Self-Bonding Boards From Plantain Fiber Bundles After Enzymatic Treatment: Adhesion Improvement of Lignocellulosic Products by Enzymatic Pre-Treatment

Transcription

1 J Polym Environ (2011) 19: DOI /s ORIGINAL PAPER Self-Bonding Boards From Plantain Fiber Bundles After Enzymatic Treatment: Adhesion Improvement of Lignocellulosic Products by Enzymatic Pre-Treatment Catalina Álvarez Benjamín Rojano Ovidio Almaza Orlando J. Rojas Piedad Gañán Published online: 2 November 2010 Ó Springer Science+Business Media, LLC 2010 Abstract Self-bonding boards were manufactured with treated fibers at different concentrations of a laccase enzyme. This enzyme induced the generation of phenoxy radicals in the fiber lignin which can generate covalent bonds and crosslinked by radical radical coupling. The effect of laccase concentration on the properties of obtained fiberboards was evaluated. The formation of free radicals and changes in the lignin macromolecule was measured using scavenging activity test, infrared spectroscopy, electron paramagnetic resonance and scanning electron microscopy. Thermal and mechanical properties of the resulting fiberboards were determined by differential scanning calorimetry, thermo gravimetric analysis and flexion tests. Increased thermal stability, modulus of elasticity and modulus of rupture and C. Álvarez (&) P. Gañán School of Engineering, Mechanical Engineering Program, New Materials Research Group, Universidad Pontificia Bolivariana, Circular 1, # 70-01, bloque 11, Medellín, Colombia alvarezl.catalina@gmail.com P. Gañán piedad.ganan@upb.edu.co C. Álvarez School of Engineering, Agro-Industrial Engineering Program, Universidad Pontificia Bolivariana, Medellín, Colombia B. Rojano Department of Chemistry, Universidad Nacional de Colombia, Medellín, Colombia O. Almaza Department of Physics, Universidad Nacional de Colombia, Bogotá, Colombia O. J. Rojas Department of Forest Biomaterials, North Carolina State University, Raleigh, NC, USA also, a reduction in thickness swelling and water absorption, were observed at higher concentrations of laccase. These results are ascribed to the effect of the free radicals that were generated during the enzymatic treatment. Keywords Self-bonding Plantain fibers Laccase Boards Free radicals Introduction An increased interest in the use of agricultural residues and by-products from agro-industries has been growing in the recent years. Non-wood plants may offer some advantages over woody biomass, since they are available in large quantities as residual, inexpensive agricultural waste [1, 2]. The use of lignocellulosic fiber bundles for structural and building materials (i.e. fiberboards) has been explored and consideration has been given to a wide variety of fiber sources [1, 3]. In the development of the above-mentioned materials, synthetic adhesives are commonly used [3]. However, due to environmental and other concerns, these adhesives, typically produced from nonrenewable resources, are expected to be of controlled use in the future [4]. Therefore, binderless or self-bonding fiberboards (free of synthetic adhesives) have received increased attention. This type of fiberboards has been developed by activating the lignin in the fiber, and generating in situ polymerization, mimicking the lignification of the cell wall in nature [5 7]. Lignin is a heterogeneous, high molecular weight polymer that forms a three-dimensional network with random linkages (ether and carbon carbon bonds) between three different types of phenolic monomers (coniferyl, sinapyl, and p-coumaryl alcohol) [8]. In plants, these

2 J Polym Environ (2011) 19: monomers can be oxidized by peroxidase or laccase enzymes (using peroxide or oxygen as oxidant agents, respectively). In both reactions high resonance chemical radicals are produced, which in the case of the laccase, occur due to the oxidation of phenolic group, reducing the oxygen and producing water (see Eq. 1) [1, 9]. 4Phe OH þ O Laccase 2! 4Phe O þ 2H 2 O ð1þ The interaction of these radicals can generate a covalent bond (radical radical coupling) and produce polymers with a complex structure [10]. Therefore, it is possible that, when the laccase reacts with fibers, fiber fiber bonding (self-bonding) can be obtained. In previous reports [11 13], the above method has been employed to develop self-bonding fiberboards and also, to produce paper with higher strength and to polymerize different phenols, among other applications. In this research work, leaf sheath fiber bundles obtained from plantain residues were treated with different laccase concentrations to form free radicals on the lignin and thereby, obtaining self-bonding among fibers to produce fiberboards. The thermal and mechanical properties of the produced fiberboards were thoroughly evaluated. Changes in the morphology of the fibers and their surface lignin were analyzed by scanning electron microscopy (SEM) and Fourier transform infrared spectroscopy (FTIR). Finally, antioxidant activity (scavenging capacity) and electron paramagnetic resonance (EPR) were used to corroborate the oxidation of fiber lignin. Experimental Materials The Corbanacol Foundation (Colombia) supplied leaf sheath fiber bundles from commercial plantain plants cultivated in Urabá region (Colombia). The samples were passed through a defibrator several times, until no greencolored features were observed on the surface of the fiber bundles. Thereafter, the fibers were rinsed with fresh water to remove any residues, and then air-dried for 3 days at room temperature. Before testing, fiber bundles were ground (target size \ 2 mm) and left to reach moisture equilibrium (approx. 7 wt%) with ambient air. For the enzymatic treatment of leaf sheath fiber bundles, fungal laccase EC from Aspergillus oryzae was used (Novozymes, México). The enzymatic activity was measured in units (U) which were taken as the amount of enzyme that oxidizes 1 lmol of 2,2 -azino-bis(3-ethylbenzthiazoline-6-sulphonic acid) (ABTS) per minute, under standard conditions of ph and temperature (5.5 and 30 C, respectively). Fiber Treatment Ground fiber bundles were treated according to Felby et al. [9, 12] with laccase in aqueous suspension at 5% consistency in four different concentrations (0, 6, 12 and 24 units per fiber gram (U/g)). The treatment conditions were approx. 30 C and ph 5.5 during 1-h reaction time and then, the fiber bundles were filtered and dried at 40 C during 24 h. Samples of the obtained fibers (referred to as F0U, F6U, F12U and F24U) were subjected to further analysis. Manufacture of Fiberboards Dry-process fiberboards with a target thickness of 3 mm and density of 1.1 kg/cm 3 were manufactured from treated fiber bundles. This was accomplished by forming them into layers and applying hot pressing. The obtained fiberboards are referred herein as B0U, B6U, B12U and B24U depending of the respective laccase pre-treatment (F0U, F6U, F12U and F24U). The pressure cycle consisted of 1,000 psi during 1 min, followed by 2,000 psi during 7 min, both at 200 C. The first step was carried out to prevent the formation of macroscopic voids within the fibers bundles that otherwise could result from steam evacuation [5]. Characterization of Fiber and Fiberboards Fiber bundles were sputter-coated with gold and SEM images were taken with a Jeol JSM 5910 LV. FTIR analyses were conducted at room temperature (23 ± 1 C) using a Nicolet 6700 ATR-FTIR spectrometer with a diamond internal reflection element. All spectra were collected in the wave number range of 4, cm -1 with a resolution of 4 cm -1 and with at least 32 scans per specimen. Variations in peak intensities of aromatic rings (1,510 and 1,425 cm -1 ) and unconjugated C = O (1,710 and 1,660 cm -1 ) were recorded. All intensity measurements were carried out using as a reference the intensity of the 895 cm -1 peak associated with the C-1 hydrogen in the glucopyranose ring of cellulose, which is stable to the oxidation treatments [14]. The scavenging activity of treated fiber bundles was measured with ABTS radicals. To oxidize the colorless ABTS to the blue-green radical cation ABTS?, 7 mmol/l of ABTS were mixed with potassium persulfate (final concentration of 2.42 mmol/l) and incubated for h at room temperature in a container protected from light. After this time, the ABTS? solution was diluted with phosphate buffer (ph 7.4) to an absorbance of 0.70 (±0.02) at 734 nm. After addition of 5.0 ml ABTS? solution to 3 mg of ground fiber bundles the mixture was stirred for

3 184 J Polym Environ (2011) 19: s and then, the absorbance was monitored continuously for 30 min total time. Data were collected at 734 nm and room temperature (approx. 25 C) using a Jenway 6405 UV vis spectrophotometer. The scavenging activity of the samples was determined from the reduction in absorbance of ABTS according to Eq. 2: scanvenging activityð%þ ¼ 1 A samples A control 100 ð2þ Detection of radicals by electron paramagnetic resonance (EPR) spectrometry was carried out on dry treated fibers bundles. The samples (approx. 20 mg each) were uniformly compressed in a quartz tube (approx. 20 mm length). EPR spectrum was collected at room temperature (23 ± 1 C) with a Bruker ESP 300 spectrometer at 9.44 GHz microwave frequency. The modulation frequency and amplitude was 100 khz and 1.95 Gauss, respectively. The modulus of elasticity (MOE), modulus of rupture (MOR), thickness swell (TS) and water absorption (WA) of the obtained fiberboards were determined for each laccase concentration, according to the ASTM standards D Twenty probes were tested for each concentration: five for mechanical properties (MOE and MOR) and fifteen for physical properties (TS and WA). Differential scanning calorimetry (DSC) and thermo gravimetric analyses (TGA) were conducted on treated fiberboards. Thermal transitions were measured using a TA Instruments Q500 DSC. The samples were heated at a heating rate of 10 C/min from -50 to 180 C in a nitrogen atmosphere. Thermal stability of the fiberboards was determined using a TA Instruments Q100 TGA at a heating rate of 10 C/min under a nitrogen atmosphere. Results and Discussion Enzymatic Treatment of Leaf Sheath Fiber Bundles The treatment of leaf sheath fiber bundles with laccase was aimed to activate the surface of the fibers by producing phenoxy radicals in a one-electron oxidation process of phenolic hydroxyl groups of lignin. Long incubation times were avoided since it has been reported that the surface of wood fibers could lose the outer surface layer, when incubated with laccase for more than 12 h [15]. As the objective of the present work was only to activate the fiber bundles by generation of free radicals, a short incubation time of 1 h was used [9]. In order to verify the integrity of the fiber bundles, SEM images were performed before and after lacasse treatment. Figure 1a and b show laccase-treated fibers (F0U and F24U) after 1 h incubation. The F24U sample showed no Fig. 1 SEM micrographs of F0U (a) and F24U (b) laccase-treated fiber bundles noticeable change in fibrillation and integrity of fiber bundle when compared to F0U. However, it is expected that for short laccase-incubation times, lignin on the fiber surfaces can be oxidized to produce stable radicals as reported elsewhere [15]. This is due to the phenolic character of lignin, which allows having antioxidant properties and maintaining stable free radicals [16]. Radicals formed during oxidative treatment (i.e. laccase or UV-irradiation) produce carboxyl groups and reduce the content of phenolic hydroxyl groups, which decreases aromatic rings [1, 14, 17]. Changes on the treated fiber bundles were examined by FTIR, EPR and by measuring the ABTS scavenging capacity. Previous experiments by Muller et al. [14], using UV light to oxidize wood, recorded considerable changes in the IR spectrum. However, treated samples F6U, F12U and F24U, did not show significant changes in the IR spectrum when compared to F0U (see Fig. 2). These results can be explained due to the laccase oxidation is limited to lignins located on the surface of the fiber bundles, as the enzyme cannot easily penetrate into their structure [18]. Barsberg and Thygesen [19] obtained

4 J Polym Environ (2011) 19: Fig. 2 FTIR spectra of laccase-treated (0, 6, 12 and 24 units) leaf sheath fiber bundles similar results by oxidizing beech fibers with laccase using photoacoustic FT-IR, diffuse reflectance FT-IR and FT-IR transmission spectroscopy, and found no detectable changes in IR spectrum. Nevertheless, in the present work, the peak heights of the aromatic rings (1,510 and 1,425 cm -1 ) (using as a reference the intensity of the 895 cm -1 peak) of the treated samples with higher concentrations of laccase decrease, while the carboxyl groups (1,710 and 1,660 cm -1 ) show an increase (see Fig. 3). Although previous studies demonstrated the antioxidant activity of different lignins [16] and black liquors from steam explosion [20], the scavenging capacity of fiber bundles has not been reported in the bibliography researched (to our knowledge). However, in the present work the antioxidant activity was measured over fiber bundles. The tests performed by ABTS scavenging capacity, qualitatively demonstrated the extent to which hydroxyl groups reacted after laccase treatment. Table 1 includes the measured scavenging capacity of the treated fibers bundles, which decreased with higher laccase concentration. Therefore, it can be suggested that higher laccase concentrations allow more phenolic hydroxyl groups to react. The F0U samples have a scavenging capacity of approx. 57.8%, while F6U, F12U and F24U have a scavenging capacity of 43.5, 40.1 and 38.2%, respectively. The lower scavenging capacity is believed to be the result of more phenolic hydroxyl groups reacting and consequently, a higher amount of free radicals generated during the oxidation process. The EPR results showed changes in the amount of free radical produced in samples F6U, F12U and F24U (measured as EPR peak height) as 151, 178, and mm, respectively. The lower radical amount in F24U could be explained by the fact of more radical radical coupling occurring before the EPR test, for example fiber fiber or fiber low molecular weight (phenolic extractives). Cao et al. [21] reported similar behavior when treating wood powder with two different laccases (laccase I and II) at concentration levels of 10, 20 and 30 U/g. Their results indicated that the maximum radical amount appeared to be at 20 U/g for both laccase I (182.0, and mm) and laccase II (96.3, and mm). Fiberboards From Laccase-Treated Fiber Bundles The physical mechanical properties of fiberboards manufactured from laccase-treated leaf sheath fiber bundles were expected to correlate with the radicals produced on the surfaces of the fibers. This is due to the fact that a larger population of free radicals could increase the probability of radical radical coupling. As such, Table 2 shows the MOE and MOR of fiberboard samples (B0U, B6U, B12U and B24U) from laccase-treated fiber bundles (F0U, F6U, F12U and F24U). The MOE of fiberboards changed with the laccase treatment. B6U, B12U and B24U had a MOE of 3.5, 3.6 and 3.6 GPa, while B0U was 1.30 GPa. With Table 1 Scavenging capacity of laccase-treated leaf sheath fiber bundles Sample Concentration (U/g) Average scavenging cap. (%) Fig. 3 Relation of absorbances of laccase-treated (0, 6, 12 and 24 units) leaf sheath fiber bundles F0U ± 1.4 F6U ± 1.7 F12U ± 1.2 F24U ± 1.5

5 186 J Polym Environ (2011) 19: respect to the MOR, B0U had a value of 13.3 MPa while B6U, B12U and B24U had a MOR of approx. 17.5, 18.6 and 18.7 MPa, respectively. While the results seem to indicate that laccase concentrations above 6 U/g do not change significantly the mechanical properties (B6U, B12U and B24U), there is a marked difference when compared to B0U. Improved performance of laccase treated fiberboards was also observed in terms of thickness swell (TS) and water absorption (WA) (see Table 2). Average WA of B0U fiberboards were 268.3% while the ones obtained for B6U, B12U and B24U were all pretty similar, between 79.7 and 82.4%. TS values showed different behavior: B0U averages were approx. 7.2 times greater than B12U and B24U averages, but only approx. 3.2 times than B6U. As seen in the case of the MOE and MOR values, WA and TS averages of B24U samples did not show significant changes when compared to the B12U. In previous works, Felby et al. [12] produced fiberboards from laccase-treated beech fibers with concentrations of 6 and 24 U/g. The MOE of these fiberboards were approx. 3.7 and 3.9 GPa, respectively, while the MOR were 40.1 and 46.0 MPa. MOE and MOR of the control fiberboard is not showed due to they were delaminated. The WA of the fiberboards showed a value of approx. 224 (control), 109 and 92% to 6 and 24 U/g, and the TS showed a value 146 (control), 69 and 46%, respectively. The better performance in physical mechanical properties (e.g., MOE, MOR, WA and TS) of beech fiberboards, when compared to the results in this present work with leaf plantain sheath fiberboards, can be ascribed to the fact that the beech has a higher lignin content and therefore, the enzymatic oxidation could generate more phenol hydroxyl radicals available to react. In the treated leaf sheath fiber bundles fiberboards, an increase in the maximum degradation temperature and a decrease in the glass transition temperature (Tg), were expected to correlate with higher laccase concentrations, due to lignin polymerization and/or cross-linking. Figure 4 shows DSC thermograms of treated fiberboards (B0U, B6U, B12U and B24U). The curves show a transition at approx. 100 C related to fiber moisture [22], while a second transition is observed at around 150 C related with glass transition temperature of lignin. Although Van Dam et al. [23] found the glass transition temperature of coconut fiber lignin also at this point, other authors reported different results and conclusions [5, 9, 24]. When B0U, B6U, B12U and B24U curves are compared, it is possible to observe a difference at B24U curve. Weight changes with temperature of the treated fiberboards were almost identical. A small weight loss is observed ( C), which can be attributed to water evaporation [25]. Also, an extensive mass loss is seen approximately C (see Fig. 5). This last change occurred in the temperature range for hemicellulose, cellulose and lignin degradation ( C). Specifically, hemicellulose breaks down at C, followed by cellulose at C and lignin at C [25, 26]. The maximum degradation temperature of treated fiberboards was 327 C for B0U, and 328, 329 and 331 C, for B6U, B12U and B24U samples. When these results are analyzed in terms of radical generation and coupling, it could be concluded that higher laccase concentrations increase thermal stability of the fiberboards. In the case of sample B24U, the higher increase in the maximum degradation temperature and glass transition temperature could be due to the coupling between fiber and low molecular weight phenols, increasing the lignin molecular weight. According to Beall [27], Fig. 4 DSC of laccase-treated fiberboards Table 2 Physic-mechanical properties of laccase-treated fiberboards B0U B6U B12U B24U MOE (GPa) 1.3 ± ± ± ± 0.2 MOR (MPa) 13.3 ± ± ± ± 1.2 Water absorption (%) ± ± ± ± 12.3 Thickness swell (%) ± ± ± ± 7.2

6 J Polym Environ (2011) 19: of grafting reactions of low molecular weight components in the fiber with the lignin, which can be more probable at higher laccase concentrations. In conclusion, plantain leaf sheath fiber bundles can be oxidized with laccase to generate phenolic hydroxyl radicals which allows developing of self-bonding fiberboards. Acknowledgments The authors would like to thank to Juan Alberto Vélez for the manuscript revision, to CIDI-UPB for their financial support and to Colciencias by grant the doctoral scholarship to Ing. Alvarez. References Fig. 5 DTG of laccase-treated fiberboards lignin has a higher thermal stability when its molecular weight is larger. This result confirm that B24U sample experimented a higher change in their structure, but it is necessary to development other analysis to clarified it. From the overall results obtained it is possible to relate the influence of free radicals in the self-bonding of lignocellulosic fibers, which is seen in the improved physical mechanical properties of the fiberboards. Moreover, these results also suggest that the graft of low molecular weight phenols could positively influence the thermal properties of composite fiberboards. Conclusions Leaf sheath fiber bundles were treated with different laccase concentrations to develop self-bonding boards. It was observed (scavenging capacity, EPR and FTIR) that fiber bundles reacted with the laccase enzyme and reduced the phenolic OH groups in lignin. This effect can be explained by the generation of free radicals during treatment. Interestingly, no surface or structural changes in the fiber bundles were observed by SEM or by FTIR. The physical mechanical properties of the treated fiberboards showed an increase in MOE and MOR and a decrease in WA and TS, when fiber bundles is treated with laccase. However, at the different laccase concentrations (B6U, B12U and B24U) the properties become almost invariant and major changes are no longer observed. Therefore no matter how much laccase is added, there would be no phenolic OH groups to interact with, and it is possible that mediators are necessary. Fiberboards obtained from laccase-treated fibers with 24 U/g (B24U) showed higher thermal stability when compared to B12U and B6U. This behavior could be the result 1. Widsten P, Kandelbauer A (2008) Adhesion improvement of lignocellulosic products by enzymatic pre-treatment. Biotechnol Adv 26: Mo X, Hu J, Sun XS, Ratto JA (2001) Compression and tensile strength of low-density straw-protein particleboard. Ind Crops Prod 14:1 3. Wang DH, Sun XZS (2002) Low density particleboard from wheat straw and corn pith. Ind Crops Prod 15:43 4. Velásquez JA, Ferrando F, Farriol X, Salvado J (2003) Binderless fiberboard from steam exploded Miscanthus sinensis. Wood Sci Techn 37:69 5. Bouajila J, Limare A, Joly C, Dole P (2005) Lignin plasticization to improve binderless fiberboard mechanical properties. Polym Eng Sci 45: Widyorini R, Higashihara T, Xu J, Watanabe T, Kawai S (2005) Self-bonding characteristics of binderless kenaf core composites. Wood Sci Techn 39: Elegir G, Bussini D, Antonsson S, Lindström ME, Zoia L (2007) Laccase-initiated cross-linking of lignocellulose fibres using ultra-filtered lignin isolated from kraft black liquor. Appl Microbiol Biotechnol 77: Barsberg S, Nielsen KA (2003) Oxidative quenching of spruce thermomechanical pulp fiber autofluorescence monitored in real time by confocal laser scanning microscopy: implications for lignin autofluorescence. Biomacromolecules 4:64 9. Felby C, Thygesen LG, Sanadi A, Barsberg S (2004) Native lignin for bonding of fiberboards-evaluation of bonding mechanisms in boards made from laccase-treated fibers of beech (Fagus sylvatica). Ind Crops Prod 20: Ikeda R, Uyama V, Kobayashi V (1996) Novel synthetic pathway to a poly (phenylene oxide). Laccase-catalyzed oxidative polymerization of syringic acid. Macromolecules 29: Chandra RP, Lehtonen LK, Ragauska AJ (2004) Modification of high lignin content kraft pulps with laccase to improve paper strength properties 1. Laccase treatment in the presence of gallic acid. Biotechnol Prog 20: Felby C, Hassingboe J, Lund M (2002) Pilot-scale production of fiberboards made by laccase oxidized wood fibers: board properties and evidence for cross-linking of lignin. Enzyme Microb Technol 31: Mai C, Schormann W, Hüttermann A, Kappl R, Hüttermann J (2002) The influence of laccase on the chemo-enzymatic synthesis of lignin graft-copolymers. Enzyme Microb Technol 8: Muller U, Ratzsch M, Schwanninger M, Steiner M, Zobl H (2003) Yellowing and IR-changes of spruce wood as result of UV-irradiation. J Photochem Photobiol B Biol 69: Huttermann A, Mai C, Kharazipour A (2001) Modification of lignin for the production of new compounded materials. Appl Microbiol Biotechnol 55:387

7 188 J Polym Environ (2011) 19: Vinardell MP, Ugartondo V, Mitjans M (2008) Potential applications of antioxidant lignins from different sources. Ind Crops Prod 27: Sarkanen KV, Ludwing CH (1971) Lignins: ocurrence, formation structure and reaction. Willey-Interscience, New York 18. Goodell B, Yamamoto K, Jellison J, Nakamura M, Fujii T, Takabe K, Hayashi N (1998) Laccase immunolabelling and microanalytical analysis of wood degraded by Lentinus edodes. Holzforschung 52: Barsberg S, Thygesen LG (1999) Spectroscopic properties of oxidation species generated in the lignin of wood fibers by a laccase catalyzed treatment: electronic hole state migration and stabilization in the lignin matrix. Biochim Biophys Acta 1472: Castro E, Conde E, Moure A, Falque E, Cara C, Ruiz E, Domınguez H (2008) Antioxidant activity of liquors from steam explosion of Olea Europea wood. Wood Sci Technol 42: Cao YJ, Duan XF, Cao YL, Lu JX, Zhu JQ, Zhou GW, Zhao BL (2008) ESR study in reactive oxygen species free radical production of Pinus kesiya var. langbianensis heartwood treated with laccase. Appl Magn Reson 35: Hon DNS, Ou NH (1989) Thermoplasticization of wood. I. Benzylation of wood. J Polym Sci Part A Polym Chem 27: JEG van Dam, MJA van den Oever, W Teunissen, ERP Keijsers, AG Peralta (2004) Process for production of high density/high performance binderless boards from whole coconut husk Part 1: Lignin as intrinsic thermosetting binder resin. Ind Crops Prod 19: Silva GG, De Souza DA, Machado JC, Hourston DJ (2000) Mechanical and thermal characterization of native brazilian coir fiber. J Appl Polym Sci 76: Byrne CE, Nagle DC (1997) Carbonization of wood for advanced materials applications. Carbon 35: Mohan D, Pittman CU, Steele PH (2006) Pyrolysis of wood/ biomass for bio-oil: a critical review. Energy Fuels 20: Beall FC (1969) Thermogravimetric analysis of wood lignin and hemicelluloses. Wood Fiber Sci 1:215