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Comparative Study of the Effects Induced by Different Laccase-Based Systems on Sisal Cellulose Fibers Elisabetta Aracri,† Agustín G. Barneto,‡ and Teresa Vidal*,† †

Textile and Paper Engineering Department, Universitat Politècnica de Catalunya, Colom 11, E-08222 Terrassa, Spain Department of Chemical Engineering, Physical Chemistry and Organic Chemistry (Campus de Excelencia Internacional Agroalimentario, ceiA3), University of Huelva, Campus El Carmen, 21071 Huelva, Spain

‡

ABSTRACT: This paper reports a comparative study of the effects induced on sisal pulp fibers by three different laccase-based systems, namely, laccase−sinapyl aldehyde, laccase−ferulic acid, and laccase−TEMPO systems, applied to perform biobleaching, biografting, and cellulose oxidation, respectively. (The abbreviations SLD, FRC, and TEMPO are used to represent sinapyl aldehyde, ferulic acid, and the 2,2,6,6-tetramethylpiperidine-1-oxyl free radical.) A novel aspect of this study was the use of thermogravimetric analysis (TGA) to monitor surface changes in cellulosic microfibrils during the enzyme treatments and gain a greater understanding of the mechanisms of action of the laccase-based systems. The different modes of action of the studied laccase-based systems reflected in the different degradation profiles of pulps after treatment. TGA showed laccase to modify the thermal degradation path of the initial pulp, increasing the proportion of cellulose degrading at low temperature. The addition of SLD resulted in virtually no change of the thermal degradation path of the initial pulp, indicating that the laccase−SLD system basically exerted its action on the lignin component of fibers. In contrast to SLD, FRC was found to significantly increase the amount of the paracrystalline fraction of cellulose, probably as a consequence of its incorporation into fibers. The presence of TEMPO, especially under those conditions boosting the oxidative functionalization, was found to cause an intense degradation of cellulose and the formation of a substantial amount of amorphous cellulose degrading at low temperature. A novel aspect of the laccase−TEMPO system was identified in this work: its ability to reduce the hexenuronic acids (HexA) content content of pulp, under specific reaction conditions.

1. INTRODUCTION In the last decades, laccases (EC 1.10.3.2) have attracted increasing attention in the pulp and paper research as a potential tool for developing cleaner processes and modifying lignocellulosic fibers to obtain novel, sustainable products, by virtue of its operational flexibility and broad substrate specificity.1 On the other hand, high-priced nonwood fibers such as those from sisal, which are typically used to manufacture specialty paper, are specially suitable for application of enzyme technologies, thanks to the increased profit margins that they provide. Ever since the discovery of chemical mediators capable of extending enzymatic oxidation to nonphenolic compounds, research interest has mainly focused on the potential of laccase−mediator systems for aiding pulp bleaching. Research has shown that the laccase−mediator systems (LMS) can substantially reduce the requirements of bleaching chemicals for chemical pulp bleaching, or allow bleaching to smaller kappa numbers and higher brightness.2,3 Despite the associated advantages of LMS, mediators are expensive and can generate toxic derivatives. There have been recent trends to use ecofriendly, potentially cost-effective alternative mediators such as naturally occurring phenols, which can be readily obtained from plants and spent pulping liquors or directly from fungal metabolism.4−7 Recently, laccases have attracted considerable attention as a means for modifying fiber chemistry and paper properties (particularly, strength-related properties). Radical coupling reactions involving phenolic compounds have been used to © 2012 American Chemical Society

enable bonding of low-molecular-weight compounds to ligninrich cellulose fibers.8 Radical coupling reactions competing with delignification represent an adverse, undesirable phenomenon in the biobleaching process.4,9 However, they have aroused increased interest as the key mechanisms behind the biograf ting of low-molecular-weight phenols onto pulp fibers. This is a versatile functionalization method by virtue of the enzyme’s nonspecific substrate requirements, which allow bonding a wide range of phenolic compounds and thus incorporating several desired properties into the fiber matrix.8,10,11 The feasibility of this approach has been demonstrated in many studies; interest, however, has focused on wood materials and lignin-rich fibers, and little research has comparatively been carried out on nonwood fibers. The laccase−TEMPO mediated system (where TEMPO represents the 2,2,6,6-tetramethylpiperidine-1-oxyl free radical) provides a potential approach to oxidatively modifying cellulose pulp fibers. TEMPO-mediated oxidation is a well-known procedure to introduce carboxyl and aldehyde functional groups into cellulose in aqueous media at room temperature.12 Although the ability to use laccase to catalyze the regenerative oxidation of TEMPO has been demonstrated,13 the reaction is commonly carried out in the presence of NaClO/NaBr as a cooxidizer system.14−16 TEMPO-mediated oxidation has been Received: Revised: Accepted: Published: 3895

December 2, 2011 February 16, 2012 February 21, 2012 February 21, 2012 dx.doi.org/10.1021/ie2028206 | Ind. Eng. Chem. Res. 2012, 51, 3895−3902

Industrial & Engineering Chemistry Research

Article

Table 1. Operating Conditions of Each Treatment Applied in the Laccase-Based Approaches for Biobleaching, Biografting, and TEMPO-Mediated Oxidation of Sisal Pulp Fibersa Biografting Treatmentsc

Bleaching Sequenceb operating conditions xylanase dose (U/g odp) laccase dose (U/g odp) phenol/TEMPO dose (% odp) Tween 80 dose (% w/v) DTPA (% odp) NaOH (% odp) H2O2 (% odp) MgSO4 (% odp) reaction time (h) PFI revolutions pulp consistency (%) pH applied oxygen temperature stirring speed (rpm) a

X

Laccase−TEMPO Treatmentsd

L20FRC1.5

L40FRC3.5

L100T2t20

L20T8t20

L60T5t14

LT5% _0rev

LT5% _4500rev

40

20

40

100

20

60

20

20

1.5

1.5

3.5

2

8

5

8

8

0.05

0.05

0.05

4

4

20 4500 1

14 4500 1

20 5

20 4500 5

5 bubbling room 60

5 bubbling room 60

5 0.6 MPa room 60

5 0.6 MPa room 60

LSLD

Q

PO

3

1.0

2

4

1

0.3 1.5 3 0.2 4

10

5

5

5

5

5

20 4500 1

7

4 0.6 MPa 50 °C 30

5−6

10−11 0.6 MPa 90 °C 30

4 0.6 MPa 50 °C 30

4 0.6 MPa 50 °C 30

5 bubbling room 60

50 °C

85 °C

Data taken from refs 21−24. bData taken from ref 23. cData taken from ref 21. dData taken from refs 22 and 24.

the amount of enzyme converting 1 μmol of xylan reducing sugar (measured as xylose equivalents) per minute at pH 5 at 50 °C. Unbleached sisal (Agave sisalana) pulp from a soda− anthraquinone cooking process was supplied by the CELESA pulp mill (Tortosa, Spain). The pulp, at 2% consistency, was conditioned with H2SO4 at pH 4 under stirring for 30 min, which was followed by passage through a glass filter funnel and extensive washing with deionized water. This step was needed to remove contaminants and metals, and also to bring the pulp to the pH required for the enzyme treatment. 2.2. Pulp Treatments. Sisal pulp was bleached by means of two different totally chlorine-free (TCF) biobleaching sequences, namely, LSLDQPO and XLSLDQPO (where L denotes the laccase-mediator treatment, Q a chelating treatment, PO an oxygen-reinforced hydrogen peroxide treatment, and X an enzyme pretreatment with xylanase).23 LSLD and PO stages were carried out in an oxygen-pressurized reactor, whereas X and Q stages occurred in polyethylene bags immersed in a thermostatic water bath, where pulps were stirred by hand. Biografting treatments were performed in an oxygenpressurized reactor similar to the L stage of the bleaching sequence.21 Three laccase−TEMPO treatments were performed in a jar testing apparatus under oxygen bubbling under different conditions of laccase dose, TEMPO dose, and reaction time.22 Prior to treatments, pulp samples were disintegrated for 30 000 revolutions, filtered through a Buchner funnel, and refined at 4500 revolutions, according to ISO Standard 5264. Two laccase−TEMPO oxidation treatments were performed at increased pulp consistency in a pressurized oxygen reactor on unrefined and refined pulps.24 At the end of each treatment, pulps were passed through a glass filter funnel and extensively washed with deionized water until a colorless, neutral filtrate was obtained. The detailed

successfully exploited to improve various physical properties of pulp fibers, including interfiber bondingand, hence, the strength-related properties of the resulting paper.17,18 Environmental concerns have recently driven research interests into the development of halide-free oxidative systems. One promising approach for this purpose is the use of laccase, together with oxygen as primary oxidants.13,19 Similarly to the NaClO/NaBr process, the oxoammonium ion is regenerated in situ, so only oxygen is consumed in the course of the reaction. In laccase-based treatments of pulp fibers, the effects produced on the cellulose microfibril surface have not or have scarcely been investigated to date.20 Thermogravimetric analysis (TGA) is a powerful tool to detect the chemical changes on the microfibril surface and determine the crystalline and amorphous cellulose contents. In this work, we compared the effects of the above-described laccase-based approaches, applied in previous studies,21−24 in terms of pulp properties and thermal degradation profiles. The studied laccase-based treatments were as follows: biobleaching, with and without a xylanase pretreatment, using the natural compound sinapyl aldehyde (SLD) as a laccase mediator; biografting, using the natural compound ferulic acid (FRC); and cellulose oxidation, mediated by TEMPO.

2. EXPERIMENTAL SECTION 2.1. Chemicals, Enzymes, and Pulp. All chemicals were purchased from Sigma−Aldrich and used as received. Laccase from Trametes villosa was supplied by Novozymes (Bagsvaerd, Denmark) and frozen until use. Laccase activity was determined via the oxidation of 2,2′-azino-bis-(3-ethylbenzylthiozoline-6sulfonate) (ABTS). One activity unit (U) was defined as the amount of laccase transforming 1 μmol/min ABTS to its cation radical (ε436 (nm) = 29 300 M−1 cm−1) in 0.1 M sodium acetate buffer at pH 5 at 25 °C. A commercial xylanase (Pulpzyme HC) supplied by Novozymes; one activity unit was defined as 3896

dx.doi.org/10.1021/ie2028206 | Ind. Eng. Chem. Res. 2012, 51, 3895−3902


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(fwhm) of the diffraction peaks was used to determine crystallite width (Bhkl), using the Scherrer equation:

operation conditions used in each treatment are reported in Table 1. 2.3. Pulp Properties. Pulps obtained from L and PO stages of the biobleaching sequences were analyzed in terms of kappa number due to lignin (KNlig), brightness, and viscosity. Pulps coming from the biografting treatments were analyzed for KNlig, brightness, Klason lignin, and surface anionic charge after Soxhlet extraction with acetone25 in order to remove the fraction of FRC that failed to couple to fibers. KNlig, hexenuronic acids content (HexA), carboxyl and aldehyde groups contents, and borohydride viscosity was determined in pulps treated with the laccase−TEMPO system. The HexA content was determined using ultraviolet (UV) spectroscopy.26,27 KNlig, providing an estimate of the actual lignin and lignin-bound ferulic acid content of the pulp,27,28 involved measuring the kappa number following removal of HexA by acid hydrolysis with mercury acetate and efficient washing with deionized water. Kappa number and brightness were determined according to ISO Standard 302 and ISO Standard 3688, respectively. Pulp viscosity was determined in accordance with ISO Standard 5351-1. Borohydride viscosity was measured after treatment with 2% NaBH4, at 5% consistency at room temperature for 30 min. The surface anionic charge of the fibers was determined by polyelectrolyte titration, using a particle charge detector (Mütek PCD 03, Germany), as described elsewhere.21 Klason lignin in each pulp was determined as the fraction of lignin insoluble in sulfuric acid resulting from acid hydrolysis, as described by Aracri and Vidal.23 The bulk acid group content was determined by conductometric titration, as described by Aracri et al.22 Pulp samples were oxidized with NaClO2 for selective conversion of aldehyde groups into carboxyl groups at room temperature for 48 h. The carboxyl content was determined with the conductometric titration method. The carboxyl groups formed via the effect of NaClO2 oxidation were assumed to be derived from aldehyde groups originally present in the pulp.29 All pulp analyses were carried out in duplicate. 2.4. Thermogravimetric Analysis. Thermogravimetric (TG) runs were carried out with a Mettler Toledo apparatus (model TGA/SDTA851e/LF1600) on samples with masses of ∼5 mg. Pyrolysis and combustion runs were carried out in nitrogen and synthetic air (N2:O2 4:1), respectively. Three heating rates (5, 10, and 20 °C/min) were applied, from 25 °C to 900 °C. Deconvolution of the TG curves was performed according to Barneto et al.30 In the present study, the pulps were modeled as a four-pseudo-component system consisting of hemicellulose (H), crystalline (C1) and amorphous (C2) celluloses, lignin (L), and their respective chars. 2.5. X-ray Diffraction. X-ray diffraction (XRD) measurements were performed with Cu Kα1 radiation (λ = 0.15418 nm) at 40 kV and 30 mA, using a Bragg−Brentano θ/2θ geometry X-ray diffractometer (Siemens, Model D-500). A divergence aperture of 0.3° and a reception aperture of 0.05° were used. The experimental XRD signal was fitted by means of Gaussian distributions, which include the amorphous background. The crystallinity of the pulps was obtained as a ratio between the area of the crystalline cellulose peaks and the total area, which includes the amorphous background contribution. The equatorial dimension of the crystallites was determined by using the (200) reflection. The full width at half-maximum

Bhkl =

0.9λ (Δ2θ)2 − (Δ2θ0)2 cos θ

where λ is the X-ray wavelength, Δ2θ the fwhm of each peak, Δ2θ0 the apparatus broadening, and θ the Bragg angle.

3. RESULTS AND DISCUSSION In previous works, laccase-aided bleaching and functionalization of pulp fibers have been studied via pulp properties (functional groups content, chemical composition, optical properties, among others). However, this approach excludes the detection of potential structural changes on the cellulose microfibril surface, which can provide further useful information concerning the mechanisms of action of the different laccasebased systems on pulp fibers. As a complementary study, and for the first time on sisal pulp, TGA was applied in this work to analyze the effects of enzyme treatments on sisal cellulose fibers from a different perspective. 3.1. Effects of Different Laccase-Based Systems on the Thermal Degradation Profile of Cellulose: Actual and Apparent Crystallinity. According to Nishiyama et al.,31 cellulose microfibril is a thin and long crystalline entity that consists of bundles of cellulose polymer chains with alternate crystalline (cellulose crystallites) and amorphous zones. The crystallite surface is ordered, and a network of hydrogen bonds protects it from external attacks (i.e., oxygen), including thermal degradation.20 It is known that crystalline cellulose thermally degrades at higher temperature than amorphous cellulose or hemicellulose. However, if the crystallite surface undergoes damage (i.e., after laccase treatment), its cellulose chains can degrade at lower temperature20 as amorphous cellulose (paracrystalline cellulose).32 The changes in the shape of the differential thermogravimetry (DTG) curves (mass loss rate versus temperature) are greater when the heating is performed in an air atmosphere. If oxygen is present, the structural degradation of the crystalline network at high temperature is accompanied by oxidation in the surface zones where the hydrogen bond network was previously weakened. Therefore, the presence of oxygen during TGA makes detection of the changes on the cellulose microfibril surface easier to identify. In an air environment, the thermal degradation of pulp consists of two mass loss stages. The first stage, similar to that observed in a nitrogen atmosphere, is related to hemicellulose and cellulose volatilizations and yields a sharp peak close to 330 °C in the DTG curve. The second stage, which is related to the oxidation of the carbonaceous residue (char), yields a peak between 400 °C and 450 °C. On this basis, Figure 1, which reports the volatilization stage of laccase-treated pulps, proves that the compound used in combination with laccase had an important effect on the thermal degradation profile of pulps. While the SLD-treated pulp volatilized in a narrow temperature interval (sharp peak) at high temperature, the TEMPO-treated pulp volatilized in a very broad temperature interval, showing an important proportion of cellulose degrading at low temperature (broad peak). Finally, the FRC-treated pulp was located at an intermediate position. Since these changes in the DTG curves are caused by changes in the microfibril surface, the compounds used can be ordered according to their aggressive behavior toward cellulose. While SLD almost did not 3897



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Figure 1. Thermal degradation profiles of sisal pulps treated with different laccase-based systems (laccase−sinapyl aldehyde (laccase− SLD), laccase−ferulic acid (laccase−FRC), and laccase−TEMPO). TG runs were performed in in air environment at a heating rate of 5 °C/min.

affect cellulose, FRC and, mainly, TEMPO damaged surface cellulose chains, increasing the proportion of paracrystalline cellulose. Deconvolution of the DTG curve allows calculation of the crystalline and amorphous cellulose contents in pulp and, consequently, its apparent crystallinity20 (Figure 2a). Since crystalline cellulose is the one crystalline component in pulp, the apparent crystallinity is its crystalline cellulose percentage. Apparent pulp crystallinity is different from actual crystallinity measured by XRD. According to the TG approach, both amorphous and paracrystalline celluloses degrade at low temperature, being considered as noncrystalline cellulose. However, although external agents such as laccase-based systems can disorder the external layer of microfibril and increase the paracrystalline cellulose content, the glucose nodes of the cellulose lattice remain in the same positions; therefore, XRD, based on the radiation reflection provoked by certain planes of lattice, does not detect crystallinity changes. Consequently, while the apparent crystallinity measured by TGA diminished when the microfibril surface became disordered, the actual crystallinity measured by XRD did not undergo any change (see Figure 2b). According to Figure 2b, XLSLDQPO and L40FRC3.5 pulps are two opposite cases. In the case of XLSLDQPO pulp, the apparent crystallinity is high (65%), achieving the same value as the actual crystallinity. This indicates the absence of paracrystalline cellulose and, consequently, the entire cellulose content (according to TGA) consists of crystalline (78%) and amorphous (intercrystallite) (22%) cellulose fractions. By contrast, in L40FRC3.5 pulp, the apparent crystallinity is significantly lower (38%) than the actual crystallinity (65%). Therefore, paracrystalline cellulose surrounds crystallites and the entire cellulose content consists of crystalline (49%), amorphous (22%), and paracrystalline (29%) cellulose fractions. Based on apparent crystallinity data and the B200 Scherrer size, a simplified microfibril model was built. For XLSLDQPO pulp, the cellulose microfibril consisted of a cylinder with consecutive crystalline (78%) and amorphous (22%) regions with a diameter of 4.1 nm. In the case of L40FRC3.5 pulp, a basic geometrical calculation showed that the surface of the

Figure 2. (a) DTG curve deconvolution for L40FRC3.5 pulp. (b) Comparison between actual crystallinity measured by XRD (red line) and apparent crystallinity obtained from deconvolution of the DTG curve (blue bars).

crystalline region was transformed to paracrystalline cellulose, until reaching a depth of 0.43 nm, leaving a crystalline core with a radius of 1.62 nm. 3.2. Biobleaching. Three TCF biobleaching sequences were applied to sisal pulp, namely, LQPO, the control sequence where the L stage was performed with 40 U/g odp laccase; LSLDQPO, where L was performed in the presence of SLD, and XLSLDQPO, which included a xylanase pretreatment as an additional stage. Because of the high content of hexenuronic acids (HexA) in soda-AQ sisal pulp and their important contribution to the kappa number, the kappa number due to lignin (KNlig) was determined after the removal of HexA, in order to assess the actual delignifying effect of the different treatments. Table 2 reports the values of KNlig, brightness, and viscosity of pulps obtained from L and PO stages of the three bleaching sequences. The analysis of pulp properties proved that laccase oxidized lignin in the absence of a mediator. The laccase-catalyzed oxidation of lignin explains the reduction in KNlig (from 3.9 to 3.4) and the increase in brightness (from 37.9 to 40.0). However, at the same time, laccase also attacked cellulose. TGA showed that laccase, by itself, modified the thermal degradation path of the initial pulp, promoting degradation at low temperature (between 280 and 300 °C), that is, transforming crystalline cellulose into paracrystalline cellulose (see Figure 3). Thus, it seems that certain damage on the external layer of the microfibril is a collateral and inevitable consequence of laccase3898



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has on cellulose. TGA showed that PO treatment increased the amount of cellulose degrading at low temperature (paracrystalline cellulose). Both L- and LSLD-treated pulps showed broader DTG peaks after the bleaching sequences (LQPO and LSLDQPO), which exposed damages on the fiber surface and loss of surface crystallinity. The increased viscosity values obtained at the end of the sequences including an X stage may have resulted from partial removal of the xylans from the pulp, increasing the average degree of polymerization of carbohydrates in the fibers.34 The xylanase effect of removing xylans deposited on the surface of the fibers and providing a cleaner microfibril surface was visible in the thermal degradation profile of the X-treated pulp after the PO stage, which showed a sharper peak than that observed in the absence of an X stage (see Figure 2b). 3.3. Biografting. Previous studies,21,25 which reported the effects of various p-hydroxycinnamic compounds combined with laccase on sisal pulp properties, revealed FRC to be the best choice for investigating biografting, because of the very pronounced tendency it showed to couple with fibers. The grafting efficiency of different laccase−FRC treatments was assessed via changes in KNlig, brightness, Klason lignin, and surface anionic charge (see Table 3). As can be seen, the

Table 2. KNlig, Brightness, and Viscosity of Initial Pulp and Pulps Obtained from L and P Stages of the Three Bleaching Sequencesa initial L LSLD XLSLD LQP LSLDQP XLSLDQP a

KNlig

brightness (% ISO)

3.9 3.4 5.1 4.0 1.3 1.0 0.3

37.9 40.0 27.0 31.5 74.5 78.5 76.9

viscosity (mL/g) 733 729 739 703 663 592 691

± ± ± ± ± ± ±

1 3 39 30 37 25 10

Data taken from ref 23.

Table 3. KNlig, Brightness, Klason Lignin, and Surface Anionic Charge of the Initial, Control, and Laccase−FRCTreated Pulps after Soxhlet Extraction with Acetonea Figure 3. Thermal degradation profiles of pulps from the L and P stages of sequences performed in the absence (and presence) of SLD, compared to that of initial pulp. TG run performed in an air environment at a heating rate of 5 °C/min.

initial laccase L20FRC1.5b L40FRC3.5c

induced degradation of lignin. However, the presence of SLD significantly modified the laccase behavior. As can be seen in Table 2, the laccase−SLD system caused an increase in KNlig (from 3.9 to 5.1) and a loss of brightness (from 37.9 to 27.0); the former may have resulted from the adsorption or partial incorporation of the phenol in pulp fibers via radical-coupling reactions,25 and the latter may be due to the polymerization of the phenolic mediator, yielding deposits that reduce brightness.33 Nevertheless, TGA proved that these chemical changes did not affect the thermal degradation path of cellulose, which remained unchanged, with respect to that of the initial pulp (see Figure 3). In a TCF laccase-based bleaching sequence, the greatest improvement is obtained after the alkaline hydrogen peroxide stage that promotes the removal of the laccase-modified lignin. Despite the adverse effects in the laccase−SLD treatment, the final bleaching stage yielded a KNlig value smaller than the control value, which suggests that the natural mediator may be simultaneously involved in coupling (homopolymerization and coupling with lignin) and oxidative reactions during the L stage, with the effect of the latter being observed at the end of the bleaching sequence. Similar to that observed for KNlig, SLDtreated pulps exhibited improved brightness, with respect to the control pulp, as a result of the oxidation and dissolution of chromophoric species and lignin degradation products, in the alkaline bleaching medium used. The positive effects produced by the PO stage were accompanied by a reduction in pulp viscosity and microfibril damage, because of the degrading effect that the hot, strongly alkaline medium used in this stage

KNlig

brightness (% ISO)

3.2 2.7 3.5 7.1

52.1 53.4 50.0 47.5

Klason lignin 1.1 1.2 1.2 1.7

± ± ± ±

0.1 0.2 0.2 0.2

surface anionic charge (μequiv/g) 60 54 60 80

± ± ± ±

5 7 0 7

a

Data taken from ref 21. Control pulp was treated with laccase alone (20 U/g odp). bL20FRC1.5 denotes pulp treated with 20 U/g odp laccase and 1.5% odp FRC. cL40FRC3.5 denotes pulp treated with 40 U/g odp laccase and 3.5% odp FRC.

analysis of pulp properties confirmed the occurrence of FRC grafting, especially in the treatment that used high doses of enzyme and phenol, which resulted in a KNlig increase from 3.2 to 7.1 and a brightness reduction from 52.1 to 47.5, compared to the initial pulp. Further evidence of the different extent of FRC grafting obtained in the two treatments was provided by the increased amount of Klason lignin and surface anionic charge of the fibers. As shown by Figure 4, the laccase−FRC system significantly modified the thermal degradation path of the initial pulp. Similar to the effect observed during the biobleaching assay, laccase degraded the surface cellulose, increasing the paracrystalline cellulose content and, consequently, making the DTG peak broader. However, in contrast to the effect produced by SLD, the presence of a similar concentration of FRC boosted the surface modification of cellulosic fibers, leading to the formation of a greater amount of paracrystalline cellulose. When the concentrations of laccase and FRC increased to 40 U/g odp and 3.5% odp, respectively, the phenomenon was intensified, and the thermal degradation profile of cellulose showed two clearly separated peaks (paracrystalline and crystalline celluloses). It is evident that FRC modified the external layer of cellulose microfibril as a collateral effect of its grafting onto fibers. 3899



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known from studies performed with lignin models36 that TEMPO selectively interacts with benzyl alcohol (or ethers) groups of lignin, oxidizing them to α-carbonyl derivatives, it was of interest to evaluate whether these modifications led to a delignification effect in pulps treated with the laccase−TEMPO system under different operating conditions. As shown in Table 4, no delignification effect was observed in pulps treated at high consistency, compared to the control pulp. The KNlig value was decreased by 11% in pulps treated at low consistency, which indicates that a slight delignification occurred under those conditions. The potential of TEMPO for HexA removal has not been investigated to date; therefore, it was of interest to determine whether laccase−TEMPO treatments of sisal pulp resulted in any change in the HexA content. The results obtained showed that laccase alone was able to eliminate 22% of HexA from the initial pulp, which is consistent with previously reported results.37,38 Laccase−TEMPO treatments performed at low consistency did not result in any decrease in HexA, while an important reduction was obtained from treatments performed at high consistency, especially after refining (44%). These results indicates that TEMPO used in combination with laccase is able to reduce the HexA content from sisal pulp, and this occurs under conditions that provide good interaction between fibers and oxidant (high consistency) and accessibility in the fiber wall (refining). Results of TGA for pulps treated at low and high consistency are shown in Figures 5a and 5b, respectively. Despite simultaneous reductions in the laccase concentration, as the TEMPO dose was increased, the thermal degradation path of pulps showed a progressively higher secondary peak at low temperature (Figure 5a). This aggressive behavior is specific to TEMPO. Under similar conditions, other mediators such as SLD or FRC did not show comparable levels of cellulose degradation. TEMPO intensely degraded cellulosic chains, causing a severe depolymerization, which yielded a significant viscosity reduction. As a result, a fraction of crystalline cellulose was transformed to amorphous cellulose, which thermally degrades at very low temperature. On the other hand, consistent with the pulp properties analysis, TGA showed that refining prior to the laccase−TEMPO treatment significantly increased the effect of the latter. As depicted in Figure 5b, after the enzyme treatment, refined pulp degraded at lower temperature, with respect to unrefined pulp, and showed a more intense degradation at low temperature. According to these data, applying a previous refining step provided more damaged fibers, because of both the mechanical action of refining and the boosted oxidation effect of the laccase−

Figure 4. Effect of laccase−FRC treatments on the thermal degradation path of sisal pulp. TG run performed in an air environment at a heating rate of 5 °C/min.

3.4. Laccase−TEMPO Oxidation. Three sisal pulp samples treated with the laccase−TEMPO system in tests of a statistical plan performed in previous work22 were selected to study their thermal degradation profile. The treatment found to maximize functionalization of fibers was performed in a reactor at an increased pulp consistency, on both refined and unrefined pulps, for the purpose of determining whether the increased fiber surface area and consistency would lead to enhanced functionalization.24 The resulting samples were also analyzed in this study via TG. As can be seen from Table 4, the increase of pulp consistency resulted in the formation of 130% and 94% higher amounts of aldehyde and carboxyl groups contents, respectively. Moreover, the treatment performed on refined pulp yielded significantly higher contents of aldehyde and carboxyl groups, with respect to that applied to unrefined pulp. Borohydride viscosity measurements were performed to assess changes in the degree of cellulose polymerization during the laccase−TEMPO treatments. In this case, TEMPO-oxidized pulp samples were treated with sodium borohydride prior to measurement in order to inactivate the carbonyl groups (via reduction to hydroxyl groups) and exclude the effect of depolymerization reactions by β-elimination promoted by the alkaline measurement medium.22,35 All laccase−TEMPO treatments caused a significant loss of borohydride viscosity, with respect to the control pulp, more markedly under those conditions resulting in the highest degree of functionalization. Two properties examined in this work in laccase−TEMPOtreated pulps were KNlig and HexA content, in order to evaluate whether this oxidative system could exert a delignification effect or reduce the content of HexA groups in sisal pulp. Since it is

Table 4. KNlig, HexA, Carboxyl and Aldehyde Groups Contents, and Borohydride Viscosity of the Initial, Control, and Laccase− TEMPO-Treated Pulpsa KNlig initial laccase L100T2t20 L20T8t20 L60T5t14 LT5%_0rev LT5%_4500rev

3.8 3.2 2.8 2.8 2.9 3.2 3.1

± ± ± ± ± ± ±

0.2 0.2 0.1 0.0 0.0 0.1 0.0

HexA (μmol/g) 38.9 30.2 29.1 30.2 32.6 26.7 21.7

± ± ± ± ± ± ±

1.3 0.7 2.4 2.7 1.4 2.1 1.5

COOH (μmol/g) 110 94 114 137 106 240 266

± ± ± ± ± ± ±

7 2 5 4 5 7 2

CHO (μmol/g)

borohydride viscosity (mL/g)

0±9 1±0 15 ± 2 83 ± 8 43 ± 5 159 ± 0 191 ± 7

736 ± 0 686 ± 8 495 ± 15 389 ± 32 403 ± 10 294 ± 18 253 ± 35

a

Data taken from refs 22 and 24. Control pulp (laccase) was treated with 60 U/g odp laccase, in the absence of TEMPO, at 1% consistency under an oxygen pressure of 0.6 MPa for 18 h. 3900



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(where TEMPO represents the 2,2,6,6-tetramethylpiperidine-1oxyl free radical) yielded increasingly degraded cellulose at increasing degrees of oxidative functionalization. A boosted functionalization of fibers was obtained by applying a laccase− TEMPO treatment at increased pulp consistency and after refining, which resulted in strongly degraded cellulosic chains and the formation of a substantial amount of amorphous cellulose, which degrades at low temperature. A novel aspect of the laccase−TEMPO system was identified in this work: its ability to reduce the HexA content of pulp under specific reaction conditions.

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AUTHOR INFORMATION

Corresponding Author

*Tel.: +34 937398180. Fax: +34 937398101. E-mail: tvidal@ etp.upc.edu. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The authors are especially grateful to Spain’s MICINN for funding this research in the framework of Projects FUNCICEL (CTQ2009-12904) and BIOFIBRECELL (CTQ2010-20238CO3-01). E.A. is additionally grateful to MICINN for the award of an FPU Research Trainee Fellowship.

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REFERENCES

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Figure 5. Thermal degradation profiles of sisal pulps treated with the laccase−TEMPO system at (a) low consistency and (b) high consistency. TG run performed in a nitrogen environment at a heating rate of 5 °C/min.

TEMPO system, which was reflected in their easier thermal degradation.

4. CONCLUSIONS The effects of three different laccase-based systems applied to sisal cellulose fibers in order to perform biobleaching, biografting, and cellulose oxidation were evaluated in terms of pulp properties and thermal degradation profile of the treated pulps. The laccase−SLD system (where SLD represents sinapyl aldehyde) was observed to aid delignification at the end of the bleaching sequence, despite its adverse effects in the L stage. Thermogravimetric analysis (TGA) showed laccase to modify the thermal degradation path of the initial pulp, increasing the proportion of cellulose that degrades at low temperature. The addition of SLD did not affect the thermal degradation path of the initial pulp, confirming that the laccase−SLD system basically acted on the lignin component of fibers. The thermal degradation paths of pulps treated with the laccase−FRC system (where FRC represents ferulic acid) were significantly modified, with respect to that of the laccase-treated pulp, and to a higher extent, under those conditions providing a higher degree of grafting. Thermogravimetric (TG) results showed that laccase−FRC treatment caused a deterioration of cellulose surface, leading to the formation of a greater amount of paracrystalline cellulose, probably as a consequence of the incorporation of FRC in fibers. Laccase−TEMPO treatments 3901



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