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Production and properties of adhesives formulated from laccase modified Kraft lignin
Industrial Crops and Products 45 (2013) 343–348
Contents lists available at SciVerse ScienceDirect
Industrial Crops and Products journal homepage: www.elsevier.com/locate/indcrop
Production and properties of adhesives formulated from laccase modified Kraft lignin Victor Ibrahim a , Gashaw Mamo a,∗ , Per-Johan Gustafsson b , Rajni Hatti-Kaul a a b
Department of Biotechnology, Center for Chemistry and Chemical Engineering, Lund University, P.O. Box 124, SE-221 00 Lund, Sweden Division of Structural Mechanics, Lund University, P.O. Box 118, SE-221 00 Lund, Sweden
a r t i c l e
i n f o
Article history: Received 27 October 2012 Received in revised form 26 December 2012 Accepted 27 December 2012 Keywords: Formaldehyde-free adhesive Lignin Laccase Fracture energy Tensile strength Water resistance
a b s t r a c t Kraft lignin was used to formulate adhesives in combination with polyethyleneimine, chitosan and soy protein, respectively. The tensile strengths of bond lines formulated with laccase-treated and untreated lignin were found to be similar. However, a remarkable difference was noted when laccase-treated lignin was reduced by sodium borohydride prior to reacting either with chitosan or soy protein – decreasing the strength in the former case while increasing it in the other. Among the formulations, laccase treated and reduced lignin–soy protein adhesive exhibited more than half the strength of commercial polyurethane adhesive and retained 70% of its initial strength after two cycles of 1 h boiling and drying. © 2013 Elsevier B.V. All rights reserved.
1. Introduction The growing public concern about environmental issues and the increasing price of fossil oil have led to an enormous interest in the use of renewable resources, especially lignocellulosic biomass, for production of energy and materials. Lignin, which accounts for 15–36% of lignocellulosic biomass, is the second most abundant renewable resource on earth (Youn et al., 1995). It is structurally complex and an amorphous polymer with randomly distributed stable linkages between the monomeric aromatic components. In nature, lignin is covalently associated with hemicellulose and serves as cement to bond together cellulose fibers in woods. It also serves as a water-proofing and antimicrobial agent. As a byproduct of the pulping process, large amount of lignin, estimated to reach over 50 × 106 tons, is produced each year (Chen et al., 2009). Except for a minor fraction, most of this lignin recovered from the pulping process is combusted to generate energy. However, there has always been interest in using this abundant renewable resource in production of value added materials (González-García et al., 2011; Hüttermann et al., 2001; Mai et al., 2000; Milstein et al., 1994).
∗ Corresponding author. Tel.: +46 46 222 47 41; fax: +46 46 222 47 13. E-mail address: [email protected] (G. Mamo). 0926-6690/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.indcrop.2012.12.051
Lignin is now being used as a raw material for various value added products such as vanillin, dimethylsulfoxide (DMSO), syringaldehyde, etc. (Priefert et al., 2001; Wu et al., 1994). Several investigations have been targeted toward using lignin in formulation of formaldehyde free wood adhesives (Li and Geng, 2005). However, the presence of abundant methyl groups makes lignin less reactive and poor in cross-linking (Okamoto et al., 1996). Hence, lignin-based adhesives never exceeded the binding quality of the conventional phenol-formaldehyde adhesives and this has hindered the large-scale application of lignin based adhesives. Thus, if lignin has to be considered for production of glue, improving its cross-linking ability seems mandatory. The reactivity of lignin can be improved by demethylating the lignin aromatic rings. Demethylation can be achieved chemically (Liu and Li, 2006; Okamoto et al., 1996) or enzymatically using laccase (Filley et al., 2002; Ibrahim et al., 2011), and the activated lignin can react with molecules bearing nucleophilic groups to form cross-linked complexes. The enzymatic treatment is milder, cleaner and safer and is preferable over the chemical demethylation. It may also result in partial/extensive degradation of lignin and release monomeric subunits, which are also reactive toward other active groups and/or polymerize in the presence of the enzyme. In this study, laccase modified lignin was reacted with other polymers including two biopolymers, chitosan and soy protein, to formulate resins that are safe and cheap, and the formulations were tested for their adhesive properties.
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Table 1 Summary of the different lignin-based adhesive formulations used in this study. The ratio of mixed formulations was 1:1 as mentioned in Section 2.1. Additive
Untreated lignin
Laccase-treated lignin
Laccase-treated and NaBH4 reduced lignin
No additive Polyethylenimine (PEI) Chitosan (C) Soy protein (SP)
L LP
LL LLP
R LL R LLP
LC LSP
LLC LLSP
R LLC R LLSP
2. Materials and methods Norway spruce (Picea abies) grown in Sweden and with an average density of 421 kg m−3 was used to study the mechanical properties of bonding adhesive layers. Hard wood Kraft lignin from Eucalyptus was kindly provided by Innventia AB (Sweden) and was used as a basic component for the formulation of adhesives. Medium molecular weight chitosan (MW ∼75,000 g mol−1 , from Fluka BioChemika, Switzerland), branched polyethylenimine (PEI, MW ∼25,000 g mol−1 by LS, from Aldrich® , Germany) and nutritional soy protein SP (88% protein concentration, from Pro Nutrition, Romania), respectively, were used as additives to the Kraft lignin in the produced composites. A commercial polyurethane (PU) wood adhesive (SikaBond® -545, Sika AB, Sweden) was tested for comparison and a two-component methyl methacrylate adhesive (X 60, from HBM, Germany) was used for gluing the wooden joints to steel parts as shown in Scheme 1. Polyvinyl acetate (PVAc) based adhesive (SikaBond® -530, Sika AB, Sweden) was used to make strong joints allowing the study of wood failure. The culture supernatant of the fungus Galerina sp. HC1 was used as source of laccase (Ibrahim et al., 2011). The enzyme activity was determined spectrophotometrically at 420 nm by the oxidation of 1 mM 2,2 -azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) diammonium salt (ABTS, Sigma–Aldrich) at pH 5. One unit of the enzyme activity was defined as the amount of the enzyme that produces 1 mol of product per minute under the reaction conditions used. 2.1. Preparation of lignin-based adhesive formulations Stock solutions of the different polymers for use in adhesive formulations were prepared: 25 mg mL−1 Kraft lignin in 80 mM sodium hydroxide, 25 mg mL−1 soy protein in water, 15 mg mL−1 chitosan in 1% acetic acid, and 25 mg mL−1 PEI solution in water with pH adjusted to 5 using 2 M hydrochloric acid. A 20 mL reaction mixture was prepared containing: 150 mg Kraft lignin, 150 mg chitosan, PEI or SP, 20 mM sodium acetate buffer pH 5, 0.75 mM ABTS and 1.5 U of laccase, and stirred in a round bottom flask at room temperature for 3 h. The mixture was then transferred to 50 mL Falcon tubes and centrifuged for 10 min at 2800 × g. The supernatant was discarded and the precipitate (with solid content ranging between 3 and 8 wt%) containing primarily insoluble- and crosslinked lignin with the other polymer was used for testing the adhesive properties. Reduced lignin was prepared by treating laccase modified lignin overnight with sodium borohydride (NaBH4 ; 1%, w/w, of the lignin dry weight) on rocking table at 22 ◦ C. After drying, 150 mg of the reduced lignin was mixed with equal weight of PEI, chitosan or soy protein in 20 mL of water for 3 h. After centrifugation, the insoluble mass was used for adhesive testing as indicated below. Formulations with lignin only, treated in the same way as the other formulations, were prepared as control. Table 1 provides a list of all the preparations made and tested.
2.2. Preparation of wood joints The wood was first cut into pieces of 3 mm × 32 mm × 32 mm. A suitable amount of the adhesive formulation with about 80 mg of the total solid content was spread all over the surfaces of the pieces to be glued. The joints were pressed at a pressure of ca. 2 MPa using hydraulic press and cured either overnight at room temperature (∼22 ◦ C) or 15 min at 120 ◦ C. Subsequently, the joints were cut into 4 pieces of ca. 15 mm × 15 mm size and used to evaluate the bond line mechanical properties. 2.3. Evaluation of bond line mechanical properties All joints were tested for their load vs. deformation performance using a conventional type of loading machine (MTS 810) with a hydraulic piston. Tensile stress vs. deformation, tensile strength and tensile fracture energy of the bond lines were determined from the recorded load vs. deformation curves. The tensile load was imposed perpendicular to the adhesive layer and the orientation of the wood was such that the grain of the wood was perpendicular to the load (Scheme 1). The specimens were attached to the loading setup as follows: First a two-component adhesive (X 60) was applied on the surface of the lower steel part onto which one side of the wooden joint was glued prior to setting it in the loading machine. After complete hardening of the adhesive, the second side of the joint was glued to the upper steel part and hardening was allowed to take place in the machine. Finally, a programmed loading sequence with a piston rate of 0.5 mm min−1 was imposed causing the specimen to delaminate as load and deformation were recorded. The recorded deformation, ı, was the relative displacement between the upper and lower steel parts. At least two tests were performed for each type of specimen. From recorded load, P, stress, , was determined at the assumption of uniform stress, i.e. simply by dividing the load by the glue area, A. The highest point of the stress-strain ( vs. ı) curve was taken as the tensile strength. The fracture energy of a bond line, Gf , is the work, W, required to completely separate the two fracture surfaces divided by the fracture area A. The fracture energy can be determined as the area under the vs. ı curve of the bond line: W 1 = Gf = A A
ı0
Pdı = 0
ı0
dı
(1)
0
where the upper integration limit for ı, ı0 , is the deformation or bond line opening corresponding to complete failure and zero stress ( = 0). 3. Results and discussion Kraft lignin was modified by laccase and used in the formulation of potential adhesives. The formulations were made by mixing the lignin samples respectively with three different polymers, all bearing amino groups. One of the polymers was PEI, a synthetic cationic polymer that has earlier been combined with lignin to give an adhesive preparation (Liu and Li, 2006). The other two polymers originate from renewable resources – chitosan, a polymer of glucosamine and N-acetylglucosamine obtained by deacetylation of chitin, and soy protein, which constitutes 40% of the total weight of the soybean that is produced in multi million metric tons yearly (Kinsella, 1979; Wolf, 1970). The preparation of the wood joints and evaluation of adhesive properties were done following the protocols used in previous reports (Li and Geng, 2005; Liu and Li, 2006; Li et al., 2010), after some modification to allow comparison of the different formulations. The tensile strength of unmodified and modified lignin
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Scheme 1. The set-up for evaluation of bond line mechanical properties.
samples blended with PEI, chitosan and soy protein, respectively, was initially assessed after curing the wood joints overnight at room temperature. The strength of unblended lignin was lower regardless of the modification (Fig. 1). Mixing of lignin with PEI, chitosan and soy protein resulted in higher tensile strength; however, the modification of lignin had varying effects when mixed with the different polymers. The tensile strength of the three formulations of lignin and PEI (LP) was similar. On the other hand, the tensile strength of lignin–chitosan (LC) formulation was slightly improved when laccase treated lignin was used but the subsequent reduction with NaBH4 drastically reduced the binding strength (Fig. 1). Chitosan and lignin have been used in the production of composite films and analysis of these films by FT-IR revealed hydrogen bonding between the two polymers (Chen et al., 2009). This may render even the untreated lignin–chitosan to have good binding strength. Peshkova and Li have earlier developed chitosan–phenolic adhesive formulations in the presence of laccase and proposed the adhesion mechanisms of these preparations to be similar to the mussel adhesive proteins (Peshkova and Li, 2003). Treatment of laccase oxidized lignin by sodium borohydride would decrease the reactive carbonyl group content which substantially lowers the hydrogen bond formation and the consequent binding force. Investigation of brown-rotted lignin has shown reduction in carbonyl groups content after sodium borohydride treatment (Li et al., 2010).
Fig. 1. Effect of different treatments on the binding strength of lignin formulations: only lignin (L), lignin–polyethylenimine (LP), lignin–chitosan (LC) and lignin–soy protein (LSP). The black, gray and white bars represent formulations in which the lignin used was untreated, treated with laccase, and treated with laccase followed by reduction with NaBH4 , respectively. The curing was carried out overnight (∼16 h) at room temperature (∼22 ◦ C).
The lignin–soy protein (LSP) formulation in which the lignin was treated with laccase and then reduced by NaBH4 exhibited an opposite trend with respect to the tensile strength which was more than fourfold higher compared to the LSP formulations with untreated or laccase treated lignin. Wei et al. have reported that addition of hydroxypropyl lignin (up to 2% by weight) to soy protein for the production of biodegradable plastics increased the tensile strength of the material by more than twofold (Wei et al., 2006). However, as the hydroxypropyl lignin concentration increased, the cross-linking was decreased due to self-aggregation of the lignin. The results in Fig. 1 indicate that the LSP with untreated lignin has lower binding strength, which could be due to the poor reactivity of the methylated lignin. However, the low binding strength of LSP with laccase treated lignin (LLSP) could be due to extensive intra- and inter-cross-linking of the polymers that potentially results in aggregation and reduces the binding of the formulation with the wood. But when the enzyme treated lignin was reduced for preparing the formulation, laccase was removed prior to mixing with the soy protein and hence oxidation and cross-linking of the protein is prevented. This allows enough interaction between the lignin and the protein, while leaving some reactive groups for binding to the wood which is expected to enhance the binding strength. In order to confirm if the laccase treatment was essential for the high tensile strength of R-LLSP, a formulation was prepared in which the lignin was subjected to treatment with NaBH4 without prior enzymatic treatment. The tensile strength of this preparation (0.8 MPa) was less than half of R-LLSP (1.7 MPa). The effect of curing temperature on the tensile strength of the formulations (except L, LL and R-LL) was studied by subjecting them to curing at different temperatures. Soy protein (SP) was also included in the study since it is commercially used as an adhesive. It was observed that curing at room temperature (∼22 ◦ C) required a longer time for removal of water and to achieve the best tensile strength. The curing time could be substantially shortened by increasing the curing temperature to above 100 ◦ C. At 120 ◦ C, except for R-LLC and LSP curing for only 10–15 min resulted in relatively higher tensile strength. A study of the fracture surfaces after tensile strength testing showed a striped appearance for almost all specimens as seen in Fig. 2. The location of the failure can be within the adhesive layer (dark color on both fracture surfaces) or at the interface between wood and adhesive (few white fibers or a very thin layer of white fibers on dark color on one side and mostly white wood on the other side) or within the wood (white wood on both sides). As it can be seen in Fig. 2a and b, the location of failure in the adhesive–wood interface was found to be dominating.
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Fig. 2. The fracture surfaces of wood joints after tensile testing. (a) Distribution of adhesive lines on each side of the opened joint; the dark brown stripes are the lignin-based adhesive on top of which white fibers from the contact surface with the wood can be observed. (b) Correlation of the adhesive stripes with the annual rings.
Moreover, the distribution of the adhesive layer into parallel lines coinciding with the “late wood” lines (dark, high density and strong part of the annual ring) is an evidence that the failure is occurring in the “early wood” (light, low density and weak) part of the annual ring (Fig. 2b). On the other hand, the SP specimens displayed failure within the adhesive layer itself while the PU samples had the failure occurring mainly at the interface but partially in the wood (∼20%) with wood strips observed on both sides of the tested joints. Besides the tensile strength, which corresponds to the peak stress value of the recorded stress (MPa) versus deformation (or relative displacement of the steel parts in mm) curve, the fracture energy (Gf ) is the second important parameter of the bond line in relation to load carrying capacity of a glued joint having the bond line stressed in tension (Eq. (1), Fig. 3 and Table 2). As can be seen in Table 2, the Gf values are close to what has been reported for resorcinol-phenol bond line (310 J m−2 ) but much lower than that of polyvinyl acetate PVAc (820 J m−2 ) (Wernersson, 1994). Moreover, the determined average fracture energy of the wood (P. abies) as used in our experiments is 404 J m−2 which is a typical value for this type of wood (Larsen and Gustafsson, 1990).
For a given tensile strength and fracture energy of bond line, the shape of the stress versus deformation curve is important for the load capacity of a structural joint. There is, however, no simple relation between bond line curve shape and joint capacity. A bond line with low strength and high fracture energy corresponds to a ductile one, while high strength and low fracture energy corresponds to a brittle bond line (Gustafsson, 1987). On comparing the lignin-based formulations which have rather close tensile strength values, we can conclude that the ones containing chitosan (LC and LLC) are more ductile than the one with soy protein (R-LLSP). On the other hand, polyurethane (PU) seems to have higher brittleness
Table 2 Calculated fracture energy of wood joints bond line made using different adhesives. The mean curves presented in Fig. 3 were used to derive the fracture energy. Abbreviations are given in text and in the legend of Fig. 1. Adhesives used to make joints
Fracture energy (J m−2 )
LC LLC R-LLSP SP PU
230 213 131 221 255
Fig. 3. Mean results of tensile tests with (––) LC, (—) LLC, (····) R-LLSP, (—) SP, and (–·–) PU. Each curve is a mathematical average of 2–4 experimental curves. Samples were heated under press for 15 min at 120 ◦ C prior to their testing.
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composite adhesive is the resistance against microbial degradation. Soy protein is susceptible to microbial degradation, particularly in humid condition. Since lignin is known to have anti-microbial effect (Zemek et al., 1979), the lignin–soy protein adhesive formulations may not need addition of antimicrobial agents. 4. Conclusion
Fig. 4. Water resistance test of the formulated adhesives. The black, gray and white bars represent the binding strength of the adhesives initially, and after 1st and 2nd rounds of placing the glued joints in boiling water, respectively. The formulations tested were lignin–chitosan (LC), laccase treated lignin–chitosan (LLC), laccase oxidized and reduced lignin–soy protein (R-LLSP), soy protein (SP) and polyurethane (PU). The wood joints glued using the different formulations were heated under press for 15 min at 120 ◦ C prior to testing.
in comparison with soy protein (SP) knowing that both have close fracture energy values. Ductility is a desired property in an adhesive when gluing brittle materials like wood, paper or glass fiber polymer composite sheets. It is also advantageous to have an adhesive with higher fracture energy than that of the adherend as it gives a higher joint load capacity and a more ductile character of failure (Gustafsson, 1987). The tensile strength and fracture energy achieved by some of the lignin based formulations (LC, LLC and R-LLSP) may be enough for applications such as in the binding of paper, cardboard boxes, etc. where very high binding strength is not necessary. For a number of indoor and outdoor applications, adhesives for making joints need to be water resistant. The water resistance of the adhesive formulations was tested by keeping the glued joints in boiling water for 1 h, subsequent drying and re-boiling for 1 h. The wood joints made using lignin–PEI formulations were separated already during the first round of boiling. Fig. 4 shows the water resistance of the joints made using lignin formulations with chitosan and soy protein in comparison with that of soy protein and polyurethane adhesives. Polyurethane adhesive exhibited the highest tensile strength and water resistance. The tensile strength of soy protein was also quite good but it was relatively less resistant to water. The R-LLSP formulation exhibited more than half of the binding strength of the polyurethane adhesive and its water resistance property seemed to be better than that of soy protein. It retained 92% of the original strength after 1 h boiling while soy protein adhesive maintained only 74% of its initial binding strength (Fig. 4). Lignin or its derivatives have been used to replace the phenol component of phenol–formaldehyde adhesives (IARC, 2006; Nielsen and Wolkoff, 2010). However, due to the unfavorable health effect of formaldehyde, such a substitution cannot lead to the achievement of the most desirable property, i.e. safe to use. In this regard, the formulation of adhesives by blending lignin with soy protein or chitosan and the adhesive property exhibited by the respective formulations is encouraging. Although soy protein adhesives are available in the market, formulation of soy protein–lignin adhesives is expected to offer economic and technical merits. When used as a composite with lignin, less amount of soy protein will be required for adhesive application, leaving more available for other applications including foods and feeds. Thus, blending may substantially cut down the glue production cost. Moreover, the observed water resistance suggests that blending can result in a better quality which may improve its marketability. The other advantage of having the lignin/soy protein
The adhesives formulated were water based which avoids exposure to solvents, prepared as ready to use single component that does not need mixing. At least the adhesive formulated from laccase modified and chemically reduced lignin, and soy protein showed a reasonable degree of water resistance. Moreover, the binding strength achieved by some of the lignin based formulations could be enough for applications where high binding strength is not necessary such as in paper binding, cardboard boxes, etc. Further investigations on improving the adhesive quality of promising formulations are ongoing in order to develop “green adhesives” that can suit different applications. Acknowledgement The Swedish Foundation for Strategic Environmental Research (MISTRA) is gratefully acknowledged for its financial support. References Chen, L., Tang, C.Y., Ning, N.Y., Wang, C.Y., Fu, Q., Zhang, Q., 2009. Preparation and properties of chitosan/lignin composite films. Chin. J. Polym. Sci. 27, 739–746. Filley, T.R., Cody, G.D., Goodell, B., Jellison, J., Noser, C., Ostrofsky, A., 2002. Lignin demethylation and polysaccharide decomposition in spruce sapwood degraded by brown rot fungi. Org. Geochem. 33, 111–124. González-García, S., Feijoo, G., Heathcote, C., Kandelbauer, A., Moreira, M.T., 2011. Environmental assessment of green hardboard production coupled with a laccase activated system. J. Cleaner Prod. 19, 445–453. Gustafsson, P.J., 1987. Analysis of generalized Volkersen joints in terms of non-linear fracture mechanics. In: Mechanical Behavior of Adhesive Joints. Proceedings of the European Mechanics Colloqium, Paris, pp. 323–338. Hüttermann, A., Mai, C., Kharazipour, A., 2001. Modification of lignin for the production of new compounded materials. Appl. Microbiol. Biotechnol. 55, 387–394. IARC, 2006. Formaldehyde, 2-Butoxyethanol and 1-tert-Butoxypropanol-2-ol. In: IARC Monographs on the Evaluation of Carcinogenic Risks to Humans. WHO Press, Lyon, pp. 37–325. Ibrahim, V., Mendoza, L., Mamo, G., Hatti-Kaul, R., 2011. Blue laccase from Galerina sp.: properties and potential for Kraft lignin demethylation. Process Biochem. 46, 379–384. Kinsella, J.E., 1979. Functional-properties of soy proteins. J. Am. Oil Chem. Soc. 56, 242–258. Larsen, H.J., Gustafsson, P.J., 1990. The fracture energy of wood in tension perpendicular to the grain – results from a joint testing project. In: CIB-W18A Meeting 23, Lisbon, Portugal. Li, G.Y., Sun, Q.N., Qin, T.F., Huang, L.H., 2010. Effect of reduction by sodium borohydride on the structural characteristics of brown-rotted lignin. Spectrosc. Spect. Anal. 30, 1930–1933. Li, K.C., Geng, X.L., 2005. Formaldehyde-free wood adhesives from decayed wood. Macromol. Rapid Commun. 26, 529–532. Liu, Y., Li, K., 2006. Preparation and characterization of demethylated lignin–polyethylenimine adhesives. J. Adhes. 82, 593–605. Mai, C., Milstein, O., Hüttermann, A., 2000. Chemoenzymatical grafting of acrylamide onto lignin. J. Biotechnol. 79, 173–183. Milstein, O., Hüttermann, A., Fründ, R., Lüdemann, H.D., 1994. Enzymatic copolymerization of lignin with low-molecular-mass compounds. Appl. Microbiol. Biotechnol. 40, 760–767. Nielsen, G.D., Wolkoff, P., 2010. Cancer effects of formaldehyde: a proposal for an indoor air guideline value. Arch. Toxicol. 84, 423–446. Okamoto, T., Takeda, H., Funabiki, T., Takatani, M., Hamada, R., 1996. Fundamental studies on the development of lignin-based adhesives 1. Catalytic demethylation of anisole with molecular oxygen. React. Kinet. Catal. Lett. 58, 237–242. Peshkova, S., Li, K.C., 2003. Investigation of chitosan–phenolics systems as wood adhesives. J. Biotechnol. 102, 199–207. Priefert, H., Rabenhorst, J., Steinbüchel, A., 2001. Biotechnological production of vanillin. Appl. Microbiol. Biotechnol. 56, 296–314. Wei, M., Fan, L.H., Huang, J., Chen, Y., 2006. Role of star-like hydroxylpropyl lignin in soy-protein plastics. Macromol. Mater. Eng. 291, 524–530. Wernersson, H., 1994. Fracture Characterization of Wood Adhesive Joints. PhD Thesis. Division of Structural Mechanics, Lund University, Lund.
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Wolf, W.J., 1970. Soybean proteins – their functional, chemical, and physical properties. J. Agric. Food Chem. 18, 969–976. Wu, G.X., Heitz, M., Chornet, E., 1994. Improved alkaline oxidation process for the production of aldehydes (vanillin and syringaldehyde) from steam-explosion hardwood lignin. Ind. Eng. Chem. Res. 33, 718–723.
Youn, H.D., Hah, Y.C., Kang, S.O., 1995. Role of laccase in lignin degradation by whiterot fungi. FEMS Microbiol. Lett. 132, 183–188. Zemek, J., Kosikova, B., Augustin, J., Joniak, D., 1979. Antibiotic properties of lignin components. Folia Microbiol. 24, 483–486.
Contents lists available at SciVerse ScienceDirect
Industrial Crops and Products journal homepage: www.elsevier.com/locate/indcrop
Production and properties of adhesives formulated from laccase modified Kraft lignin Victor Ibrahim a , Gashaw Mamo a,∗ , Per-Johan Gustafsson b , Rajni Hatti-Kaul a a b
Department of Biotechnology, Center for Chemistry and Chemical Engineering, Lund University, P.O. Box 124, SE-221 00 Lund, Sweden Division of Structural Mechanics, Lund University, P.O. Box 118, SE-221 00 Lund, Sweden
a r t i c l e
i n f o
Article history: Received 27 October 2012 Received in revised form 26 December 2012 Accepted 27 December 2012 Keywords: Formaldehyde-free adhesive Lignin Laccase Fracture energy Tensile strength Water resistance
a b s t r a c t Kraft lignin was used to formulate adhesives in combination with polyethyleneimine, chitosan and soy protein, respectively. The tensile strengths of bond lines formulated with laccase-treated and untreated lignin were found to be similar. However, a remarkable difference was noted when laccase-treated lignin was reduced by sodium borohydride prior to reacting either with chitosan or soy protein – decreasing the strength in the former case while increasing it in the other. Among the formulations, laccase treated and reduced lignin–soy protein adhesive exhibited more than half the strength of commercial polyurethane adhesive and retained 70% of its initial strength after two cycles of 1 h boiling and drying. © 2013 Elsevier B.V. All rights reserved.
1. Introduction The growing public concern about environmental issues and the increasing price of fossil oil have led to an enormous interest in the use of renewable resources, especially lignocellulosic biomass, for production of energy and materials. Lignin, which accounts for 15–36% of lignocellulosic biomass, is the second most abundant renewable resource on earth (Youn et al., 1995). It is structurally complex and an amorphous polymer with randomly distributed stable linkages between the monomeric aromatic components. In nature, lignin is covalently associated with hemicellulose and serves as cement to bond together cellulose fibers in woods. It also serves as a water-proofing and antimicrobial agent. As a byproduct of the pulping process, large amount of lignin, estimated to reach over 50 × 106 tons, is produced each year (Chen et al., 2009). Except for a minor fraction, most of this lignin recovered from the pulping process is combusted to generate energy. However, there has always been interest in using this abundant renewable resource in production of value added materials (González-García et al., 2011; Hüttermann et al., 2001; Mai et al., 2000; Milstein et al., 1994).
∗ Corresponding author. Tel.: +46 46 222 47 41; fax: +46 46 222 47 13. E-mail address: [email protected] (G. Mamo). 0926-6690/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.indcrop.2012.12.051
Lignin is now being used as a raw material for various value added products such as vanillin, dimethylsulfoxide (DMSO), syringaldehyde, etc. (Priefert et al., 2001; Wu et al., 1994). Several investigations have been targeted toward using lignin in formulation of formaldehyde free wood adhesives (Li and Geng, 2005). However, the presence of abundant methyl groups makes lignin less reactive and poor in cross-linking (Okamoto et al., 1996). Hence, lignin-based adhesives never exceeded the binding quality of the conventional phenol-formaldehyde adhesives and this has hindered the large-scale application of lignin based adhesives. Thus, if lignin has to be considered for production of glue, improving its cross-linking ability seems mandatory. The reactivity of lignin can be improved by demethylating the lignin aromatic rings. Demethylation can be achieved chemically (Liu and Li, 2006; Okamoto et al., 1996) or enzymatically using laccase (Filley et al., 2002; Ibrahim et al., 2011), and the activated lignin can react with molecules bearing nucleophilic groups to form cross-linked complexes. The enzymatic treatment is milder, cleaner and safer and is preferable over the chemical demethylation. It may also result in partial/extensive degradation of lignin and release monomeric subunits, which are also reactive toward other active groups and/or polymerize in the presence of the enzyme. In this study, laccase modified lignin was reacted with other polymers including two biopolymers, chitosan and soy protein, to formulate resins that are safe and cheap, and the formulations were tested for their adhesive properties.
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Table 1 Summary of the different lignin-based adhesive formulations used in this study. The ratio of mixed formulations was 1:1 as mentioned in Section 2.1. Additive
Untreated lignin
Laccase-treated lignin
Laccase-treated and NaBH4 reduced lignin
No additive Polyethylenimine (PEI) Chitosan (C) Soy protein (SP)
L LP
LL LLP
R LL R LLP
LC LSP
LLC LLSP
R LLC R LLSP
2. Materials and methods Norway spruce (Picea abies) grown in Sweden and with an average density of 421 kg m−3 was used to study the mechanical properties of bonding adhesive layers. Hard wood Kraft lignin from Eucalyptus was kindly provided by Innventia AB (Sweden) and was used as a basic component for the formulation of adhesives. Medium molecular weight chitosan (MW ∼75,000 g mol−1 , from Fluka BioChemika, Switzerland), branched polyethylenimine (PEI, MW ∼25,000 g mol−1 by LS, from Aldrich® , Germany) and nutritional soy protein SP (88% protein concentration, from Pro Nutrition, Romania), respectively, were used as additives to the Kraft lignin in the produced composites. A commercial polyurethane (PU) wood adhesive (SikaBond® -545, Sika AB, Sweden) was tested for comparison and a two-component methyl methacrylate adhesive (X 60, from HBM, Germany) was used for gluing the wooden joints to steel parts as shown in Scheme 1. Polyvinyl acetate (PVAc) based adhesive (SikaBond® -530, Sika AB, Sweden) was used to make strong joints allowing the study of wood failure. The culture supernatant of the fungus Galerina sp. HC1 was used as source of laccase (Ibrahim et al., 2011). The enzyme activity was determined spectrophotometrically at 420 nm by the oxidation of 1 mM 2,2 -azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) diammonium salt (ABTS, Sigma–Aldrich) at pH 5. One unit of the enzyme activity was defined as the amount of the enzyme that produces 1 mol of product per minute under the reaction conditions used. 2.1. Preparation of lignin-based adhesive formulations Stock solutions of the different polymers for use in adhesive formulations were prepared: 25 mg mL−1 Kraft lignin in 80 mM sodium hydroxide, 25 mg mL−1 soy protein in water, 15 mg mL−1 chitosan in 1% acetic acid, and 25 mg mL−1 PEI solution in water with pH adjusted to 5 using 2 M hydrochloric acid. A 20 mL reaction mixture was prepared containing: 150 mg Kraft lignin, 150 mg chitosan, PEI or SP, 20 mM sodium acetate buffer pH 5, 0.75 mM ABTS and 1.5 U of laccase, and stirred in a round bottom flask at room temperature for 3 h. The mixture was then transferred to 50 mL Falcon tubes and centrifuged for 10 min at 2800 × g. The supernatant was discarded and the precipitate (with solid content ranging between 3 and 8 wt%) containing primarily insoluble- and crosslinked lignin with the other polymer was used for testing the adhesive properties. Reduced lignin was prepared by treating laccase modified lignin overnight with sodium borohydride (NaBH4 ; 1%, w/w, of the lignin dry weight) on rocking table at 22 ◦ C. After drying, 150 mg of the reduced lignin was mixed with equal weight of PEI, chitosan or soy protein in 20 mL of water for 3 h. After centrifugation, the insoluble mass was used for adhesive testing as indicated below. Formulations with lignin only, treated in the same way as the other formulations, were prepared as control. Table 1 provides a list of all the preparations made and tested.
2.2. Preparation of wood joints The wood was first cut into pieces of 3 mm × 32 mm × 32 mm. A suitable amount of the adhesive formulation with about 80 mg of the total solid content was spread all over the surfaces of the pieces to be glued. The joints were pressed at a pressure of ca. 2 MPa using hydraulic press and cured either overnight at room temperature (∼22 ◦ C) or 15 min at 120 ◦ C. Subsequently, the joints were cut into 4 pieces of ca. 15 mm × 15 mm size and used to evaluate the bond line mechanical properties. 2.3. Evaluation of bond line mechanical properties All joints were tested for their load vs. deformation performance using a conventional type of loading machine (MTS 810) with a hydraulic piston. Tensile stress vs. deformation, tensile strength and tensile fracture energy of the bond lines were determined from the recorded load vs. deformation curves. The tensile load was imposed perpendicular to the adhesive layer and the orientation of the wood was such that the grain of the wood was perpendicular to the load (Scheme 1). The specimens were attached to the loading setup as follows: First a two-component adhesive (X 60) was applied on the surface of the lower steel part onto which one side of the wooden joint was glued prior to setting it in the loading machine. After complete hardening of the adhesive, the second side of the joint was glued to the upper steel part and hardening was allowed to take place in the machine. Finally, a programmed loading sequence with a piston rate of 0.5 mm min−1 was imposed causing the specimen to delaminate as load and deformation were recorded. The recorded deformation, ı, was the relative displacement between the upper and lower steel parts. At least two tests were performed for each type of specimen. From recorded load, P, stress, , was determined at the assumption of uniform stress, i.e. simply by dividing the load by the glue area, A. The highest point of the stress-strain ( vs. ı) curve was taken as the tensile strength. The fracture energy of a bond line, Gf , is the work, W, required to completely separate the two fracture surfaces divided by the fracture area A. The fracture energy can be determined as the area under the vs. ı curve of the bond line: W 1 = Gf = A A
ı0
Pdı = 0
ı0
dı
(1)
0
where the upper integration limit for ı, ı0 , is the deformation or bond line opening corresponding to complete failure and zero stress ( = 0). 3. Results and discussion Kraft lignin was modified by laccase and used in the formulation of potential adhesives. The formulations were made by mixing the lignin samples respectively with three different polymers, all bearing amino groups. One of the polymers was PEI, a synthetic cationic polymer that has earlier been combined with lignin to give an adhesive preparation (Liu and Li, 2006). The other two polymers originate from renewable resources – chitosan, a polymer of glucosamine and N-acetylglucosamine obtained by deacetylation of chitin, and soy protein, which constitutes 40% of the total weight of the soybean that is produced in multi million metric tons yearly (Kinsella, 1979; Wolf, 1970). The preparation of the wood joints and evaluation of adhesive properties were done following the protocols used in previous reports (Li and Geng, 2005; Liu and Li, 2006; Li et al., 2010), after some modification to allow comparison of the different formulations. The tensile strength of unmodified and modified lignin
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Scheme 1. The set-up for evaluation of bond line mechanical properties.
samples blended with PEI, chitosan and soy protein, respectively, was initially assessed after curing the wood joints overnight at room temperature. The strength of unblended lignin was lower regardless of the modification (Fig. 1). Mixing of lignin with PEI, chitosan and soy protein resulted in higher tensile strength; however, the modification of lignin had varying effects when mixed with the different polymers. The tensile strength of the three formulations of lignin and PEI (LP) was similar. On the other hand, the tensile strength of lignin–chitosan (LC) formulation was slightly improved when laccase treated lignin was used but the subsequent reduction with NaBH4 drastically reduced the binding strength (Fig. 1). Chitosan and lignin have been used in the production of composite films and analysis of these films by FT-IR revealed hydrogen bonding between the two polymers (Chen et al., 2009). This may render even the untreated lignin–chitosan to have good binding strength. Peshkova and Li have earlier developed chitosan–phenolic adhesive formulations in the presence of laccase and proposed the adhesion mechanisms of these preparations to be similar to the mussel adhesive proteins (Peshkova and Li, 2003). Treatment of laccase oxidized lignin by sodium borohydride would decrease the reactive carbonyl group content which substantially lowers the hydrogen bond formation and the consequent binding force. Investigation of brown-rotted lignin has shown reduction in carbonyl groups content after sodium borohydride treatment (Li et al., 2010).
Fig. 1. Effect of different treatments on the binding strength of lignin formulations: only lignin (L), lignin–polyethylenimine (LP), lignin–chitosan (LC) and lignin–soy protein (LSP). The black, gray and white bars represent formulations in which the lignin used was untreated, treated with laccase, and treated with laccase followed by reduction with NaBH4 , respectively. The curing was carried out overnight (∼16 h) at room temperature (∼22 ◦ C).
The lignin–soy protein (LSP) formulation in which the lignin was treated with laccase and then reduced by NaBH4 exhibited an opposite trend with respect to the tensile strength which was more than fourfold higher compared to the LSP formulations with untreated or laccase treated lignin. Wei et al. have reported that addition of hydroxypropyl lignin (up to 2% by weight) to soy protein for the production of biodegradable plastics increased the tensile strength of the material by more than twofold (Wei et al., 2006). However, as the hydroxypropyl lignin concentration increased, the cross-linking was decreased due to self-aggregation of the lignin. The results in Fig. 1 indicate that the LSP with untreated lignin has lower binding strength, which could be due to the poor reactivity of the methylated lignin. However, the low binding strength of LSP with laccase treated lignin (LLSP) could be due to extensive intra- and inter-cross-linking of the polymers that potentially results in aggregation and reduces the binding of the formulation with the wood. But when the enzyme treated lignin was reduced for preparing the formulation, laccase was removed prior to mixing with the soy protein and hence oxidation and cross-linking of the protein is prevented. This allows enough interaction between the lignin and the protein, while leaving some reactive groups for binding to the wood which is expected to enhance the binding strength. In order to confirm if the laccase treatment was essential for the high tensile strength of R-LLSP, a formulation was prepared in which the lignin was subjected to treatment with NaBH4 without prior enzymatic treatment. The tensile strength of this preparation (0.8 MPa) was less than half of R-LLSP (1.7 MPa). The effect of curing temperature on the tensile strength of the formulations (except L, LL and R-LL) was studied by subjecting them to curing at different temperatures. Soy protein (SP) was also included in the study since it is commercially used as an adhesive. It was observed that curing at room temperature (∼22 ◦ C) required a longer time for removal of water and to achieve the best tensile strength. The curing time could be substantially shortened by increasing the curing temperature to above 100 ◦ C. At 120 ◦ C, except for R-LLC and LSP curing for only 10–15 min resulted in relatively higher tensile strength. A study of the fracture surfaces after tensile strength testing showed a striped appearance for almost all specimens as seen in Fig. 2. The location of the failure can be within the adhesive layer (dark color on both fracture surfaces) or at the interface between wood and adhesive (few white fibers or a very thin layer of white fibers on dark color on one side and mostly white wood on the other side) or within the wood (white wood on both sides). As it can be seen in Fig. 2a and b, the location of failure in the adhesive–wood interface was found to be dominating.
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Fig. 2. The fracture surfaces of wood joints after tensile testing. (a) Distribution of adhesive lines on each side of the opened joint; the dark brown stripes are the lignin-based adhesive on top of which white fibers from the contact surface with the wood can be observed. (b) Correlation of the adhesive stripes with the annual rings.
Moreover, the distribution of the adhesive layer into parallel lines coinciding with the “late wood” lines (dark, high density and strong part of the annual ring) is an evidence that the failure is occurring in the “early wood” (light, low density and weak) part of the annual ring (Fig. 2b). On the other hand, the SP specimens displayed failure within the adhesive layer itself while the PU samples had the failure occurring mainly at the interface but partially in the wood (∼20%) with wood strips observed on both sides of the tested joints. Besides the tensile strength, which corresponds to the peak stress value of the recorded stress (MPa) versus deformation (or relative displacement of the steel parts in mm) curve, the fracture energy (Gf ) is the second important parameter of the bond line in relation to load carrying capacity of a glued joint having the bond line stressed in tension (Eq. (1), Fig. 3 and Table 2). As can be seen in Table 2, the Gf values are close to what has been reported for resorcinol-phenol bond line (310 J m−2 ) but much lower than that of polyvinyl acetate PVAc (820 J m−2 ) (Wernersson, 1994). Moreover, the determined average fracture energy of the wood (P. abies) as used in our experiments is 404 J m−2 which is a typical value for this type of wood (Larsen and Gustafsson, 1990).
For a given tensile strength and fracture energy of bond line, the shape of the stress versus deformation curve is important for the load capacity of a structural joint. There is, however, no simple relation between bond line curve shape and joint capacity. A bond line with low strength and high fracture energy corresponds to a ductile one, while high strength and low fracture energy corresponds to a brittle bond line (Gustafsson, 1987). On comparing the lignin-based formulations which have rather close tensile strength values, we can conclude that the ones containing chitosan (LC and LLC) are more ductile than the one with soy protein (R-LLSP). On the other hand, polyurethane (PU) seems to have higher brittleness
Table 2 Calculated fracture energy of wood joints bond line made using different adhesives. The mean curves presented in Fig. 3 were used to derive the fracture energy. Abbreviations are given in text and in the legend of Fig. 1. Adhesives used to make joints
Fracture energy (J m−2 )
LC LLC R-LLSP SP PU
230 213 131 221 255
Fig. 3. Mean results of tensile tests with (––) LC, (—) LLC, (····) R-LLSP, (—) SP, and (–·–) PU. Each curve is a mathematical average of 2–4 experimental curves. Samples were heated under press for 15 min at 120 ◦ C prior to their testing.
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composite adhesive is the resistance against microbial degradation. Soy protein is susceptible to microbial degradation, particularly in humid condition. Since lignin is known to have anti-microbial effect (Zemek et al., 1979), the lignin–soy protein adhesive formulations may not need addition of antimicrobial agents. 4. Conclusion
Fig. 4. Water resistance test of the formulated adhesives. The black, gray and white bars represent the binding strength of the adhesives initially, and after 1st and 2nd rounds of placing the glued joints in boiling water, respectively. The formulations tested were lignin–chitosan (LC), laccase treated lignin–chitosan (LLC), laccase oxidized and reduced lignin–soy protein (R-LLSP), soy protein (SP) and polyurethane (PU). The wood joints glued using the different formulations were heated under press for 15 min at 120 ◦ C prior to testing.
in comparison with soy protein (SP) knowing that both have close fracture energy values. Ductility is a desired property in an adhesive when gluing brittle materials like wood, paper or glass fiber polymer composite sheets. It is also advantageous to have an adhesive with higher fracture energy than that of the adherend as it gives a higher joint load capacity and a more ductile character of failure (Gustafsson, 1987). The tensile strength and fracture energy achieved by some of the lignin based formulations (LC, LLC and R-LLSP) may be enough for applications such as in the binding of paper, cardboard boxes, etc. where very high binding strength is not necessary. For a number of indoor and outdoor applications, adhesives for making joints need to be water resistant. The water resistance of the adhesive formulations was tested by keeping the glued joints in boiling water for 1 h, subsequent drying and re-boiling for 1 h. The wood joints made using lignin–PEI formulations were separated already during the first round of boiling. Fig. 4 shows the water resistance of the joints made using lignin formulations with chitosan and soy protein in comparison with that of soy protein and polyurethane adhesives. Polyurethane adhesive exhibited the highest tensile strength and water resistance. The tensile strength of soy protein was also quite good but it was relatively less resistant to water. The R-LLSP formulation exhibited more than half of the binding strength of the polyurethane adhesive and its water resistance property seemed to be better than that of soy protein. It retained 92% of the original strength after 1 h boiling while soy protein adhesive maintained only 74% of its initial binding strength (Fig. 4). Lignin or its derivatives have been used to replace the phenol component of phenol–formaldehyde adhesives (IARC, 2006; Nielsen and Wolkoff, 2010). However, due to the unfavorable health effect of formaldehyde, such a substitution cannot lead to the achievement of the most desirable property, i.e. safe to use. In this regard, the formulation of adhesives by blending lignin with soy protein or chitosan and the adhesive property exhibited by the respective formulations is encouraging. Although soy protein adhesives are available in the market, formulation of soy protein–lignin adhesives is expected to offer economic and technical merits. When used as a composite with lignin, less amount of soy protein will be required for adhesive application, leaving more available for other applications including foods and feeds. Thus, blending may substantially cut down the glue production cost. Moreover, the observed water resistance suggests that blending can result in a better quality which may improve its marketability. The other advantage of having the lignin/soy protein
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