Structural Changes in Lignin During the Deinking of Old Newsprint With

http://www.paper.edu.cn

Enzyme and Microbial Technology 39 (2006) 969–975

Structural changes in lignin during the deinking of old newsprint with laccase–violuric acid system Qinghua Xu a,b, Menghua Qin a,∗, Shulan Shi c, Liqiang Jin a, Yingjuan Fu a a Shandong Key Laboratory of Pulp and Paper Engineering, Shangdong Institute of Light Industry, Jinan 250100, China b State Key Laboratory of Pulp and Paper Engineering, South China University of Technology, Guangzhou 510640, China c Tianjin University of Science and Technology, Tianjin 300222, China Received 24 June 2005; received in revised form 11 January 2006; accepted 11 January 2006

Abstract Old newsprint (ONP) could be deinked with laccase–violuric acid system (LVS). To study the mechanism of LVS deinking, lignins from the control and LVS-deinked pulp were isolated and characterized by chemical, spectroscopic and chromatographic methods. The molecular weight change of lignin was tracked by gel permeation chromatogram (GPC), and structural changes in lignin extracted from the control and LVS- deinked pulps were characterized by elemental analysis, functional groups measurement, FT-IR, 1H NMR and 1H–13C correlation 2D NMR. The experimental results showed that the molecular weight of lignin from LVS-deinked pulp was lower than that of lignin from the control pulp. The contents of free phenolic hydroxyl groups and methoxyl groups of lignin from LVS-deinked pulp decreased, while the contents of carboxyl and carbonyl groups increased. Guaiacyl units in lignin were oxidized by LVS. Stilbene and 5–5 substructures in lignin were stable to LVS treatment, while ␤-O-4 and ␤–␤ substructures were partially destroyed during the LVS deinking. According to structural changes in lignin, the mechanism of LVS deinking was deduced. © 2006 Elsevier Inc. All rights reserved.

Keywords: Old newsprint; Laccase–violuric acid system; Deinking; Lignin; Structural changes

1. Introduction Ligninases, which can catalyze the removal of surface lignin, are promising for the deinking of ONP that contains lignin- In the traditional deinking process, large quantities of chem- rich mechanical pulp [5]. Ligninases include mainly oxidative ical products are used, which make the method environmentally enzymes such as lignin peroxidase, manganese peroxidase, and damaging. In order to overcome the disadvantages of chemical laccase. Thereinto laccase (EC1.10.3.2) is a kind of polyphenol deinking, enzymatic deinking has been attracted a great deal of oxidase. When laccase was used in the presence of chemical attention because it can detach ink particles from fibers without mediator and oxygen, it could delignify pulps [6,7]. Call and additional discharge of pollutants [1]. Strittmatter [8] reported that comparing with chemical deink- During the process of deinking, enzymes can attack either ink ing, deinking of old newsprint with laccase could increase the or fiber surfaces [2]. In generally, the vegetable-oil based inks brightness and bleachability of the deinked pulps. can be directly degraded by lipase and esterase, while the fiber In our previous work, the deinking of ONP with LVS was surfaces or bonds in the vicinity of ink particles can be altered by explored [9]. The effective residual ink concentration (ERIC, cellulases, hemicellulases, pectinases and ligninolytic enzymes, 700 nm) value was lower in the LVS-deinked pulp than in thereby freeing ink particles for removal by washing or flota- the control pulp, which indicated that more ink particles were tion [2]. Although deinking with cellulase and hemicellulase detached from fibers during the LVS deinking. The breaking has been industrialized [3,4], it is desirable to search for more length and tearing index of the LVS-deinked pulp were 20% effective enzymes for deinking. and 13% higher than those of the control pulp, respectively. The brightness of the LVS-deinked pulp was 4.2% ISO higher than that of the control pulp after they were both bleached with ∗ Corresponding author. Fax: +86 531 88619806. H2O2. All these results suggested that ONP could be effectively E-mail address: [email protected] (M. Qin). deinked by LVS.

970 Q. Xu et al. / Enzyme and Microbial Technology 39 (2006) 969–975

Deinking of ONP with LVS would be improved if the fun- 2.2.5. Elemental analysis damental chemical reactions contributing to this process were Elemental analysis for carbon, hydrogen and nitrogen of lignins were con- understood. In this study, lignins extracted from the control ducted on a VARIO E1 III elemental analyzer. and LVS-deinked pulps were characterized by GPC, elemental analysis, functional groups determination, FT-IR, 1H NMR, 2.2.6. Functional groups determination 1 13 Methoxyl groups were determined according to the literature [13]. Phenolic and H– C correlation 2D NMR (HMQC) in order to obtain a hydroxyl groups were determined by ultraviolet spectrum [14]. Carboxyl groups better understanding of structural changes in lignin during the were determined by non-aqueous conductometric titration [15]. Carbonyl groups deinking of ONP with LVS, from which the mechanism of LVS were determined by reduction difference ultraviolet spectrum [16]. deinking was deduced. 2.2.7. Spectroscopy 2. Materials and methods Fourier transform infrared (FT-IR) spectra were recorded in a Nicolet Nexus 470 FT-IR spectrometer using KBr pellets. The resolution is 4 cm−1, and the number of scans is 32. 2.1. Materials 1 For H NMR, acetylated lignins were dissolved in CDCl3, and the spectra were recorded in a Bruker Avance 600 spectrometer. The conditions included a Laccase NS51003 with an activity of 1000 LAMU/g was provided by TD of 32k, a 2 s delay, and a NS of 128. Novozymes. 1H–13C correlation 2D NMR (HMQC) spectra were recorded in a Bruker Violuric acid (Fluka, analytical reagents) was used as a mediator. Avance 600 spectrometer. One hundred milligrams of lignin was dissolved in 0.5 ml of DMSO-d6. The solvent signal was used as a standard. The spectra 2.2. Methods were recorded with a standard program at 300 K. In order to detect the minor structures in lignin, 12485 scans were accumulated. 2.2.1. Pulping and flotation Pulping was carried out in a Formax Micro-Maelstrom Laboratory Pulper 3. Results and discussion (H-272M) at 55–60 ◦C. The pulping conditions were: 10 LAMU/g of laccase, 0.5% of violuric acid, time of pulping 30 min, keeping the pulp at the optimum temperature for 15 min. The control experiment was done in the same manner 3.1. Molecular weights as the LVS deinking, except that 10 LAMU/g heat-inactivated laccase was used. Flotation was carried out in a Formax Deink Cell (L-100) at 1% consistency The weight-average molecular weight (M¯ w), the number- for 15 min. average molecular weight (M¯ n) and the polydispersity (M¯ w/M¯ n) of lignins were summarized in Table 1. 2.2.2. Preparation of dioxane lignins As can be seen from Table 1, comparing with those of Lc, M¯ w, From the control and LVS-deinked pulps, the corresponding residual lignins M¯ n and polydispersity of Ld decreased, which indicated that the Lc and Ld were isolated according to the procedure described by Evtuguin et al. [10]. Air-dried pulp was soxhlet extracted with ethanol/toluene (1:1 v/v) lignin was partially degraded during the enzymatic treatment. for 24 h. Then an alkaline extraction of extractive-free pulp was performed in nitrogen atmosphere with 0.3% (0.075 mol/l) NaOH solution during 1 h under 3.2. Elemental analysis and methoxyl groups content reflux (liquid-to-pulp ratio is 50:1). Extracted pulp was thoroughly washed with hot distilled water until the filtrate was neutral, then dried at room temperature Table 2 listed the elemental composition and methoxyl con- under P2O5. Lignin was isolated by acidolysis from the alkaline pre-extracted pulp in tents of Lc and Ld, together with the approximate C9 formula a nitrogen atmosphere by the dioxane method described as follows. 500 ml of calculated therefrom. The lignins obtained were of considerably dioxane/water (9:1 v/v) containing 0.2 M HCl solution was added to 50 g of pulp, high purity (klason and acid-soluble lignin contents are 98.4% and this mixture was refluxed for 40 min, then the liquid phase was decanted after ◦ for Lc and 98.2% for Ld), which was indicative of low carbo- the mixture was cooled to 50 C in a nitrogen atmosphere. The solid residue was subjected to the next extraction with 400 ml of acidic dioxane/water solution for hydrate and protein contamination. So, the impurities were not 30 min. Two more extractions were done in the same manner. The last extraction taken into account during the calculations. The carbon content was performed without addition of HCl, only in the dioxane/water mixture. Each was higher and oxygen content was lower in Lc than in Ld. portion of extract was concentrated separately and the concentrates were com- Methoxyl content was higher in Lc than in Ld. These results bined and lignin was precipitated by addition of the dioxane solution in cooled water. Filtered isolated lignin was extracted with diethyl ether, then washed with water, and freeze-dried. Table 1 The lignin was purified according to the method of Lundquist et al. [11]. Average molecular weights of lignin preparations

Lignin M¯ n M¯ w M¯ w/M¯ n 2.2.3. Acetylation of lignins L 7770 14173 1.824 One gram of lignin was dissolved in 3 ml of pyridine and then 3 ml of acetic c L 7079 11951 1.688 anhydride was added. The mixture was left for 48 h at room temperature and d then poured into 100 ml of ice water with stirring. After 1 h, the precipitate was ﬁltered out and washed with 1% of AcOH and water, respectively, and ﬁnally lyophilized [12]. Table 2 Element composition, methoxyl contents and C9 formula of lignin preparations

2.2.4. Estimation of molecular weight Lignin C (%) H (%) N (%) O (%) OCH3 C9 formula Average molecular weights and their distribution of acetylated lignins were (%) estimated by GPC on an HPLC system (Waters) with Waters Styragel HT3, HT4 L 62.90 6.06 0.17 30.87 16.00 C H O (OCH ) and HT5 columns. The columns were calibrated with authentic polystyrene. c 9 8.34 2.68 3 0.98 L 62.82 5.51 0.18 31.27 14.95 C H O (OCH ) Tetrahydrofuran (THF) was used as eluent at a ﬂow rate of 0.5 ml/min. d 9 7.66 2.78 3 0.91 中国科技论文在线 http://www.paper.edu.cn

Q. Xu et al. / Enzyme and Microbial Technology 39 (2006) 969–975 971

Table 3 bonding. Perhaps it was one of the reasons that the strength Functional groups contents of lignin preparations properties of LVS-deinked pulp were higher than those of the lilignin PhOH (%) C O (/100C9) COOH (%) control. Lc 3.24 2.48 1.36 Ld 2.81 3.08 1.88 3.4. FT-IR spectra

FT-IR spectra of Lc and Ld were shown in Fig. 1, and the suggested that lignin demethylation took place during the LVS assignments of the observed bands [19] and their relative inten- treatment. Similar lignin demethylation was previously shown sity were listed in Table 4. in the presence of ABTS as a mediator by Bourbonnais and Paice As shown in Fig. 1,Lc and Ld were structurally similar. The [17]. most notable change was the increase in the relative intensity of the band at 1666–1659 cm−1 due to conjugated carbonyl groups, 3.3. Functional groups which showed that some new conjugated carbonyl groups were formed during the LVS deinking. This result was in accor- Functional groups contents of Lc and Ld were shown in dance with the above functional groups analysis. The band at −1 Table 3.Ld had fewer phenolic hydroxyl groups than Lc, which 1718–1715 cm was assigned to carboxylic acid and unconju- indicated that the contents of phenolic hydroxyl groups were gated carbonyl groups. Its relative intensity increased after the reduced during the LVS deinking. However, it was illustrated LVS deinking, indicating that residual lignin after an LVS treat- that some new carbonyl groups were formed during the LVS ment was enriched in these types of functional groups. The band −1 treatment because Ld had more carbonyl groups than Lc. Maybe at 1421 cm was assigned to aromatic skeletal vibrations com- it was the reason that the brightness of LVS-deinked pulp was bined with –OCH3 in-plane deformations. The relative intensity lower than that of the control pulp. Carboxyl groups of Ld were decrease after LVStreatment showed that some methoxyl groups −1 also higher than those of Lc. According to the study of Laine were removed during the LVS treatment. The band at 1365 cm and Stenius [18], the acid groups drew water into the pulp by was assigned to aliphatic C–H stretching in CH3 (not in –OCH3) the osmotic pressure of their counter ions, resulting in improved and phen–OH. The decrease of its relative intensity illustrated

Fig. 1. FT-IR spectra of lignin preparations Lc (left) and Ld (right).

Table 4 FT-IR absorption bands and their relative intensitya of lignins [18] Band (cm−1) Assignment Relative intensity

Lc Ld 1718–1715 Carboxylic acid and unconjugated carbonyl groups in ketone and aldehydes 0.73 0.76 1666–1659 C O stretching in conjugated p-substituted aryl ketones – 0.80 1602–1594 Aromatic skeletal vibrations plus C O stretching 0.93 0.81 1510–1507 Aromatic skeletal vibrations 1.00 1.00 1421 Aromatic skeletal vibrations combined with –OCH3 in-plane deformations 0.95 0.92 1365 Aliphatic C–H stretching in CH3 (not in –OCH3) and phen–OH 0.28 0.25

a The relative intensity was calculated as the ratio of intensity of the band to the intensity of band at 1510 cm−1. 中国科技论文在线 http://www.paper.edu.cn

972 Q. Xu et al. / Enzyme and Microbial Technology 39 (2006) 969–975

1 Fig. 2. H NMR spectrum of lignin preparations Lc (left) and Ld (right).

that either the side chains or phen–OH of lignin decreased after ing to 1H NMR were a little higher than the results determined the LVS treatment. by functional groups analysis, the trend of change was similar. This probably was caused by overlapping of protons signals in 3.5. 1H NMR spectra different structures.

The 1H NMR signals and average numbers for each pro- 3.6. 1H–13C correlation 2D NMR (HMQC) spectra tons type per C9 unit of the acetylated lignins, as assigned by 1 13 Lundquist [20,21], were listed in Table 5, and the spectra were H– C correlation 2D NMR spectra of Lc and Ld were shown in Fig. 2. shown in Figs. 3–6, and the analysis results were shown in Comparing with the proton numbers of Lc, the proton num- Table 7. Heteronuclear multiple quantum coherence (HMQC) bers of Ld decreased. The numbers of methoxyl groups, phenolic and aliphatic hydroxyl groups per C9 unit for each lignin, as calculated from the above proton numbers, were listed in Table 6. Methoxyl groups, phenolic and aliphatic hydroxyl contents of Ld were lower than those of Lc. although results calculated accord-

Table 5 1 H signal assignments of lignins and average numbers of protons per C9 unit [20,21]

δ (ppm) Assignment Protons (/C9)

Lc Ld 7.27 Chloroform (solvent) 7.2–6.28 Aromatic protons 2.39 2.28 6.28–5.75 H␣ in ␤-O-4 and ␤-1 structures 0.41 0.34 5.75 H␣ in ␤ 0.35 0.29 5.00 H␤ and H␥ in ␤ 0.66 0.63 4.55 H␥ in some structures 1.17 1.16 4.05 Protons in methoxyl groups 4.02 3.56 3.10 H␤ in ␤–␤ structures 0.35 0.31 2.50–2.25 Aromatic acetates 0.71 0.56 2.25–1.58 Aliphatic acetates 3.70 3.46 0.00–1.58 Hydrocarbon contaminant 0.50 0.45

Table 6 Content of lignin functional groups calculated by 1H NMR

Functional groups/C9 Lc Ld Methoxyl 1.34 1.18 OHphenolic 0.24 0.18 OHaliphatic 1.23 1.15 OH 1.47 1.33 total 1 13 13 1 Fig. 3. 2D H– C NMR (HMQC) of Lc in C (100–130)– H (6.2–7.8) range. 中国科技论文在线 http://www.paper.edu.cn

Q. Xu et al. / Enzyme and Microbial Technology 39 (2006) 969–975 973

1 13 13 1 Fig. 4. 2D H– C NMR (HMQC) of Lc in C (40–100)– H (2.5–6.0) range. is a common method for qualitative and today also for quan- titative analysis of the structural changes in lignin [22]. This 13 1 13 13 1 spectra provide good resolution of signals that overlap in C Fig. 5. 2D H– C NMR (HMQC) of Ld in C (100–130)– H (6.0–8.0) range. NMR spectra, and more reliable assignments of the signals are possible. 1 The signal at δ (6.37, 116.9 ppm) corresponding to H–C2 of Syringyl units could not be distinguished in the spectra of H diquinone structure disappeared after LVS treatment [22], which NMR because of the signal overlapping, which showed that showed that lignin with this structure was destroyed due to ring HMQC was an effective method to analyze the lignin struc- opening. Maybe it was one of the reasons of the increase of ture. carboxyl group content. Signals at δ (7.48, 125.4 ppm), δ (7.38, 124.0 ppm) corre- The signals at δ (7.55, 120.7 ppm), δ (7.40, 113.0 ppm) cor- sponded to H–C6 of phenolic 5–5 with ␣–C O substructure responded to H–C6 and H–C2 in G–CO–R structure generated [23]. These signals became weaker obviously after the LVS by oxidizing of guaiacyl units during the LVS treatment [23]. treatment. The signal at δ (7.45, 113.0 ppm) corresponding to These signals were not found in the spectrum of Lc, which sug- H–C6 of nonphenolic 5–5 substructure changed little. The gested that lignins were oxidized and ␣–C O increased during results showed that the phenolic substructures in lignin could the LVS treatment. be degraded more easily than nonphenolic substructure. Signals at δ (7.23, 128.3 ppm) and (6.87, 128.9 ppm) were The signal at δ (4.90, 84.0 ppm) for H–C␤ of ␤-O-4 and assigned to H–C␣ of stilbene in lignin [22]. These signals the signal at δ (3.00, 54.0 ppm) for H–C␤ of ␤–␤ disappeared changed little during the LVS treatment, indicating that stilbene after the LVS treatment [25]. This may indicate the cleavage was relatively stable to LVS. of ␤-O-4 and ␤–␤ bonds and the depolymerization of lignin The signal at δ (6.80, 105.0 ppm) corresponded to the H–C2/6 during the LVS treatment. It was accordant with the molecular of syringyl units [24]. The signal became weaker after LVS weight measurement. treatment, which indicated that syringyl units were degraded. Signals at δ (5.50, 87.0 ppm), δ (3.50, 53.0 ppm), δ (4.16, Whatever in the lignin from the control pulp, or in the lignin 68.0 ppm) corresponding to H–C␣, H–C␤ and H–C␥ of from the LVS-deinked pulp, the signal of syringyl units was ␤-5 changed little during the LVS treatment [24], which much weaker than that of guaiacyl units. It was indicated showed that ␤-5 substructure in lignin was stable to the LVS that the proportion of syringyl units in lignin was very small. oxidation. 中国科技论文在线 http://www.paper.edu.cn

974 Q. Xu et al. / Enzyme and Microbial Technology 39 (2006) 969–975

Table 7 1 13 Analysis of 2D H– C NMR spectra for Lc and Ld [22–25] 1H (ppm) 13C (ppm) Assignment

7.23 128.3 H–C␣in G–CH CH–G 6.87 128.9 H–C␣in G–CH CH–G 6.77 126.4 H–C␤ in coniferylaldehyde 7.48 125.4 H–C6 of 5–5 with ␣–C O (phenolic) 7.38 124.0 H–C6 of 5–5 , G, with ␣–C O (phenolic) 6.70 121.3 G, H–C6 6.91 120.5 G, H–C6 7.55 120.7 H–C6 of G with ␣–C O 6.37 116.9 H–C2 of diquinone 7.01 115.8 G, H–C5 6.72 114.0 G, H–C5 7.45 113.0 H–C6 of 5–5 with ␣–C O (non-phenolic) 7.01 112.7 G, H–C2 6.91 110.7 G, H–C2 7.40 113.0 H–C2 of G with ␣–C O 6.80 105.0 S, H–C2/6 5.1 92.9 H–C1 in carbohydrates 5.50 87.3 H–C␣ in ␤-5 4.70 85.3 H–C␣ in ␤–␤ 4.38 84.7 H–C␤ in ␤-O-4 (threo) 4.60 84.9 H–C␤ in ␤-O-4 (erythro) 4.80 72.5 H–C␣ in ␤-O-4 4.16 71.9 H–C␥ in ␤–␤ (equatorial) 3.76 71.2 H–C␥ in ␤–␤ (axial) 3.40 70.8 Xylose 4.16 68.5 H–C␥ in ␤-5 3.76 63.2 H–C5 in carbohydrates 3.70 60.4 H–C␥ in ␤-O-4 3.80 55.9 Methoxyl 1 13 13 1 3.00 53.9 H–C␤ in ␤–␤ Fig. 6. 2D H– C NMR (HMQC) of Ld in C (40–100)– H (2.7–5.7) range. 3.50 53.4 H–C␤ in ␤-5

3.7. Probable mechanism of LVS deinking Because laccase could not diffuse into pulp ﬁbers due to size limitation, many attempts to circumvent these limitations have Combining the structural changes in lignin during the LVS focused on using chemical mediators which could transmit elec- deinking with surface analysis of deinked pulp ﬁbers [26], prob- tron between laccase and lignin [27]. In our experiment, violuric able deinking mechanism could be deduced as shown in Fig. 7. acid was used as a mediator. Firstly, violuric acid was oxidized

Fig. 7. Probable mechanism of LVS deinking schematic diagram. 中国科技论文在线 http://www.paper.edu.cn

Q. Xu et al. / Enzyme and Microbial Technology 39 (2006) 969–975 975 by laccase in the presence of oxygen to be VIO complex with [5] Bajpai P, Bajpai PK. Deinking with enzymes: a review. Tappi J strong oxidation ability which easily diffused into pulp fibers 1998;81(12):111–7. and removed lignin via a series of oxidative degradation reac- [6] Sealey J, Ragauskas AJ. Residual lignin studies of laccase-delignified kraft pulps. Enzyme Microb Technol 1998;23:422–6. tion. With the removal of lignin, the bondings between fiber and [7] Wong KKY, Anderson KB, Kibblewhite RP. Effects of the laccase- ink particles attached on the fiber became loose. At the aid of mediator system on the handsheets properties of two high kappa kraft shear forces provided by the deinking equipment, ink particles pulps. Enzyme Microb Technol 1999;25:125–31. could be detached from the fibers. [8] Call HP, Strittmatter G. Application of lignolytic enzymes in the paper and pulp industry—recent results. Papier 1992;46(10):32. [9] Xu QH, Qin MH, Shi SL, Zhang AP, Xu Q. Deinking of old newsprint 4. Conclusions with laccase-mediator system. Trans China Pulp Pap 2004;19(2):48–51. [10] Evtuguin DV, Neto CP, Silva AMS, Domingues PM, Amado FML, Structural changes in lignin during the deinking of ONP with Robert D, et al. Comprehensive study on the chemical structure of diox- LVS were studied. The experimental results showed the molec- ane lignin from plantation eucalyptus globlus. Wood J Agric Food Chem ular formula of lignin from the control and LVS-deinked pulps 2001;49:4252–61. [11] Lundquist K, Ohlsson B, Simonson R. Isolation of lignin by means of were C9H8.34O2.68 (OCH3)0.98 and C9H7.66O2.78(OCH3)0.91, liquid–liquid extraction. Svensk Paperstidning 1977;80(5):143–4. respectively. The molecular weight of lignin from the LVS- [12] Pan XJ, Sano Y. Atmospheric acetic acid pulping of rice straw deinked pulp were lower than those of lignin from the control IV: physico-chemical characterization of acetic acid lignins from pulp, which showed the lignin was partially degraded during rice straw and woods. Part 1. Physical characteristics. Holzforschung the LVS treatment. Comparing with lignin from the control 1999;53(5):511–8. [13] Lin SY, Dence CW. Methods in lignin chemistry, vol. 5. Springer-Verlag; pulp, the phenolic hydroxy and methoxyl contents of lignin 1992. pp. 465–472. from the LVS-deinked pulp decreased, while the contents of [14] Goldschmid O. Determination of phenolic hydroxyl content of carboxyl and carbonyl groups increased. FT-IR results showed lignin preparations by ultraviolet spectrophotometry. Anal Chem that some new conjugated carbonyl groups were formed during 1954;26:1421–3. the LVS deinking. The contents of methoxyl, phenolic hydroxy, [15] Pobiner H. Improved inflection points in the non-aqueous potentiometric 1 titration of acid functionalities in lignin chemicals by using internal aliphatic hydroxy of lignin calculating according to H NMR standardization and ion exchange. Anal Chim Acta 1983;155:57–65. all decreased after LVS treatment. HMQC results showed that [16] Alder E, Marton J. Zur kenntnis der carbonyl-gruppen in lignin. Acta the guaiacyl units were oxidized by LVS. Stilbene, dibenzene Chem Scand 1959;13:75–96. methane and 5–5 type substructure in lignin were stable to LVS [17] Bourbonnais R, Paice MG. Enzymatic delignification of kraft pulp using treatment, while ␤-O-4 and ␤–␤ substructures were partially laccase and a mediator. Tappi J 1996;79:199–204. [18] Laine J, Stenius P. Effect of charge on the fiber and paper properties of destroyed. According to structural changes in lignin, the mech- bleached industrial kraft pulps. Paper Ja Puu 1997;79:257–66. anism of LVS deinking was deduced. [19] Faix O. Classification of lignins from different botanical originated by FTIR spectroscopy. Holzforschung 1991;45(Suppl):21–7. 1 Acknowledgements [20] Lundquist K. NMR studies of lignins. 2. Interpretation of the HNMR spectrum of acetylated birch lignin. Acta Chem Scand B 1979;33:27–9. [21] Lundquist K. NMR studies of lignins. 3. 1H NMR spectroscopic data We are grateful to Novozymes for providing the laccase sam- for lignin model compounds. Acta Chem Scand B 1979;33:418–20. ple and the National Natural Science Foundation of China for [22] Kishimoto T, Ueki A, Tsksmori H, Uraki Y, Ubukata M. Delignifica- their support (grant Nos: 20576065 and 30471359). tion mechanism during high-boiling solvent pulping. Part 6. Changes in lignin structure analyzed by 1H–13C correlation 2D NMR spectroscopy. Holzforchung 2004;58(10):355–62. References [23] Fu SY, Zhan HY, He W. Degradation of residual lignin in kraft pulp by laccase and mediator system. J South China Univ Technol (Nat Sci Ed) [1] Welt T, Dinus RJ. Enzymatic deinking: effectiveness and mechanisms. 2002;30(12):30–6. Wochenblatt fur Papierfabrikation 1998;126(9):396–407. [24] Fukagawa N, Meshitsuka G, Ishizu A. A two-dimensional NMR study [2] Harkki, Tiina. Oxidative enzymes for fiber modification. In: Proceedings of birch milled wood lignin. J Wood Chem Technol 1991;11(3):373–96. of the Seventh International Conference on Biotechnology of Pulp and [25] Fukagawa N, Meshitsuka G, Ishizu A. Isolation of a syringyl-␤-O-4 rich Paper Industry. Mintreal, Canada: Canadian Pulp and Paper Association end-wise type lignin fraction from birch periodate lignin. J Wood Chem (CPPA); 1998. p. A121–4. Technol 1992;12(1):91–109. [3] Heise OU, Unwin JP, Klungness JH, Fineran WG, Sykes M, Abubark S. [26] Xu QH, Qin MH, Shi SL, Zhang AP, Xu Q. Surface chemical compo- Industrial scale up of enzyme-enhanced deinking of nonimpact printed sition and morphology of the fibers of old newsprint laccase-mediator toners. Tappi J 1996;79(3):207–12. system deinked pulp. Trans China Pulp Pap 2004;20(1):78–82. [4] Borchardt JK, Matalamaki DW, Lott VG, Grimes DB. Pilot plant studies: [27] Levlin KP, Wang W, Tamminen T, Hortling B, Viikari L, Paavola MLN. two methods for deinking sorted office paper. Tappi J 1997;80(10):269– Effects of laccase/HBT treatment on pulp and lignin structures. JPPS 78. 1999;25(3):90–5.