Structural Changes in Lignin During the Deinking of Old Newsprint With
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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. 0141-0229/$ – see front matter © 2006 Elsevier Inc. All rights reserved. doi:10.1016/j.enzmictec.2006.01.031 转载 中国科技论文在线 http://www.paper.edu.cn 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, elemen- tal 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.