Effects offlaxligninadditiononenzymaticoxidationofpoly(ethyleneadipate)

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NDCRO-5657; No. of Pages 12

Industrial Crops and Products xxx (2011) xxx–xxx

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Industrial Crops and Products

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ffects of flax lignin addition on enzymatic oxidation of poly(ethylene adipate)rethanes

. Ignata,∗, M. Ignata, C. Ciobanua, F. Doroftei a, V.I. Popab

“Petru Poni” Institute of Macromolecular Chemistry, Grigore Ghica Voda Alley 41 A, Iasi 700487, RomaniaDepartment of Cellulose, Paper, Fibers and Polygraphy, “Gh. Asachi” Technical University, Mangeron Avenue 71, Iasi 700050, Romania

r t i c l e i n f o

rticle history:eceived 24 December 2010eceived in revised form 5 March 2011ccepted 9 March 2011vailable online xxx

eywords:igninolyurethane

a b s t r a c t

Lignin use in polymer industry has recently become very attractive from both economical and environ-mental reasons. In the specific case of blending, the addition of low lignin amounts was found to improvethe properties and extends the application field of other natural or synthetic polymers, but the effects ofoxidative enzymes on resulting blends are widely unknown. Thereby, the current study was carried outto determine some potential effects of small flax lignin concentrations (4.2 and 9.3 wt%) on the enzymaticdegradation of a poly(ethylene adipate) urethane (PU). Thin cast films of PU and its lignin blends wereincubated for 3 days at 30 ◦C with buffered solutions of fungal peroxidase and laccase extracted fromAspergillus sp., and compared with the untreated ones. Changes in surface structure and morphology

lenderoxidaseaccasenzymatic oxidation

were investigated by attenuated total reflectance–Fourier transform infrared spectroscopy (ATR–FTIR)and scanning electron microscopy (SEM), while the impact on bulk was assessed from tensile tests andthermogravimetry analysis (TGA). Although the addition of flax lignin reduces the surface structural mod-ifications after enzyme treatment, the morphology, tensile and thermo-oxidative characteristics are stillaffected, with laccase showing the higher degradative efficiency. The lignin concentration, its high impacton the resulting blends morphology and relative low resistance to laccase and peroxidase degradation

actor
was the most important f
. Introduction

The use of degradable materials derived from annually renew-ble resources, including lignin and lignocellulose derivatives, inhe preparation of multi-component systems with synthetic poly-

ers are currently receiving an increased interest because of theconomic and environmental concerns (Belgacem and Gandini,008; Yu, 2009). Different types of lignin-based compounds result

n high amounts as by-products of pulp and paper industry, andre considered of great potential as raw compounds for obtainingew engineering plastics due to the low cost, relative numerouseactive functional groups, biodegradability, negligible toxicity andacile chemical modification (Notley and Norgren, 2009; Stewart,008). However, the lignins applicability is strongly limited by

ts complex three-dimensional structures, which consist fromhenylpropane units interconnected through various, statistically

Please cite this article in press as: Ignat, L., et al., Effects of flax lignin additCrops Prod. (2011), doi:10.1016/j.indcrop.2011.03.010

istributed chemical bonds. Likewise, its structure and molar massaries as function of vegetable sources, environmental crop condi-ions, extraction and processing techniques, whereas the isolationnd purification techniques may alter the natural lignin and hardly

∗ Corresponding author. Fax: +40 232 211299.E-mail address: [email protected] (L. Ignat).

926-6690/$ – see front matter © 2011 Elsevier B.V. All rights reserved.oi:10.1016/j.indcrop.2011.03.010

s proved to driven the enzymatic oxidation.© 2011 Elsevier B.V. All rights reserved.

conduct to high purity degrees (Buranov and Mazza, 2008; Lu andRalph, 2010).

Regardless of these factors that interfere with the industrialapplication, many outcomes have been made by using the lignincompounds either as precursors in polymer synthesis or as individ-ual components incorporated into polymer matrices (Hatakeyamaand Hatakeyama, 2010). It was found for example that blend-ing of different natural and synthetic polymers like polyesters(Mousavioun et al., 2010), natural rubber (Kramárová et al., 2007),polyvinyl alcohol (Fernandes et al., 2006) and polyolefins (Cazacuet al., 2004) with low amounts of lignin imparts better tensile prop-erties and modifies the oxidative resistance and thermal stabilityof the resulting materials. We have also reported the synthe-sis and thermo-mechanical behavior of a series of blends basedon a polyester-type polyurethane and lignin extracted from flax(Ciobanu et al., 2004).

The increasing demand for implementation of environmental-friendly technologies in pulp and paper industry together withthe broaden use of polyurethanes as biomaterials have attracted

ion on enzymatic oxidation of poly(ethylene adipate) urethanes. Ind.

numerous studies on enzymatic degradation of lignin and respec-tive polyurethane polymers (Cullen and Kersten, 2004; Howard,2002). Although the oxidoreductases, and in particular the fun-gal peroxidases and laccases, have been intensively researchedfor successful oxidation of lignin and polyphenol derivatives

dx.doi.org/10.1016/j.indcrop.2011.03.010


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Bajpai et al., 2006; Sánchez, 2009), their effects on polyester-typeolyurethanes and lignin blends are widely unknown.

The susceptibility of polyurethanes to decomposition in biolog-cal media was mainly tested with hydrolytic enzymes (Umare andhandure, 2008; Yamamoto et al., 2007), and only a few reports areelated to degradation under the action of fungal strains (Barrattt al., 2003; Cosgrove et al., 2007). The degradative potential ofhemical oxidative media like H2O2/CoCl2, either alone or followedy treatment with hydrolytic enzymes, was also investigated (Fengnd Li, 2006; Hsu and Huang, 2000), but the oxidative effects andechanisms are still unclear (McBane et al., 2007). However, a

ariety of soft- and hard-segment chemistries were employed andifferent oxidative degradation mechanisms have been proposedChristenson et al., 2004; Sarkar and Lopina, 2007).

In this context, the current work aims to fill a gap in the degra-ation studies related to polyurethanes and lignin blends, and to

dentify some effects of polyurethane–lignin blending with directpplicative implications. The peroxidase and laccase were cho-en because are obtained from industrial grown fungi, catalyzehe oxidation of a wide range of substrates containing electroncceptor groups like phenols, carboxyls and aromatic amines, andfford a comparative investigation due to their different oxidativeechanisms. Peroxidases act in two one-electron steps reactionsith the involvement of a peroxyl molecule (Torres and Ayala,

010), whereas laccases perform in one-electron processes cou-led with the four-electron reduction of molecular oxygen to water,nd without hydrogen peroxide formation (Alcalde, 2007). More-ver, the radicals formed on substrate during oxidation determineubsequent enzymatic and non-enzymatic degradative chain reac-ions and could participate, especially in the presence of laccase, atrosslinking and polymerization processes (Mikolasch and Schauer,009) which may involve both lignin and polyurethane chains.

Considering the lack of information related to the behavior ofolyurethane and lignin–polyurethane blends at enzymatic oxi-ation and the practical significance of these kinds of materialsnd procedures, we have studied in this paper the effects ofretreatment with fungal peroxidase and laccase on the surfacetructure, morphology, tensile properties and thermal stability ofoly(ethylene adipate) urethane blends as compared with the purelastomer, properties that are relevant in terms of processing, envi-onmental stability, outdoor applications and waste recycling.

. Experimental

.1. Materials

Lignin was industrially produced by flax/soda pulping andindly provided by Granit SA, Switzerland (type PF 3035, appar-nt density 0.4 g/cm3, humidity 6.2%, mineral ash 7.36%, phenolicH 1.58 mmol/g, COOH 1.75 mmol/g). The lignin received was usedithout further modifications.

The segmented polyurethane elastomer (PU) was synthesizedn our group as previously described (Ciobanu et al., 2004).riefly, poly(ethylene adipate) (PEA, number-average moleculareight of 2000, hydroxyl number 56 mg KOH/g, and acid number

.2 mg KOH/g) was obtained through the condensation of adipiccid and ethylene glycol (1:1.1 molar ratio), vacuum dried at0 ◦C for 1 h, and subsequently reacted with 4,4′-diphenylmethaneiisocyanate (MDI). The resulted polyurethane prepolymer washen reacted with the ethylene glycol used as chain extender


PEA:MDI:EG = 1:6:5 molar ratio) until the resulted polymer wasully free of NCO groups. The polyurethane synthesis was carriedut without addition of catalysts.

The fungal peroxidase (E.C. 1.11.1.7, NS 51004, 10,000 POXU/g)nd laccase (E.C. 1.10.3.2, NS 51003, 1000 LAMU/g) isolated from

PRESSProducts xxx (2011) xxx–xxx

Aspergillus sp. were provided by Novozymes AS, Denmark. As spec-ified by provider, one POXU unit represent the amount of enzymethat catalyzes the conversion of 1 �mol H2O2 per minute in asystem where 2,2′-azinobis [3-ethylbenzothiazoline-6-sulfonate](ABTS) is oxidized. Laccase solution (50 �g/mL; 0.05 LAMU/mL) wasprepared in acetate buffer 200 mM (pH 5.5), where one LAMU unitis the amount of enzyme which oxidize 1 mmol syringaldazine perminute. H2O2 solutions (∼30%) were purchased from Merck. Allother reagents were 99% purity or higher.

2.2. Blend preparation

Solutions of polyurethane and lignin in dimethylformamide(DMF) were separately prepared and proper volumes were mixedto obtain blends by casting as described elsewhere (Ciobanu et al.,2004). Lignin content was varied between 4.2 and 23.2 wt%. Theresulting brown-colored materials were casted on a clean pre-heated glass at room temperature, dried for 12 h in an oven at 105 ◦Cand for another 24 h under vacuum, at 60 ◦C. Since the blends withlignin content beyond 10% were given non-homogeneous and rel-ative brittle films, with little relevance from applicative point ofview, we have used only those with lower lignin amounts (4.2% and9.3%) for testing the effect of oxidative enzymatic pretreatment.

2.3. Degradation of blend samples

The polyurethane and polyurethane–lignin casted films werecut into 1 cm × 5 cm strips with a thickness of approximately0.6 mm and immersed in individual glass vials containing 10 mL ofan enzyme solution. Peroxidase solution (50 �g/mL; 0.5 POXU/mL)was prepared in phosphate buffer 200 mM (pH 7.0) containingH2O2 5 mM.

The glass vials were placed in a constant temperature waterbath and incubated at 30 ◦C for 3 days with occasional shaking.The period of incubation has been evaluated by taken into accountthe increasing decay of enzymes activity and the need for enoughtime to afford the occurrence of measurable changes in the struc-ture, morphology, thermal degradation and mechanical behaviorof samples. In particular, the incubation time has been establishedby monitoring the samples transformation through the means ofinfrared spectroscopy and scanning electron microscopy. More-over, the degradation time of 3 days is low enough to have anyquantifiable effects on the hydrolytic decomposition of samples insuch experimental conditions (Loh et al., 2006) as compared withenzymatic oxidation. The temperature and pH conditions were setup considering the optimum range of enzymatic activity. After incu-bation all film samples were rinsed in double distilled H2O, washedwith ethanol (70%, v/v), vacuum dried at room temperature, andstored at dark in a desiccator until further analysis.

2.4. Characterization

2.4.1. Fourier transform infra-red spectroscopy (FTIR)The FTIR spectra were collected by attenuated total reflectance

(ATR) method with a DIGILAB Scimitar Series spectrometerequipped with solid cell accessories and a ZnSe crystal with a refrac-tion index of 2.4. The spectra were recorded over 600–4000 cm−1

domain at room temperature and a resolution of 4 cm−1. The pen-etration degree was in the range of about 2–3 �m.


2.4.2. Scanning electron microscopy (SEM)The morphology of lignin–polyurethane blends was examined

before and after enzymatic pretreatment by using a SEM/ESEMFEI Quanta 200 microscope equipped with EDAX Si(Li) X-raydetector and Gatan Alto Cyro stage, operating at 20 kV. Samples


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due to O–H stretching from hydroxyl and respective carboxylgroups (Kadla and Kubo, 2004; Kim et al., 2009). In the C O stretch-ing region, the two intense peaks observed at 1737 and 1702 cm−1

correspond to free and hydrogen bonded carbonyl groups, whereas

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ere mounted on graphite supports and observed under differentegrees of magnifications.

.4.3. Tensile testingStress–strain measurements were carried out with a TIRATEST

161 Model Universal Testing machine equipped with a data acqui-ition module, at room temperature and 120 mm/min crossheadpeed. The specimens for tensile testing were dumbbell cut fromoth treated and untreated films. A 40 mm benchmark and theriginal cross-sectional area were used to calculate their tensileroperties. Three identical specimens were tested and results aver-ged.

.4.4. Thermogravimetric analysisThe thermogravimetric (TG) and derivative thermogravimetry

DTG) measurements were performed on an MOM-Budapest Q-500 D derivatograph. Samples of 50 mg were heated under airtmosphere at a rate of 12 ◦C/min, and �-Al2O3 was used as refer-nce material. A second run was made for each sample. The thermalharacteristics determined for each decomposition stage include:emperature corresponding to a mass loss of 10%, respective 50%T10%, T50%), temperature corresponding to the maximum mass lossTm), onset and offset temperature (Ti, Tf), and mass loss (Wm). Theetermination errors were of ±2 ◦C and ±1% mass loss.

. Results and discussion

The lignin–polyurethane blends are characterized by a complexupramolecular architecture owing to the specific properties ofheir components. Thus, polyurethanes used in this work are linearegmented polymers comprised from soft, amorphous PEA-basedegments, and hard, crystalline MDI-EG based segments (Fig. 1).hese segments conduct to a phase separated morphology with dif-erent degrees of reciprocal miscibility, and it is well known (Yilgort al., 2006) that a better microphase separation, obtained for exam-le by lowering the competitive hydrogen bonding between hardnd soft segments, enhances the mechanical properties. On thether hand, lignin is a rigid, amorphous three-dimensional poly-er, which exhibits relative numerous hydroxyl functional groups

ctive in hydrogen bonding.As we earlier report (Ciobanu et al., 2004), blending of

olyurethanes with low amounts of lignin has improved theechanical properties of resulting materials. In this case ligninas finely dispersed and incorporated into the polymer amor-hous phase mainly, acting as an emulsifier or compatibilizer forolyurethane soft and hard segments. Because of its structural char-cteristics, lignin may also increase the volume and reinforce theoft phase at molecular level.

Further addition of lignin (usually beyond approximately0–12%) was found to generate a strong decay in the tensile prop-


rties of resulting blends. These results were explained based onignin macromolecules tendency to self-associate, agglomerate andnevenly distribute at higher concentrations instead of physically

nteract with the polyurethane chains (Ciobanu et al., 2004; Cui etl., 2008). Moreover, the lignin’s associations disrupt the hydrogen

Fig. 1. PU str

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bonds between the high ordered urethane moieties, affecting theoverall cohesion of polyurethane chains.

An important aspect related to the applicability of thepolyurethane blends with low lignin content is related to theirresponse to environmental biodegradation. In this respect, we havechosen two representative blends with low lignin concentrations(4.2% for LPU 1; 9.3% for LPU 2) and two oxidative enzymes of fungalorigin (peroxidase and laccase) for studying the potential impact ofbiological oxidative media on their structure, morphology, tensileproperties and thermal decomposition behavior.

3.1. Surface structure and morphology

The enzymatic degradation of polymer films and membranesusually starts with a process of bioerosion limited to the externallayers, whereas the bulk properties are affected later, dependingon the surface structure, composition and roughness, phase sepa-ration, porosity, and other factors (Lucas et al., 2008; Santerre et al.,2005). Changes that occur during enzyme treatment in the charac-teristics of external material layers may trigger bulk degradationand were monitored by SEM imagery and ATR–FTIR.

The representative ATR–FTIR absorbance bands (arbitrary units,a.u.) for flax lignin (Fig. 2) and PU (Fig. 3) show frequent overlappingsignatures.

Flax lignin spectrum contains a very intense band between3000 and 2800 cm−1 (with major peaks at 2916 and 2848 cm−1)attributed to the C–H stretching from aliphatic chains, and twoweak broad bands centered at approximately 3400 and 2660 cm−1


Fig. 2. ATR–FTIR spectrum of flax lignin.

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romatic ring stretch gives rise to the 1594 and 1503 cm−1 weakignals (Kaparaju and Felby, 2010). The major peaks related to the–H bending of aliphatic chains appear at 1463 and 1472 cm−1. Aomplex pattern composed from different bands and peaks risesn the range 1314–1034 cm−1 due to the contributions of varioustretching and bending vibrations like C O, C–O and aliphatic C–HGosselink et al., 2004). At lower wavelengths, the –C–H rockingnd C–H wagging are the major sources for weak signals like thoserom 944, 728, 720 and 689 cm−1.

The infrared signatures of PU are common for polyester-typeolyurethanes and result from different contributions of urethane,ethylene, carbonyl and ester groups (Thompson et al., 2006;ang et al., 2002). Thus, urethane hard segments vibrations are

epresented by N–H stretching (3321 cm−1; hydrogen bonded)nd wagging (768 cm−1), C–N stretching coupled with N–H bend-ng (1309 cm−1), C–N stretching coupled with N–H bending and–O stretching (1531, 1217 cm−1; amide II and III), C C stretching1598 cm−1; MDI phenyl rings) and C–H twisting in plane and outf plane (1021 and 817 cm−1; MDI phenyl rings). MDI also presentsdistinct and specific C C vibration at 1413 cm−1. Two strongly

verlapped bands centered at 1730 and 1707 cm−1 correspond tohe stretching of free and hydrogen bonded carbonyls from esternd urethane groups, whereas methylene asymmetric and sym-etric stretching vibrations appear at 2955 and 2872 cm−1 (adipate

esidues from soft segments), respective 2922 and 2854 cm−1

ethylene residues from hard and soft segments). In addition, the455 and 1443 cm−1 peaks are attributable to the CH2 bending, andhose at 1381 and 1352 cm−1 to the CH2 wagging mode. Further-

ore, the 1163 and 1136 cm−1 bands result due to symmetricaltretching of polyester C(O)–O groups, while the 1064 cm−1 ones given by stretching of C–C–O from polyurethane soft and hardegments.

Since many strong bands mask weaker signals, it must be notedhat bands attribution was made by taken into account only the

ajor corresponding contributions. For comparison purposes, allU and LPU spectra were baseline corrected and normalized tohe peak height of the absorbance band at 1413 cm−1 (Christensont al., 2007; Wiggins et al., 2004), which is the most stable major


ignal at both lignin addition and enzymatic degradation.A key element in the understanding of lignin effects on

olyurethane enzymatic degradation consists in the way of inter-ction between blend components at material surface, which maye very well depicted by ATR–FTIR examination (Fig. 3).


No major changes have been expected in the polyurethane spec-tra after blending with 4.2% lignin (LPU 1) because of the lignin lowamounts, relative weak signals (except the C–H and C O stretch-ing domains) and resembling absorbance active regions. However,the presence of lignin at or near blend surface was certified by thesignificant increase of peaks attributed to the C–H stretching fromits aliphatic chains, coupled with a modest increase of bands corre-sponding to CH2 bending (1455 cm−1) and wagging (1352 cm−1). Ageneral reduction in the intensities of polyurethane specific bandswas also observed, and is partial determined by the increase in theabsolute intensity of reference peak due to the minor contributionof CH3 bending vibrations from lignin. Anyway, the reduction inthe relative intensities of peaks is much higher for hard than forsoft segments, which indicates that lignin tends to associate pre-ponderant with the polyester phase at blends surface. The peaksat 1309 and 1217 cm−1 made an exception because of the contri-bution of lignin carboxyl and hydroxyl groups; the amide III bandalso contains the vibrations rising from polyester C(O)–O asym-metrical stretching. A very slight increase was also observed in thecarbonyl stretching region due to lignin addition and increasingester concentration at surface.

A closer look to LPU 1 spectrum shows more distinct peaksand a series of less broad bands, like those of all amide andester groups, suggesting a better material organization. This isalso confirmed by the fact that narrowing affects especially theright, low wavenumber side of bands. Furthermore, the reduc-tion in strongly hydrogen bonded carbonyls from around 1640to 1670 cm−1 is accompanied by a diminished absorbance of thefree N–H and O–H (∼3400–3600 cm−1), despite the addition ofhydroxyl-containing lignin. Also, the N–H stretching peak migratefrom 3321 to 3316 cm−1, which means stronger hydrogen bondedurethanes. Thus, lignin macromolecules interferes with hydrogenbond formation between polyurethane soft and hard segments, andmakes its own physical bonds with each of them, but mostly withthe polyester moieties.

The miscibility of lignin especially with the polyurethane softsegment is also confirmed by the intermediate wavenumbers ascompared with the pure polymers obtained for hydrogen-bondedcarbonyls, CH2 stretching and bending. On the other hand, thesplit of amide II band with formation of a new, less intense peakat 1513 cm−1, argues a sharp increase of phase separation. Suchapparent opposite behavior is determined by the structure of bothpure polymers. It is known that formation of strong inter-associatedhydrogen bonds between components enhances the miscibility ofthe polymer blends and depends on factors like the number andefficacy of proton donors and acceptors, chain length and flexibility,steric hindrance (He et al., 2004). Lignin, besides its relative numer-ous and available carboxyl, alcoholic, phenolic and etheric groups, isa rigid, three-dimensional polymer, and also contains bulky struc-tures. As consequence, it tends to simultaneously act like a phaseseparation and miscibility promoter. Moreover, the polyurethanesare already phase separated polymers due to the association ofurethane hard segments. Since lignin–polyester associations byhydrogen bonding are more facilitated than lignin–urethane onesat least at surface level, as showed by ATR spectra, results that ligninwill enhance the preexistent polyurethane phase separation.

The furthermore increase in lignin concentration (9.3%; LPU 2)is reflected in ATR–FTIR spectra by a new increase in the intensitiesof C–H stretching, CH2 bending and CH2 wagging peaks as com-pared with LPU 1. However, the reduction of NH peak intensityis much smaller and comparable with that of ester groups. Car-


bonyl peak corresponding to hydrogen bonded groups migratesto 1703 cm−1, becomes more distinct and is only slightly reducedcomparative with the peak of unbounded carbonyl as an effect ofincreased lignin concentration. The free N–H and O–H region showsincreased absorbance values, whereas C(O)–O vibrations are more


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ffected than C–C–O and amide III. These surface and subsurfacebservations indicate that polyester phase becomes “saturated”ith lignin, and lignin excess determines an incipient formation

f lignin aggregates and disruption of hard segments. The occur-ence of such processes may also explain why the increase of ligninoncentration beyond 12–15% conducts to non-homogeneous andrittle materials.

Polyurethane oxidative pretreatments with peroxidase (PU) and laccase (PU L) show significant structural changes inhe external polymer layers (Fig. 4). As can be observed fromTR–FTIR spectra, the degradation patterns are different from onenzyme to another, which was expected since peroxidases andaccases have distinct oxidation mechanisms. Furthermore, per-xidase degrades the polyphenol aromatic rings in the presencef hydrogen peroxide, while laccase activity is enhanced in theresence of nitrogen-containing aromatic compounds and transi-ional metal complexes (Xu et al., 2007). So, albeit both enzymes

ay generate radicals and subsequent degradative chain reactionsn each macromolecular substrate, laccase may supplementarynvolves the urethane moieties as chemical mediators, amplifyinghe depolymerization and crosslinking processes. Despite the com-lexity and specificity of these degradative patterns, the ATR–FTIR

nvestigations could be a source of very useful informations forqualitative appreciation of transformations occurring in a given

ubstrate.In the case of PU P peroxidase treatments produce a high sur-

ace enrichment in methylene groups, whereas the intensities ofeaks corresponding to secondary amine (3321, 1531, 767 cm−1)re substantially lowered. The hydrogen bonded N–H becameentered at 3316 cm−1 and is followed by an intensified broadand (∼3400–3600 cm−1) which should contains newly formedree and associated O–H vibrations. The carbonyl bands move toower wavelengths (1726, 1703 cm−1) and decrease in intensity,he unassociated more than hydrogen bonded ones. Moreover,he carbonyl bonded region between 1670 and 1630 cm−1 grow

uch more intense, indicating not only the supplementaryresence of much stronger associated carbonyls, but also the struc-ural modification of some carbonyl adjacent groups. Since theariations of 1670–1630 cm−1 band intensities are always corre-


ated in all enzyme treated samples with a similar behavior of600–3400 cm−1 band, their simultaneous increase should be arove of carboxyl group formation. Ester symmetrical stretchingand from 1163 cm−1 shifts to 1174 cm−1 and strongly decays,hile the 1136 cm−1 ones shifts to 1124 cm−1 maintaining its


intensity. It must be noted that the 1136 cm−1 peak contains sec-ondary contributions like those added by methylene vibrationsfrom polymer backbone and, since are highly enhanced, thesecould mask the second band decays. Meantime, the amide III peak(1217 cm−1) which contains the weaker asymmetrical vibrations ofC(O)–O remains almost unchanged and C–C–O peak (1064 cm−1)intensifies, suggesting a higher presence of hard segments atdegraded polymer surface and, taken into account the lower N–Habsorbance values, a possible formation of tertiary amines withoutloosing of urethane segments. Decarboxylation reactions involv-ing the broken ends of soft segments are also possible, but inlimited extent. No significant changes have been observed in theMDI phenyl rings absorbances (1598 cm−1, 817 cm−1). All thesetransformations indicate that fungal peroxidase obtained fromAspergillus sp. is effective in the degradation of polyester-typepolyurethanes. The oxidative attacks, although nonspecific, seemsto be oriented on the scission of ester groups and in a lower extenton substitution of urethane hydrogen.

Laccase treatment (PU L) also determines higher methylenegroup absorbances, but amplitudes are generally lowers thanthose observed for peroxidase treated polyurethanes. An impor-tant exception is given by the 1381 cm−1 peak which stronglyincrease and shifts to 1388 cm−1, a characteristic wavenumber forPEA methylene groups. Likewise, the shape of 3000–2800 cm−1

band shows a comparative more involvement of adipate seg-ments in the surface architecture of PU. Furthermore, the estersymmetric vibrations move closer (from 1163 and 1136 cm−1 to1157 and 1140 cm−1, respectively) and intensifies, the urethanegroup absorbance shifts to 1219 cm−1 and sharply diminishes,while C–C–O peak keeps its intensity constant but moves to1060 cm−1. The former masked polyester unsymmetrical C(O)–O(1274, 1255 cm−1) and methylene rocking (748, 735 cm−1) vibra-tions becomes visible because of increased ester absorbances andlowered amide III, respective N–H wagging intensities. These obser-vations demonstrate an important change in surface compositionin the favor of PEA soft segments. Moreover, the sharp increaseof unbounded C O band, its shift to higher wavelengths (from1730 to 1733 cm−1), and the approaching between enhanced estersymmetric vibrations indicate not only an increased PEA concen-tration, but also weaker associations formed by polyester segments.In addition, the urethane carbonyls and virtually all secondaryamine related peaks are more affected by laccase, whereas the O–Hand strongly bonded C O stretching regions show much moderatechanges (i.e. carboxyl formation) than after peroxidase treatment.Unlike peroxidase, laccase treatment reduces the absorbance ofaromatic C C and C–N stretching (1598 and 1309 cm−1). Basedon these data, it can be supposed that laccase degrades the ure-thane segments, may be involved in crosslinking and aromatic ringreactions and have a lower influence on polyester chains.

The ATR–FTIR spectra of samples taken before and after enzy-matic treatment (Figs. 5 and 6) clearly demonstrate the importanceof lignin presence, concentration and physical interactions withpolyurethane in the resistance of resulting blends against short-term enzymatic degradation. According to infrared observationsthe LPU 1 blend surfaces remain surprisingly stable after 3 daysof enzymatic oxidation as compared with PU. The most impor-tant changes are related to the absorbances from aliphatic C–Hstretching region, which give very good indirect clues on thedegradation of each blend component. In this case the C–H stretch-ing intensities are suppressed instead of increased, indicatingthe occurrence of different processes than those involved in PUdegradation.


Peroxidase lowers the absorbance heights of all aliphaticC–H vibrations (LPU 1P) even slightly under those of untreatedpolyurethane, which means that LPU 1 loss its lignin from exter-nal layers together with small amounts of PU aliphatic chains. The


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microscopy (Figs. 7 and 8). The pure polyurethanes usually show

Fig. 6. ATR–FTIR spectra of enzyme treated and untreated LPU 2 blends.

eak decrease of C(O)–O peaks and compensatory faint increasef urethane related peaks observed at 3319 and 1531 cm−1 alsoonfirm a small reduction in the surface polyester chains.

Unlike peroxidase, the laccase treatment (LPU 1L) amplifiesery slightly all C O related peaks, whereas the decrease ofethylene absorbances are less important and can be actually

bserved only for C–H stretching (3000–2800 cm−1) and C–H bend-ng (1470–1430 cm−1) regions. Another change is related to thetretching region of hydrogen bonded carbonyls, where a single dis-inct peak remains (1703 cm−1), hinting to a possible modificationf some urethane groups. A very slight increase may be observedn the 3600–3400 cm−1 region and N–H stretching peak shifted to321 cm−1, whereas the 1531 cm−1 peak remains unchanged. So, iteems that laccase still affects the urethane segments, but in a veryow extent.


Although these spectral informations indicate a strong to almostomplete decay of exposed lignin, there are little evidences forolyurethane soft or hard segment degradation induced by per-xidase and laccase in the LPU 1 blends.


When lignin concentration growths to 9.3% (LPU 2), the aliphaticC–H stretching intensities vary after enzymatic treatment in andifferent way as compared with LPU 1, pointing out to a higherdegradation of polyurethane chains.

Peroxidase determines only a slight increase of aliphatic C–Hstretching, while the bending vibrations from 1458 to 1437 cm−1

which contain much moderate lignin methylene contributions donot change, and all other aliphatic and aromatic C–H absorbanceswith minor lignin contamination are actually reduced. Further-more, except the 1670–1630 cm−1 region, all other C O relatedintensities are decreased after peroxidase treatment; the mostaffected being the ester and unbounded carbonyls. In addition, theN–H absorbances (3317, 768 cm−1) remain almost unchanged andthe 1670–1630 cm−1 carbonyl band have a very modest increases.All these observations suggest that lignin decay is strongly inhibitedand polyester chains are more sensible to peroxidase degradationin LPU 2 compared with LPU 1 blends. Anyway, the low intensityof spectral changes apparently shows that PU is much more stableat peroxidase attacks after blending with 9.3% lignin than in nativeform.

The laccase treatment (LPU 2L) conduct to an increase ofall aliphatic and aromatic C–H absorbances, and in particular ofthose related to methylene stretching and bending vibrations.The urethane group absorbances are somewhat diminished, theN–H (3314, 768 cm−1) less than C–N related ones (1530, 1309,1218 cm−1). Also, the C–C–O peak (1065 cm−1) slightly increases.These variations suggest a minor degradation of urethane groupsand a surface enrichment in non-degraded lignin and polyesterchains. However, there are an important reduction in the intensi-ties of ester (1163, 1134 cm−1), free and associated carbonyl (1728,1703 cm−1) bands, and a significant increase of C O and O–Hstretching domains attributed to carboxyl group formation. So, itappears that even if polyester backbones are more present at blendsurface, their ester groups are partially scissored by laccase andnewly formed carboxyl groups are engaged in hydrogen bonding.Some urethane groups could be also affected by ester scission, andlimited decarboxylation events cannot be ruled out. These trans-formations indicate that lignin presence and concentration highlyaffect the specificity of laccase in polyurethane oxidative degrada-tion. Thus, the degradation of LPU 2, which contains higher ligninamount (9.3%), was focused more on polyester than on urethanesegments, whereas the lignin decay seems to be low. Anyway, thecomparison of spectra shows that LPU 2 blend is more stable againstlaccase than unblended polyurethane.

Although the oxidative activities of peroxidase and laccase arebased on different mechanisms, the ATR–FTIR spectra illustratesthat both are effective in PU degradation. On the other hand, ligninaddition seems to inhibit the enzymatic attack especially at lowconcentrations. However, the better resistance of LPU 1 blend sur-face at enzymatic deterioration as compared with LPU 2 appearsto be also influenced by the effects of lignin content on the inter-actions between blend components, which in turn affect the phaseseparation and surface externalization of different segments andchains. Crosslinking reactions and physical interactions betweenpartially degraded lignin and scissored polyester chains may alsomask the ATR–FTIR spectral evidence of true degradation extent inblends, especially in the case of better structured and miscible LPU1.

The morphological changes that accompany both polyurethaneblending and oxidative enzymatic treatment were furthermoreevaluated from images obtained through scanning electron


a high degree of miscibility between hard urethane and soft,polyester-based segments, with phase separations that occur inrelative low extent and are too limited in size to be seen in theSEM images. Blending with lignin highly increases the PU phase



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Fig. 7. SEM micrographs of pure and blended polyurethane films: (a) PU, (b) LPU 1and (c) LPU 2. Magnification ×3000.


separation and domains size. As consequence, the material surfacehas developed a typical spongious structure with numerous pores,clear evidenced in Fig. 7b and c. Furthermore, the surface porosityand granulation increase when lignin content attains 9.3%.

These findings strongly suggest that while lignin is primarilyincorporated within the soft phase, it also behave like a compat-ibilizer until a critical concentration is reached. Thus, the lignindense and relative voluminous macromolecules may interact byphysical forces like hydrogen bond formation not only with esterand urethane groups from the soft phase, but also with thenearby hard segments. Such processes should result in formationof network-like globular structures composed from lignin cov-ered with flexible soft segments, and linked through the residuallinear chains that are preponderant involved in hard segment archi-tectures. The subsequent supramolecular arrangement of thesestructures through physical interactions may conduct to the rough,sponge-like surfaces observed in the SEM images. When lignin con-tent exceeds 10–12%, it can be supposed that its tendencies toassociate, the lower cohesion between hydrogen bonded three-dimensional lignin molecules compared with that between ligninand linear polyurethane chains, and other factors like steric hin-drance, molecular geometry and increased volume of aggregates,will not afford a proper blending and film formation.

The surface bioerosion have occurred for all treated samples butin different manners, as shown in Fig. 8. Morphological changesindicate that films degradation was modulated by lignin presenceand depends on the type of oxidative enzyme.

Peroxidase treatment generally leads to uneven surfaces, domi-nated by channels and small holes. The initially smooth PU surfacebecomes rough, with numerous small clusters separated by holesthat eventually hang together in irregular cracks. On the other hand,the sponge-like surface of LPU 1P shows a more compact topogra-phy, with less pores but larger structures and crevices, whereasLPU 2P features are somewhat intermediate between those pre-sented by PU P and LPU 1P. The rough, irregular surfaces showedafter enzymatic treatment are the result of non-uniform erosionof external layers due to preferential degradation of amorphousregions based on lignin and polyester chains. The SEM images alsoconfirm that peroxidase action was facilitated by spongious surfaceformation and better exposure of polyester segments, morpholog-ical changes generated through addition of low lignin amounts. Inaddition, the slow-eroding regions which appear more pregnant atLPU 1P than LPU 2P should be represented by the less amorphousblend domains (Sarkar et al., 2007; Spyros et al., 1997) structuredunder the presence of lignin macromolecules. Furthermore, themore compact aspect of these regions as compared with untreatedblends strongly suggests that simultaneous degradation of ligninand polyester components must be accompanied by crosslinkingreactions.

The laccase surface erosion proceeded as well in a non-uniformmanner, but leads to large, semi-spherical structures, which arebigger and better evidenced in the case of blends. These structures,which have also appeared in a less distinct shape after peroxidasetreatment, were supposed to be crystal spherulites eroded at areduced rate in comparison to the amorphous regions (Skarja andWoodhouse, 2001), and are an indication of both laccase degrada-tive specificity and increased size of crystalline domains after ligninaddition. Besides these structures, the enzymatic treated PU showscomparable textures, but much coarser and with bigger clusterswhen laccase was involved in degradation. On the other hand, thetexture under the spherulites is smooth and continuous at LPU 1L


and LPU 2L, indicating that laccase is more effective during PUdegradation and generates superior amounts of crosslinks in thelignin presence as compared with peroxidase. The lignin additionseems to also facilitate a more uniform degradation of less crys-talline and amorphous blend regions.


Please cite this article in press as: Ignat, L., et al., Effects of flax lignin addition on enzymatic oxidation of poly(ethylene adipate) urethanes. Ind.Crops Prod. (2011), doi:10.1016/j.indcrop.2011.03.010

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Fig. 8. SEM micrographs of samples after degradation with peroxidase: (a) PU P, (b) LPU 1P and (c) LPU 2P; and laccase: (d) PU L, (e) LPU 1L and (f) LPU 2L. Magnification×3000.


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The surface analysis based on SEM and ATR–FTIR data revealshe complex influence of small lignin amounts on the characteris-ics of LPU blends, both before and after oxidative treatments. The

ajor implications related to lignin addition seem to be related tots strong impact on blends morphologies and ordering at molecularnd supramolecular levels. Even if lignin can act as an antioxidantGregorová et al., 2005), partially preventing the oxidative degra-ation of polyurethane chains, this process is overwhelmed by theaster lignin decomposition and its effects on blends morphologynd crosslinking reactions. The examination of some relevant bulkroperties like tensile and thermo-oxidative behavior was further-ore carried out in order to confirm the results obtained by surface

nalysis and better evidenced the extent of enzymatic degradation.

.2. Tensile behavior

The mechanical properties of polymeric materials and in par-icular the tensile strength, elongations at break and elasticity

odulus are very sensitive to degradation processes. Therefore,ensile tests were carried out on all materials before and afternzymatic treatment and results compared. Data obtained are sum-arized in Table 1, whereas stress–strain curves are shown in Fig. 9

or PU and in Fig. 10 for its corresponding blends.The enzymatic treatments have lowered the elongation at break


ith approximately 30–31% and increased with about 35–38% theensile strength of PU samples. Modulus of elasticity also rises by.3–2.5 times. The concomitant increases in tensile strength andlasticity modulus confirms that active species formed on polymer

able 1epresentative mechanical properties of initial and enzymatic treated PU and LPUlends.

Sample Modulus ofelasticity (MPa)

Elongation atbreak (%)

Ultimate tensilestrength (MPa)

PU 16.53 249.97 5.83PU P 41.19 172.40 9.03PU L 37.77 175.02 9.45LPU 1 25.99 346.93 21.64LPU 1P 28.04 215.47 10.23LPU 1L 28.13 212.27 11.05LPU 2 26.45 328.41 18.80LPU 2P 40.27 148.78 7.99LPU 2L 61.56 131.40 7.57

ig. 9. Stress–strain curves for polymer samples before (PU) and after degradationith peroxidase (PU P) and laccase (PU L).

Fig. 10. Stress–strain curves for LPU blends before (LPU 1 and LPU 2) and afterdegradation with peroxidase (LPU 1P and LPU 2P) and laccase (LPU 1L and LPU 2L).

chains during oxidative degradation of surfaces are subsequentlyinvolved in crosslinking reactions. Since PU elasticity is given byits soft segments, the participation of poly(ethylene adipate) atboth degradation and reticulation processes justifies the observedreduction in ultimate strain. There are relative minor differencesbetween mechanical properties of peroxidase and laccase degradedPU. However, the evolution of stress–strain curves for PU P indi-cates that peroxidase treatment conducts to more plastic and brittlematerials, whereas laccase do not change the overall viscoelasticpattern.

The superior mechanical properties of LPU 1 and LPU 2 blendsare strongly affected by oxidative enzymatic degradation, as shownin Fig. 10. In the case of LPU 1, the slight increase in elasticity mod-ulus (∼8%) is accompanied by a major reduction of extensibility(38–39%) and tensile strength (47–51%). This behavior confirms themorphological changes induced by lignin addition, including thebetter exposure of polyester chains and therefore their increasedsusceptibility to enzymatic degradation. Since modulus of elastic-ity only slight increases and ultimate tensile strength is at abouta half of initial values, the crosslinks that should also occur seemto take place preponderantly between polyester and lignin macro-molecules. Moreover, lignin is sensitive to peroxidase and laccase,determines spongious morphologies and favors the surface bio-erosion. All these lignin characteristics contribute to the higherdegradability of its polyurethane blends. Despite their extensivedecay, the ultimate tensile strengths and elongations are still higherthan those of degraded PU. The tensile behaviors of degraded LPU1 are comparable, with LPU 1L retaining a little higher tensilestrength.

The ultimate tensile strength and elongation of LPU 2 blendsshows an even higher reduction than LPU 1 ones after enzy-matic oxidative treatments, attaining less than a half of initialvalues. On the other hand, the modulus of elasticity increasesby about 1.5 times after peroxidase and by 2.3 times after lac-case treatments. These results mean that interactions betweenblends components are weaker at LPU 2 and lignin is lesscovered by polyester segments, and thus more available toenzymatic degradation. As consequence, lignin degrades faster,and crosslinks between polyurethane segments are favored.


The increased lignin concentration also appears to enhance thelaccase efficiency in blend degradation as compared with perox-idase.


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.3. Thermal stability

Besides mechanical properties, the thermal stability is anothermportant bulk characteristic for potential end uses of materials.he thermoplastic polyurethanes show a relative limited thermaltability, especially because of the presence of thermal labile ure-hane bonds. Thermo-oxidative degradation is typical initiated byrethane bonds scission at temperatures around 200 ◦C, continuesith hard segments decomposition which becomes complete at

pproximately 300 ◦C, and follows with the breakdown of polyesternd some of the previously formed secondary products up to50–500 ◦C (Chattopadhyay and Webster, 2009; Petrovic et al.,994). Polyurethane decomposition ends up beyond 500 ◦C withhe decomposition of char formed in the previous stages (Herrerat al., 2002). On the other hand, lignin degradation initiates at about50–200 ◦C and continues at moderate rates up to 500–550 ◦CCanetti et al., 2006; Ciobanu et al., 2004). Furthermore, ligninecomposes with moderate fluctuations in weight and its concen-ration in blends is quite low, so the mass loss of blends will be

ainly discussed as the result of polyurethane decomposition.Since the first decomposition events are influenced by addition

f other compounds and depend on the structure and compara-ive amounts of hard and soft segments (Cervantes-Uc et al., 2009;outinho and Delpech, 2000), the thermogravimetric analysis canrovide useful information regarding the effects of both ligninlending and enzymatic treatments on the initial PU. The deriva-ive weight loss curves (DTG) are shown in Figs. 11 and 12 for initialnd enzymatic treated PU and respectively LPU blends, whereas theharacteristic decomposition temperatures and mass loss valuesesulted from TG/DTG analysis are presented in Table 2.

As it can be seen in Fig. 11, decomposition initiates earlierfter PU enzymatic treatments, accelerates at much lower tem-eratures and Tm values are reduced with about 80–90 ◦C, thentire main decomposition region being virtually shifted down-ard. The individual degradative steps become less distinctive and

accase treatment shows a greater impact than peroxidase ones.hese results confirm that both enzymes have been effective inolyurethane degradation.

It must be mentioned that thermal oxidation determineshe formation of macromolecular radicals, peroxy radicals and


ydroperoxides which interact with PU through chain reactions.hese reactions favor the polymer scission, but do not influ-nce the decomposition rate of PU to diisocyanates and polyolsChattopadhyay and Webster, 2009). However, the previous enzy-

ig. 11. DTG curves of polymers before (PU) and after peroxidase (PU P) and laccasePU L) treatments.

Fig. 12. DTG curves of blends before (LPU 1; LPU 2) and after peroxidase (LPU 1P;LPU 2P) and laccase (LPU 1L; LPU 2L) treatments.

matic treatments have already fragmented the exposed polyesterchains, making them more available to subsequent thermal oxida-tion. Furthermore, the substantial reduction in mass losses mainlyresults due to pores formation and enzymatically driven crosslink-ing reactions. The presence of pores also facilitates an earlier andfaster mass loss especially after peroxidase treatment, which seemsto have only a limited effect on hard segment degradation.

The importance of film morphology together with the additionof chemical species having higher susceptibility to form radicalsand participate at chain reactions during thermo-oxidative pro-cesses is evidenced by LPU 1 and LPU 2 blends, which show anoverall decomposition pattern more comparable with that of PU Pand PU L as lignin concentration increases. Two important param-eters in the assessment of hard and soft segment degradation arerepresented by the temperatures corresponding to a mass loss of10% (T10%), respective 50% (T50%) (Yeganeh et al., 2006). Lignin addi-tions have significantly lowered the T10% values, while enzymatictreatments have provoked a comparable less reduction for PU andno significant changes for LPU 1. The T10% increased for LPU 2P andLPU 2L up to the LPU 1 range, and all laccase pretreated materi-als gave similar or slightly lower T10% values relative to peroxidasedegraded samples. On the other hand, the T50% values have beeninfluenced in a higher extent by the enzymatic treatment of blendsand increased lignin amounts. Thus, these decomposition temper-atures have been less lowered for LPU 1 and PU P than for LPU


2 and LPU 1P, and substantially increased for LPU 2P as comparedwith LPU 2. Also, laccase pretreatments have conducted to 10–14 ◦Clower T50% values than peroxidase.

Table 2Thermal decomposition data for studied samples.

Sample T10% T50% Ti Tf Tm Wm

PU 286 422 184 519 392 68.32PU P 272 406 165 495 311 60.90PU L 268 393 161 473 304 59.63LPU 1 255 410 165 482 380 59.85LPU 1P 258 380 171 455 286 52.11LPU 1L 246 366 159 436 283 56.91LPU 2 234 348 154 449 287 69.93LPU 2P 256 395 177 464 284 57.64LPU 2L 256 385 167 458 289 58.40

T10%, T50%, temperature of 10%, respective 50% mass loss (◦C); Tm , temperature cor-responding to the maximum rate of mass loss (◦C); Ti , Tf , onset and offset stagetemperature (◦C); Wm , mass loss (%).


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Considering the low lignin concentrations as well as the mod-rate and less variable degradation rates as compared with PU, itas expected that its direct effects on decomposition temperatures

nd mass losses are rather limited. However, the morphologicalhanges induced by blending, like spongious structure, increasedolume and phase separation, together with its high radical form-ng potential at relative low temperatures, strongly interfere witholyurethane decompositions, accounting for the major impact of

ignin observed during thermo-oxidative degradation.The characteristics of thermal decomposition can be well

xplained and correlated with the preliminary enzymatic oxida-ions by taking into account the results obtained from tensile testsnd surface analysis. The enzymatic treatment partially degradeshe exposed lignin macromolecules from external polymer layernd eliminates some of the active species that can participate toadical processes. In the same time, some of the radical activatedpecies formed on lignin macromolecules may participate at cou-ling and crosslinking reactions, generating a protective networkgainst thermal oxidation of both hard and soft segments. More-ver, the optimum structural organization attributed to LPU 1,ith lignin rigid macromolecules finely incorporated within flexi-

le polyester segments and linked through hydrogen bonding withrethane moieties, explains very well why the polyester chainsre more readily lost after enzymatic treatment whereas the ure-hane hard segments decomposition is not significantly affected.hese phenomena also occur in the case of LPU 2, but supplemen-ary lignin macromolecules seem to surpass the saturation limitf soft phases and aggregate. The less compact morphology andncreased number of radical forming species should be thereforeesponsible for the lowest T10% and T50% and highest mass loss val-es obtained for untreated LPU 2. The same causes allow randometiculation processes during enzymatic action, which increase theecomposition temperatures up to similar or even higher valuess compared with LPU 1 treated samples. Despite its strong reduc-ion, the mass loss still remains higher for enzymatic oxidized LPU, indicating a lower lignin–polyurethane crosslinking extent than

n LPU 1 analogues samples. Laccase has been proved to be a betterxidative degradation agent than peroxidase for all used samples.n the other hand, the thermo-oxidative data indicates that LPUwas more efficient degraded than PU and LPU 2 despite its bet-

er ordered molecular architecture and laccase higher affinity forolymerization and crosslinking reactions. As consequence, laccaseeems to be also capable to use the urethane groups as mediatorsr activators of its enzymatic activity.

. Conclusions

Additions of low lignin amounts were found to have a majormpact on the bulk and surface properties of resulting polyurethanelends, highly enhancing the mechanical behavior and over-ll macromolecular organization, and lowering the resistance athermal oxidation. We have reported that both polyester-typeolyurethanes and corresponding lignin–polyurethane blends areegraded by laccase and peroxidase obtained from industrial grownungi, and certain effects of enzymatic oxidation could be exploitedn the design and application of new biodegradable materials. Flaxignin has influenced the polyurethane enzymatic oxidation byomplex and different ways. It was shown that morphological andrdering effects generated by lignin addition highly depends onts concentration and have enhanced the polyurethane degrada-


ion. Lignin also act as an antioxidant, partially preventing thexidative degradation of polyurethane chains, but this process isverwhelmed by the faster lignin decomposition and its effects onlends morphology. Owing its relative numerous functional groupsnd dense, three-dimensional structure, lignin can readily deter-


mine the formation of crosslinks and physical associations withboth intact and scissored soft segments during oxidation, increas-ing the blends exposure to enzymatic degradation. From enzymesused, laccase has been proved to have a better degradation effect.

This study shows the high potential of lignin amounts lowerthan 10 wt% and preferably around 4–5 wt% in the increasing ofboth mechanical properties and biodegradability of common usedpolyurethanes based on MDI/polyester. At these low concentra-tions, lignin may be used to successfully modulate the polyurethaneelasticity, tensile strength and decomposition in oxidative envi-ronments, properties that are relevant in terms of industrialprocessing, environmental stability, waste management, and out-door applications like packaging.

Acknowledgements

One of the authors (L. Ignat) acknowledges the financialsupport of European Social Fund – “Cristofor I. Simionescu”Postdoctoral Fellowship Programme (ID POSDRU/89/1.5/S/55216),Sectoral Operational Programme Human Resources Development2007–2013. The authors are also grateful to Granit (Switzerland)and Novozymes (Denmark) for kindly providing the flax lignin,respective peroxidase and laccase enzymes.

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