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Pilot-scale production of fiberboards made by laccase oxidized wood fibers: board properties and evidence for cross-linking of lignin
Enzyme and Microbial Technology 31 (2002) 736–741
Pilot-scale production of fiberboards made by laccase oxidized wood fibers: board properties and evidence for cross-linking of lignin Claus Felby a,b,∗ , Jens Hassingboe a , Martin Lund c a c
Plant Fibre Laboratory, The Royal Veterinary and Agricultural University, Hoejbakkegaard Alle 1, DK-2630 Taastrup, Denmark b Novozymes A/S, Krogshoejvej 36, DK-2880 Bagsvaerd, Denmark Chemistry Department, The Royal Veterinary and Agricultural University, 40 Thorvaldsensvej, DK-1871 Frederiksberg, Denmark Received 9 July 2001; accepted 12 March 2002
Abstract Oxidoreductases can be applied for bonding of fiberboards, particle boards, paper boards and kraft-liner boards. In this work we report on pilot-scale production of laccase bonded fiberboards made from fibers of beech (Fagus sylvatica). Dioxane extractable lignin from fibers and boards are isolated and the molecular mass estimated by gel permeation chromatography. The strength properties of the enzyme bonded boards are comparable to boards bonded by an urea–formaldehyde adhesive, whereas the dimensional stability properties of the enzyme bonded boards are not at the same level. Wax treatment of the fibers, in order to improve dimensional stability of boards, is not compatible with the enzyme treatment. Cross-linking of the lignin can be observed in enzyme treated fibers and boards. Hot pressing of enzyme treated fibers results in a substantial cross-linking of lignin in boards. The enzymatic bonding effect may be caused by covalent bonds between fibers or an adhesive effect of polymerized loosely associated lignin. Laccase catalyzed bonding requires higher pressing temperatures and longer pressing times, and the concept may not be economically feasible as it is. However, it shows promise and possibilities in the use of oxidative enzymes for industrial bonding and modification of lignin. © 2002 Published by Elsevier Science Inc. Keywords: Laccase; Fiberboards; Lignin; Oxidation; Pilot-scale production
1. Introduction Bonding of wood fibers and particles by adhesives can be accomplished by forming a resinous matrix in which the particles or fibers are bonded together, e.g. by mechanical entanglement or covalent cross-linking. The bonding is caused not only by the added adhesive but also by the auto-adhesive properties of the wood components. Oxidoreductases such as laccase and peroxidase may be used for polymerization or cross-linking of wood components in order to bond these components. The concept of using lignin-oxidizing enzymes for bonding applications is based on the reactivity of phenoxy radicals in the plant cell wall. In vivo, oxidoreductases catalyze polymerization of lignin through cross-linking of phenoxy radicals, and thus it may be possible to utilize a similar type of reaction for bonding of lignocellulosic materials in vitro. Previous work reported the use of oxidoreductases for bonding of fiberboards, particle boards, paper boards and ∗
Corresponding author. Tel.: +45-35-28-22-18; fax: +45-35-28-22-16. E-mail address: [email protected] (C. Felby).
0141-0229/02/$ – see front matter © 2002 Published by Elsevier Science Inc. PII: S 0 1 4 1 - 0 2 2 9 ( 0 2 ) 0 0 1 1 1 - 4
kraft-liner boards [1–7]. So far, the work reported has been done on a laboratory-scale, and the results suggest the possibility of a new technology for bonding of lignocellulosic materials. In this work we report on pilot-scale production of laccase bonded fiberboards made from fibers of beech (Fagus sylvatica). The physicomechanical properties of the boards are presented and discussed. A new method for isolation of fiber surface lignin from boards is presented, and the molecular mass of lignin in boards and fibers is characterized by gel permeation chromatography (GPC).
2. Materials and methods 2.1. Materials Myceliophtera thermophila laccase was obtained from Novozymes A/S, Bagsvaerd, Denmark. Enzyme activity was measured in units (U), with 1 U defined as the amount of enzyme that oxidizes 1 mol syringaldazine per minute in potassium phosphate buffer pH 7.0, 30 ◦ C.
C. Felby et al. / Enzyme and Microbial Technology 31 (2002) 736–741
Urea–formaldehyde (UF)-resin was obtained from Casco-Nobel, Stockholm, Sweden. Mobilcer 072 liquid wax for improving the water tolerance of fiberboards was obtained from Mobil, London, UK. Beech wood chips for preparation of fiberboards were supplied by Junckers Industrier, Koege, Denmark. 1,4-Dioxane from Fisher Scientific, Leicestershire, UK for lignin extraction was distilled over sodium borhydride prior to use. Tetrahydrofuran (THF) as eluent for GPC was supplied by Merck, Darmstadt, Germany. Polystyrene standards with nominal molecular mass of 2500, 5000, 17500, and 30000 Da were supplied by Supelco, Bellafonte, PA and standards with nominal molecular mass of 3000 and 10000 Da were supplied by Crompak, Church Stretton, UK. 2.2. Preparation of fiberboards The fiberboards were made at Sunds Defibrator pilot-plant, Sundsvall, Sweden. This facility is capable of fully simulating a medium density fiberboard production from refining of chips to mat forming of fibers. Pressing was done in an electrically heated daylight type press using a dynamic press cycle. Fiberboards were prepared from five different treatments: Control—untreated UF-resin (8% w/w) + wax (1% w/w) Laccase treated 6 U/g fiber Laccase treated 24 U/g fiber Laccase + wax treated 6 U/g fiber + wax (1% w/w). Note that no control treatment using deactivated laccase is included as it was shown in [4], that there is no adhesive effect of deactivated laccase. For each treatment a minimum of six boards (500 mm × 500 mm) were pressed. Target thickness and density of the boards were 8 mm and 850 kg/m3 , i.e. that each board contained approximately 1700 g of fibers. The chips were preheated for 4 min and refined in a twin-disk refiner at 8.5 bar. The target moisture content before adding enzyme was set to 55%, water was added in
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the blow-line in order to reach this level. UF-resin and wax was added at the end of the blow line. An aqueous solution (pH 7) of the enzyme was applied to the fibers in an atmospheric refiner preheated to 60 ◦ C. The atmospheric refiner only served as a mixer for enzymes and fibers and caused no further mechanical separation of the fibers. In order to allow sufficient time for the enzymatic reaction, fibers blended with enzyme were transferred to a 8 m3 rotary bin for 30 min at 50 ◦ C. Fibers from all treatments were dried in a flash drier to a moisture content (MC) of 14–18%. From the dried fibers, air-laid mats were formed and pre-pressed in a cold press. Note that the air layering of the mats lowered the moisture content to 11–13%. Hot pressing was done for 150 s at 180 ◦ C for UF-resin + wax treatment, and 203 s at 200 ◦ C for control and enzyme treatments. The full process is outlined in Fig. 1, and the process parameters are listed in Table 1. 2.3. Board properties For each treatment the boards were tested for modulus of elasticity (MOE), modulus of rupture (MOR), internal bond strength (IB), thickness swell (TS), and water absorption (WA) following a 24 h cold water soak. All testing was done according to ASTM D1037-78, except that the board samples were equilibrated at 55% RH and 20 ◦ C prior to testing. 2.4. Electron spin resonance analysis Quantification of radicals by electron spin resonance (ESR) spectrometry was done by preparing the samples in Wilmad, Buena, NJ 710-SQ quartz tubes. Each tube contained approximately 400 mg of fibers corresponding to a sample height of 25 mm. Fiber samples were frozen in liquid nitrogen to stop further reactions of residual laccase. A Bruker, Karlsruhe, Germany ECS 106 X-band ESR spectrometer equipped with an X-band ER 403TM cavity was used for the measurements. Radicals on frozen fibers were quantified at −73 ◦ C by double integration of the first
Fig. 1. Schematic plot of pilot-scale process for production of laccase bonded fiberboards.
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Table 1 Processing parameters for pilot-scale production of fiberboards Parameter Preheating pressure (bar) Specific energy excluding idle load (kWh/ton) True disc clearance (mm) Production rate (kg/h) Preheating time (min) Preheating temperature (◦ C) Defibrator house pressure (bar) Differential pressure (bar) Main motor speed (rpm) Drier inlet temperature (◦ C) Drier outlet temperature (◦ C) Pressing temperature (◦ C) Pressing time (s)
UF-resin (8% w/w) + wax (1% w/w)
Laccase treated 6 U/g fiber
Laccase treated 24 U/g fiber
Laccase + wax treated 6 U/g fiber + wax (1% w/w)
Control
8.5 55
8.5 55
8.5 55
8.5 55
8.5 55
1.0 60 4 170 8.4 −0.1 1500 94 45 180 150
1.0 60 4 170 8.4 −0.1 1500 86 38 200 203
1.0 60 4 170 8.4 −0.1 1500 78 39 200 203
1.0 60 4 170 8.4 −0.1 1500 106 50 200 203
1.0 60 4 170 8.4 −0.1 1500 108 53 200 203
derivative ESR signal and compared to a weak pitch sample (Bruker) containing a known amount of unpaired spins [8]. Quantification of radicals was done for all treatments except for fibers treated with UF-resin + wax. Samples were prepared on site following the enzyme reaction in the rotary bin (wet) and after the drying of the fibers (dry). 2.5. Determination of lignin molecular mass by GPC Samples of MDF boards were disintegrated into fibers or bundles of fibers by gently grating the board. Lignin was extracted from a 5% suspension of the grated sample in 90% dioxane and 10% water at 40 ◦ C for 24 h. Subsequently, lignin was isolated from this solution by removing dioxane and then lowering pH to 2.5 with 1 M HCl, thereby precipitating the lignin. The suspension was frozen to facilitate coalescing of lignin. Remaining carbohydrate, extractives, and other water-soluble components were removed from the isolated lignin by washing the precipitate three times with water acidified to pH 2.4. Finally, water was removed from the isolated lignin by freeze-drying. For determination of molecular mass the isolated lignin samples were derivatized by acetylation following the procedure in [9]. The derivatized sample was then dissolved in 1.5 ml THF, filtered through a 0.45 m PTFE filter and injected into the HPLC. Molecular mass was determined relative to polystyrene standards by separation on Styragel HR 6, HR 4, and HR 3 columns (Waters, Milford, MA) connected in series [10]. Flow rate was set at 1 ml/min. Absorbance was monitored at 280 nm for lignin samples and at 254 nm for the polystyrene standards. Mass and number average molecular mass (Mw and Mn ) were calculated according to [11]. 3. Results and discussion In each of the individual treatments the results for MOE, MOR, and IB had a coefficient of variance of less than
6% within board samples and less than 9% between board samples. For TS and WA the figures were 11 and 16%, respectively. During the pilot-scale trials each treatment was run as a continuous process from preheating of chips to the final board pressing. The processing parameters (Tables 1 and 2) showed that only little variation in some process parameters occurred between the treatments. This is supported by the high repeatability of the tested physical board properties mentioned above. The UF-resin treated fibers were pressed at 180 ◦ C according to the specification of the adhesive, whereas the control and enzyme treated fibers were pressed at 200 ◦ C. Optimum pressing time as determined from the back pressure/curing profile for the enzyme and control treated boards was found to be 203 s, whereas only 150 s were needed for boards bonded by UF-resin. As lignin is believed to be the active component in enzymatic bonding, a higher temperature in the board is necessary to cure and bond the fibers. Thus, for enzyme treated boards, longer pressing times will be necessary in order to reach temperatures above the glass transition point of lignin in the center of the board. Due to the relatively small batch sizes it was difficult to fully stabilize the drying process and fluctuations occurred in the final MC (cf. Table 2). The MC of the fiber mats before hot pressing were 11–13%. This variation in MC of the fiber mats is believed not to have any significant impact upon the board properties. However, initial testing showed that a moisture content in the mat prior to hot pressing of at least 10% is important for the bonding process. This is probably related to the correlation between the moisture content and the glass transition temperature of lignin [12]. In these experiments the reaction time at 50 ◦ C for the enzyme treatment was approximately 30 min. The time needed for the enzyme treatment may be even shorter, however, no clear indication of the time needed can be obtained from these experiments, since only one time period was applied. As the maximum temperature for M. thermophila laccase
C. Felby et al. / Enzyme and Microbial Technology 31 (2002) 736–741
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Table 2 Temperature, pH and moisture parameters during the process flow
Pressurized refiner pH Pressurized refiner fiber outlet temperature (◦ C) Pressurized refiner MC (%) Atmospheric refiner inlet fiber temperature (◦ C) Atmospheric refiner outlet fiber temperature (◦ C) Atmospheric refiner outlet MC (%) Tumble mixer temperature (◦ C) Dried fibers MC (%)
UF-resin (8% w/w) + wax (1% w/w)
Laccase treated 6 U/g fiber
Laccase treated 24 U/g fiber
Laccase + wax treated 6 U/g fiber + wax (1% w/w)
Control
– 55 48 – – – – 14
4.15 59 50 47 45–50 61 47 18
4.13 48–58 50 41 50–58 59 48 17
4.15 52–58 50 42 50–55 60 48 16
4.14 55–60 50 41 50–55 58 46 16
is approximately 70 ◦ C according to the enzyme supplier (Novozymes A/S), it should be possible to increase the temperature by 15–20 ◦ C, thereby increasing the rate of reaction correspondingly. The strength and dimensional stability of the boards as shown in Table 3 are improved by the laccase treatment as compared to the untreated control. This is in accordance with previous findings at laboratory-scale [4,5]. The mechanical properties for the enzyme treated boards are at the same level as for boards made with UF-resin (cf. Table 3). For an enzyme dosage of 24 U/g nearly all mechanical properties are significantly higher than treatment at 6 U/g. The addition of wax seems to lower the bonding effect of the enzyme treatment (cf. Table 3). However, this does not correlate with the amount of laccase generated radicals as the level of radicals is similar for all enzyme treatments (Fig. 2). Detection of stable free radicals has been found to correlate well with the degree of laccase catalyzed oxidation [4]. Apparently the “wax coating” of fiber surfaces reduces the fiber–fiber interaction in the enzyme treated boards, and thereby the bonding effect. Similar observations on bonding mechanisms have been made [13,14]. For the combined wax and enzyme treatment the dimensional stability is not reduced as seen for the strength properties, this is most likely caused by the effect of the wax and to a lesser extent by the enzyme treatment. The “wax coating” in enzymatic bonding may be solved by changing the wax composition, e.g. applying it as granules or using a wax having a different surface energy, and thereby reducing the wetting of the lignin on fiber surfaces. Alternatively the possibility of using other methods for dimensional stabilization of the boards could
be investigated, e.g. the wet-strength technology applied in the paper industry. ESR-measurements show a high number of radicals in the laccase treated samples compared to untreated control fibers (Fig. 2). Drying of laccase treated fibers in the flash drier lowered the level of radicals, but the number of radicals was still significantly higher compared to the untreated control. The level of radicals shows that even though no free liquid water was present during the process, the enzyme was active and catalyzed a thorough oxidation of the fibers. This is an important observation as it shows that the enzyme can be applied under industrial type conditions, with a moisture content of 50–60%. In a previous work [15], laccase was added to fibers at the end of the blow line in order to use the
Fig. 2. The number of free radicals measured on laccase treated fibers before and after drying. The figures are reported as spins/C9 lignin unit assuming an approximate lignin content of 20% and an average C9 unit molecular mass of 200 Da.
Table 3 Board properties (delam. indicates delaminated samples) Treatment
Density (kg/m3 )
MOE (GPa)
MOR (MPa)
IB (MPa)
T.S (%)
W.A. (%)
Control UF-resin + wax 6U 24 U 6 U + wax
820 835 858 868 811
delam. 4.18 a 3.70 a 3.95 a 3.44 b
delam. 42.8 a 40.1 b 46.0 a 32.3 c
0.33 0.99 0.82 0.93 0.28
146 34 69 46 53
224 52 109 92 108
Homogenous groups at 95% LSD are shown by identical letters.
a b c b a
a b c d d
a b c d c
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existing processing equipment for MDF-boards, and to facilitate adaptation to an industrial process. The properties of the fiber boards were not improved and no excess radicals could be detected, probably because the high temperature, pressure and shear forces in the blow line inactivated the enzyme. An important question in laccase catalyzed bonding is whether covalent bonding of the lignin component is actually taking place. The difficulty in analyzing the lignin properties in hot-pressed fiberboards is among others, that a number of process parameters such as temperature, time, pressure, and moisture content will influence the lignin flow and degree of cross-linking. As the series of boards were obtained from a continuous process using consistent processing parameters, it was possible to do a valid comparison of the lignin molecular mass in untreated and laccase treated boards. Fig. 3 shows GPC chromatograms of lignin isolated from fibers and MDF boards treated with and without laccase, respectively. The corresponding values for Mw and Mn are shown in Table 4. It was assumed that during disintegration of the boards for preparing samples, rupture of inter-fiber bonds dominated over cleavage of fibers so the exposed surface resembled the fiber surface. This is supported by the similar retention time of the peak maximum found for lignin isolated from pulp and boards.
Table 4 Molecular mass and polydispersity for untreated and laccase treated fibers and MDF boards Treatment
Mw Mn Mw /Mn
Lignin isolated from fibers (Da)
Lignin isolated from MDF (Da)
Untreated
Laccase
Untreated
Laccase
14550 2395 6.1
19960 2855 7.0
26195 2230 11.7
44435 2745 16.2
Upon oxidation of fibers with laccase, Mw and Mn of the dioxane-soluble lignin increased 40 and 20%, respectively, giving only a minor change in polydispersity. Hot pressing of laccase treated fibers into fiberboards causes a substantial lignin polymerization, compared to pressing of untreated fibers. From the shoulder in the chromatogram in Fig. 3B, the Mw was increased about 70%, whereas Mn increased about 20%. The polydispersity increased from 11.7 to 16.2. For boards only, a small residue of the dioxane-extractable lignin from the enzyme- and control-treatments turned out to be insoluble in THF. The decreased solubility could be caused by a heat-induced polymerization of lignin, perhaps by cross-linking to carbohydrates.
Fig. 3. GPC chromatograms of lignin isolated from pulp (A) and fiberboards (B): control, laccase treated at dosage of 24 U/g pulp. Samples were acetylated and separated THF at 1.0 ml/min. Eluate monitored at 280 nm.
C. Felby et al. / Enzyme and Microbial Technology 31 (2002) 736–741
The increased molecular mass of isolated surface lignin from the laccase treated boards strongly suggests that polymerization of lignin is part of the increased bonding, i.e. covalent bonding of the lignin is taking place. The question is whether lignin tightly associated to fibers is involved in the polymerization, or if the strength is gained solely from polymerization of loosely associated lignin. Since technical lignin is known to have significant improved adhesive properties when polymerized through a laccase/peroxidase catalyzed oxidation [1], obviously the polymerization of lignin loosely associated to fibers would be expected to have a similar effect. This may also be supported from the fact that equal levels of radicals are seen for 6 and 24 U, but the strength of boards treated with 24 U is higher. This could indicate that, e.g. precipitation of lignin on the fiber surfaces and thereby loosely associated lignin is partly responsible for the bonding effect. Given that the dimensional properties of the boards using laccase as bonding catalyst can be improved to a level comparable to synthetic adhesives, the described process may be scaled up for full industrial application. The nature of laccase catalyzed bonding requires higher pressing temperatures and longer pressing times, two reasons why the concept may not be economically feasible as it is. However, the concept shows promise and possibilities in the use of oxidative enzymes for industrial bonding and modification of lignin.
4. Conclusion Fiberboards bonded by laccase catalyzed oxidation can be made in a pilot-scale process simulating continuous full-scale industrial production. The strength properties of the enzyme bonded boards are comparable to boards bonded by a conventional UF-resin and wax, whereas the dimensional stability properties of the enzyme bonded boards are not at the same level. The latter cannot be improved using a wax as this inhibits the bonding effect of the enzyme. This is most likely caused by reduced fiber–fiber interactions caused by a wax coating of the fibers. In enzyme bonded boards a cross-linking of the lignin can be observed. It cannot be concluded if this cross-linking is forming covalent bonds between fibers or if the polymerized loosely associated lignin works as an adhesive.
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Acknowledgments This work was financed by the Danish Ministry of Environment, the grant committee for product development in the forestry and timber industries, and Novozymes A/S.
References [1] Haars A, Kharazipour A, Zanker H, Hüttermann A. Roomtemperature curing adhesives based on lignin and phenoloxidases. In: Adhesives from renewable resources, ACS Symposium Series 385. Washington, DC: ACS, 1989. p. 126–34. [2] Hüttermann A, Milstein O, Nicklas B, Trojanowski J, Haars A, Kharazipour A. Enzymatic modification of lignin for technical use. Strategies and results. In: Lignin: properties and materials, ACS symposium series 397. ACS: Washington, DC, 1989. p. 361–70. [3] Yamaguchi H, Maeda Y, Sakata I. Application of phenol dehydrogenative polymerization by laccase to binding among woody-fibers. Mokuzai Gakkaishi 1992;38:931–7. [4] Felby C, Pedersen LS, Nielsen BR. Enhanced auto adhesion of wood fibers using phenol oxidases. Holzforschung 1997;51:281–6. [5] Kharazipour A, Hüttermann A, Luedemann HD. Enzymatic activation of wood fibers as a means for the production of wood composites. J Adhes Sci Technol 1997;11:419–27. [6] Felby C, Olesen PO, Hansen TT. Laccase catalyzed bonding of wood fibers. In: Enzymes in fiber processing, ACS Symposium Series 687. Washington, DC: ACS, 1998. p. 88–98. [7] Lund M, Felby C. Wet strength improvement of kraft pulp through laccase catalyzed oxidation. Enzyme Microb Technol 2001;28:760–5. [8] Felby C, Nielsen BR, Olesen PO, Skibsted LH. Identification and quantification of radical reaction intermediates by ESR-spectrometry of laccase catalyzed oxidation of wood fibers from beech (Fagus sylvatica). Appl Microbiol Biotechnol 1997;48:459–64. [9] Pellinen J, Salkinoja-Salonen M. High-performance size-exclusion chromatography of lignin and its derivatives. J Chromatogr 1985;328:299–308. [10] Glasser WG, Dave V, Frazier CE. Molecular weight distribution of (semi-) commercial lignin derivatives. J Wood Chem Technol 1993;13:545–59. [11] Yau WW, Kirkland JJ, Bly DD. Data handling. In: Modern size exclusion chromatography: practice of gel permeation and gel filtration chromatography. New York: John Wiley & Sons Inc, 1979. p. 315–26. [12] Goring DAI. Thermal softening of lignin, hemicellulose and cellulose. Pulp Paper Mag Can 1963;64:517–27. [13] Back EL. The bonding mechanism in hardboard manufacture. Holzforschung 1987;41:247–58. [14] Back EL. Oxidative activation of wood surfaces for glue bonding. Forest Prod J 1991;41:30–6. [15] Felby C. Enzymatic bonding of lignocellulosic materials. In: Environmental project report No. 442 (ISBN 87-7090-198-9). Denmark: Ministry of Environment and Energy, 1999. p. 68.
Pilot-scale production of fiberboards made by laccase oxidized wood fibers: board properties and evidence for cross-linking of lignin Claus Felby a,b,∗ , Jens Hassingboe a , Martin Lund c a c
Plant Fibre Laboratory, The Royal Veterinary and Agricultural University, Hoejbakkegaard Alle 1, DK-2630 Taastrup, Denmark b Novozymes A/S, Krogshoejvej 36, DK-2880 Bagsvaerd, Denmark Chemistry Department, The Royal Veterinary and Agricultural University, 40 Thorvaldsensvej, DK-1871 Frederiksberg, Denmark Received 9 July 2001; accepted 12 March 2002
Abstract Oxidoreductases can be applied for bonding of fiberboards, particle boards, paper boards and kraft-liner boards. In this work we report on pilot-scale production of laccase bonded fiberboards made from fibers of beech (Fagus sylvatica). Dioxane extractable lignin from fibers and boards are isolated and the molecular mass estimated by gel permeation chromatography. The strength properties of the enzyme bonded boards are comparable to boards bonded by an urea–formaldehyde adhesive, whereas the dimensional stability properties of the enzyme bonded boards are not at the same level. Wax treatment of the fibers, in order to improve dimensional stability of boards, is not compatible with the enzyme treatment. Cross-linking of the lignin can be observed in enzyme treated fibers and boards. Hot pressing of enzyme treated fibers results in a substantial cross-linking of lignin in boards. The enzymatic bonding effect may be caused by covalent bonds between fibers or an adhesive effect of polymerized loosely associated lignin. Laccase catalyzed bonding requires higher pressing temperatures and longer pressing times, and the concept may not be economically feasible as it is. However, it shows promise and possibilities in the use of oxidative enzymes for industrial bonding and modification of lignin. © 2002 Published by Elsevier Science Inc. Keywords: Laccase; Fiberboards; Lignin; Oxidation; Pilot-scale production
1. Introduction Bonding of wood fibers and particles by adhesives can be accomplished by forming a resinous matrix in which the particles or fibers are bonded together, e.g. by mechanical entanglement or covalent cross-linking. The bonding is caused not only by the added adhesive but also by the auto-adhesive properties of the wood components. Oxidoreductases such as laccase and peroxidase may be used for polymerization or cross-linking of wood components in order to bond these components. The concept of using lignin-oxidizing enzymes for bonding applications is based on the reactivity of phenoxy radicals in the plant cell wall. In vivo, oxidoreductases catalyze polymerization of lignin through cross-linking of phenoxy radicals, and thus it may be possible to utilize a similar type of reaction for bonding of lignocellulosic materials in vitro. Previous work reported the use of oxidoreductases for bonding of fiberboards, particle boards, paper boards and ∗
Corresponding author. Tel.: +45-35-28-22-18; fax: +45-35-28-22-16. E-mail address: [email protected] (C. Felby).
0141-0229/02/$ – see front matter © 2002 Published by Elsevier Science Inc. PII: S 0 1 4 1 - 0 2 2 9 ( 0 2 ) 0 0 1 1 1 - 4
kraft-liner boards [1–7]. So far, the work reported has been done on a laboratory-scale, and the results suggest the possibility of a new technology for bonding of lignocellulosic materials. In this work we report on pilot-scale production of laccase bonded fiberboards made from fibers of beech (Fagus sylvatica). The physicomechanical properties of the boards are presented and discussed. A new method for isolation of fiber surface lignin from boards is presented, and the molecular mass of lignin in boards and fibers is characterized by gel permeation chromatography (GPC).
2. Materials and methods 2.1. Materials Myceliophtera thermophila laccase was obtained from Novozymes A/S, Bagsvaerd, Denmark. Enzyme activity was measured in units (U), with 1 U defined as the amount of enzyme that oxidizes 1 mol syringaldazine per minute in potassium phosphate buffer pH 7.0, 30 ◦ C.
C. Felby et al. / Enzyme and Microbial Technology 31 (2002) 736–741
Urea–formaldehyde (UF)-resin was obtained from Casco-Nobel, Stockholm, Sweden. Mobilcer 072 liquid wax for improving the water tolerance of fiberboards was obtained from Mobil, London, UK. Beech wood chips for preparation of fiberboards were supplied by Junckers Industrier, Koege, Denmark. 1,4-Dioxane from Fisher Scientific, Leicestershire, UK for lignin extraction was distilled over sodium borhydride prior to use. Tetrahydrofuran (THF) as eluent for GPC was supplied by Merck, Darmstadt, Germany. Polystyrene standards with nominal molecular mass of 2500, 5000, 17500, and 30000 Da were supplied by Supelco, Bellafonte, PA and standards with nominal molecular mass of 3000 and 10000 Da were supplied by Crompak, Church Stretton, UK. 2.2. Preparation of fiberboards The fiberboards were made at Sunds Defibrator pilot-plant, Sundsvall, Sweden. This facility is capable of fully simulating a medium density fiberboard production from refining of chips to mat forming of fibers. Pressing was done in an electrically heated daylight type press using a dynamic press cycle. Fiberboards were prepared from five different treatments: Control—untreated UF-resin (8% w/w) + wax (1% w/w) Laccase treated 6 U/g fiber Laccase treated 24 U/g fiber Laccase + wax treated 6 U/g fiber + wax (1% w/w). Note that no control treatment using deactivated laccase is included as it was shown in [4], that there is no adhesive effect of deactivated laccase. For each treatment a minimum of six boards (500 mm × 500 mm) were pressed. Target thickness and density of the boards were 8 mm and 850 kg/m3 , i.e. that each board contained approximately 1700 g of fibers. The chips were preheated for 4 min and refined in a twin-disk refiner at 8.5 bar. The target moisture content before adding enzyme was set to 55%, water was added in
737
the blow-line in order to reach this level. UF-resin and wax was added at the end of the blow line. An aqueous solution (pH 7) of the enzyme was applied to the fibers in an atmospheric refiner preheated to 60 ◦ C. The atmospheric refiner only served as a mixer for enzymes and fibers and caused no further mechanical separation of the fibers. In order to allow sufficient time for the enzymatic reaction, fibers blended with enzyme were transferred to a 8 m3 rotary bin for 30 min at 50 ◦ C. Fibers from all treatments were dried in a flash drier to a moisture content (MC) of 14–18%. From the dried fibers, air-laid mats were formed and pre-pressed in a cold press. Note that the air layering of the mats lowered the moisture content to 11–13%. Hot pressing was done for 150 s at 180 ◦ C for UF-resin + wax treatment, and 203 s at 200 ◦ C for control and enzyme treatments. The full process is outlined in Fig. 1, and the process parameters are listed in Table 1. 2.3. Board properties For each treatment the boards were tested for modulus of elasticity (MOE), modulus of rupture (MOR), internal bond strength (IB), thickness swell (TS), and water absorption (WA) following a 24 h cold water soak. All testing was done according to ASTM D1037-78, except that the board samples were equilibrated at 55% RH and 20 ◦ C prior to testing. 2.4. Electron spin resonance analysis Quantification of radicals by electron spin resonance (ESR) spectrometry was done by preparing the samples in Wilmad, Buena, NJ 710-SQ quartz tubes. Each tube contained approximately 400 mg of fibers corresponding to a sample height of 25 mm. Fiber samples were frozen in liquid nitrogen to stop further reactions of residual laccase. A Bruker, Karlsruhe, Germany ECS 106 X-band ESR spectrometer equipped with an X-band ER 403TM cavity was used for the measurements. Radicals on frozen fibers were quantified at −73 ◦ C by double integration of the first
Fig. 1. Schematic plot of pilot-scale process for production of laccase bonded fiberboards.
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C. Felby et al. / Enzyme and Microbial Technology 31 (2002) 736–741
Table 1 Processing parameters for pilot-scale production of fiberboards Parameter Preheating pressure (bar) Specific energy excluding idle load (kWh/ton) True disc clearance (mm) Production rate (kg/h) Preheating time (min) Preheating temperature (◦ C) Defibrator house pressure (bar) Differential pressure (bar) Main motor speed (rpm) Drier inlet temperature (◦ C) Drier outlet temperature (◦ C) Pressing temperature (◦ C) Pressing time (s)
UF-resin (8% w/w) + wax (1% w/w)
Laccase treated 6 U/g fiber
Laccase treated 24 U/g fiber
Laccase + wax treated 6 U/g fiber + wax (1% w/w)
Control
8.5 55
8.5 55
8.5 55
8.5 55
8.5 55
1.0 60 4 170 8.4 −0.1 1500 94 45 180 150
1.0 60 4 170 8.4 −0.1 1500 86 38 200 203
1.0 60 4 170 8.4 −0.1 1500 78 39 200 203
1.0 60 4 170 8.4 −0.1 1500 106 50 200 203
1.0 60 4 170 8.4 −0.1 1500 108 53 200 203
derivative ESR signal and compared to a weak pitch sample (Bruker) containing a known amount of unpaired spins [8]. Quantification of radicals was done for all treatments except for fibers treated with UF-resin + wax. Samples were prepared on site following the enzyme reaction in the rotary bin (wet) and after the drying of the fibers (dry). 2.5. Determination of lignin molecular mass by GPC Samples of MDF boards were disintegrated into fibers or bundles of fibers by gently grating the board. Lignin was extracted from a 5% suspension of the grated sample in 90% dioxane and 10% water at 40 ◦ C for 24 h. Subsequently, lignin was isolated from this solution by removing dioxane and then lowering pH to 2.5 with 1 M HCl, thereby precipitating the lignin. The suspension was frozen to facilitate coalescing of lignin. Remaining carbohydrate, extractives, and other water-soluble components were removed from the isolated lignin by washing the precipitate three times with water acidified to pH 2.4. Finally, water was removed from the isolated lignin by freeze-drying. For determination of molecular mass the isolated lignin samples were derivatized by acetylation following the procedure in [9]. The derivatized sample was then dissolved in 1.5 ml THF, filtered through a 0.45 m PTFE filter and injected into the HPLC. Molecular mass was determined relative to polystyrene standards by separation on Styragel HR 6, HR 4, and HR 3 columns (Waters, Milford, MA) connected in series [10]. Flow rate was set at 1 ml/min. Absorbance was monitored at 280 nm for lignin samples and at 254 nm for the polystyrene standards. Mass and number average molecular mass (Mw and Mn ) were calculated according to [11]. 3. Results and discussion In each of the individual treatments the results for MOE, MOR, and IB had a coefficient of variance of less than
6% within board samples and less than 9% between board samples. For TS and WA the figures were 11 and 16%, respectively. During the pilot-scale trials each treatment was run as a continuous process from preheating of chips to the final board pressing. The processing parameters (Tables 1 and 2) showed that only little variation in some process parameters occurred between the treatments. This is supported by the high repeatability of the tested physical board properties mentioned above. The UF-resin treated fibers were pressed at 180 ◦ C according to the specification of the adhesive, whereas the control and enzyme treated fibers were pressed at 200 ◦ C. Optimum pressing time as determined from the back pressure/curing profile for the enzyme and control treated boards was found to be 203 s, whereas only 150 s were needed for boards bonded by UF-resin. As lignin is believed to be the active component in enzymatic bonding, a higher temperature in the board is necessary to cure and bond the fibers. Thus, for enzyme treated boards, longer pressing times will be necessary in order to reach temperatures above the glass transition point of lignin in the center of the board. Due to the relatively small batch sizes it was difficult to fully stabilize the drying process and fluctuations occurred in the final MC (cf. Table 2). The MC of the fiber mats before hot pressing were 11–13%. This variation in MC of the fiber mats is believed not to have any significant impact upon the board properties. However, initial testing showed that a moisture content in the mat prior to hot pressing of at least 10% is important for the bonding process. This is probably related to the correlation between the moisture content and the glass transition temperature of lignin [12]. In these experiments the reaction time at 50 ◦ C for the enzyme treatment was approximately 30 min. The time needed for the enzyme treatment may be even shorter, however, no clear indication of the time needed can be obtained from these experiments, since only one time period was applied. As the maximum temperature for M. thermophila laccase
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Table 2 Temperature, pH and moisture parameters during the process flow
Pressurized refiner pH Pressurized refiner fiber outlet temperature (◦ C) Pressurized refiner MC (%) Atmospheric refiner inlet fiber temperature (◦ C) Atmospheric refiner outlet fiber temperature (◦ C) Atmospheric refiner outlet MC (%) Tumble mixer temperature (◦ C) Dried fibers MC (%)
UF-resin (8% w/w) + wax (1% w/w)
Laccase treated 6 U/g fiber
Laccase treated 24 U/g fiber
Laccase + wax treated 6 U/g fiber + wax (1% w/w)
Control
– 55 48 – – – – 14
4.15 59 50 47 45–50 61 47 18
4.13 48–58 50 41 50–58 59 48 17
4.15 52–58 50 42 50–55 60 48 16
4.14 55–60 50 41 50–55 58 46 16
is approximately 70 ◦ C according to the enzyme supplier (Novozymes A/S), it should be possible to increase the temperature by 15–20 ◦ C, thereby increasing the rate of reaction correspondingly. The strength and dimensional stability of the boards as shown in Table 3 are improved by the laccase treatment as compared to the untreated control. This is in accordance with previous findings at laboratory-scale [4,5]. The mechanical properties for the enzyme treated boards are at the same level as for boards made with UF-resin (cf. Table 3). For an enzyme dosage of 24 U/g nearly all mechanical properties are significantly higher than treatment at 6 U/g. The addition of wax seems to lower the bonding effect of the enzyme treatment (cf. Table 3). However, this does not correlate with the amount of laccase generated radicals as the level of radicals is similar for all enzyme treatments (Fig. 2). Detection of stable free radicals has been found to correlate well with the degree of laccase catalyzed oxidation [4]. Apparently the “wax coating” of fiber surfaces reduces the fiber–fiber interaction in the enzyme treated boards, and thereby the bonding effect. Similar observations on bonding mechanisms have been made [13,14]. For the combined wax and enzyme treatment the dimensional stability is not reduced as seen for the strength properties, this is most likely caused by the effect of the wax and to a lesser extent by the enzyme treatment. The “wax coating” in enzymatic bonding may be solved by changing the wax composition, e.g. applying it as granules or using a wax having a different surface energy, and thereby reducing the wetting of the lignin on fiber surfaces. Alternatively the possibility of using other methods for dimensional stabilization of the boards could
be investigated, e.g. the wet-strength technology applied in the paper industry. ESR-measurements show a high number of radicals in the laccase treated samples compared to untreated control fibers (Fig. 2). Drying of laccase treated fibers in the flash drier lowered the level of radicals, but the number of radicals was still significantly higher compared to the untreated control. The level of radicals shows that even though no free liquid water was present during the process, the enzyme was active and catalyzed a thorough oxidation of the fibers. This is an important observation as it shows that the enzyme can be applied under industrial type conditions, with a moisture content of 50–60%. In a previous work [15], laccase was added to fibers at the end of the blow line in order to use the
Fig. 2. The number of free radicals measured on laccase treated fibers before and after drying. The figures are reported as spins/C9 lignin unit assuming an approximate lignin content of 20% and an average C9 unit molecular mass of 200 Da.
Table 3 Board properties (delam. indicates delaminated samples) Treatment
Density (kg/m3 )
MOE (GPa)
MOR (MPa)
IB (MPa)
T.S (%)
W.A. (%)
Control UF-resin + wax 6U 24 U 6 U + wax
820 835 858 868 811
delam. 4.18 a 3.70 a 3.95 a 3.44 b
delam. 42.8 a 40.1 b 46.0 a 32.3 c
0.33 0.99 0.82 0.93 0.28
146 34 69 46 53
224 52 109 92 108
Homogenous groups at 95% LSD are shown by identical letters.
a b c b a
a b c d d
a b c d c
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existing processing equipment for MDF-boards, and to facilitate adaptation to an industrial process. The properties of the fiber boards were not improved and no excess radicals could be detected, probably because the high temperature, pressure and shear forces in the blow line inactivated the enzyme. An important question in laccase catalyzed bonding is whether covalent bonding of the lignin component is actually taking place. The difficulty in analyzing the lignin properties in hot-pressed fiberboards is among others, that a number of process parameters such as temperature, time, pressure, and moisture content will influence the lignin flow and degree of cross-linking. As the series of boards were obtained from a continuous process using consistent processing parameters, it was possible to do a valid comparison of the lignin molecular mass in untreated and laccase treated boards. Fig. 3 shows GPC chromatograms of lignin isolated from fibers and MDF boards treated with and without laccase, respectively. The corresponding values for Mw and Mn are shown in Table 4. It was assumed that during disintegration of the boards for preparing samples, rupture of inter-fiber bonds dominated over cleavage of fibers so the exposed surface resembled the fiber surface. This is supported by the similar retention time of the peak maximum found for lignin isolated from pulp and boards.
Table 4 Molecular mass and polydispersity for untreated and laccase treated fibers and MDF boards Treatment
Mw Mn Mw /Mn
Lignin isolated from fibers (Da)
Lignin isolated from MDF (Da)
Untreated
Laccase
Untreated
Laccase
14550 2395 6.1
19960 2855 7.0
26195 2230 11.7
44435 2745 16.2
Upon oxidation of fibers with laccase, Mw and Mn of the dioxane-soluble lignin increased 40 and 20%, respectively, giving only a minor change in polydispersity. Hot pressing of laccase treated fibers into fiberboards causes a substantial lignin polymerization, compared to pressing of untreated fibers. From the shoulder in the chromatogram in Fig. 3B, the Mw was increased about 70%, whereas Mn increased about 20%. The polydispersity increased from 11.7 to 16.2. For boards only, a small residue of the dioxane-extractable lignin from the enzyme- and control-treatments turned out to be insoluble in THF. The decreased solubility could be caused by a heat-induced polymerization of lignin, perhaps by cross-linking to carbohydrates.
Fig. 3. GPC chromatograms of lignin isolated from pulp (A) and fiberboards (B): control, laccase treated at dosage of 24 U/g pulp. Samples were acetylated and separated THF at 1.0 ml/min. Eluate monitored at 280 nm.
C. Felby et al. / Enzyme and Microbial Technology 31 (2002) 736–741
The increased molecular mass of isolated surface lignin from the laccase treated boards strongly suggests that polymerization of lignin is part of the increased bonding, i.e. covalent bonding of the lignin is taking place. The question is whether lignin tightly associated to fibers is involved in the polymerization, or if the strength is gained solely from polymerization of loosely associated lignin. Since technical lignin is known to have significant improved adhesive properties when polymerized through a laccase/peroxidase catalyzed oxidation [1], obviously the polymerization of lignin loosely associated to fibers would be expected to have a similar effect. This may also be supported from the fact that equal levels of radicals are seen for 6 and 24 U, but the strength of boards treated with 24 U is higher. This could indicate that, e.g. precipitation of lignin on the fiber surfaces and thereby loosely associated lignin is partly responsible for the bonding effect. Given that the dimensional properties of the boards using laccase as bonding catalyst can be improved to a level comparable to synthetic adhesives, the described process may be scaled up for full industrial application. The nature of laccase catalyzed bonding requires higher pressing temperatures and longer pressing times, two reasons why the concept may not be economically feasible as it is. However, the concept shows promise and possibilities in the use of oxidative enzymes for industrial bonding and modification of lignin.
4. Conclusion Fiberboards bonded by laccase catalyzed oxidation can be made in a pilot-scale process simulating continuous full-scale industrial production. The strength properties of the enzyme bonded boards are comparable to boards bonded by a conventional UF-resin and wax, whereas the dimensional stability properties of the enzyme bonded boards are not at the same level. The latter cannot be improved using a wax as this inhibits the bonding effect of the enzyme. This is most likely caused by reduced fiber–fiber interactions caused by a wax coating of the fibers. In enzyme bonded boards a cross-linking of the lignin can be observed. It cannot be concluded if this cross-linking is forming covalent bonds between fibers or if the polymerized loosely associated lignin works as an adhesive.
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Acknowledgments This work was financed by the Danish Ministry of Environment, the grant committee for product development in the forestry and timber industries, and Novozymes A/S.
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