciocalteau’s phenol reagent (2 N), hydroiodic acid (57%), vanillin, and methyl iodide were supplied by Sigma-Aldrich (Shanghai, China). All other chemicals were of analytical grade. Laccase and xylanase was supplied by Denykem Co., Ltd. (Shanghai, China), and Ruicong Technology Co., Ltd. (Shanghai, China), respectively, and were kept at −20 °C. In order to support the feasibility of industrial applications of laccase incubation, the laccase was used in its commercial form without purification. The commercial laccase might include additives and even mediators. Therefore, the observed influence was ascribed to the commercial laccase solution. The laccase activity (based on 1 μmol/min of ABTS converted)21 was 120 U/g. The xylanase activity was 19 357 U/g based on 1 μmol/min of xylose produced.22 Titanium dioxide (TiO2) was a commercial product in powder form from Kermel Chemistry Co. Ltd. (Tianjin, China) with purity of 99%. 1249

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Table 1. Influence of Different Dosages of Laccase/Xylanase and Different Incubation Times on Molecular Weight Distribution and Sulfonic Group Content of SAL dosage of laccase (U/g)

dosage of xylanase (U/g)

incubation time (h)

SAL products

0 5 5 5 5 10 10 10 10 20 20 20 20 10 10 10 10

0 0 0 0 0 0 0 0 0 0 0 0 0 20 20 20 20

0 3 13 24 36 3 13 24 36 3 13 24 36 3 13 24 36

SAL SLAL5-3 SLAL5-13 SLAL5-24 SLAL5-36 SLAL10-3 SLAL10-13 SLAL10-24 SLAL10-36 SLAL20-3 SLAL20-13 SLAL20-24 SLAL20-36 SLXAL10-20-3 SLXAL10-20-13 SLXAL10-20-24 SLXAL10-20-36

spectrometer (Bruker Corp., Germany). The spectra were obtained at 30 °C after 32 scans. A 60° pulse angle and 6 s pulse time were used. Xylan Content Measurement. The AL and enzymatic incubated AL were hydrolyzed using sulfuric acid in two steps as described previously.25 Xylose content in the hydrolysate was analyzed using a Dionex HPLC system (Ultimate 3000) equipped with an RI (RI-101) detector and BioRad Aminex HPX-87P column operated at 80 °C. Double distilled water was used as eluent at a flow rate of 0.6 mL/min. All samples were diluted in distilled water, and filtered by a 0.22 μm filter before injection. The injection volume was 20 μL. Dispersion Properties. The dispersion properties of lignin on TiO2 slurry was measured by Turbiscan Lab Expert (Formulaction, France). The TiO2 powder (3 wt %) was slowly mixed with 0.5 wt % lignin in distilled water and stirred at 1000 rpm for 5 min before measurements. The Turbiscan technology consists of measuring backscattering and transmission intensities versus the sample height for detecting particle size changes and phase separation.

Mn (Da) Mw (Da) 2260 2240 2330 2650 2300 2220 2230 2400 2130 2140 2100 2400 2210 2190 2440 2470 2410

2940 2940 3150 3400 3150 2980 2970 3480 2890 2840 2860 3460 3050 2800 3540 3500 3470

Mw/Mn

sulfonic group content (mmol/g)

1.30 1.31 1.35 1.47 1.37 1.34 1.33 1.45 1.36 1.33 1.36 1.44 1.38 1.28 1.45 1.42 1.44

1.76 1.76 1.99 2.26 2.05 1.79 2.00 2.34 2.07 1.88 2.01 2.36 2.08 2.18 2.28 2.35 2.28

sulfomethylation reactivity was improved by 24%. It indicated that LXS could significantly decrease the incubation time or enhance the incubation rate of laccase. Nevertheless, the maximum sulfonic group content was almost the same, 2.34 and 2.35 mmol/g for SLAL10-24 and SLXAL10-20-24, respectively. It was reported that the molecular weight of lignosulfonate also has a great influence on its properties.7 The lignosulfonate with higher molecular weight has better dispersion performance. Table 1 recorded the weight-average (Mw) and numberaverage (Mn) molecular weights and the polydispersity (Mw/ Mn) of sulfomethylated lignin. After the incubation of laccase and LXS, the molecular weights of most sulfomethylated lignins increased, and some of them decreased. Generally, the sulfomethylated lignin incubated for 24 h had the highest molecular weight. Compared with SAL, the Mw of SLAL10-24 increased by 18%. Functional Group Content of AL. The influence of laccase and LXS incubation on the methoxyl and phenolic groups of AL was shown in Table 2. With increasing incubation time, the methoxyl group content of AL decreased, which might be due to the demethylation of laccase. Moreover, the decrease range increased with the increase of laccase dosages. When using LXS incubation, the demethylation rate increased. However, the terminal methoxyl group content was almost the same. With the increase of incubation time, the phenolic group content decreased first, then increased, and reached a maximum at 24 h, before it decreased again. However, when the laccase dosage was 20 U/g or with LXS incubation, there was no initial decrease process. With increasing laccase dosages, the phenolic group content of LAL increased. The demethylation and ether linkage cleavage by laccase incubation resulted in the increase of phenolic group content. Meanwhile, laccase could oxidize phenolic groups into phenoxy radicals, which subsequently underwent radical−radical couplings, leading to the decrease of phenolic group content and increase of Mw. When laccase dosage was less (5 and 10 U/g laccase), laccase oxidized phenolic groups in priority during the initial stage, resulting in the decrease of phenolic group content. Besides, partial laccase oxidized methoxyl groups and other linkages, leading to the

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RESULTS AND DISCUSSION Influence of Laccase and LXS Incubation on Sulfomethylation Reactivity of AL. The diagram of enzymatic incubation and sulfomethylation of AL was shown in Figure 1. The sulfomethylation reactivity was described by sulfonic group content in sulfomethylated lignin. As shown in Table 1, when using laccase incubation, the sulfonic group content increased first and then decreased with the increase of incubation time. When the incubation time was 24 h, the sulfonic group content achieved a maximum. For instance, when laccase dosage was 5 U/g, the sulfonic group content of sulfomethylated AL (SLAL5-24) reached 2.26 mmol/g at laccase incubation for 24 h. With increasing laccase dosages, the sulfonic group content increased. However, the increased range was limited, especially for the 10 U/g laccase incubation of AL, which was not very different from the 20 U/g laccase incubation. When AL was modified with 10 U/g laccase for 24 h, the sulfomethylation reactivity could be improved by 33%. Therefore, the laccase dosage of 10 U/g was chosen for the LXS incubation. With the increase of xylanase dosages (Figure S1, Supporting Information), the sulfonic group content continuously increased until the xylanase addition was 20 U/ g. As observed in Table 1, when AL was modified with 10 U/g laccase and 20 U/g xylanase for 3 h, the sulfonic group content could achieve 2.18 mmol/g. Compared with SLAL10-3, the 1250

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Table 3. Signal Assignment in 1H NMR Spectra of AL with Laccase and LXS Incubation and Results from Quantification of Functional Groups

Table 2. Influence of Different Dosages of Laccase/Xylanase and Different Incubation Times on Methoxyl and Phenolic Group Content of AL sample

methoxyl group content (%)

phenolic group content (mmol/g)

AL LAL5-3 LAL5-13 LAL5-24 LAL5-36 LAL10-3 LAL10-13 LAL10-24 LAL10-36 LAL20-3 LAL20-13 LAL20-24 LAL20-36 LXAL10-20-3 LXAL10-20-13 LXAL10-20-24 LXAL10-20-36

12.65 12.42 12.11 11.94 11.49 12.00 11.91 11.77 11.27 11.80 11.66 11.28 11.18 11.82 11.75 11.57 11.38

1.989 1.809 1.857 2.026 1.897 1.903 2.025 2.145 2.003 2.110 2.115 2.291 2.212 2.034 2.030 2.156 2.095

amount (DMSO−1) δ (ppm)

assignment

AL

LAL10-24

LXAL10-20-24

7.20−6.80

H2, H5, H6 in guaiacyl units H2, H6 in syringyl units Hα, Hβ in β-5′ structures H in xylan residues Hα, Hβ in β-O-4′ structures Hα in β−β′ structures and H in xylan residues H in methoxyls Hβ in β−β′ structures DMSO H in aromatic acetates H in aliphatic acetates aliphatic H

1.64

1.91

1.93

1.96

1.52

1.44

0.43

0.56

0.49

0.52 0.73

0.52 0.71

0.31 0.53

0.72

0.68

0.64

6.68 0.38 1.00 0.51 0.71 2.33

5.58 0.32 1.00 0.56 0.66 2.32

5.09 0.31 1.00 0.58 0.42 1.49

6.80−6.20 5.75−5.25 5.20−4.90 4.90−4.50 4.40−4.10 4.00−3.50 3.10−3.00 2.60−2.30 2.30−2.10 2.10−1.80 1.80−0.80

of laccse might result in the cleavage of β-O-4′ and β−β′ structures. In addition, the intensities of β-5′ increased, indicating the repolymerization of lignin molecule.15,32 When using LXS incubation, the most remarkable feature was the decrease of xylan content, as observed in Table 3. This feature would be further investigated using HPLC measurement. Sulfomethylation Reactivity Mechanism Analysis. The key of sulfomethylation of AL lies in the hydroxymethylation of AL. The hydroxymethylation is an electrophilic addition reaction, which occurs more easily on the carbon atom rich in electron cloud. The methoxyl, etherfied, and nonetherified hydroxyl groups are electron-donating groups. Our previous study showed that the grant electron effect of hydroxyl was more than methoxyl group.9 The analysis of functional group content and 1H NMR showed that, after enzymatic incubation, there was more content of guaiacyl and hydroxyl, and less content of syringyl and methoxyl, which could enhance the sulfomethylation reactivity of AL. The steric hindrance was also an important factor affecting the chemical reactivity of lignin. When the laccase dosage was small, although there was some demethylation and linkages cleavage, the phenolic group content decreased. In this situation, the improvement of sulfomethylation reactivity was mainly due to the decrease of steric hindrance. Compared with laccase incubation only, the sulfonic group content of SLXAL10-20-3 (LXS incubated for 3 h) was close to that of SLAL10-24 (laccase incubated for 24 h) (Table 1). However, the maximum sulfonic group content was almost the same, 2.34 and 2.35 mmol/g for SLAL10-24 and SLXAL10-2024, respectively. It indicated that the addition of xylanase could enhance the reaction rate. The role of xylanase was likely to be one of the following: (1) The xylanase was compounded with laccase, which might increase the enzymatic activity and extend the half-life of laccase. (2) The xylanase could result in the cleavage of lignin−carbohydrate complexes (LCCs) and the opening up of lignin, which was beneficial to the laccase incubation and sulfomethylation of AL. In order to investigate if there was a synergetic effect between xylanase and laccase, the influence of xylanase on laccase

slight decrease of methoxyl groups. With increasing incubation time, the methoxyl group content decreased gradually, while the phenolic group content started to increase. When more laccase was added (20 U/g laccase), the demethylation was relatively enhanced, resulting in the direct increase of phenolic groups. Compared to the same laccase dosage (10 U/g) system, there was no phenolic group content decrease process in LXS incubation, which might be due to the faster demethylation during LXS incubation. In order to clarify the relationship between the demethylation and phenolic groups’ oxidation during laccase and LXS incubation, the phenolic group content was plotted against the methoxyl group content. As shown in Figure S2, there was no correlation between them. This suggested that laccase could oxidize the phenolic groups; meanwhile, the demethylation was taking place. Therefore, the changes of phenolic groups were dynamic. 1 H NMR Analysis. For the in-depth elucidation of structural characterization of AL by laccase and LXS incubation, AL, LAL10-24, and LXAL10-20-24 were subjected to 1H NMR analysis, as shown in Figure S3. The resonance at 7.20−6.80 ppm was assigned to H2, H5, H6 in guaiacyl units, while the resonance at 6.80−6.20 ppm was attributed to H2, H6 in syringyl units.26,27 The signal between 5.75 and 5.25 ppm was related to Hα, Hβ in β-5′ structures, and the signal between 5.20 and 4.90 ppm was assigned to H in xylan residues.26,28,29 The resonance at 4.90−4.50 ppm was attributed to Hα, Hβ in β-O-4′ structures.27,30 The resonance at 4.00−3.50 ppm was assigned to methoxyl protons.30,31 The signal at 3.10−3.00 ppm was related to Hβ in β−β′ structures.26,27 The resonance at 2.30− 2.10 ppm was assigned to H in aromatic acetates, while the resonance at 2.10−1.80 ppm was assigned to H in aliphatic acetates. The signal between 1.80 and 0.80 ppm was attributed to aliphatic H. The intensities of different regions were normalized by the DMSO-d6 cross-signal (2.60−2.30 ppm), as shown in Table 3. After laccase and LXS incubation, syringyl unit content decreased, while guaiacyl unit content increased. Moreover, the methoxyl groups decreased. It indicated that the oxidation 1251

DOI: 10.1021/acssuschemeng.5b01291 ACS Sustainable Chem. Eng. 2016, 4, 1248−1254

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Figure 2. Influence of xylanase on the laccase activity.

incubation conditions. As observed in Figure 2, the activity of laccase with xylanase addition was higher than laccase alone, especially before 24 h. When the soaking time was 12 h, the activity of laccase with xylanase addition was almost twice that of laccase alone. Although the laccase activity increased after xylanase addition, this was not the only reason for the improvement of sulfomethylation reactivity by LXS incubation. When the laccase dosage was 20 U/g, the sulfonic group content of SLAL was still less than that of SLXAL, 1.88 and 2.18 mmol/g for SLAL20-3 and SLXAL10-20-3, respectively (Table 1). The analysis of 1H NMR showed that xylan residue content decreased after the addition of xylanase. In order to verify this conclusion, the xylan content was determined by HPLC. As observed in Table 4, the xylan content of LXAL reduced from

Figure 3. Relationship between phenolic group in AL and (a) Mw of SAL, and (b) sulfonic group content in SAL with laccae and LXS incubation.

Table 4. Xylan Content in AL by Laccase and LXS Incubation samples

xylan content (%)

AL LAL10-24 LXAL10-20-24

3.03 ± 0.04% 2.85 ± 0.12% 1.67 ± 0.08%

depolymerization.33,34 Laccase initiated oxidation of phenolic end-groups into stabilized radicals, which subsequently underwent radical−radical couplings. The Mw increased, and the phenolic group content decreased. However, laccase could also disrupt the linkages between lignin molecules and attack the carboxyl and methoxyl groups through decarboxylation and demethylation, resulting in the decrease of Mw.14,15 In the current study, the partial linkage cleavages and demethylation made an increase in the phenolic groups; meanwhile, laccase was able to polymerize the lignin molecules through radicals’ couplings. When the phenolic group content increased, the formaldehyde reacted more easily with the lignin molecules, resulting in increscent Mw of sulfomethylated lignin. Nevertheless, there was no linear relationship between phenolic group content of AL and Mw of SLXAL. Except for the aforementioned process in laccase incubation alone, the addition of xylanase could disrupt the linkages between LCCs and degrade the xylan, causing the decrease of Mw. Therefore, the changes of Mw were not only related to the phenolic group content in AL, but also related to the degradation of xylan. For the in-depth elucidation of the influence of phenolic group content on sulfomethylation reactivity of AL, the correlation between sulfonic group content of sulfomethylated lignin and phenolic group content of AL was shown in Figure 3b. In terms of laccase incubation, the sulfonic group content and the phenolic group content presented a good linear relationship (R2 > 90%). However, the linearly dependent

3.03% to 1.67%. The degradation of xylan resulted in the cleavage of bonds between LCCs. Lignin was more accessible to laccase and the chemical reagents in subsequent sulfomethylation. Therefore, the addition of xylanase was beneficial to enhance the reaction rate. The phenolic group is an important active functional group during the chemical modification of lignin. Our previous research9 showed that the increase of phenolic group content was beneficial to the hydroxymethylation of lignin. Moreover, using microwave oxidation, ultrasound and phenolation modification could increase the phenolic group content of lignin, improving the reactivity of lignin. In order to investigate the relationship between the phenolic group content and the extent of polymerization, the correlation between Mw of sulfomethylated lignin and phenolic group content of AL was shown in Figure 3a. It was obvious that laccase incubation presented a better linear relationship (R2 > 93%) than that of LXS incubation (no linear relationship). As reported, laccase was able to promote both polymerization and 1252

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Figure 4. Model scheme of xylanase hydrolysis of the lignin−carbohydrate complexes.

coefficient was only 30% for the LXS incubation. For laccase incubation, the linearly dependent coefficient between phenolic group content and sulfonic group content was smaller than that between phenolic group content and Mw (R2 > 93%). When the phenolic group content increased, the hydroxylation was enhanced. Nevertheless, it did not mean the improvement of sulfomethylation reactivity. Not all the hydroxymethyl groups were involved in the subsequent sulfonation; some of them coupled directly. Therefore, the linearly dependent coefficient between phenolic group content and sulfonic group content was smaller. The hydroxymethylation competed with the sulfonation, which was also the reason that the lignosulfonate with high Mw and high sulfonic group content could not be easily obtained by sulfomethylation. As shown in Figure 4, the role of xylanase addition lay in the degradation of xylan residues and the cleavages of bonds between LCCs. Due to the action of xylanase, lignin was exposed to laccase, therefore, leading into the improved reaction rate. In conclusion, the sulfonation and polymerization were not only dependent on the phenolic group content, but also on the cleavages of LCCs. Dispersion Properties. As known, TiO2 is one of the most widely used mineral oxides in the industry, including paints, plastics, ceramics, papermaking, fibers, and inks.35,36 The dispersion stabilization of TiO2 is very important for these industries. However, TiO2 particles often tend to form agglomerates during the industrial processes because of their large surface area and surface properties. Therefore, the end-use properties are highly affected. In this study, the influence of laccase and LXS incubation on dispersion properties of sulfomethylated lignin on TiO2 slurry was investigated. The lower value of particle size indicates a more stable suspension and fewer agglomerates. As observed in Figure 5, the dispersion properties of SLAL10-24 and SXLAL10-20-24 were better than SAL, indicating that laccase and LXS incubation could improve the dispersion properties of sulfomethylated lignin. The particle size of TiO2 particles with addition of SXLAL10-20-24 was a little higher than that with SLAL10-24 addition. As observed in Table 1, the sulfonic group content and Mw were almost the same for these two samples. With the addition of xylanase, the linkages between LCCs were disrupted, resulting in the decrease of steric hindrance. Therefore, the dispersion properties of SLXAL10-20-24 on TiO2 slurry slightly decreased. In conclusion, the laccase and LXS incubation could improve the sulfomethylation reactivity of AL significantly. When AL was modified with 10 U/g laccase for 24 h, the sulfonation and polymerization reactivity increased by 33% and 18%, respectively. LXS incubation was beneficial to increase the laccase incubation rate of AL. The improvement of

Figure 5. Influence of SAL with laccase and LXS incubation on the particle size of TiO2 slurry.

sulfomethylation reactivity was mainly due to the increase of phenolic group and the decrease of steric hindrance. In LXS incubation, xylanase hydrolyzed xylan, enhancing reaction rate by exposing lignin for the action of laccase. After the laccase or LXS incubation, the dispersion properties of sulfomethylated lignin on TiO2 slurry were enhanced.

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ASSOCIATED CONTENT

* Supporting Information S

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acssuschemeng.5b01291. Figures demonstrating the influence of dosages of xylanase on the sulfonic group content of SAL, the relationship between content of methoxyl group and phenolic group in AL with laccase and LXS incubation, and the 1H NMR spectra of KL with laccase and LXS incubation (PDF)

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AUTHOR INFORMATION

Corresponding Author

*Phone: +86-20-87114722. Fax: +86-20-87114721. E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The authors would like to acknowledge the financial support of the National Natural Science Foundation of China (21506117, 21576106), Scientific Research Foundation of Shandong University of Science and Technology for Recruited Talents 1253

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(2015RCJJ008), and the International Science and Technology Cooperation Program of China (2013DFA41670).

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DOI: 10.1021/acssuschemeng.5b01291 ACS Sustainable Chem. Eng. 2016, 4, 1248−1254