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Wang, H., Liu, Y., Wang, Z., Yang, G., and Lucia, L. A. (2016). "Structural analysis of fast-growing aspen alkaline peroxide mechanical pulp lignin: A post-enzymatic treatment," BioRes. 11(1), 2723-2733.


An enzymatic mild acidic hydrolysis was used to separate and purify residual lignin from alkaline peroxide mechanical pulp (APMP). Using the optimum conditions for the laccase treatment (pH 4.5, temperature 50 °C, lignin consistency of 1%, a reaction time of 60 min, and a laccase dosage of 8 μ/g), oven-dried lignin was treated with laccase and in a laccase mediator system (LMS) to explore the mechanism for laccase and the LMS modification of APMP. The changes of functional groups in lignin were analyzed using nuclear magnetic resonance (31P-NMR and 13C-NMR). The molecular weight distributions of the lignin samples were confirmed by gel permeation chromatography (GPC). The 31P-NMR and 13C-NMR spectra revealed that the lignin structure changed significantly with the laccase and the LMS treatments. Meanwhile, GPC demonstrated that laccase without a mediator could lead to the polymerization of lignin, while the LMS could degrade the lignin. Hence, it was concluded that laccase is an attractive enzyme for lignin modification.

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Structural Analysis of Fast-Growing Aspen Alkaline Peroxide Mechanical Pulp Lignin: A Post-Enzymatic Treatment

Huimei Wang,a Yu Liu,a,* Zhen Wang,a GuihuaYang,a and Lucian A. Lucia b

An enzymatic mild acidic hydrolysis was used to separate and purify residual lignin from alkaline peroxide mechanical pulp (APMP). Using the optimum conditions for the laccase treatment (pH 4.5, temperature 50 °C, lignin consistency of 1%, a reaction time of 60 min, and a laccase dosage of 8 μ/g), oven-dried lignin was treated with laccase and in a laccase mediator system (LMS) to explore the mechanism for laccase and the LMS modification of APMP. The changes of functional groups in lignin were analyzed using nuclear magnetic resonance (31P-NMR and 13C-NMR). The molecular weight distributions of the lignin samples were confirmed by gel permeation chromatography (GPC). The 31P-NMR and 13C-NMR spectra revealed that the lignin structure changed significantly with the laccase and the LMS treatments. Meanwhile, GPC demonstrated that laccase without a mediator could lead to the polymerization of lignin, while the LMS could degrade the lignin. Hence, it was concluded that laccase is an attractive enzyme for lignin modification.

Keywords: Lignin; 31P-NMR; 13C-NMR; GPC; Laccase; LMS

Contact information: a: Key Laboratory of Pulp & Paper Science and Technology, Ministry of Education, Qilu University of Technology, Jinan, Shandong Province 250353; b: Department of Forest Biomaterials, North Carolina State University, Box 8005, Raleigh, NC27695-8005 USA;

* Corresponding author:


Currently, high yield pulp (HYP) is a profitable material to produce because of its diversified advantages, such as a higher yield, environmental friendliness, low production costs, higher bulk, and good opacity. However, HYP fibers display poor flexibility, low whiteness, susceptibility to photo-yellowing, thermal reversion, and low contribution to paper strength because of the high lignin content (Liu et al. 2012). Therefore, modifications to HYP are needed to improve the quality of the paper and hence broaden its application (Lachenal et al. 1995; Henriksson and Gatenhalm 2002). The common modification methods for fiber include mechanical modification, chemical modification (Gruber et al. 2002), and physical modification (Goring 1967; Carlsson and Ström 1995). With the development of biotechnology, enzymes are of great interest in studies for the modification of pulp (Sigoillot et al. 1997; Richardson et al. 1998; Henriksson and Gatenhalm 2002). Enzymes do not generate the pollution that other methods generate. In addition, they provide a low energy consumption method.

Cellulase is an example of an enzyme that has the ability to modify fibers by improving their printability and smoothness (Mansfield et al. 1997). Additional examples include hemicellulase, which is capable of reducing the beating energy and improving the quality of paper (Stork et al. 1995), peroxidase, which plays an important role in the degradation process of lignin, and laccase, which is the most promising lignin-degrading and lignin-polymerizing enzyme (Thurston 1994). This is because laccase is an environmentally friendly enzyme, which works in the presence of oxygen and produces water as its only byproduct (Kalia et al. 2014). In addition, laccase can be used for many technical processes (Call and Mücke 1997; Couto and Toca-Herrera 2006; Riva 2006; Mikolasch and Schauer 2009), such as bio-bleaching in the pulp and paper industry (Balakshin et al. 2001; Zhang et al. 2007), decreasing the refining energy (Mansfield 2002), deinking of waste paper pulp (Welt and Dinus 1998; Ibarra et al. 2012), lignin degradation (Rico et al. 2014), and dyeing in the textile industry (Galante and Formantici 2003).

However, the redox ability of laccase is unfavorable. Moreover, laccase requires a free hydroxyl group on the substrate for the reaction to occur (Euring et al. 2012). Bourbonnais and Paice (1990) were the first researchers to discover that laccase can attack a non-phenolic lignin and accelerate the scope of the reaction after adding low molecular weight redox compounds as mediators. Therefore, a laccase mediator system (LMS) plays an important role in indirectly governing and amplifying the low redox ability of laccase (Euring et al. 2012). The role of laccase in lignin modification has been explored, and the mechanism of the LMS has been established (Bourbonnais and Paice 1990).

A pulp with a higher lignin content often equates to a more efficient enzymatic process. Therefore, laccase modification with HYP is a potential avenue for improving the quality of HYP and their ultimate usage. Mansfield et al. (1997), Wong et al. (1999, 2000), and Mansfield (2002) showed that the tensile strength of paper was improved after the laccase treatment of chips processed by steam and a screw extruder. Furthermore, Yamaguchi et al. (1994) was successful in finding that after laccase treatment on thermo-mechanical pulp, the tensile strength of paper was greatly improved due to the formation of chemical bonding between pulp lignin and dehydrogenation polymer. When pretreated in a LMS, Jujop (1991) found that the breaking length and tear index of the unbleached and bleached Masson pine ground wood pulp was markedly improved.

This study aims to explore the mechanism of laccase and the LMS for the modification of alkaline peroxide mechanical pulp (APMP). Lignin, separated from the APMP, was treated with laccase and the LMS. Nuclear magnetic resonance (NMR) and gel permeation chromatography were used to analyze the structural changes of lignin and its molecular weight to provide insights into the mechanism of laccase and the LMS on modifying the APMP.



The APMP was provided by Shandong Zhongmaoshengyuan Group, China. The commercial laccase and the 1-hydroxybenzotriazole (HBT; mediator) were supplied by Novozymes, Beijing, China. Enzyme activity was measured at 2400 μ/g at a pH value of 4.5.

Lignin Isolation

The residual lignin in the APMP was isolated and purified by the enzymatic mild acidic hydrolysis method (Argyropoulos et al. 2002; Baumberger et al. 2002; Wu and Argyropoulos 2003). The isolation process has the ability to produce a higher yield, higher purity, and minimize structural changes (Keiichi et al. 2005).

First, extractives were eliminated using an acetone-based 8 h extraction process. The extractive-free pulp was washed repeatedly with deionized water and air dried. Then, the air-dried pulp was milled in a ball mill.

The enzymatic mild acidic hydrolysis was done in two separate stages. In the first stage, cellulase and hemicellulase were added to the milled pulp to remove cellulose and hemicellulose. The slurry was agitated in a Rocking Incubator (HZQ-F100), which was manufactured by Shanghai FUMA Equipment Co., Ltd for 48 h at 40 °C at 5% consistency. After the enzyme treatment, the slurry was centrifuged at 5000 rpm for 15 min in the GL-20G-II High-speed Centrifuge produced by Shanghai Anting Scientific Instrument Factory. The sediment was washed with acidified water (pH 2) prior to freeze-drying to obtain a crude lignin sample.

In the second stage, crude lignin was added to a dioxane-water solution (dioxane: water, 81:15 v/v). Under a nitrogen atmosphere, the mixture was heated and refluxed for 2 h. The resulting mixture was filtered, and the solid component was washed with dioxane-water. The filtrate was neutralized with sodium bicarbonate and concentrated under reduced pressure at 40°C.

The concentrated mixture was added to the acidified, deionized water (pH 2) in order to isolate the lignin by letting it stand for 8 h, centrifuging, and freeze-drying. Lastly, the lignin was washed with hexane to obtain a purified sample.

Lignin Modification

Modification with laccase

The isolated lignin from APMP was directly treated with laccase. The optimum conditions for laccase treatment were: pH 4.5, temperature 50 °C, consistency of lignin 1%, reaction time 1 h, and concentration 8 μ/g (referring to the oven-dried lignin) (Wang and Liu 2010).

Modification with laccase mediator system

1-hydroxy benzotriazole (HBT) was used as mediator for the LMS at a 2% concentration of oven-dried lignin (Wang and Liu 2010).

31P-NMR Spectroscopy

Approximately 40 mg of oven-dried lignin was added to 250 µL of pyridine-d5 and chloroform (1.6:1, v/v) in a small test-tube and stirred to dissolve. Then, 100 µL of N-hydroxy naphlthalimide (internal standard) and 50 µL of chromium acetylacetonate (relaxation regent) were added. Lastly, O-4, 4, 5, 5-tetramethyl-1, 3, 2-dioxaphospholane (TMDP) was added to produce phosphitylation. The sample was thoroughly mixed and analyzed using an Avance II 400 MHz spectrometer (BRUKER, Germany).


Under a nitrogen atmosphere, 15 mL of the acetylated reagents pyridine and acetic anhydride (1:2, v/v) were added to 300 mg of lignin sample in a 50 mL conical flask for 72 h. After the reaction was run to completion, the mixture was slowly poured into 200 mL of diethyl ether, which produced a white precipitate. The sample was filtered through a glass filter, and then washed with diethyl ether until no pyridine odor was detected; the product formed was acetylated lignin. Finally, the lignin samples were dried in a vacuum drying oven.

13C-NMR Spectroscopy

Approximately 200 mg of oven-dried acetylated lignin and 500 μL of dimethyl sulphoxide-d6 (DMSO-d6) were added and stirred to fully dissolve. The lignin sample was scanned for 16 h by adopting inverse gate decoupling sequence (C13IG sequence) from Bruker Standard Pulse Library.

Lignin Molecular Weight Distribution

The molecular weight distribution of the acetylated lignin samples was obtained using gel permeation chromatography (GPC). Approximately 10 mg of oven-dried acetylated lignin sample was fully dissolved in 10 mL of tetrahydrofuran (THF). The temperature was maintained at 40 °C and the flow velocity was 0.22 mL/min.


31P-NMR Spectroscopy

From the 31P-NMR spectra (Fig. 1), changes in the functional groups of lignin were detected following the treatment with laccase and the LMS.

Fig. 1. The 31P-NMR spectrum of lignin samples

As shown in Fig. 1 and Table 1, the concentration of aliphatic OH exhibited reductions of 10.1% and 12.5% after modification of laccase and LMS, respectively. Similarly, the total phenolic hydroxyl content decreased by 15.4% and 22.3%, respectively, demonstrating partial or complete removal of phenolic OH. The content of condensed phenolic OH was reduced by 19.2% and 30.8%, respectively. Similarly, after the laccase treatment, syringyl OH decreased by 12.5%. However, after the LMS, the change in syringyl OH content was not obvious. Meanwhile, guaiacyl and demethylated OH groups were reduced by 17.9% and 28.2%, respectively. Additionally, the content of p-hydroxyl-phenyl OH also decreased after the laccase and the LMS treatment by12.5% and 22.5%, respectively. Because of the laccase treatment, the total COOH content increased by 10.5%. When the mediator, HBT, was added there was no obvious variation in COOH content, which was in agreement with the result by Crestini et al. (2003).

Table 1. Functional Group Frequency and Integration Regions for Quantitative Analysis of 31P-NMR Spectra of Lignin and Relative Absorption Strength (Liu et. al. 2012)

13C-NMR Spectroscopy

13C-NMR spectra of each sample are compared in Fig. 2, in which chemical shifts in the range 102.0 to 162.0 are the absorption peaks of the benzene ring carbon.

Fig. 2. The 13C-NMR spectra of lignin samples

The integration area of these peaks can be employed as the reference standard to calculate the content of each functional group and connecting structure in lignin structure. However, this integration area is integrated area of 6 benzene rings, so the number of each functional group and connecting structure can be obtained by multiplying by 6. The specific calculation formula is shown as Eq. 1 (Chen 1998),


where Ai-j is the number of functional groups and connecting structure, of which the chemical shifts are between i to j, Ii-j are the integration areas between i to j, and I102.0-162.0 are the integration areas between 102.0 to162.0 of chemical shifts.

Table 2. Functional Group Frequency and Integration Regions for Qualitative Analysis of 13C-NMR Spectra of Lignin and Relative Absorption Strength

Figure 2 and Table 2 show that the laccase and the LMS treatments affected the structure of lignin. After the treatments with laccase and LMS, the content of C3-OH changed greatly, while the LMS-treated group had the highest content (an increase of 19.1%).

Meanwhile, compared to the control group, the C1-C content of the laccase-treated group increased by 52.7%, whereas the C1-C content of the LMS-treated group decreased by 13.7%. In addition, Cβ of β-O-4 was reduced by 19.2% after the treatment with LMS. This change was not obvious after the modification with laccase. The changes to the Cα of β-O-4 were inconspicuous; however, the overall content decreased after the treatment with LMS.

These results showed that the LMS could degrade lignin. Moreover, laccase and LMS could diminish the contents of OCH3 by 5.8% and 14.3%, respectively (Fig. 5).

Average Molecular Weight Changes of Lignin

The molecular weight distribution curves (Fig. 3) of the lignin samples were obtained by GPC. In general, GPC analysis was always done after lignin acetylation.

Fig. 3. The average molecular weight distribution curves of the three lignin samples

Table 3. Average Molecular Weight and Polydispersity of Lignin

*Values are an average of #samples for each group. Mn: number-average molecular weight, which can be averaged according to the number of molecules; Mw: weight-average molecular weight, which can be averaged according to the weight of molecules; Mz: Z- average molecular weight; Mw/Mn: polydispersity

As shown in Table 3, the average molecular weight of lignin changed after the laccase and the LMS treatments. The Mn of lignin increased for laccase and LMS by 76.7% and 26.4%, respectively. Meanwhile, during the modification reaction with laccase, the Mw of lignin increased 69.6%, while the Mw of lignin decreased after modifying with the LMS (47.2%). These changes in the Mw of lignin can possibly explain why the Cβ of β-O-4 was reduced after modifying with the LMS. This also means that the LMS treatment was beneficial for degrading β-O-4 (Kawai et al. 2002; Wei et al. 2004).

The polydispersities of lignin changed, which may have occurred because the lignin was treated directly with laccase and LMS. During the reaction with laccase, the polydispersities of lignin decreased slightly from 6.26 to 6.01. However, during the reaction with LMS, the polydispersities of lignin decreased from 6.26 to 2.61, with a reduction of 58.3%.


  1. The results of this study revealed that the laccase and LMS treatments could greatly influence the structure of isolated lignin from APMP. After the modification reaction with laccase and LMS, the phenolic OH and aliphatic OH contents decreased. This study also provided a better understanding for the mechanism of laccase and the LMS modification of APMP. The laccase and LMS treatments can improve the quality of fibers through catalytic oxidation of lignin.
  2. After the modification by the LMS, the content of the Cβ of the β-O-4 functional group decreased, which suggests that the LMS can lead to the degradation of lignin.
  3. The31P-NMR and 13C-NMR spectra analysis indicated that the influence of the LMS modification on lignin was greater than with laccase modification.
  4. The GPC analysis provided evidence that the reactions of lignin with laccase and with the LMS were different. For the laccase modification process, the Mw of lignin was greatly influenced and increased, while for the LMS, the Mw decreased dramatically. Meanwhile, after the laccase and the LMS treatments, the Mn of lignin increased.


The authors are grateful for the financial support from the National Science Foundation of China (Grant NO. 31270626) and Taishan Scholars Project Special Funds. In addition, the Life Sciences Laboratory of Shandong University is gratefully acknowledged for the generous use of its laboratory equipment.


Argyropoulos, D. S., Sun, Y., and Palus, E. (2002). “Isolation of residual kraft lignin in high yield and purity,” Journal of Pulp and Paper Science 28(2), 50-54.

Balakshin, M., Capanema, E., Chen, C. L., Gratzl, J., Kirkman, A., and Gracz, H. (2001). “Biobleaching of pulp with dioxygen in the laccase-mediator system-reaction mechanisms for degradation of residual lignin,” Journal of Molecular Catalysis B: Enzymatic 13(1), 1-16. DOI: 10.1016/s1381-1177(00)00225-3

Baumberger, S., Dole, P., and Lapierre, C. (2002). “Using transgenic poplars to elucidate the relationship between the structure and the thermal properties of lignins,” Journal of Agricultural and Food Chemistry 50(8), 2450-2453. DOI: 10.1021/jf0113530

Bourbonnais, R., and Paice, M. G. (1990). “Oxidation of non-phenolic substrates: An expanded role for laccase in lignin biodegradation,” FEBS Letters 267(1), 99-102. DOI: 10.1016/0014-5793(90)80298-w

Call, H. P., and Mücke, I. (1997). “History, overview and applications of mediated lignolytic systems, especially laccase-mediator-systems (Lignozym®-process),” Journal of Biotechnology 53(2), 163-202. DOI: 10.1016/s0168-1656(97)01683-0

Carlsson, G., and Ström, G. (1995). “Water proportion and surface composition of untreated or oxygen plasma-treated chemical pulps,” Nordic Pulp& Paper Res. J. 10(7), 17-23. DOI: 10.3183/npprj-1995-10-01-p017-023

Chen, C. L. (1998). “Characterization of milled wood lignins and dehydrogenative polymerisates from monolignols by carbon-13 NMR spectroscopy,” Lignin and Lignan Biosynthesis, ACS Symposium Ser. 697, Ch. 18, pp. 255-275.

Couto, S. R., and Toca-Herrera, J. L. (2006). “Laccases in the textile industry,” Biotechnology Molecular Biology Review 1(4), 115-120.

Euring, M., Trojanowski, J., Horstmann, M., and Kharazipour, A. (2012). “Studies of enzymatic oxidation of TMP-fibers and lignin model compounds by a laccase-mediator-system using different 14C and 13C techniques,” Wood Science and Technology 46(4), 699-708. DOI: 10.1007/s00226-011-0439-6

Galante, Y. M., and Formantici, C. (2003). “Enzyme applications in detergency and in manufacturing industries,” Current Organic Chemistry 7(13), 1399-1422. DOI: 10.2174/1385272033486468

Goring, D. A. I. (1967). “Surface modification of cellulose in a corona discharge,” Pulp & Paper Magazine Canada 68(6), 372-376. DOI: 10.1007/bf02705895

Gruber, E. (2002). “Is there a future for chemically modified pulp?” Das Paper 87(6), 73-85.

Henriksson, A., and Gatenhalm, P. (2002). “Surface properties of CTMP fibers modified with xylans,” Cellulose 9(1), 55-64. DOI: 10.1023/A: 1015826713109

Ibarra, D., Monte, M. C., Blanco, A., Martínez, A. T., and Martínez, M. J. (2012). “Enzymatic deinking of secondary fibers: Cellulases/ hemicellulases versus laccase-mediator system,” Journal of Industrial Microbiology & Biotechnology 39(1), 1-9. DOI: 10.1007/s10295-011-0991-y

Jujop, P. (1991). “Enzymes are breaking into paper,” Pulp and Paper International 33(9), 81-83.

Kalia, S., Thakur, K., Kumar, A., and Celli, A. (2014). “Laccase-assisted surface functionalization of lignocellulosics,” Journal of Molecular Catalysis B: Enzymatic 102(7), 48-58. DOI: 10.1016/j.molcatb.2014.01.014

Kawai, S., Nakagawa, M., and Ohashi, H. (2002). “Degradation mechanisms of a nonphenolic β-O-4 lignin model dimer by Trametes versicolor laccase in the presence of 1-hydroxybenzotriazole,”Enzyme and Microbial Technology 30(4), 482-489. DOI: 10.1016/s0141-0229(01)00523-3

Lachenal, D., Fernandes, J. C., and Froment, P. (1995). “Behavior of residual lignin in kraft pulp during bleaching,” Journal of Pulp and Paper Science 21(5), J173.

Liu, Y., Wang, Z., Wang, J., Yang, G., Huang, F., and Lucia, L. (2012). “Evaluation of the structural and molecular weight changes of lignin during the treatment of hardwood alkaline peroxide mechanical pulp with laccase and a laccase-mediator-system,” BioResources 7(3), 4284-4293. DOI: 10.15376/biores.7.3.4284-4293

Mansfield, S. D. (2002). “Laccase impregnation during mechanical pulp processing: Improved refining efficiency and sheet strength,” Appita Journal 55(1), 49-53.

Mansfield, S. D., Jong, E. D., Stephens, R. S., and Saddler, J. N. (1997). “Physical characterization of enzymatically modified kraft pulp fibers,” Journal of Biotechnology 57(1), 205-216. DOI: 10.1016/s0168-1656(97)00100-4

Mikolasch, A., and Schauer, F. (2009). “Fungal laccases as tools for the synthesis of new hybrid molecules and biomaterials,” Applied Microbiology and Biotechnology 82(4), 605-624. DOI: 10.1007/s00253-009-1869-z

Richardson, J. D., Wong, K. K. Y., and Clark, T. A. (1998). “Modification of mechanical pulp using carbohydrate-degrading enzymes,” Journal of Pulp and Paper Science 24(4), 125-129.

Rico, A., Rencoret, J., Del Río, J. C., Martínez, A. T., and Gutiérrez, A. (2014). “Pretreatment with laccase and a phenolic mediator degrades lignin and enhances saccharification of Eucalyptus feedstock,” Biotechnology Biofuels 7(6), 1-14. DOI: 10.1186/1754-6834-7-6

Riva, S. (2006). “Laccases: Blue enzymes for green chemistry,” Trends in Biotechnology 24(5), 219-226. DOI:10.1016/j.tibtech.2006.03.006

Sigoillot, J. C., Petit-Conil, M., Ruel, K., Moukha, S., Comtat, J., Laugero, C., and Asther, M. (1997). “Enzymatic treatment with manganese peroxidase from Phanerochaete chrysosporium for enhancing wheat straw pulp characteristics,” Holzforschung-International Journal of the Biology, Chemistry, Physics and Technology of Wood 51(6), 549-556. DOI: 10.1515/hfsg.1997.51.6.549

Stork, G., Pereira, H., Wood, T. M., Dusterhoft, E. M., Toft, A., and Puls, J. (1995). “Upgrading recycled pulps using enzymatic treatment,” TAPPI Journal 78(2), 79-88.

Thurston, C. F. (1994). “The structure and function of fungal laccases,” Microbiology 140(1), 19-26. DOI: 10.1099/13500872-140-1-19

Wang, J., and Liu, Y. (2010). “Studies on alkali peroxide mechanical pulp of triploid of Populus tomentosa by laccase treatment,” Paper & Papermaking 31(4), 51-54. DOI: 0.4028/

Wei, H. L., Shi, S. L., and Pei, J. C. (2004). “Analysis of laccase treated lignin structure by 13C-NMR,” Transactions of China Pulp and Paper 19(2), 025. DOI: 10.3321/j.issn:1000-6842.2004.02.026.

Welt, T., and Dinus, R. J. (1998). “Enzymatic deinking: Effectiveness and mechanisms,” Wochenblatt fuer Papierfabrikation 126(9), 396-407. DOI: 10.1016/s0065-2164(08)70265-x

Wong, K. K., Anderson, K. B., and Kibblewhite, R. P. (1999). “Effects of the laccase mediator system on the hand sheet properties of two high kappa kraft pulps,” Enzyme and Technology 25(1), 125-131. DOI: 10.1016/s0141-0229(99)00022-8

Wong, K. K., Richardson, J. D., and Mansfield, S. D. (2000). “Enzymatic treatment of mechanical pulp fibers for improving papermaking properties,” Biotechnology Progress 16(6), 1025-1029. DOI: 10.1021/bp000064d

Wu, S., and Argyropoulos, D. S. (2003). “An improved method for isolating lignin in high yield and purity,” Journal of Pulp and Paper Science 29(7), 235-240.

Yamaguchi, H., Maeda, Y., and Sakata, I. (1994). “Bonding among woody fibers by use of enzymatic phenol dehydrogenative polymerization: Mechanism of generation of bonding strength,” Mokuzai Gakkaishi 40(2), 185-190.

Zhang, A. P., Qin, M. H., Sun, R. C., Xu, Q. H., Fu, Y. J., and Liu, C. F. (2007). “Characterization of Masson pine stone ground wood pulp modified with laccase/mediator system,” Cell Chem Technol41(1), 63-75. DOI: 10.1016/j.biortech.2011.12.120

Article submitted: March 18, 2015; Peer review completed: June 20, 2015; Revised version received and accepted: January 11, 2016; Published: February 1, 2016.

DOI: 10.15376/biores.11.1.2723-2733