A new biobleaching sequence for kenaf pulp: Influence of the chemical nature of the mediator and thermogravimetric analysis of the pulp

Bioresource Technology 130 (2013) 431–438

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A new biobleaching sequence for kenaf pulp: Influence of the chemical nature of the mediator and thermogravimetric analysis of the pulp Glòria Andreu a, Agustín G. Barneto b, Teresa Vidal c,⇑ a

Chemical Engineering Department, ETSEIAT, Universitat Politècnica de Catalunya, Colom 11, E-08222 Terrassa, Spain Chemical Engineering Department, Physical Chemistry and Organic Chemistry, University of Huelva (ceiA3), Spain c Textile and Paper Engineering Department, ETSEIAT, Universitat Politècnica de Catalunya, Colom 11, E-08222 Terrassa, Spain b

h i g h l i g h t s " We report a novel laccase-mediator treatment for kenaf pulp biobleaching. " Relevance of phenoxy radicals formed in the enzymatic stage. " Kenaf increases oxidative efficiency of laccase. " Thermogravimetric analysis realizes cellulose surface changes.

a r t i c l e

i n f o

Article history: Received 12 June 2012 Received in revised form 14 November 2012 Accepted 5 December 2012 Available online 14 December 2012 Keywords: Kenaf Trametes villosa laccase Mediator chemical nature Optical pulp properties Thermogravimetric analysis

a b s t r a c t This paper evaluates five phenolic compounds as mediators for kenaf pulp biobleaching by laccase. The results have been compared with the treatment using a non-phenolic mediator, 1-hydroxybenzotriole and laccase alone. The influence of the nature of the chemical mediators used on various pulp properties is discussed. In addition to oxidizing lignin, the phenolic radicals formed in the process take part in condensation and grafting reactions in enzymatic stage. After biobleaching sequence (LP), syringaldehyde was shown to be the best phenolic mediator, allowing a delignification of 43% and 72% ISO brightness. These results were similar to the use of laccase alone due to the role as mediators of syringyl units resulting from oxidative lignin degradation. As a novelty, the study was supplemented with thermogravimetric analysis, with emphasis on the crystallinity degree of the cellulose surface and the aim of elucidating the action mechanisms of laccase-mediator systems on fiber. Ó 2012 Elsevier Ltd. All rights reserved.

1. Introduction Kenaf (Hibiscus cannabinus) is an annual dicotyledonous plant which grows in temperate and tropical areas. Kenaf adapts easily to various types of soil and requires only minimal chemical treatment to grow effectively (Elsaid et al., 2011). Kenaf plants can adsorb approximately 1.5 times their weight in carbon dioxide, which is an increased level of adsorption relative to other plants (Mohanty et al., 2005). This fact, its rapid growth and its high yield makes kenaf one of the most promising nonwood plants. In the developed world, fiber from nonwood plants has a growing market for producing paper with high added value (Moore, 1996). The contents in long (bast) and short (core) fibers of kenaf are in fact suitable for manufacturing paper and various other products (Ahmed et al., 1998); however, kenaf bast fibers are especially suitable for producing high-quality paper. ⇑ Corresponding author. Tel.: +34 937 398 180; fax: +34 937 398 101. E-mail address: [email protected] (T. Vidal). 0960-8524/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.biortech.2012.12.014

The use of laccases in combination with various natural phenolic compounds is receiving increasing attention for various purposes including pulp delignification, wood fiber modification, dye or stain bleaching, contaminated water purification and soil remediation (Widsten and Kandelbauer, 2007). Laccases hold much promise for the paper and pulp industry in its search for ways to avoid the environmental impact of the chlorine-based oxidants currently in use in delignification and bleaching processes (Cañas and Camarero, 2010). Laccases are multi-copper oxidases catalyzing the oxidation of phenolic substrates with the concomitant reduction of oxygen (Leonowicz et al., 2001). However, these enzymes have a moderate oxidizing power and can only attack phenolic moieties in lignin polymers (Xu et al., 1996), so they require the assistance of a natural or synthetic mediator to efficiently degrade nonphenolic lignin (Morozova et al., 2007). Mediators are low-molecular weight compounds that form radicals upon oxidation by laccase; such radicals are indeed capable of oxidizing lignin linkages. Laccase-mediator systems (LMS) have been successfully used to oxidize lignin in sisal

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(Aracri et al., 2009), flax (Fillat et al., 2010) and kenaf (Andreu and Vidal, 2011). However, the effect of an LMS depends on the balance between oxidative degradation and other reactions taking place during the enzymatic stage (Moldes et al., 2008; Barneto et al., 2012). Thermogravimetric analysis is a powerful tool for detecting chemical changes in microfibril surfaces, as well as for quantifying crystalline and amorphous cellulose in fiber. Clean and crystalline cellulose are thermally degraded at high temperatures spanning a narrow range because ordered cellulose chains yield crystallites that are protected from external attack by a surface hydrogen bond network (Barneto et al., 2011). For example, reaching the highest possible mass loss rate for crystalline cellulose requires heating at about 360 °C in a nitrogen environment and at only 20 °C less in the air because oxygen reacts much more easily with volatiles evolved during heating than with cellulose (Mamleev et al., 2007). Amorphous and paracrystalline cellulose respond differently to heating (Ioelovich et al., 2010); thus, they are degraded at lower temperatures over a broad range. Amorphous cellulose is located between cellulose crystallites in cellulose microfibrils; by contrast, paracrystalline cellulose is unordered crystalline cellulose located on crystallite surfaces, onto which it is chemically or enzymatically adsorbed. The aim of the present work is to evaluate the efficiency of five phenolic mediators on a kenaf bleaching sequence. These treatments will be compared with laccase alone and a non phenolic mediator, 1-hydroxybenzotriole (HBT). The results will be evaluated in terms of delignification, brightness and viscosity. Their effect on optical properties of the treated pulp will be emphasized. Finally, thermogravimetric analysis will be employed by first time to kenaf treated pulp.

lone), and 1-hydroxybenzotriole (HBT), were all purchased from Sigma–Aldrich. 2.2. Bleaching sequence (LP) Each laccase-mediator treatment (L stage) was performed by using an amount of 40 g of pulp in 50 mM sodium tartrate buffer at pH 4, 20 U/g TvL and a proportion of 1.5% HBT or natural mediator (all relative to pulp dry weight). The treatments were carried out in a reactor under O2 pressure (0.6 MPa) at 30 rpm at 50 °C for 4 h. Pulp consistency was 5% in all treatments. Pulp samples treated under identical conditions except for the absence of a mediator were used as controls. After the enzyme treatment, samples were filtered and extensively washed with deionized water. The L stage was followed by an alkaline peroxide bleaching treatment (P stage) that was performed in an Ahiba Spectradye dyeing apparatus from Datacolor equipped with closed vessels of 150 mL volume; the vessels were loaded with 5 g odp (oven-dried pulp) at 5% consistency, 3% odp H2O2, 1.5% odp NaOH, 1% odp DTPA (diethylenetriaminepentaacetic acid) and 0.2% odp MgSO4 at 90 °C for 2 h. Then, each treated sample was filtered and extensively washed with deionized water (García et al., 2003). 2.3. Soxhlet extraction (LSox) After enzymatic treatment, pulps were extracted with acetone in a Soxhlet apparatus for 2 h and 15 min in order to remove residual phenolic compounds adsorbed in the pulp (Aracri et al., 2010). The pulp samples after this extraction are named LSox in the text. 2.4. Analysis of pulp properties

2. Methods 2.1. Raw materials, laccase and natural phenols Kenaf (Hibiscus cinnabinus) alkaline pulp samples were obtained by soda–anthraquinone cooking in the CELESA pulp mill (Tortosa, Spain). Prior to the enzyme treatments, the pulp was washed with acidified water (pH 2) at 3% pulp consistency for 30 min, followed by filtration and extensive washing with deionized water. This procedure was needed to remove contaminants and metals, as well as to adjust the pulp to the pH required for the enzyme treatments. The properties of the washed pulp were as follows: kappa number 12.9 ± 0.1, ISO brightness 35.0%, viscosity 925 ± 23 mL g–1, glucan content 83.5% ± 0.2, xylan content 14.3% ± 0.06 and klason lignin content 2.1% ± 0.1. The pulp was treated with laccase and either HBT or one of the natural mediators shown in Table 1. Laccase (EC 1.10.3.2) from Trametes villosa (TvL) was supplied by Novozymes (Bagsvaerd, Denmark). Its activity was assessed by monitoring the oxidation of ABTS in 0.1 M sodium acetate buffer (pH 5) at 436 nm (e436 = 29 300 M–1 cm–1). One activity unit was defined as the amount of laccase converting 1 lmol min–1 ABTS at 25 °C. All absorbance measurements were made with a Shimadzu UV–Vis 1603 Spectrophotometer. The natural laccase mediators (syringaldehyde, acetosyringone, p-coumaric acid, vanillin and acetovanil-

The treated pulp samples were characterized in terms of kappa number, brightness and viscosity according to ISO 302:2004, 2470–1:2009 and 5351:2011, respectively. Analyses were performed in duplicate for kappa number (errors associated to measurement were lower than 0.5 standard deviation) and viscosity, and in quadruplicate for brightness (standard deviation = 0.1). The optical properties of each pulp were determined by using a Technidyne Colour Touch reflectometer in accordance with ISO 11475. Reflectance spectra and the k/s ratio for paper sheets made from the pulp were obtained by following the Kulbelka–Munk theory (Andreu and Vidal, 2011). The area decrease rate (ADR) is a measure of the area decrease in the k/s plot with respect to the ini1Þ tial pulp: ADR ¼ ðA0AA  100, where A0 is the initial area and A1 the 0 final area (Andreu and Vidal, 2011). The area difference represents the amount of chromophoric groups removed (positive values) or introduced (negative values) by the treatment. The initial kenaf pulp was used as reference. Sample color was described in terms of the CIE L⁄a⁄b⁄ color space. L⁄, a⁄ and b⁄ are the coordinates of the space, which is based on the assumption that color is perceived as L⁄ (lightness, which ranges from 100 for perfect white to 0 for absolute black), a⁄ (which ranges from greenness to redness), and b⁄(which ranges from blueness to yellowness, from negative to positive values) (Hunt, 1998).

Table 1 Natural mediators used in the enzymatic stage: name mediator (abbreviation). Substituents in ortho-phenol position

Mediators

Two methoxy groups One methoxy group Without substituents

Syringyl derivatives: Coniferyl derivatives: Guaiacyl derivatives:

Acetosyringone (AS) Acetovanillone (AV) p-coumaric acid (PC)

Syringaldehyde (SA) Vainillin (V)

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We additionally used the optical parameter chroma [C⁄ = (a⁄2 + b ) )], which represents the normal distance from the lightness axis and measures color saturation (Moldes et al., 2010). The standard deviation was 0.1 for all optical parameters. Carbohydrate composition of the initial pulp was characterized by quantitative acid hydrolysis; the resulting hydrolysates were analyzed for glucan (HPLC determination of glucose) and xylan (HPLC determination of xylan), and the solid residue was used to quantify klason lignin. Analyses were performed in duplicate. ⁄2 1/2

2.5. Thermogravimetric analysis Thermogravimetric analyses were carried out on a Mettler Toledo TGA/SDTA851e/LF1600 thermobalance, using about 5 mg of sample in each run. Pyrolysis and combustion runs were performed in nitrogen and synthetic air (4:1 N2/O2), respectively. The temperature was raised from 25 to 900 °C at three different heating rates: 5, 10 and 20 °C/min. 3. Results and discussion We have recently reported the results of a screening on five natural phenols (Table 1) and HBT in laccase-mediator system to bleach kenaf pulp (Andreu and Vidal, 2011) Further research was needed in order to improve the results obtained with the natural mediators in this application. 3.1. Effect of the chemical structure of the mediator on the L stage Delignification and brightness are parameters to monitor during the stages of a pulp bleaching sequence. Fig. 1a (results presented in this figure were published by Barneto et al., 2012)

Fig. 1. Kappa number and brightness of kenaf pulp after the enzyme treatment (a) and subsequent Soxhlet extraction with acetone (b). Laccase denotes the control pulp (viz. pulp that was treated with the enzyme alone).

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illustrates these parameters for the L stage. After the enzyme treatment, the pulp samples treated with the natural mediators exhibited an increased kappa number (KN) relative to the control samples: PC increased KN (by 3.7 points), and so did the other mediators, in the following sequence: V > AV > SA > AS. Brightness was improved by PC and AV (2.7 and 1.7 ISO percent points, respectively) with respect to laccase alone (32.8% ISO). By contrast, AS and SA decreased brightness by 2.4% and 2.1% ISO points relative to the control. HBT was the only mediator simultaneously decreasing kappa number (0.9 points) and increasing brightness (1.8% ISO points) respect to laccase control treatment. Soxhlet extraction with acetone was used to remove adsorbed low molecular-mass phenols contributing to kappa number in the pulp and facilitated discrimination of the action of each mediator. Acetone extraction (Fig. 1b) reduced KN in all samples —those treated with laccase and HBT or the enzyme alone included—, which is consistent with the removal of the phenolic compounds adsorbed in the pulp (Aracri et al., 2010). However, KN for the PC-treated pulp was still greater than that for the laccase-treated pulp by 1.9 points. In addition to a decrease in kappa number, the treatments with AS and SA provided lower pulp brightness (34.0%ISO both) than laccase alone (35.5%ISO); also, the PC-, V- and AV-treated samples exhibited higher brightness than the control pulp. Table 2 shows the k/s ratio for the pulp samples after the enzyme treatment, as well as the calculated ADR values. As can be seen from Table 2a, a substantial amount of chromophoric groups remained in the pulp after the enzyme treatments (L) with AS and SA as mediators judging by the ARD values obtained (–25.5% and – 22.2%, respectively). Also, only the samples treated with HBT and AV exhibited color removal. These results suggest a dissimilar behavior of the mediators in the enzyme treatment. During this stage, the oxidation of N–OH compounds (synthetic mediator) or Ph–OH compounds (phenol mediators) involves H+ abstraction and electron transfer to generate an intermediate radical (N–O or Ph–O) (Xu et al., 2000; Díaz-Gonzalez et al., 2011). The results obtained with the natural mediators were found to depend on their chemical nature and, ultimately, on the reactivity of the intermediate radical formed (Ph–O). Based on the chemical properties of phenols (Morrison and Boyd, 1998), the presence of electron-donor groups (e.g. methoxy) in ortho facilitates the formation of a phenoxy radical; whereas steric effects (occupied ortho positions) prevent grafting reactions. The treatments with AS and SA —mediators with two methoxy substituents in ortho on the phenol ring— provided lower pulp brightness, which is indication of a coloring process. The amount of chromophoric groups present could be the result of condensation reactions between phenoxy radicals. Thus, these reactive radicals may undergo an intermolecular nucleophilic attack and produce condensation compounds, possibly via Cphenol–O links (Bollag et al., 1992). These condensed phenolic compounds can be further oxidized to extended quinones, the complex mixture being responsible for color in the pulp (Jeon et al., 2010). In fact, AS–O and SA–O (syringyl derivatives) exhibit a substantial tendency to condensation reactions; this reflected in the decreased brightness, kappa number and ADR values obtained after the enzyme treatments. On the other hand, the decreased ADR values obtained after Soxhlet extraction with acetone when using AS and SA mediators relative to the other samples, were consistent with the decreased brightness also obtained and testifies again the tendency of the syringyl mediators (AS and SA) to undergo condensation reactions (Table 2b). This behavior of AS and SA may be accentuated in the enzyme treatment of kenaf pulp: the ratio of syringyl units to guaiacyl units in kenaf lignin is Section 3.3 (De la Rosa, 2003) and this additional concentration of the two radicals —a result of oxidative

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Table 2 k/s area and area decrease rate (ADR) of kenaf pulp samples after the enzyme treatment (L) and acetone extraction (LSox). Kenaf

Laccase

HBT

AS

SA

PC

V

AV

(a) L stage k/s area ADR

112.43 –

121.10 7.72

108.50 3.49

141.05 25.46

137.43 22.24

101.15 10.03

126.22 12.27

108.85 3.18

(b) LSox stage k/s area ADR

112.43 –

103.50 7.94

96.21 14.39

108.53 3.47

108.41 3.60

87.49 22.17

95.18 15.30

83.72 25.55

degradation of lignin (Cañas and Camarero, 2010)— provides more capacity to condensation reactions. By contrast, PC–O, which is a less easily formed radical and has no appreciable steric effects (its ortho positions are unoccupied), undergoes grafting reactions preferentially; as a result, it increased kappa number after L by 3.7 points with respect to laccase and also after LSox (1.9 points) (see Fig. 1a and b). In fact, this result confirms the occurrence of grafting reactions in kenaf fibers during the enzyme treatment, consistent with previous findings with the same mediator as applied to flax pulp (Fillat, 2011). Additional optical parameters including C⁄ (color saturation), L⁄ (amount of color) and the color coordinates a⁄ and b⁄ were determined in order to confirm the production of colored species and the dissimilar action of the natural mediators on the underlying reactions. The highest C⁄, a⁄ and b⁄ values were those for the pulp samples treated with AS and SA (Fig. 2, L and LSox stages); this confirms the tendency of these two mediators to engage in condensation reactions and in the subsequent formation of quinones (Bollag et al., 1992). The optical properties of the pulp confirmed differences in the development of the enzyme process due to differences in the role played by the mediators and in the production of colored species. The reactivity and stability of N–O radicals are seemingly better balanced than in phenoxy radicals, which may account for the better performance of the former radicals as mediators for laccase-

based delignification (Moldes et al., 2008). Thus, the final brightness, kappa number and optical properties of the pulp depend on the following competitive reactions: oxidation of lignin, grafting and condensation of phenoxy radicals during the enzymatic stage. 3.2. Chemical bleaching: hydrogen peroxide stage (P) The tendency of natural mediators to bind to fibers requires inserting a P stage after the enzyme treatment (Fillat and Roncero, 2010). This bleaching stage has a twofold effect on pulp; thus, the hydrogen peroxide oxidizes residual lignin in the pulp and the alkaline pH used facilitates dissolution of lignin. Hydrogen peroxide has also been used to remove nonfibrous dark particles from kenaf pulp (Ström et al., 1998; De la Rosa, 2003). In all cases, substantial delignification (reduction in kappa number) and a marked improvement in brightness with respect to untreated kenaf pulp (KN = 12.9, brightness = 35.0%ISO), were observed after the LP sequence (Fig. 3). HBT proved more effective than the natural mediators; thus, it reduced KN by 6 points and provided a pulp brightness of 69.7%ISO). Syringaldehyde was the most effective natural mediator (Fig. 3, empty squares). At the end of the sequence, the pulp treated with this mediator had a kappa number of 7.3% and 71.8%ISO brightness. The enzymatic treatment of kenaf pulp with laccase alone gave similar value of kappa number than the laccase-syringaldehyde system. This fact

Fig. 2. Optical parameters and color coordinates of kenaf pulp treated with various laccase-mediator systems, after the enzyme treatment (L stage: a1 and b1) and subsequent acetone extraction (LSox stage: a2 and b2).

G. Andreu et al. / Bioresource Technology 130 (2013) 431–438

Fig. 3. Plot of brightness versus kappa number after the LP (e) and LSoxP (j) sequences.

might be due to the role as mediators of syringyl units resulting from oxidative lignin degradation of kenaf pulp (syringyl units/ guaiacyl units = 3.3). On the other hand, the LSoxP sequence, which included Soxhlet extraction (Fig. 3, filled diamonds), increased brightness with respect to LP with all mediators except SA. This suggests that some hydrogen peroxide was consumed during P stage to oxidize chromophoric groups present in the pulp after the enzyme treatment, which affected brightness in the final pulp (Andreu and Vidal, 2011). Therefore, the operating conditions for the chemical bleaching stage should be optimized in order to improve the properties of the pulp at the end of the biobleaching process. The optical analysis allowed the whole bleaching process to be characterized. Table 3 shows the L⁄, C⁄, a⁄ and b⁄ values obtained after the LP sequence with each mediator. As can be seen, L⁄ was increased and C⁄ (chroma) decreased relative to unbleached kenaf pulp. The fact that C⁄ was always reduced indicates that some color was removed from the pulp at the end of the bleaching sequence. a⁄ was reduced until all redness was suppressed (negative a⁄ values), simultaneously with a reduction in yellowness (a decrease in b⁄). The optical parameters of the CIE L⁄a⁄b⁄ color system correlated well with kappa number and brightness. 3.3. Effect of the biobleaching sequence on pulp viscosity The pulp samples were subjected to viscosity measurements in order to assess the effect of each treatment on cellulose integrity. The results obtained after the L stage (Fig. 4) suggest that HBT acted differently from the natural mediators. Thus, the enhanced bleaching obtained with HBT was accompanied by a drop in viscosity. This mediator degrades and oxidizes carbohydrate chains, which affects pulp viscosity (García et al., 2003). By contrast, the pulp samples treated with the natural mediators retained their viscosity or even gained some during the enzymatic stage. After the P stage, all samples except SA-treated pulp exhibited a viscosity loss

Table 3 Optical parameters (L⁄ and C⁄), color coordinates (a⁄ and b⁄) and area decrease rate (ADR) of kenaf pulp after bleaching. Sequence: LP

L⁄

C⁄

a⁄

b⁄

ADR

Unbleached kenaf Laccase HBT AS SA PC V AV

73.83 92.42 92.36 92.08 93.60 93.60 93.60 93.60

15.00 11.02 10.52 11.46 10.33 11.94 12.53 11.52

4.44 0.60 0.56 0.65 0.96 0.36 0.28 0.62

14.33 11.00 10.51 11.45 10.28 11.94 12.53 11.47

0.00 89.62 89.84 88.61 92.09 86.06 85.32 88.39

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Fig. 4. Viscosity of kenaf pulp treated with various laccase-mediator systems (grey bars) and after the biobleaching sequence (dotted bars).

relative to the control. This may have resulted from carbonyl groups formed during the L stage (Camarero et al., 2004) facilitating degradation of the pulp by the strong alkaline medium used in the bleaching stage (Fillat and Roncero, 2009). In any case, viscosity was retained (especially in the pulp treated with SA, with 884 mL g–1); this suggests that the biobleaching sequence has a mild effect on cellulose integrity, so it allows highly efficient biodelignification without decreasing pulp viscosity (Martín-Sampedro et al., 2011). 3.4. Thermogravimetric analysis 3.4.1. Effect of laccase and the laccase-HBT system on the thermal degradation of kenaf pulp As can be seen in Fig. 5a, the thermal degradation of the initial pulp (In, which denotes unbleached kenaf pulp) exhibited two well-defined steps at 310 and 340 °C due to the degradation of amorphous–paracrystalline and crystalline cellulose, respectively (Barneto et al., 2011). The split peak for cellulose indicates that cellulose microfibrils were dirty, possibly as consequence of the deposition of xylans and lignin during cooking of the kenaf biomass (Barneto et al., 2012). Subsequent treatment with laccase or the laccase-HBT system [L and L(HBT) in Fig. 5a and b, respectively] scarcely altered the thermal degradation of the pulp. Therefore, the enzyme treatment cannot remove surface cellulose deposits in the initial pulp. On the other hand, chemical bleaching with alkaline hydrogen peroxide had a significant cleaning effect. Initial pulp treated with alkaline hydrogen peroxide (InP) led to a sharper peak than the laccase and laccase-mediator pulps after a similar chemical treatment [compare InP with LP and L(HBT)P in Fig. 5a and b]. Obtaining fiber as clean as that in InP requires subjecting laccase-treated pulps to Soxhlet extraction (Sox stage) prior to bleaching with hydrogen peroxide. Once Soxhlet extraction removes deposits on the cellulose surface, a P stage can lead clean fiber surfaces in laccase-treated pulp (LSoxP pulp). Changes in fiber surfaces as derived from the thermogravimetric curves were consistent with the basic trends in kappa number and brightness during bleaching. Thus, the absence of significant changes in the thermal degradation profile of the pulp after the laccase treatment (L pulp) was consistent with the slight changes in kappa number and brightness during this stage (Table 4). In addition, the presence of sharper peaks in the profile after the alkaline hydrogen peroxide (InP pulp) treatment was accompanied by a significant increase in brightness (nearly 35.0 ISO percent points) and a reduction in kappa number close to 5 points. There were, however, two divergences between the results of the thermal and chemical analyses, namely: (a) after hydrogen peroxide bleaching,

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must be related with some type of deposit caused by laccase on pulp. Cellulose surface in L pulp is significantly dirtier than that in the initial pulp. 3.4.2. Effect of the laccase-natural mediator and laccase-HBT treatments on the thermal degradation of kenaf pulp In addition to laccase, the thermal degradation profile of each pulp was dependent on the particular mediator used (Vila et al., 2011). As can be seen from Fig. 6a, the specific mediator for laccase had a very significant effect on the thermal degradation profile of the pulp after the subsequent alkaline hydrogen peroxide treatment (P stage). On the one hand, mediators such as vanillin (V) and HBT facilitated the subsequent action of H2O2 and led to cleaner, more crystalline cellulose surfaces than in the control pulp (LP, without mediator) after chemical bleaching; on the other, mediators such as syringaldehyde (SA), p-coumaric acid (PC), acetovanillone (AV) and acetosyringone (AS), in this sequence, hindered subsequent bleaching of the pulp with hydrogen peroxide. The poorest mediator in this respect was AS, which virtually prevented subsequent chemical-based cleaning of the cellulose surface. In thermogravimetric terms, kenaf pulp treated with the laccase-AS system was almost refractory to chemical bleaching with alkaline hydrogen peroxide and deposits on the cellulose surface remained nearly unchanged. Obviously, the interaction between laccase, the mediator and the pulp can yield different results depending on the oxidative degradation of lignin, grafting of the mediator onto lignin or cellulose

Fig. 5. Thermal degradation profiles for kenaf pulps: (a) comparison between the initial pulp (In) and laccase-treated pulp (L) after hydrogen peroxide bleaching and Soxhlet extraction, (b) comparison between the initial pulp (In), bleached initial pulp (InP) and laccase-HBT treated pulp. TG runs were performed at 10 °C/min in an air atmosphere.

Table 4 Pulp properties after the L, LP and LSoxP sequences of kenaf pulp samples treated with laccase alone and the laccase-HBT mediator system. In denotes unbleached pulp and InP the same pulp after the hydrogen peroxide stage.

Unbleached pulp (In) InP Laccase LaccaseP LaccaseSoxP LHBT LHBTP LHBTSoxP

Kappa Number

Brightness (% ISO)

12.9 ± 0.1 7.5 ± 0.2 11.6 ± 0.4 7.3 ± 0.1 7.2 ± 0.2 10.7 ± 0.2 6.5 ± 0.4 6.6 ± 0.1

35.0 64.3 32.8 68.5 69.0 34.6 69.7 72.0

the initial pulp (InP) and the laccase-treated pulp (LP) exhibited different TG profiles, but similar kappa number and brightness; and (b) after Soxhlet extraction the laccase-treated pulp (L) leads significant TG changes, but only slight changes in kappa number and brightness (comparing with L pulp). In both cases, small changes in chemical properties (kappa number and brightness) had significant effects on the thermal degradation profile of pulps. Inefficacy of alkaline hydrogen peroxide bleaching to clean cellulose surface of laccase-treated pulps (L) and to lead similar narrow peaks in TG curves of both InP and LP pulps (see Fig. 5a),

Fig. 6. Influence of the mediator on the thermal degradation profile for kenaf pulp after hydrogen peroxide bleaching (a); and after Soxhlet extraction and hydrogen peroxide bleaching (b).

G. Andreu et al. / Bioresource Technology 130 (2013) 431–438

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Scheme 1. Changes in surface cellulose as derived from the thermogravimetric analysis.

fibers, mediator condensation and laccase adsorption on pulp, among other processes (Barneto et al., 2012). These can favor the formation of new deposits and hindering subsequent bleaching as a result. However, we found these shortcomings to be effectively suppressed by Soxhlet extraction with acetone. Fig. 6b compares the thermal degradation profiles (in an inert atmosphere) of the studied pulp samples when Soxhlet extracted between the enzymatic and chemical bleaching steps. As can be seen, extracting the laccase-treated pulp samples allowed alkaline hydrogen peroxide to provide clean cellulose surfaces (similarly clean to the initial pulp, which was subjected to no laccase treatment), whichever the mediator. However, these significant changes in TG profiles were accompanied by small changes in brightness and kappa number. Soxhlet extraction may remove xylans linked to the mediator onto the pulp (Cadena et al., 2011), thereby affecting surface cellulose crystallinity without appreciably altering the content in chromophoric groups.

(43.4%). This suggests that phenolic fragments resulting from oxidative lignin degradation may act as mediators and oxidize nonphenolic substrates of lignin despite the moderate oxidation power of laccase. On the other hand, 71.8% (SA) and 68.5% (laccase) ISO brigthness were obtained. Optical properties and ADR are useful indicators of biobleaching efficiency in addition to kappa number and brightness. Thermogravimetric analyses showed that laccase-mediator systems, instead of removing the initial deposits on the cellulose surface, can reinforce their strength and hinder subsequent chemical bleaching.

3.4.3. Summary of the effects of the laccase-mediator systems on kenaf pulp as determined by thermogravimetry Scheme 1 summarizes the effects of enzymatic and chemical bleaching on pulp in thermogravimetric terms. The initial pulp was completely covered with a layer of xylans and lignin which turned the surface of cellulose crystallites into paracrystalline cellulose as reflected in splitting of the cellulose TG peak. Chemical bleaching of the initial pulp removed most of the outer layer and left a clean cellulose surface which degraded at a high temperature over a narrow range as reflected in the sharp peak for InP. On the other hand, applying a laccase-mediator system to the initial pulp failed to remove the outer layer; rather, it increased its thickness with news deposits to an extent dependent on the particular mediator used (L pulp). Chemical bleaching of the laccase-treated pulp samples was less efficient owing to the presence of that reinforced layer around cellulose fibers. Acetosyringone (AS) had an especially adverse effect on the subsequent bleaching with alkaline hydrogen peroxide, which it prevented from cleaning the cellulose surface. On the other hand, vanillin (V) and HBT facilitated the formation of more crystalline cellulose relative to the control pulp. The problems derived from the use of a laccase-mediator system can be circumvented by inserting a Soxhlet extraction step before the hydrogen peroxide treatment (LSoxP pulp). In fact, acetone efficiently removed deposits on the cellulose surface, thereby allowing subsequent chemical bleaching to yield crystalline cellulose which was thermally degraded similarly to the initial pulp (InP).

References

4. Conclusions The ability of natural mediators to act as delignifying agents is dependent on their chemical nature. Thus, syringaldehyde (SA) afforded delignification to a similar extent as with laccase alone

Acknowledgements The authors are especially grateful to Spain’s MICINN for funding this research in the framework of Projects FUNCICEL (CTQ200912904) and BIOFIBRECELL (CTQ2010-20238-CO3-01).

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