Enzymatic Deinking of Secondary Fibers: Carbohydrate Hydrolases 2 Versus Laccase-Mediator System 3 4 David Ibarra1, A, *, M
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View metadata, citation and similar papers at core.ac.uk brought to you by CORE provided by EPrints Complutense 1 Enzymatic deinking of secondary fibers: Carbohydrate hydrolases 2 versus laccase-mediator system 3 4 David Ibarra1, a, *, M. Concepción Monte2, Angeles Blanco2, Angel T. Martínez1 and 5 María J. Martínez1 6 1 7 Centro de Investigaciones Biológicas, Environmental Biology Department, CSIC. Ramiro de 8 Maeztu 9, E-28040 Madrid, Spain 9 2 Universidad Complutense, Chemical Engineering Department, Avenida Complutense s/n, 10 E-28040 Madrid, Spain 11 12 13 a Present address: CIEMAT, Renewable Energy Division, Biofuels Unit, Avenida 14 Complutense 22, E-28040 Madrid, Spain 15 16 * Corresponding author: CIEMAT, Renewable Energy Division, Biofuels Unit. Avenida 17 Complutense 22, E-28040 Madrid, Spain; E-mail: [email protected]; Tel: 34 18 913466388; Fax: 34 913466005 19 20 21 Abbreviated title: Enzymatic deinking of secondary fibers 22 1 Abstract 2 The use of enzymes has been suggested as an environmentally-friendly alternative to 3 complement conventional chemical deinking in the recycling of recovered paper. This study 4 compares the use of carbohydrate hydrolases versus laccase-mediator system for deinking 5 printed fibers from newspapers and magazines. For this purpose, two commercial enzyme 6 preparations with endoglucanase and endoxylanase activities (Viscozyme Wheat from 7 Aspergillus oryzae and Ultraflo L from Humicola insolens) and a commercial laccase 8 (NS51002 from Trametes villosa), the latter in the presence of synthetic or natural (lignin- 9 related) mediators, were evaluated. The enzymatic treatments were studied at laboratory scale, 10 using a standard chemical deinking sequence consisting of pulping, alkaline deinking and 11 peroxide bleaching stages. Then, handsheets were prepared and their brightness, residual ink 12 concentration, and strength properties were measured. Among the different enzymatic 13 treatments assayed, both carbohydrate hydrolases were found to deink the secondary fibers 14 more efficiently. Brightness increased up to 3-4% ISO on newspaper fibers, being Ultraflo 15 20% more efficient in the ink removal. Up to 2.5% ISO brightness increase was obtained 16 when magazine fibers were used, being Viscozyme 9% more efficient in the ink removal. As 17 regards laccase-mediator system, alone or combined with carbohydrate hydrolases, it was 18 ineffective deinking both newspaper and magazine fibers, resulting in pulps with worse 19 brightness and residual ink concentration values. However, pulp deinking by laccase-mediator 20 system was displayed when secondary fibers rich in lignin, i.e. printed cardboard, were used, 21 obtaining up to 3% ISO brightness increase and lower residual ink concentrations. 22 23 Keywords: Cellulases; deinking; laccase-mediator system; recycling; secondary fibers; 24 xylanases 25 2 1 1. Introduction 2 Recovered paper is an important source of raw material for the pulp and paper 3 industry. Indeed, the utilization of these secondary fibers is increasing all over the world, 4 being the deinking an important step in the recycling process for white grade papers. In the 5 traditional deinking process, large quantities of chemicals are used (Shrinath et al. 1991), 6 which makes the method expensive, environmentally damaging and increases the release of 7 contaminants. In this context, enzymes could reduce the demand of chemicals and would also 8 lower the process costs and the environmental impact (Thomas, 1994; Bajpai and Bajpai, 9 1998). These enzymes include cellulases, hemicellulases, pectinases, amylases, lipases, 10 esterases and laccases (Call and Stritmatter, 1992; Gübitz et al. 1998; Zollner and Schroeder, 11 1998; Morkbak et al. 1999; Pleach et al. 2003; Pala et al. 2004). 12 Among the different enzymes assayed, the enzymatic deinking using carbohydrate 13 hydrolases, with activities involved in cellulose and hemicellulose hydrolysis, has been 14 widely demonstrated on different secondary fibers (Jeffries et al. 1994; Gübitz et al. 1998; 15 Marques et al. 2003; Pleach et al. 2003; Vyas and Lachke, 2003; Pala et al. 2004; Lee et al. 16 2007; Zhang et al. 2008; Kuhad et al. 2010) being applied in several paper mills (Heise et al. 17 1996). The mechanism of deinking by these enzymes has not been completely elucidated, 18 although some hypotheses have been described (Jeffries et al. 1994; Heise et al. 1996). The 19 cellulose and hemicellulose hydrolysis on the surface of the fibers leads to a removal of small 20 fibrils, phenomenon known as “peeling-off fibers”, which facilitates ink detachment from the 21 surface. It has also been suggested that the alteration of the ink particles hydrophobicity 22 because of the removal of small fibrils enhances their separation (fiber/ink separation) in the 23 flotation/washing step. 24 The interest in the use of laccases, alone or in combination with carbohydrate 25 hydrolases, for deinking secondary fibers has been greatly increased in the last years (Knutson 26 and Ragauskas, 2004; Xu et al. 2004; Kapoor et al. 2007; Mohandass et al. 2008; Qinghua et 27 al. 2009; Kuhad et al. 2010). Laccases in the presence of redox mediators, the so-called 28 laccase-mediator system described 20 years ago (Bourbonnais and Paice, 1990), has been 29 largely used to delignify, and bleach, different types of pulps (Bourbonnais and Paice, 1996; 30 Sealey et al. 1999; Herpoël et al. 2002; Camarero et al. 2004a; Ibarra et al. 2006). This fact 31 offers the possibility for deinking secondary fibers rich in lignin, such as fibers based on 32 mechanical pulps (Bajpai and Bajpai, 1998). With the removal of lignin, the bonds between 33 fiber and ink particles became loose, facilitating ink detachment. Moreover, successful 34 transformation of recalcitrant dyes by laccase-mediator system has been recently described 35 (Claus et al. 2002; Camarero et al. 2004b), which has supposed a new way for secondary 36 fibers deinking by direct decolorization of the inks. 37 In the present study, two different strategies for enzymatic deinking of secondary 38 fibers were evaluated: one using carbohydrate hydrolases for removing inks in an indirect 39 way by peeling-off fibers; and other using the laccase-mediator system for removing inks in 40 an indirect way by the removal of surface lignin or in a direct way by decolorizing the inks. 41 For this purpose, different raw materials (secondary fibers from newspapers and magazines) 42 were used. Brightness, residual ink concentration and strength properties of the handsheets 43 formed from the resulting deinked pulps were tested. 44 45 2. Materials and Methods 46 2.1 Recovered paper 47 Secondary fibers from newspaper and magazines were used. Prior to the treatments, 48 the dried raw materials were shredded and maintained in distilled water for 24 h. In addition, 49 secondary fibers rich in lignin, i.e. printed cardboard, were also used to evaluate the laccase- 50 mediator system. 3 1 2.2 Enzymes and mediators 2 Carbohydrate hydrolases used in this work, Viscozyme Wheat (from Aspergillus 3 oryzae) and Ultraflo L (from Humicola insolens) were obtained from Novozymes. Their 4 activities involved in cellulose hydrolysis (exoglucanase, E.C.3.2.1.91; endoglucanase, 5 E.C.3.2.1.4; and -glucosidase, E.C.3.2.1.21) were studied on: Avicel (Merck) for total 6 cellulose activity (exoglucanase); and the specific substrates carboxymethylcellulose (Serva) 7 and p-nitrophenyl glucoside (Sigma) for endoglucanase and -glucosidase activities, 8 respectively (Wood and Bhat, 1988). Their activities involved in hemicellulose hydrolysis 9 (endoxylanase, EC 3.2.1.8; and -xylosidase, EC 3.2.1.37) were studied on birch xylan 10 (Sigma) and p-nitropheyl xyloside (Sigma) for endoxylanase and -xylosidase activities, 11 respectively (Wood and Bhat, 1988). Their activities involved in starch hydrolysis (- 12 amylase, E.C. 3.2.1.1; and glucoamylase, E.C.3.2.1.3) were studied on soluble starch 13 (Calbiochem) and p-nitrophenyl glucoside (Sigma) for -amylase and glucoamylase 14 activities, respectively (Wood and Bhat, 1988). The endoglucanase, exoglucanase, 15 endoxylanase and -amylase activities, determined in 50 mM sodium acetate buffer (pH 5) at 16 50 ºC, were followed by the release of reducing sugars estimated as glucose or xylose at 540 17 nm (Nelson 1944). The -glucosidase, -xylosidase and glucoamylase activities, determined 18 in 50 mM sodium acetate buffer (pH 5) at 50 ºC, were followed by the release of p- -1 -1 19 nitrophenol (412 15 200 M cm ). Their optimum pH was investigated on the different 20 substrates in 100 mM citrate-phosphate-borate buffer (pH range from 3 to 8) at 24 ºC, 21 determining the activities as described above. 22 A Novozymes commercial laccase NS51002 (from Trametes villosa) was used. Laccase 23 activity was determined by measuring the oxidation of 5 mM 2,2’-azino-bis(3- 24 ethylbenzothiazoline-6-sulphonic acid) (ABTS) buffered with 100 mM sodium acetate (pH 5) -1 -1 25 at 24 ºC. Formation of the ABTS cation radical was monitored ( 436 29 300 M cm ). 26 Proteins were determined according to Bradford method (1976), using bovine albumin as 27 standard and Bio-Rad kit assay. 28 One unit of enzyme activity was defined as the amount of enzyme that transforms 1 29 mol of substrate per minute. All spectrophotometric measurements were carried out on a 30 Shimadzu UV–vis 160. 31 The compounds used as redox mediators 1-hydroxybenzotriazole (HBT), violuric acid 32 (VIO), sinapic acid (SIN), ferulic acid (FER) and p-coumaric acid (PCO) were purchased 33 from Sigma. 34 2.3 Deinking pulp sequences 35 A standard (industrial type) deinking sequence P−D−B (Fig. 1) was performed in 36 laboratory scale including: (1) pulping stage (P) carried out in a lab disintegrator ENJO-D- 37 33.73/D at 3% consistency, room temperature and 3000 rev/min, according to the ISO 38 standard 5263-1:2004; (2) alkaline deinking stage (D) using 1.5% NaOH, 3% Na2Si2O3 and 39 0.06% of soap for 60 min at 60ºC and 5% consistency; and (3) alkaline peroxide bleaching 40 stage (B) using 3% H2O2 and 1.5% NaOH for 120 min at 90 ºC and 5% consistency (above 41 percentages referred to dry weight pulp).