Bioresource Technology 155 (2014) 418–421
Contents lists available at ScienceDirect
Bioresource Technology journal homepage: www.elsevier.com/locate/biortech
Short Communication
Polymerization reactivity of sulfomethylated alkali lignin modified with horseradish peroxidase Dongjie Yang, Xiaolei Wu, Xueqing Qiu ⇑, Yaqi Chang, Hongming Lou State Key Laboratory of Pulp and Paper Engineering, South China University of Technology, Guangzhou, China School of Chemistry and Chemical Engineering, South China University of Technology, Guangzhou, China
h i g h l i g h t s A new technology to prepare high molecular weight soluble alkali lignin is proposed. The molecular weight of lignin increases over 20 times after HRP modification. The structure changes of lignin during HRP modification are investigated. Sulfonation and HRP modification were mutually promoted. HRP modification can improve the adsorption quantity of lignin.
a r t i c l e
i n f o
Article history: Received 11 October 2013 Received in revised form 2 December 2013 Accepted 4 December 2013 Available online 14 December 2013 Keywords: Sulfomethylated alkali lignin Horseradish peroxidase Polymerization Reactivity
a b s t r a c t Alkali lignin (AL) was employed as raw materials in the present study. Sulfomethylation was conducted to improve the solubility of AL, while sulfomethylated alkali lignin (SAL) was further polymerized by horseradish peroxidase (HRP). HRP modification caused a significant increase in molecular weight of SAL which was over 20 times. It was also found to increase the amount of sulfonic and carboxyl groups while decrease the amount of phenolic and methoxyl groups in SAL. The adsorption quantity of self-assembled SAL film was improved after HRP modification. Sulfonation and HRP modification were mutually promoted. The polymerization reactivity of SAL in HRP modification was increased with its sulfonation degree. Meanwhile, HRP modification facilitated SAL’s radical-sulfonation reaction. Ó 2014 Published by Elsevier Ltd.
1. Introduction Alkali lignin (AL), the major byproduct obtained from black liquor in kraft pulping process, accounts for more than 85% of technical lignin in the world. The utilization of AL contributes considerable economic, social and environmental benefits. However, the low reactivity of AL limits its chemical modification and further practical application, owing to the aryl ether linkages cleavage, the disappearance of the reactive functional groups and condensation of polyphenyl propene units during violent kraft pulping process (Sun et al., 2013; Ouyang et al., 2009). Sulfonation degree and molecular weight are the two main factors influencing the application performance of AL. Extensive studies have focused on improving these two main factors of AL by activation, including chemical, physical or biological modifica⇑ Corresponding author at: State Key Laboratory of Pulp and Paper Engineering, South China University of Technology, Guangzhou, China. Tel.: +86 02087114722. E-mail address: [email protected] (X. Qiu). http://dx.doi.org/10.1016/j.biortech.2013.12.017 0960-8524/Ó 2014 Published by Elsevier Ltd.
tions (Zhou et al., 2007; Yang et al., 2013). Generally, physical modification process is inefficient and chemical modification is environmentally unfriendly. Moreover, the increase of sulfonation degree and molecular weight of AL by physical or chemical modifications is limited. Horseradish peroxidase (HRP) is a kind of high catalytic activity peroxidase extracted from the root of horseradish, which is efficient to polymerize phenols, anilines and their derivatives (Kersten et al., 1990; Hong et al., 2006). HRP is also efficient in lignin modification. Grönqvist et al. (2005) discovered that HRP shows a higher catalytic activity than laccase in lignin polymerization. Blinkovsky and Dordick (1993) found that lignin and phenol could be polymerized by HRP to synthetize phenolic resins. However, the reactivity of lignin during HRP modification is barely investigated to date. The aim of this work is to modify AL by both chemical and biological treatment. AL was first sulfomethylated with sulfite to obtain sulfomethylated alkali lignin (SAL), and then further incubated with HRP to gain HRP polymerized sulfomethylated alkali
D. Yang et al. / Bioresource Technology 155 (2014) 418–421
lignin (HSAL).The polymerization reactivity and catalytic mechanism of SAL by HRP/H2O2 incubation in aqueous system were also investigated. 2. Methods 2.1. Lignin, enzyme and reagents AL was isolated from pine pulping black liquor in sulfate acid treatment. It was supplied and purified by Jilin Paper Co. Ltd. (Jilin, China). HRP was supplied by Xueman Biotech Co. Ltd. (Shanghai, China). The enzyme activity of HRP (Robert and Bardsley, 1975) was 12,708 U g1. All other chemicals were of analytical grade. 2.2. Preparation of SAL by sulfomethylation 10 g of AL was firstly dissolved in sodium hydroxide solution (pH 13) in a reaction vessel (Carousel 6, Radleys Corp., England) and the solution was heated to 70 °C. Subsequently, 0.41 g aqueous formaldehyde solution of 37% concentration was added and stirred for 1 h. Then it was heated to 95 °C, to which 0.5–6 g of sodium sulfite was added and stirred for 3 h. Finally, the product was adjusted to pH 6 and filtered by Büchner funnel. SAL was obtained by freeze-drying and SAL1–SAL6 with different sulfonic group contents were obtained by adjusting the dosage of sodium sulfite. 2.3. Preparation of HSAL by HRP modification 1 g of SAL was dissolved in 50 mL phosphate buffer (0.1 M, pH 6.0) in a reaction vessel and the incubation was started by addition of 0.88 mmol L1 H2O2 and 6 g L1 HRP. The reaction was maintained at 30 °C and lasted for 2 h. At last, HSAL was collected by freeze-drying. HSAL1–HSAL6 were prepared by HRP modification of SAL1–SAL6 respectively. 2.4. Gel permeation chromatography (GPC) The molecular weights of lignin samples were determined by aqueous GPC as described by Yang et al. (2013). 2.5. 1H NMR spectra The 1H NMR spectra of lignin samples were determined by DRX400 (400 MHz 1H frequency, Bruker Corp., Germany) with 30 mg of each sample dissolved in 0.5 mL DMSO-d6. 2.6. Functional group content measurements All samples were purified by ion-exchange before functional group content measurements. The sulfonic group content (Ouyang et al., 2009) and carboxyl group content (Zhou et al., 2012) of lignin samples were measured by automatic potentiometric titrator (905 Titrando, Metrohm Corp., Switzerland). The phenolic hydroxyl content of lignin samples was detected by FC method (de Sousa et al., 2001). The methoxyl content of lignin samples was determined by the head-space gas chromatography (HS-GC) as described by Li et al. (2012).
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3. Results and discussion 3.1. Sulfonation and polymerization reactivity of SAL during HRP modification The sulfonation reactivity of SAL during HRP modification was evaluated by the sulfonation degree of corresponding HSAL. With the increasing dosage of sodium sulfite, the sulfonation degree of HSAL increased from 1.51 to 2.87 mmol g1 (Table 1). Under the same dosage of sodium sulfite, the sulfonation degree of HSAL was much higher than that of SAL. These results indicated that the sulfonation reactivity of SAL was both improved by the increasing dosage of sodium sulfite and HRP modification. The polymerization reactivity of SAL during HRP modification was evaluated by the Mw of corresponding HSAL. After further modification by HRP, the Mw of HSAL is obviously increased, which was over 20 times (Table 1) higher than that of corresponding SAL (expect for HSAL1). Moreover, Mw of HSAL was improved with the increasing dosage of sodium sulfite. It exhibited a linear relationship (y = 33272x – 31395; R2 = 0.913) between sulfonic group contents and Mw of HSAL. Yang’s et al. study Yang et al. (2013) showed that laccase modification was able to increase the sulfomethylation reactivity of AL by 35% and promoted its polymerization, but the change of Mw is limited (from 2900 to 3500 Da at maximum). Compared with laccase, HRP modification is more efficient in improving the Mw and sulfonation reactivity of lignin as outlined above. For example, the increase of the Mw and the sulfonation degree of SAL6 were 26.95 times and 39%, respectively. These results were similar to the results reported by Grönqvist et al. (2005).
3.2. 1H NMR spectra analyses The 1H NMR spectra of lignin samples were recorded and the proton signal intensities of lignin samples (Supplementation data Table 2) were benchmarked through the DMSO proton intensity (taken as 1.00). The proton signal intensity of H, G, S unit (Fan et al., 2008; Jahan et al., 2007) all decreased obviously after HRP modification compared with those of SAL, and it was noteworthy that the signal of G unit decreased from about 1.5–0.8 (Supplementation data Table 2). The drop of H, G and S protons may be due to the destruction of phenyl structure during HRP modification. Compared with corresponding SAL, all the proton signal intensities of HSAL of b-O-40 , b-10 , b-50 and b–b0 structures (Jahan et al., 2007; Yang et al., 2013) increased after HRP modification and increased with the increase of sodium sulfite dosage, especially for those of b-O-40 and b–b0 structures (Supplementation data Table 2). This suggested that SAL formed different inter-unit linkages during HRP modification, most of which was b-O-4 and b–b types. The proton signal intensities of methoxyl groups (Jahan et al., 2007; Yang et al., 2013) were both weakened after sulfomethylation and HRP modification and they also decreased with the increasing sodium sulfite dosage (Supplementation data Table 2). Moreover, the proton signal intensity of hydrocarbon (Yang et al., 2013; Xu et al., 2006) increased obviously after HRP modification owing to the enhancement of shielding effect caused by the increase of Mw.
2.7. Self-assembled film preparation and characterization 3.3. The functional group contents of SAL and HSAL The layer-by-layer (LBL) self-assembled films were prepared according to the method of Deng et al. (2010). The adsorption quantity of the assembled film was detected by UV–Vis spectra.
As shown in Fig. 1a, with the increasing sulfonation degree of SAL samples, the carboxyl group contents of SAL increased while
420
D. Yang et al. / Bioresource Technology 155 (2014) 418–421
Table 1 The effect of dosage of Na2SO3 on Mw and sulfonation degree of SAL and HSAL. Dosage of Na2SO3 (g/100 g AL)
5 20 30 40 50 60
SAL
HSAL
Sample
Sulfonation degree (mmol g
SAL1 SAL2 SAL3 SAL4 SAL5 SAL6
1.15 1.34 1.51 1.67 1.98 2.06
1
)
Mw (Da)
Mw Mn
1100 1700 1800 1800 2000 2100
3.66 6.06 4.88 5.91 5.85 6.47
1
Sample
Sulfonation degree (mmol g1)
Mw (Da)
Mw Mn1
HSAL1 HSAL2 HSAL3 HSAL4 HSAL5 HSAL6
1.51 1.96 2.16 2.46 2.60 2.87
14,600 39,100 44,500 46,700 51,800 56,600
4.61 7.39 7.52 7.63 8.26 8.47
SAL, sulfomethylated alkali lignin. HSAL, HRP polymerized sulfomethylated alkali lignin. Mw, weight-average molecular weight. Mn, number-average molecular weight.
the methoxyl group contents decreased. In contrast, the change of phenolic hydroxyl group contents was negligible. Fig. 1b showed that all of the functional group contents of HSAL samples showed a linear correlation with their Mw values. With the increasing Mw, the sulfonic group and carboxyl group contents of HSAL increased markedly while the methoxyl group contents decreased rapidly. The linear regression equation between Mw and sulfonic group contents, carboxyl group contents and methoxyl group contents of HSAL were as following: y = 0.091x + 5.239 105 (expect for HSAL1, R2 = 0.9537), y = 6.592 105x 0.237 (expect for HSAL1, R2 = 0.7674) and y = 1.648 105x + 2.376 (R2 = 0.9061). However, with the increasing Mw, the phenolic hydroxyl group contents of HSAL nearly unchanged, which was about 2.15 mmol g1. Comparing the functional group contents of HSAL with those of SAL under the same dosage of sodium sulfite, the carboxyl group contents of HSAL significantly increased by 36–115% and the
2.5
phenolic hydroxyl group and methoxyl group contents of HSAL decreased by 50% and 40% respectively. These results can be explained as follows. HRP initiated the reaction by oxidizing phenol hydroxyl groups into phenoxy radicals (Gong et al., 2012), which were easily delocalized and activated the C5, C3 and b positions of SAL. By radical–radical coupling, the lignin polymer chains propagated and formed linkages like b-O-40 , b-b0 , b-10 , b-50 , etc. (Brunow et al., 1979; Setälä et al., 1999; Yue, 2012). Moreover, the activation also caused the previously formed free radicals sulfonation of SAL with the rest sodium sulfite and caused the further increase of the sulfonation degree of HSAL. HRP also oxidized hydroxyl and aldehyde groups of SAL into carboxyl groups and oxidized methoxy benzene structures into benzoquinones (Kersten et al., 1990). To sum up, sulfonation and HRP modification were mutually promoted. On one hand, the increase of the sulfonation degree was conducive to increase the water-solubility of SAL, which made it easier to combine with the active center of HRP and produced more phenoxy radicals. Moreover, sulfomethylation decreased the methoxyl group content of SAL, which was helpful to improve its polymerization reactivity. On the other hand, radicalization during HRP modification caused activation of ortho, para and Cb positions of SAL, which improve the radical-sulfonation reactivity of SAL.
2.0
3.4. Adsorption characteristics
SAL1
SAL2
SAL3 SAL4
SAL5 SAL6
-1
Functional group contents (mmol g )
(a) 3.5 3.0
1.5
phenolic hydroxyl group methoxyl group carboxyl group
1.0 1.2
1.4
1.6
1.8
2.0
2.2 -1
Functional group contents (mmol/g)
3.5 3.0
H SA L2 H SA L H 3 SA L4 H SA L5 H SA L6
(b)
H SA L1
Sulfonation degree of SAL samples (mmol g )
phenolic hydroxyl group methoxyl group sulfonic group carboxyl group
4. Conclusion HRP modification of SAL caused a markedly increase of its molecular weight which was over 20 times. HRP modification was also found to increase the amount of sulfonic and carboxyl groups while decrease the amount of phenolic and methoxyl groups in SAL. Sulfonation and HRP modification were mutually promoted. Moreover, a significant improvement in SAL’s adsorption quantity after HRP modification was also detected.
2.5 2.0 1.5 1.0 20000
30000
40000
The absorbance of self-assembled films at 280 nm was applied to evaluating the adsorption characteristics of lignin samples. The absorbance of SAL6/PDAC or HSAL6/PDAC assembled films had a significant linear correlation with the number of layers and the absorbance of HSAL6 layer was obviously higher than that of SAL6 (Supplementation data Fig. 2). Namely, HRP modification could effectively improve the adsorption performance of SAL.
50000
60000
Mw of HSAL (Da) Fig. 1. Functional group contents of SAL and HSAL under different dosage of sodium sulfite (a) SAL, (b) HSAL.
Acknowledgements The authors would like to acknowledge the financial support of the National Science Foundation for Distinguished Young Scholars of China (20925622), the National Natural Science Foundation of
D. Yang et al. / Bioresource Technology 155 (2014) 418–421
China (21176096, 21376100), International S&T Cooperation Program of China (2013DFA41670), the National Basic Research Program of China 973 (2012CB215302), the Fundamental Research Funds for the Central Universities. 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.2013.12. 017. References Brunow, G., Rönnberg, M., Teien, G., Pakkanen, T., Servin, R., Sternerup, H., Wistrand, L., Nørskov, L., Schroll, G., 1979. The peroxidatic oxidation of some phenolic lignin model compounds. Acta Chim. Sinica B 33, 22–26. Blinkovsky, A.M., Dordick, J.S., 1993. Peroxidase-catalyzed synthesis of lignin– phenol copolymers. J. Polym. Sci., Part A Polym. Chem. 31 (7), 1839–1846. de Sousa, F., Reimann, A., Björklund, J. M., Nilberbrant, N. O., 2001. Estimating the amount of phenolic hydroxyl groups in lignins. In: 11th International Symposium on Wood and Pulping Chemistry. Nice, France, 11–14 June 2001, Vol III, 649–653. Deng, Y.H., Wu, Y., Qian, Y., Ouyang, X.P., Yang, D.J., Qiu, X.Q., 2010. Adsorption and desorption behaviors of lignosulfonate during the self-assembly of multilayers. BioResources 5 (2), 1178–1196. Fan, Y.M., Xu, A.Q., Shen, Y.N., Li, M.F., Xie, Y.M., Zhang, Z.Y., 2008. Alcoholysis of lignin with ethylene glycol. Chem. Ind. Forest Prod. 28 (2), 105–109. Grönqvist, S., Viikari, L., Niku-Paavola, M.L., Orlandi, M., Canevali, C., Buchert, J., 2005. Oxidation of milled wood lignin with laccase, tyrosinase and horseradish peroxidase. Appl. Microbiol. Biotechnol. 67, 489–494. Gong, R., Lv, Sh.H., Hou, M.M., 2012. Enzymatic synthesis and characterization of copolymer of resorcinol and 3,5-dihydroxy benzoic acid. Chem. Ind. Eng. Prog. 31 (2), 412–416.
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Hong, F., Jönsson, L.J., Lundquist, K., Wei, Y.J., 2006. Oxidation capacity of laccases and peroxidases as reflected in experiments with methoxy-substituted benzyl alcohols. Appl. Biochem. Biotechnol. 129, 303–319. Jahan, M.S., Chowdhury, D.A.N., Islam, M.K., Moeiz, S.M.I., 2007. Characterization of lignin isolated from some nonwood available in Bangladesh. Bioresour. Technol. 98, 465–469. Kersten, P.J., Kalyanaraman, B., Hammel, K.E., Reinhammar, B., Kirk, T.K., 1990. Comparison of lignin peroxidase, horseradish peroxidase and laccase in the oxidation of methoxybenzenes. Biochem. J. 268, 475–480. Li, H.L., Chai, X.S., Liu, M.R., Deng, Y.H., 2012. Novel method for the determination of the methoxyl content in lignin by headspace gas chromatography. J. Agric. Food Chem. 60, 5307–5310. Ouyang, X.P., Ke, L.X., Qiu, X.Q., Guo, Y.X., Pang, Y.X., 2009. Sulfonation of alkali lignin and its potential use in dispersant for cement. J. Dispersion Sci. Technol. 30, 1–6. Robert, E.C., Bardsley, W.G., 1975. The steady-state kinetics of peroxidase with 2,20 azino-di-(3-ethylbenzthiazoline-6-sulphonicacid) as chromogen. Biochem. J. 145, 93–103. Setälä, H., Pajunen, A., Rummakko, P., Sipilä, J., Brunow, G., 1999. A novel type of spiro compound formed by oxidative cross coupling of methyl sinapate with a syringyl lignin model compound. A model system for the b-1 pathway in lignin biosynthesis. J. Chem. Soc. Perkin Trans. 1 (4), 461–464. Sun, Y., Qiu, X.Q., Liu, Y.Q., 2013. Chemical reactivity of alkali lignin modified with laccase. Biomass Bioenergy 55, 198–204. Xu, F., Sun, J.X., Sun, R.C., Fowler, P., Baird, M.S., 2006. Comparative study of organosolv lignins from wheat straw. Ind. Crops Prod. 23, 180–193. Yue, F. X., 2012. Syntheses of new lignin-related model compounds and their uses for structural characterization of lignin. A Dissertation submitted for the Degree of Doctor of Philosphy. South China University of Technology, China. Yang, D.J., Zhou, H.F., Xie, S.Q., Wu, X.L., Qiu, X.Q., 2013. Sulfomethylation reactivity of alkali lignin with laccase modification. Acta Chim. Sinica. 2, 232–240. Zhou, M.S., Qiu, X.Q., Yang, D.J, Lou, H.M., Ouyang, X.P., 2007. High-performance dispersant of coal–water slurry synthesized from wheat straw alkali lignin. Fuel Process. Technol. 88, 375–382. Zhou, M.S., Huang, K., Qiu, X.Q., Yang, D.J., 2012. Content determination of phenolic hydroxyl and carboxyl in lignin by aqueous phase potentiometric titration. CIESC J. 63 (1), 258–265.
Contents lists available at ScienceDirect
Bioresource Technology journal homepage: www.elsevier.com/locate/biortech
Short Communication
Polymerization reactivity of sulfomethylated alkali lignin modified with horseradish peroxidase Dongjie Yang, Xiaolei Wu, Xueqing Qiu ⇑, Yaqi Chang, Hongming Lou State Key Laboratory of Pulp and Paper Engineering, South China University of Technology, Guangzhou, China School of Chemistry and Chemical Engineering, South China University of Technology, Guangzhou, China
h i g h l i g h t s A new technology to prepare high molecular weight soluble alkali lignin is proposed. The molecular weight of lignin increases over 20 times after HRP modification. The structure changes of lignin during HRP modification are investigated. Sulfonation and HRP modification were mutually promoted. HRP modification can improve the adsorption quantity of lignin.
a r t i c l e
i n f o
Article history: Received 11 October 2013 Received in revised form 2 December 2013 Accepted 4 December 2013 Available online 14 December 2013 Keywords: Sulfomethylated alkali lignin Horseradish peroxidase Polymerization Reactivity
a b s t r a c t Alkali lignin (AL) was employed as raw materials in the present study. Sulfomethylation was conducted to improve the solubility of AL, while sulfomethylated alkali lignin (SAL) was further polymerized by horseradish peroxidase (HRP). HRP modification caused a significant increase in molecular weight of SAL which was over 20 times. It was also found to increase the amount of sulfonic and carboxyl groups while decrease the amount of phenolic and methoxyl groups in SAL. The adsorption quantity of self-assembled SAL film was improved after HRP modification. Sulfonation and HRP modification were mutually promoted. The polymerization reactivity of SAL in HRP modification was increased with its sulfonation degree. Meanwhile, HRP modification facilitated SAL’s radical-sulfonation reaction. Ó 2014 Published by Elsevier Ltd.
1. Introduction Alkali lignin (AL), the major byproduct obtained from black liquor in kraft pulping process, accounts for more than 85% of technical lignin in the world. The utilization of AL contributes considerable economic, social and environmental benefits. However, the low reactivity of AL limits its chemical modification and further practical application, owing to the aryl ether linkages cleavage, the disappearance of the reactive functional groups and condensation of polyphenyl propene units during violent kraft pulping process (Sun et al., 2013; Ouyang et al., 2009). Sulfonation degree and molecular weight are the two main factors influencing the application performance of AL. Extensive studies have focused on improving these two main factors of AL by activation, including chemical, physical or biological modifica⇑ Corresponding author at: State Key Laboratory of Pulp and Paper Engineering, South China University of Technology, Guangzhou, China. Tel.: +86 02087114722. E-mail address: [email protected] (X. Qiu). http://dx.doi.org/10.1016/j.biortech.2013.12.017 0960-8524/Ó 2014 Published by Elsevier Ltd.
tions (Zhou et al., 2007; Yang et al., 2013). Generally, physical modification process is inefficient and chemical modification is environmentally unfriendly. Moreover, the increase of sulfonation degree and molecular weight of AL by physical or chemical modifications is limited. Horseradish peroxidase (HRP) is a kind of high catalytic activity peroxidase extracted from the root of horseradish, which is efficient to polymerize phenols, anilines and their derivatives (Kersten et al., 1990; Hong et al., 2006). HRP is also efficient in lignin modification. Grönqvist et al. (2005) discovered that HRP shows a higher catalytic activity than laccase in lignin polymerization. Blinkovsky and Dordick (1993) found that lignin and phenol could be polymerized by HRP to synthetize phenolic resins. However, the reactivity of lignin during HRP modification is barely investigated to date. The aim of this work is to modify AL by both chemical and biological treatment. AL was first sulfomethylated with sulfite to obtain sulfomethylated alkali lignin (SAL), and then further incubated with HRP to gain HRP polymerized sulfomethylated alkali
D. Yang et al. / Bioresource Technology 155 (2014) 418–421
lignin (HSAL).The polymerization reactivity and catalytic mechanism of SAL by HRP/H2O2 incubation in aqueous system were also investigated. 2. Methods 2.1. Lignin, enzyme and reagents AL was isolated from pine pulping black liquor in sulfate acid treatment. It was supplied and purified by Jilin Paper Co. Ltd. (Jilin, China). HRP was supplied by Xueman Biotech Co. Ltd. (Shanghai, China). The enzyme activity of HRP (Robert and Bardsley, 1975) was 12,708 U g1. All other chemicals were of analytical grade. 2.2. Preparation of SAL by sulfomethylation 10 g of AL was firstly dissolved in sodium hydroxide solution (pH 13) in a reaction vessel (Carousel 6, Radleys Corp., England) and the solution was heated to 70 °C. Subsequently, 0.41 g aqueous formaldehyde solution of 37% concentration was added and stirred for 1 h. Then it was heated to 95 °C, to which 0.5–6 g of sodium sulfite was added and stirred for 3 h. Finally, the product was adjusted to pH 6 and filtered by Büchner funnel. SAL was obtained by freeze-drying and SAL1–SAL6 with different sulfonic group contents were obtained by adjusting the dosage of sodium sulfite. 2.3. Preparation of HSAL by HRP modification 1 g of SAL was dissolved in 50 mL phosphate buffer (0.1 M, pH 6.0) in a reaction vessel and the incubation was started by addition of 0.88 mmol L1 H2O2 and 6 g L1 HRP. The reaction was maintained at 30 °C and lasted for 2 h. At last, HSAL was collected by freeze-drying. HSAL1–HSAL6 were prepared by HRP modification of SAL1–SAL6 respectively. 2.4. Gel permeation chromatography (GPC) The molecular weights of lignin samples were determined by aqueous GPC as described by Yang et al. (2013). 2.5. 1H NMR spectra The 1H NMR spectra of lignin samples were determined by DRX400 (400 MHz 1H frequency, Bruker Corp., Germany) with 30 mg of each sample dissolved in 0.5 mL DMSO-d6. 2.6. Functional group content measurements All samples were purified by ion-exchange before functional group content measurements. The sulfonic group content (Ouyang et al., 2009) and carboxyl group content (Zhou et al., 2012) of lignin samples were measured by automatic potentiometric titrator (905 Titrando, Metrohm Corp., Switzerland). The phenolic hydroxyl content of lignin samples was detected by FC method (de Sousa et al., 2001). The methoxyl content of lignin samples was determined by the head-space gas chromatography (HS-GC) as described by Li et al. (2012).
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3. Results and discussion 3.1. Sulfonation and polymerization reactivity of SAL during HRP modification The sulfonation reactivity of SAL during HRP modification was evaluated by the sulfonation degree of corresponding HSAL. With the increasing dosage of sodium sulfite, the sulfonation degree of HSAL increased from 1.51 to 2.87 mmol g1 (Table 1). Under the same dosage of sodium sulfite, the sulfonation degree of HSAL was much higher than that of SAL. These results indicated that the sulfonation reactivity of SAL was both improved by the increasing dosage of sodium sulfite and HRP modification. The polymerization reactivity of SAL during HRP modification was evaluated by the Mw of corresponding HSAL. After further modification by HRP, the Mw of HSAL is obviously increased, which was over 20 times (Table 1) higher than that of corresponding SAL (expect for HSAL1). Moreover, Mw of HSAL was improved with the increasing dosage of sodium sulfite. It exhibited a linear relationship (y = 33272x – 31395; R2 = 0.913) between sulfonic group contents and Mw of HSAL. Yang’s et al. study Yang et al. (2013) showed that laccase modification was able to increase the sulfomethylation reactivity of AL by 35% and promoted its polymerization, but the change of Mw is limited (from 2900 to 3500 Da at maximum). Compared with laccase, HRP modification is more efficient in improving the Mw and sulfonation reactivity of lignin as outlined above. For example, the increase of the Mw and the sulfonation degree of SAL6 were 26.95 times and 39%, respectively. These results were similar to the results reported by Grönqvist et al. (2005).
3.2. 1H NMR spectra analyses The 1H NMR spectra of lignin samples were recorded and the proton signal intensities of lignin samples (Supplementation data Table 2) were benchmarked through the DMSO proton intensity (taken as 1.00). The proton signal intensity of H, G, S unit (Fan et al., 2008; Jahan et al., 2007) all decreased obviously after HRP modification compared with those of SAL, and it was noteworthy that the signal of G unit decreased from about 1.5–0.8 (Supplementation data Table 2). The drop of H, G and S protons may be due to the destruction of phenyl structure during HRP modification. Compared with corresponding SAL, all the proton signal intensities of HSAL of b-O-40 , b-10 , b-50 and b–b0 structures (Jahan et al., 2007; Yang et al., 2013) increased after HRP modification and increased with the increase of sodium sulfite dosage, especially for those of b-O-40 and b–b0 structures (Supplementation data Table 2). This suggested that SAL formed different inter-unit linkages during HRP modification, most of which was b-O-4 and b–b types. The proton signal intensities of methoxyl groups (Jahan et al., 2007; Yang et al., 2013) were both weakened after sulfomethylation and HRP modification and they also decreased with the increasing sodium sulfite dosage (Supplementation data Table 2). Moreover, the proton signal intensity of hydrocarbon (Yang et al., 2013; Xu et al., 2006) increased obviously after HRP modification owing to the enhancement of shielding effect caused by the increase of Mw.
2.7. Self-assembled film preparation and characterization 3.3. The functional group contents of SAL and HSAL The layer-by-layer (LBL) self-assembled films were prepared according to the method of Deng et al. (2010). The adsorption quantity of the assembled film was detected by UV–Vis spectra.
As shown in Fig. 1a, with the increasing sulfonation degree of SAL samples, the carboxyl group contents of SAL increased while
420
D. Yang et al. / Bioresource Technology 155 (2014) 418–421
Table 1 The effect of dosage of Na2SO3 on Mw and sulfonation degree of SAL and HSAL. Dosage of Na2SO3 (g/100 g AL)
5 20 30 40 50 60
SAL
HSAL
Sample
Sulfonation degree (mmol g
SAL1 SAL2 SAL3 SAL4 SAL5 SAL6
1.15 1.34 1.51 1.67 1.98 2.06
1
)
Mw (Da)
Mw Mn
1100 1700 1800 1800 2000 2100
3.66 6.06 4.88 5.91 5.85 6.47
1
Sample
Sulfonation degree (mmol g1)
Mw (Da)
Mw Mn1
HSAL1 HSAL2 HSAL3 HSAL4 HSAL5 HSAL6
1.51 1.96 2.16 2.46 2.60 2.87
14,600 39,100 44,500 46,700 51,800 56,600
4.61 7.39 7.52 7.63 8.26 8.47
SAL, sulfomethylated alkali lignin. HSAL, HRP polymerized sulfomethylated alkali lignin. Mw, weight-average molecular weight. Mn, number-average molecular weight.
the methoxyl group contents decreased. In contrast, the change of phenolic hydroxyl group contents was negligible. Fig. 1b showed that all of the functional group contents of HSAL samples showed a linear correlation with their Mw values. With the increasing Mw, the sulfonic group and carboxyl group contents of HSAL increased markedly while the methoxyl group contents decreased rapidly. The linear regression equation between Mw and sulfonic group contents, carboxyl group contents and methoxyl group contents of HSAL were as following: y = 0.091x + 5.239 105 (expect for HSAL1, R2 = 0.9537), y = 6.592 105x 0.237 (expect for HSAL1, R2 = 0.7674) and y = 1.648 105x + 2.376 (R2 = 0.9061). However, with the increasing Mw, the phenolic hydroxyl group contents of HSAL nearly unchanged, which was about 2.15 mmol g1. Comparing the functional group contents of HSAL with those of SAL under the same dosage of sodium sulfite, the carboxyl group contents of HSAL significantly increased by 36–115% and the
2.5
phenolic hydroxyl group and methoxyl group contents of HSAL decreased by 50% and 40% respectively. These results can be explained as follows. HRP initiated the reaction by oxidizing phenol hydroxyl groups into phenoxy radicals (Gong et al., 2012), which were easily delocalized and activated the C5, C3 and b positions of SAL. By radical–radical coupling, the lignin polymer chains propagated and formed linkages like b-O-40 , b-b0 , b-10 , b-50 , etc. (Brunow et al., 1979; Setälä et al., 1999; Yue, 2012). Moreover, the activation also caused the previously formed free radicals sulfonation of SAL with the rest sodium sulfite and caused the further increase of the sulfonation degree of HSAL. HRP also oxidized hydroxyl and aldehyde groups of SAL into carboxyl groups and oxidized methoxy benzene structures into benzoquinones (Kersten et al., 1990). To sum up, sulfonation and HRP modification were mutually promoted. On one hand, the increase of the sulfonation degree was conducive to increase the water-solubility of SAL, which made it easier to combine with the active center of HRP and produced more phenoxy radicals. Moreover, sulfomethylation decreased the methoxyl group content of SAL, which was helpful to improve its polymerization reactivity. On the other hand, radicalization during HRP modification caused activation of ortho, para and Cb positions of SAL, which improve the radical-sulfonation reactivity of SAL.
2.0
3.4. Adsorption characteristics
SAL1
SAL2
SAL3 SAL4
SAL5 SAL6
-1
Functional group contents (mmol g )
(a) 3.5 3.0
1.5
phenolic hydroxyl group methoxyl group carboxyl group
1.0 1.2
1.4
1.6
1.8
2.0
2.2 -1
Functional group contents (mmol/g)
3.5 3.0
H SA L2 H SA L H 3 SA L4 H SA L5 H SA L6
(b)
H SA L1
Sulfonation degree of SAL samples (mmol g )
phenolic hydroxyl group methoxyl group sulfonic group carboxyl group
4. Conclusion HRP modification of SAL caused a markedly increase of its molecular weight which was over 20 times. HRP modification was also found to increase the amount of sulfonic and carboxyl groups while decrease the amount of phenolic and methoxyl groups in SAL. Sulfonation and HRP modification were mutually promoted. Moreover, a significant improvement in SAL’s adsorption quantity after HRP modification was also detected.
2.5 2.0 1.5 1.0 20000
30000
40000
The absorbance of self-assembled films at 280 nm was applied to evaluating the adsorption characteristics of lignin samples. The absorbance of SAL6/PDAC or HSAL6/PDAC assembled films had a significant linear correlation with the number of layers and the absorbance of HSAL6 layer was obviously higher than that of SAL6 (Supplementation data Fig. 2). Namely, HRP modification could effectively improve the adsorption performance of SAL.
50000
60000
Mw of HSAL (Da) Fig. 1. Functional group contents of SAL and HSAL under different dosage of sodium sulfite (a) SAL, (b) HSAL.
Acknowledgements The authors would like to acknowledge the financial support of the National Science Foundation for Distinguished Young Scholars of China (20925622), the National Natural Science Foundation of
D. Yang et al. / Bioresource Technology 155 (2014) 418–421
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