Bioresource Technology 152 (2014) 253–258

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An improved TCF sequence for biobleaching kenaf pulp: Influence of the hexenuronic acid content and the use of xylanase Glòria Andreu a,⇑, Teresa Vidal b a b

Chemical Engineering Department, ETSEIAT, Universitat Politècnica de Catalunya, Colom 11, E-08222 Terrassa, Spain Textile and Paper Engineering Department, ETSEIAT, Universitat Politècnica de Catalunya, Colom 11, E-08222 Terrassa, Spain

h i g h l i g h t s  LMS system was incorporated to an industrial-type bleaching sequence, LmediatorQPo.  LHBTQPo sequence was very effective in bleaching kenaf pulp.  LHBTQPo-treated kenaf fibers retained high cellulose content.  Recalcitrant HexA had negative effects in bleaching kenaf pulp.  Xylanase applied after LMS system moderately removed HexA content.

a r t i c l e

i n f o

Article history: Received 20 August 2013 Received in revised form 31 October 2013 Accepted 7 November 2013 Available online 16 November 2013 Keywords: Kenaf TCF bleaching sequence Laccase-mediator system (LMS) Hexenuronic acids Xylanase

a b s t r a c t Enzymatic delignification with laccase from Trametes villosa used in combination with chemical mediators (acetosyringone, acetovanillone and 1-hydroxybenzotriazole) to improve the totally chlorine-free (TCF) bleaching of kenaf pulp was studied. The best final pulp properties were obtained by using an LHBTQPo sequence developed by incorporating a laccase-mediator stage into an industrial bleaching sequence involving chelation and peroxide stages. The new sequence resulted in increased kenaf pulp delignification (90.4%) and brightness (77.2%ISO) relative to a conventional TCF chemical sequence (74.5% delignification and 74.5% brightness). Also, the sequence provided bleached kenaf fibers with high cellulose content (pulp viscosity of 890 g  mL1 vs 660 g  mL1). Scanning electron micrographs revealed that xylanase altered fiber surfaces and facilitated reagent access as a result. However, the LHBTX (xylanase) stage removed 21% of hexenuronic acids in kenaf pulp. These recalcitrant compounds spent additional bleaching reagents and affected pulp properties after peroxide stage. Ó 2013 Elsevier Ltd. All rights reserved.

1. Introduction The global demand for natural fibers is steadily increasing through an also increasing demand for cleaner, greener industrial products. Kenaf plants absorb CO2 from the atmosphere to a greater extent than any other crop. Thus, each hectare of kenaf crop consumes as much CO2 during a single growing cycle as is released by 20 car exhausts in whole year (Kenaf Green Industries Ltd., 2013). Kenaf is also an eco-friendly nonwood plant as it requires no chemical pest control during its growth cycle. This makes it an ideal source of natural fibers for various industries (pulp and paper mills included). Enzymatic biobleaching with laccase-mediator systems (LMS) has been used with various now-wood plants. Including a biobleaching stage in some totally chlorine free (TCF) sequences has been found to facilitate bleaching and fiber functionalization (Aracri ⇑ Corresponding author. Tel.: +34 937398174; fax: +34 937398101. E-mail address: [email protected] (G. Andreu). 0960-8524/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.biortech.2013.11.014

et al., 2009; Fillat and Roncero, 2010; Fillat et al., 2010; Fillat et al., 2011). Kenaf pulp has been highly successfully processed in terms of delignification and brightness with TCF processes using a laccase–mediator system (Andreu and Vidal, 2011; Andreu et al., 2013). However, some chemical and morphological properties of the pulp have a decisive influence on the delignification efficiency obtained and require deeper investigation with a view to improving biobleaching results. Kenaf pulp has a high content in hexenuronic acids (HexA). These unsaturated compounds formed during pulp cooking by effect of methanol elimination from 4-O-methylglucuronic acid groups bonded to xylans as side groups (Johansson and Germgard, 2006). HexA are known have adverse effects on pulp bleaching and the properties of the resulting pulp. With regard to bleaching, hexenuronic acids contribute to kappa number (Costa and Colodette, 2007), retain metal ions by chelation and increase the consumption of bleaching agents (Vuorinen et al., 1999).

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Recent studies have shown that a xylanase/LMS treatment used as a bleaching stage can have a bleach boosting effect and reduce the HexA content of pulp (Valls and Roncero, 2009; Valls et al., 2010a; Fillat et al., 2011). The latter, interesting effect can be ascribed to xylan elimination: the xylanase pretreatment (X stage) removes xylan depositing onto fibers during cooking and increases fiber wall accessibility as a result. The main objective of this work was to develop an industrially applicable biobleaching sequence, which could compete with existing conventional TCF sequences in terms of delignification and brightness. Also, changes in residual lignin and HexA content in kenaf pulp during a TCF bleaching sequence including an LMS treatment are for the first time examined, and the effectiveness of using xylanase after an LMS treatment for the same purpose is assessed. 2. Methods 2.1. Raw material Unbleached pulp from kenaf (Hibiscus cinnabinus) was produced by soda–anthraquinone cooking at CELESA, S.A. (Spain). The pulp was washed with acidified (H2SO4) water at 3% consistency at pH 2 for 30 min to reduce its content in metal ions and remove impurities. The properties of the washed pulp were as follows: kappa number 12.9 ± 0.1, ISO brightness 35.0%, viscosity 925 ± 23 mL g1, according to ISO 302:2004, 2470-1:2009 and 5351:2011, respectively. Analyses were performed in duplicate for kappa number (measurement errors were all less than 0.5 times the standard deviation) and viscosity, and in quadruplicate for brightness (standard deviation = 0.1). Carbohydrate composition of the initial pulp was: 83.5 ± 0.2% glucan, 14.3 ± 0.06% xylan and 2.1 ± 0.1% klason lignin. After quantitative acid hydrolysis, the resulting hydrolysates were analyzed for glucan (HPLC determination of glucose) and xylan (HPLC determination of xylose), and the solid residue was used to quantify klason lignin. Analyses were performed in duplicate. The hexenuronic acid (HexA) content of unbleached pulp (55.6 ± 2.5 lmol  g1 oven-dried pulp) was determined by UV spectroscopy following the method described by Chai et al. (2001). The pulp additionally contained substantial amounts of metal ions and, especially, Fe2+ (937.0 ± 20.0 ppm) (de la Rosa, 2003). 2.2. Laccase and the L stage Laccase (EC 1.10.3.2) from Trametes villosa (Novozymes, Bagsvaerd, Denmark) was used in combination with three different mediators (i.e. in laccase–mediator systems), namely: acetosyringone (AS), acetovanillone (AV) and 1-hydroxybenzotriazole (HBT), which were all purchased from Sigma–Aldrich. Laccase activity was monitored by measuring the absorbance at 436 nm (e436 = 29 300 M1  cm1) during ABTS oxidation in 0.1 M sodium acetate buffer (pH 5). The L stage (viz. the laccase-mediator treatment) was carried out with 60 g of unbleached pulp at 10% odp consistency in an oxygen pressurized (0.6 MPa) reactor containing 50 mmol  L1 sodium tartrate buffer at pH 4, 20 U  g1 laccase (TvL) and 1.5% mediator, all relative to pulp dry weight. Pulp samples were also processed in the absence of mediator (laccase treatment). All mixtures were shaken at 60 rpm at 50 °C for 4 h. After the enzyme treatment, the samples were filtered and washed with deionized water. 2.3. Bleaching sequences: LQPo and LXP Two bleaching sequences were studied. In the LQPo sequence, the L stage was followed by a chelating step (Q stage) and a subse-

quent hydrogen peroxide stage (Po). The Q stage was conducted in the presence of 1% DTPA (diethylenetriaminepentaacetic acid) at 10% consistency, pH 5–6 and 85 °C for 1 h. The hydrogen peroxide treatment (Po stage) was conducted by using 3% H2O2 in 1.5% NaOH, 0.3% DTPA and 0.2% MgSO4 at 10% consistency in an oxygen pressurized (0.6 MPa) reactor with stirring at 30 rpm at 90 °C for 4 h. This stage was performed in three steps (t1 = 1 h, t2 = 1 h and t3 = 2 h) each including the addition of 1% odp H2O2. Then, the pulp samples were washed with acidified (H2SO4) water at 3% consistency at pH 2 for 30 min (Wa stage) and, finally, with deionized water. The residual liquors from each stage were collected for subsequent analysis. The other sequence (LXP) included an X stage after L. The xylanase enzyme used in this stage was PulpzymeÒ HC from Novozymes. The treatment was performed in polyethylene bags, using 3 U  g1 odp xylanase in 50 mmol L1 Tris–HCl buffer (pH 7) at 5% consistency at 50 °C for 2 h. The LX sequence was followed by an alkaline peroxide bleaching treatment. The P stage was conducted as a Po stage for 2 h in a Datacolor Ahiva Spectradye apparatus equipped with 150 mL closed vessels and without oxygen pressure. 2.4. Pulp properties Treated pulp samples were characterized in terms of kappa number (KN), brightness (%ISO) and viscosity. Delignification, brightness increase and HexA removal were calculated according to the following equations:

Delignification ð%Þ ¼

KNi  KNf  100 KNi

ð1Þ

Brightness increase ¼ Bð%ISOÞf  Bð%ISOÞi

ð2Þ

HexAi  HexAf  100 HexAi

ð3Þ

HexA removal ð%Þ ¼

where the subscript i refers to the unbleached pulp value, f to the final pulp after LQPo, LQPoWa and LXP treatments, and NK, B (%ISO) and HexA denote kappa number, brightness and HexA content, respectively. An estimate of the residual lignin content of each sample was obtained from the kappa number due to lignin (KNlig) (Li et al., 2002). NKlig was determined following removal of HexA by hydrolysis with mercury acetate. The contribution of HexA to kappa number (KNHexA) was calculated from

NKHexA ¼ NK  NKlig

ð4Þ

2.5. TCF chemical sequence (OpPoP) A previously assayed conventional TCF treatment (de la Rosa, 2003) was used as reference for the proposed biobleaching sequence. The conventional chemical sequence was an OpPoP where Op was a delignification stage with oxygen and hydrogen peroxide, Po a bleaching stage with pressurized hydrogen peroxide and P a bleaching stage with hydrogen peroxide. Kenaf pulp bleached with the OpPoP sequence exhibited 74.5%ISO brightness, 74.5% ‘‘real’’ delignification and 660 mL  g1 viscosity. 2.6. Scanning electron microscopy Handsheets made from treated pulp (ISO 3688: 1999) were cut into small pieces 2 mm long and examined for surface morphology at variable magnification with a JEOL JSM-6400 Scanning Electron Microscope (SEM).

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3.1. LQPo biobleaching sequence

from Table 2, acid washing additionally increased brightness by effect of neutralization of the pulp converting colored quinones into colorless phenols.

Table 1 shows the results obtained with the LQPo and LQPoWa sequences – the latter involved acidified water washing at the end of the process. Based on these results, and on Eqs. (1) and (2), the increase in delignification and brightness (Table 2) with respect to the unbleached initial pulp were calculated to be KNi = 12.9 and B (%ISO)i = 35.0, respectively. A comparison of a previously reported LP sequence (Andreu et al., 2013) and the proposed LQPo sequence revealed that the latter was more efficient in terms of delignification and brightness. The chelating stage included in the biobleaching sequence facilitated delignification by effect of DTPA chelating some transition metals in the pulp and facilitating their dissolution and removal by subsequent washing (Lapierre et al., 1995). The removal of metal ions prevented hydrogen peroxide decomposition and led to more efficient bleaching judging by the results. The pulp samples obtained after the Po stage were washed with acidified water (Wa stage) and, finally, with deionized water. As can be seen

3.1.1. HexA content: ‘‘Real’’ delignification and brightness As noted in the Introduction, one adverse effect of HexA in pulp is their interference with the standard method for determining kappa number, which involves titration with KMnO4 in an acid medium (Costa and Colodette, 2007). Kappa number is a combination of the contributions of residual lignin (NKlig), HexA (KNHexa) and other minor structures (Valls and Roncero, 2013). HexA, KN and NKlig in all samples were measured after each enzymatic stage and bleaching sequence and KNHexA was calculated from Eq. (4). Figs. 1a and 2a show their values for the L stage and LQPoWa sequence. As can be seen from Fig. 1a, HexA were removed during the L stage – to the extent shown in Fig. 1c. HexA may have been removed by effect of the oxidation of their double bonds by radicals formed during the enzymatic stage (Valls and Roncero, 2013). As can be seen from Fig. 1 and inferred from the standard deviation of measurements, the HexA content remained

3. Results and discussion

Table 1 Kappa number and brightness obtained after L and LX stages and LQPo/LQPoWa, LXP biobleaching sequences, with laccase alone and the three laccase-mediator systems. L

Laccase AS AV HBT

LQPo

LQPoWa

LX

LXP

KN

B%ISO

KN

B%ISO

KN

B%ISO

KN

B%ISO

KN

B%ISO

11.6 11.7 14.1 10.5

35.1 30.8 34.3 34.7

6.6 6.9 7.1 5.8

74.4 70.4 72.6 74.0

6.3 7.1 6.9 5.8

75.7 73.3 74.0 77.2

10.8 11.0 12.1 9.3

36.5 33.9 37.7 37.4

6.8 6.2 6.7 5.8

62.3 66.6 61.4 70.0

Table 2 Percent increase in delignification, brightness and viscosity resulting from the LQPo/LQPoWa and LXP biobleaching sequences for laccase alone and the three laccase-mediator systems. LQPo

LQPoWa LXP LQPo

Delignification increase (%)

Viscosity (mL  g1)

Brightness increase (%ISO)

Laccase

AS

AV

HBT

Laccase

AS

AV

HBT

Laccase

AS

AV

HBT

48.8 51.2 47.3

46.5 50.1 51.9

45.0 46.5 48.1

55.2 55.1 55.0

39.4 40.7 27.3

35.4 38.3 31.6

37.6 39.0 26.4

39.0 42.0 35.0

– 861 ± 59 863 ± 27

– 922 ± 31 866 ± 36

– 941 ± 7 892 ± 11

– 890 ± 16 863 ± 14

Fig. 1. Pulp HexA content (lmol/g odp) after (a) L/QPoWa and (b) LX/LXP. Percent HexA removal by xylanase after (c) L/LQPoWa and (d) LX/LXP.

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(a)

(b)

Fig. 2. Comparison of KNlig and KNHexA of kenaf pulp after (a) L/LQPoWa and (b) LX/ LXP enzymatic sequence.

unchanged after the LQPoWa sequence. This is consistent with the similarity of NKHexA after L and LQPoWa (Fig. 2a). Fig. 2a also shows the kappa number due to residual lignin (NKlig) after LQPoWa; as can be seen, KNlig was substantially reduced from its initial value (7.3). These results were used to estimate the real delignification after treatment with the different laccase–mediator systems by using Eq. (1) with NKi = 7.3 and NKf as the corresponding value for NKlig (Fig. 2a). Based on the results, the proposed bleaching sequence boosts delignification (90%) with the laccase–HBT system. There were no substantial differences between using laccase alone or in combination with AS or AV as mediator (delignification amounted to about 75% in all cases). As can be seen from Fig. 3 (line a), kappa number and brightness for the LQPoWa sequence (Table 1) were linearly related. The the-

oretical brightness of each sample as calculated from the kappa number due to lignin (NKlig) was estimated by extrapolation from the linear relationship and is shown as B⁄ in Fig. 3. The theoretical brightness exceeded the experimental brightness, which suggests that hexenuronic acids in the pulp limited its bleachability, possibly by effect of HexA binding metals and causing their retention in pulp fibers. In fact, metal cations are known to catalyze the decomposition of hydrogen peroxide (Vuorinen et al., 1999) and detract from bleaching efficiency as a result. 3.1.2. Viscosity Viscosity after the LQPoWa sequence was not significantly different from the initial value, i.e. it was not appreciably reduced by the enzymatic treatment (Table 2). The fact that viscosity was essentially retained indicates that cellulose in the pulp was scarcely decomposed. This result is a substantial improvement in relation to the TCF no enzymatic treatment (OpPoP), which provided a pulp viscosity of 660 mL  g1. Based on the foregoing, the proposed TCF treatment using a laccase-mediator system led to increased delignification relative to no enzymatic treatment (viz. 90.4% with the laccase-HBT, 76.7% with the laccase-AS and laccase-AV and 75.3% with laccase compared to the 74.5% with no enzymatic treatment). Also, brightness was increased by 2.7%ISO with the laccase-HBT treatment and 1.2%ISO by laccase alone in relation to the use of OpPoP treatment. Finally, the enzymatic treatment resulted in efficient delignification while preserving the integrity of cellulose chains. One added advantage of the proposed enzymatic treatment (LQPoWa) is that it is industrially feasible. In fact, its chemical stages (Q and Po) and the final acidified washing are used at present by the pulp and paper industry (e.g. by CELESA, S.A.). 3.1.3. Effluent properties The effluents produced by the proposed sequence were analyzed for chemical oxygen demand (COD), color and toxicity according to Fillat et al. (2011). Table 3 shows the COD and color values for the effluents from the enzymatic stage (L) and the final bleaching treatment (LQPoWa), as well as their toxicity. As can be seen, the main source of organic environmental pollutants was the L stage with the different laccase-mediator systems and laccase alone. Regarding effluents of L stage, (i) COD increases for the laccase-mediator systems, save for HBT; it should be noted that COD was ascribed partly to the presence of sodium tartrate buffer and the formation of certain products during the reaction process (Fillat et al., 2010) (ii) effluent color can be due to the presence of chromophoric groups formed by oxidation and/or degradation of lignin and the mediators (Andreu et al., 2013), and (iii) effluent toxicity was found to be significantly increased by the phenolic mediator AS. Using an LHBT stage produced an effluent of acceptable characteristics, which, however, would require further processing to comply with existing pollutant emission limits. Finally, the COD and color values for the effluents from the different LQPoWa treatments were consistent with the use of hydrogen peroxide as the chemical bleaching agent. 3.2. Use of xylanase after the laccase-mediator treatment

Fig. 3. Plot of brightness versus total kappa number (KN/N, j) and kappa number due to lignin (KNlig/4, h) in pulps subjected to LQPoWa sequence (line a) and LXP sequence (line b). The dashed square bounds the values of theoretical brightness (B⁄) based on the kappa number due to lignin (NKlig).

As stated in Section 3.1.1, the HexA content of kenaf pulp affects the outcome of its bleaching. In this work, xylanase was used after the laccase–mediator treatment (LXP sequence) to assess the enzyme efficiency in removing HexA from kenaf pulp. Table 1 shows the KN and brightness values for xylanase-treated kenaf pulp samples after the LX stage and LXP sequence. Increase in delignification and brightness, as well as viscosity values for LXP sequence are presented in Table 2. The results are discussed in detail below.

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G. Andreu, T. Vidal / Bioresource Technology 152 (2014) 253–258 Table 3 COD, color and toxicity of the effluents from L stage and LQPoWa biobleaching sequence with laccase alone and the three laccase-mediator systems. L

COD (kg O2/tpulp) Color (kg Pt/tpulp) Toxicity (T.U.)

LQPoWa

Laccase

AS

AV

HBT

Laccase

AS

AV

HBT

51 ± 9 11 ± 1 36 ± 3

151 ± 24 74 ± 14 396 ± 91

81 ± 13 27 ± 6 5±1

40 ± 7 105 ± 20 59 ± 6

23 ± 3 3±4 4±0

16 ± 2 3±4 1±0

17 ± 3 3±4 3±1

21 ± 1 3±4 3±1

3.2.1. HexA removal The HexA content of kenaf pulp fibers subjected to LX was similar to or lower (with AV and HBT as mediators) than that of fibers subjected to an L treatment alone (Fig. 1a and b). The LX stage boosted HexA removal (Fig. 1d), probably through elimination of xylans from fiber surfaces (Shatalov and Pereira, 2007, 2009). SEM images (presented as Supplementary material, Fig. S1) showed that the xylanase treatment cleaned the cellulose surface and increased external fibrillation relative to the L stage. Based on the data of Fig. 1b and d, the greatest effect of xylanase on HexA removal occurred at the P stage. This is consistent with extraction of HexA bound to xylan–lignin complexes under the alkaline conditions used (Li et al., 2002; Costa and Colodette, 2007). In fact, a xylanase treatment modifies pulp fiber surfaces and produce alterations (grooves) in some fiber regions that must have facilitated reagent penetration (Fig. S1). 3.2.2. Effect of L and LX on kappa number and brightness Table 1 shows the kappa number and brightness values obtained after the L and LX stages. As can be seen, xylanase decreased kappa number and increased brightness. These results are consistent with enhanced removal of lignin leading to smaller NKlig values after LX (Fig. 2a and b). The increased removal of lignin led to also increased pulp brightness. As noted earlier, xylanase modified fiber surfaces, thereby facilitating the removal of previously oxidized lignin in the L stage. 3.2.3. Effect of xylanase As can be seen from Table 1, the LXP sequence reduced kappa number relative to LP (Andreu et al., 2013). This result emphasizes the effect of xylanase on the process: the enzyme facilitates access of NaOH inside fibers and subsequent dissolution of modified lignin, thereby reducing kappa number. However, the increased removal of lignin resulted in no increase in brightness, probably as a result of a reduced efficiency of the hydrogen peroxide stage in LXP. The increased accessibility of fibers may have accelerated H2O2 catalyzed decomposition by metal ions still retained by chelation by HexA. As can be seen from Fig. 1d, the acids were inadequately removed (maximum 35%) by the LXP treatment and remained in more inner fibers. Fig. 2a and b illustrate the changes in NKlig and NKHexA caused by the LQPoWa and LXP treatments. As can be seen, HexA removal by the LXP treatment reduced NKHexA; also, however, it increased NKlig, which suggests reduced extraction of modified lignin by the LMS treatment. A fraction of the NaOH used was spent on removing additional amounts of HexA bound to xylan–lignin complexes, which seemingly reduced extraction of modified lignin at the xylanase stage. The decreased ‘‘real’’ delignification obtained is consistent with increased consumption of bleaching reagents by effect of the presence of HexA as found in previous studies (Vuorinen et al., 1999). The brightness values obtained after LXP can be ascribed to a reduced efficiency of P relative to Po and, especially, to the use of no chelating treatment in the sequence. Line b in Fig. 3 presented a linear relationship between kappa number and brightness (see KN and brightness values after LXP in Table 1). The theoretical brightness of each sample, based on

the kappa number due to lignin (NKlig), was estimated by extrapolation from the linear relationship and is denoted by B⁄ in the figure. As can be seen, the theoretical brightness exceeded both the experimental brightness and the B⁄ value after the LQPoWa sequence. This suggests that xylanase could be an effective enhancer of brightness in kenaf pulp, but after an effective bleaching stage. 3.2.4. Viscosity As can be seen from Table 2, pulp viscosity was only slightly reduced by the LXP bleaching sequence. The commercial xylanase used was specific for xylans and cellulose was unaffected. Similar effects on fiber viscosity were previously observed with some other xylanases (Valls et al., 2010b). 3.2.5. Changes in kenaf fiber morphology Thermogravimetric analysis (Andreu et al., 2013) demonstrated that the cellulose surface of unbleached kenaf pulp was modified, possibly as a consequence of the deposition of short-chain xylans, lignin and nonfibrous materials during cooking of the kenaf biomass. Changes in fiber morphology in laccase, xylanase and laccase/xylanase treated pulp were examined by SEM. Xylanase modified kenaf fiber surfaces (Fig. S2 Supplementary material), removing surface compounds, including short-chain xylans, lignin and nonfibrous material remaining after application of laccase. Also, the laccase/xylanase treatment removed the previous compounds and caused more marked changes in fiber surfaces relative to laccase alone. The removal of surface compounds by xylanase decreased kappa number and increased brightness (Fig. S2). However, no synergistic effect was observed after LX, which suggests that laccase caused some deposit onto the pulp and hindered subsequent chemical bleaching as a result (Andreu et al., 2013). The findings highlighted, using xylanase after a laccase-mediator treatment has three clear-cut effects: (i) modifying fiber surfaces; (ii) facilitating access of bleaching reagents to fibers; and (iii) slightly enhancing HexA removal. As stated in Section 3.2.3, additional bleaching reagents were consumed during the LXP treatment by residual HexA and the metal ions they retained. This resulted in lower than expected delignification and bleaching efficiency. However, HexA in bleached kenaf pulp can be advantageous with a view to developing specialty papers by virtue of the antioxidant properties of TCF-treated pulp containing residual HexA (Valls and Roncero, 2013). 4. Conclusion Trametes villosa laccase was used in combination with various mediators in a TCF bleaching sequence to assess the delignification potential of laccase-mediator systems (LMS). Based on the results, the laccase-HBT system was especially effective for bleaching kenaf pulp. The bleaching sequence used provided kenaf fibers with a high cellulose content and brightness above 77%ISO. An LX stage was used to assess the ability of xylanase to remove HexA from kenaf pulp. The results obtained after an LXP treatment revealed the presence of recalcitrant HexA in the pulp. As shown here, HexA in kenaf pulp restricts its bleachability.

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Acknowledgements The authors would like to acknowledge Spain’s MICINN for funding this research in the framework of Projects AGAUR2009GR00327, BIOFIBRECELL (CTQ2010-20238-CO3-01) and BIOSURFACEL (CTQ2012-34109). The authors are also grateful to CELESA, S.A. (Tortosa, Spain) for supplying the starting kenaf pulp. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.biortech.2013.11. 014. References Andreu, G., Vidal, T., 2011. Effects of laccase-natural mediator systems on kenaf pulp. Bioresource Technology 102, 5932–5937. Andreu, G., Barneto, Agustín, G., Vidal, T., 2013. A new biobleaching sequence for kenaf pulp: influence of the chemical nature of the mediator and thermogravimetric analysis of the pulp. Bioresource Technology 130, 431–438. Aracri, E., Colom, J.F., Vidal, T., 2009. Application of laccase-natural mediator systems to sisal pulp: an effective approach to biobleaching or functionalizing pulp fibres? Bioresource Technology 100, 5911–5916. Chai, X.S., Zhu, J.Y., Li, J., 2001. A simple and rapid method to determine hexenuronic acid groups in chemical pulps. Journal of Pulp and Paper Science 27, 165–170. Costa, M.M., Colodette, J.L., 2007. The impact of kappa number composition on eucalyptus kraft pulp bleachability. Brasil J. Chem. Eng. 24, 61–71. de la Rosa A., 2003. Utilización papelera de fibras no madereras (kenaf y Miscanthus sinensis) Estudio de les secuencias ECF y TCF. PhD Thesis. Departament

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