Spectroscopic properties of oxidation species generated in the lignin of wood fibers by a laccase catalyzed treatment: electronic hole state migration and stabilization in the lignin matrix

Biochimica et Biophysica Acta 1472 (1999) 625^642 www.elsevier.com/locate/bba

Spectroscopic properties of oxidation species generated in the lignin of wood ¢bers by a laccase catalyzed treatment: electronic hole state migration and stabilization in the lignin matrix SÖren Barsberg

a;

*, Lisbeth G. Thygesen

b

a

b

Plant Fibre Laboratory, The Royal Veterinary and Agricultural University, Agrovej 10, DK-2630 Taastrup, Denmark Food Technology, Department of Dairy and Food Science, The Royal Veterinary and Agricultural University, Rolighedsvej 30, DK-1958 Frederiksberg C, Denmark Received 30 June 1999; received in revised form 23 September 1999; accepted 28 September 1999

Abstract A laccase catalyzed oxidative treatment of wood pulp fibers has been found to induce unusual modifications of these fibers that are qualitatively different from those encountered when more severely degraded fibers are subjected to similar enzymatically catalyzed oxidative treatments. These results suggest that the physical/conformational state of the lignin of wood fibers determines which oxidation pathways dominate in a given oxidative treatment, leading to different lignin modifications depending on both the chemical and the physical structure of the lignin polymer. Spectroscopic measurements (ESR, IR, UV-Vis and fluorescence) show that the laccase treatment results in the formation of two different species in the dried fibers: one is interpreted as chemically transformed (via oxygen) lignin products, and the other as initial oxidation radicals which have gained stabilization against transformation into the first mentioned products via a migration mechanism. It is argued that these initial radicals may likely be cation radical (or hole state) parts in lignin. The migration mechanism is identified with site-to-site transfer or `hopping' via electron transfer and it is postulated that this mechanism `carries' cation radical parts of the lignin, produced at the surface of the fiber, into parts of the lignin where chemical transformation pathways are suppressed due to the lignin conformational state. The possible existence of such a migration mechanism, the relative dominance of which should depend sensitively on the polymer conformational state, may have implications for the biogeneration and biodegradation of lignin as well as for oxidative treatments of non-natural conjugated polymers. ß 1999 Published by Elsevier Science B.V. All rights reserved. Keywords: Oxidation; Wood ¢ber; Spectroscopy; Lignin; Radical migration; Electron transfer

1. Introduction We have examined the initial e¡ect of an enzymatically catalyzed oxidative treatment on wood ¢bers by various spectroscopic measurements of dried ¢bers

* Corresponding author. Fax: +45-35-28-22-16; E-mail: [email protected]

obtained from such a treatment. A partial result is the generation of long lived (half-life of several weeks at ambient conditions) free radicals [1,2] as shown by electron spin resonance (ESR). All results reported here are governed by a similar time invariance. We believe these to be qualitatively di¡erent from results obtained with enzyme treatments of more degraded ¢ber types (see below), possibly re£ecting important consequences of the native-like physico-chemical

0304-4165 / 99 / $ ^ see front matter ß 1999 Published by Elsevier Science B.V. All rights reserved. PII: S 0 3 0 4 - 4 1 6 5 ( 9 9 ) 0 0 1 9 2 - 0

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state of the lignin towards the ¢ber modi¢cation which, to the best of the authors' knowledge, has not been emphasized before: namely the matrix stabilization of primary lignin oxidation radical products, possibly accomplished via an electron transfer migration mechanism which transfers them into regions of the lignin where they are stabilized. The existence of such stabilization, in combination with a migration mechanism, will play an important role in the oxidation of native lignin or mildly processed lignin and may have analogous counterparts in, e.g., electrochemical doping of non-natural conjugated polymers. Wood ¢bers are a composite material of lignin, cellulose and hemicelluloses in which lignin is an amorphous, branched and cross-linked disordered aromatic polymer situated as a `¢ller' together with hemicelluloses between the highly ordered cellulose micro¢brils. The monomeric units of the native lignin polymer derive from three di¡erent types of phenolic units which by a complex biogenetic system are coupled into the native polymer via the transformation of these units into phenoxy radicals. This process is believed to be largely random [3] resulting in a complex heterogeneous polymer containing di¡erent inter-unit bond types where a lignin structure as such is not a meaningful concept. Lignin chemical structures are thus mostly presented tentatively in the wood chemistry literature [4^6] (Fig. 1). The demand for environmentally friendly and cost minimizing pulping and bleaching processes on wood

¢bers has in recent years given rise to research on the utilization of enzymes as catalysts for oxidative treatments of wood pulp ¢bers [7^9]. Most of these treatments rely on the addition of a low molecular weight compound, a mediator, which is a substrate for the enzyme and which, once oxidized to its radical form, is able to oxidize wood ¢ber lignin, itself being reduced. A mediator has, contrary to the enzyme, a high accessibility to the ¢ber surface [10]. Enzyme-mediator oxidative treatments modify the ¢ber lignin in various ways depending on the treatment conditions (i.e., type and amount of enzyme and mediator, temperature, pH, duration of treatment etc.). Kraft pulp ¢bers (where most of the native lignin has been removed chemically from the ¢bers), the enzyme laccase and an appropriate mediator are commonly chosen for the study of such treatments [11], and generally bleaching (decreased absorbance at 457 nm) of the ¢bers is observed due to degradation of the residual lignin. The degradation is initiated by one-electron oxidation of the lignin by the mediator, a process which has been extensively examined in systems where the ¢bers have been replaced by appropriate lignin model compounds [12^14]. A simpli¢ed model of these systems is described by the equations 4M ‡ O2

L

! 4M=‡ ‡ 2H2 O

4M=‡ ‡ lignin ! 4M ‡ lignin=‡

…2†

lignin=‡ ! products

…3†

in which M symbolizes the mediator, L the laccase enzyme and / + the fact that either neutral or cation radicals may be involved depending on the mediator. The system used in this work di¡ers mainly from that mentioned above (1) by the fact that the used ¢bers stem from a high yield thermo-mechanical pulping process as used in the MDF (medium density ¢berboard) manufacturing industry, the surface lignin of which is likely to be closer to the physicochemical state of native lignin than the residual lignin of Kraft pulp [15,16], and (2) by the fact that no mediator is required. The di¡erence in ¢ber type we believe is decisive for obtaining such ¢ber modi¢cations as are observed in this work contrary to what is observed for Kraft pulp systems. The treatment used in this b b

Fig. 1. (a) The three precursor phenolic units of lignin (R1 = R2 = H, R1 = OCH3 and R2 = H, or R1 = R2 = OCH3 ). (b) A stilbene lignin moiety. (c) A biphenyl lignin moiety. (d, e) Carbonyl containing lignin moieties. The moieties (b), T, (e) exemplify structures in lignin with low energy electronic transitions (V350 nm) and the R1, T, R6 substitutions in these represent either the precursor methoxyl substitution, possible interunit bonds or H atoms.

…1†

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work depends on the presence of low molecular weight compounds adsorbed to the ¢bers prior to the treatment. These (unknown) compounds are either natural wood extractives, or they are generated during the mechanical pulping process which produces the raw material (¢bers, ¢ber bundles). They are likely to play the same role as the mediators (ABTS, HBT, etc.) commonly employed in enzyme catalyzed oxidative treatments of wood Kraft pulp ¢bers [17]. This is further supported by the observation that the aqueous solution, in which the ¢bers are suspended during the treatment, sustains a roughly constant ESR signal strength throughout the residence time of the treatment (1 h), and that these radicals decay within a few minutes upon removal of the ¢bers [1,2]. The change in time of the ¢ne structure of this signal showed the involvement of several (possibly) mediating radical species. The present work is, however, limited to the study of the modi¢cation of the ¢ber part of the laccase¢ber suspension system which is observable after the ¢bers have been removed and dried. 2. Materials and methods 2.1. Wood ¢bers To demonstrate that the modi¢cation is not speci¢c to the species, but a general phenomenon, two very di¡erent wood species were chosen, namely a softwood (spruce) and a hardwood (beech). The main di¡erence between the lignin of the two species lies in the relative content of the three lignin monomers, where hardwoods contain a larger fraction of dimethoxy substituted lignin monomers than softwood. Beech (Fagus sylvatica) TMP ¢bers were supplied by the MDF-board plant of Junckers Industries, KÖge, Denmark, where ¢bration is carried out using an Asplund process. The ¢bers were frozen after ¢bration, thawed and conditioned to the ambient environment just before usage. Norway spruce (Picea abies) TMP ¢bers were supplied by Sunds De¢brator, Sundsvall, Sweden, where ¢bration is carried out using an Asplund process, and handled as the beech ¢bers before usage. Fibration is achieved by pre-heating the wood

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chips to 140³C in a steam saturated environment at 4 atm. The wood chips are then fed into a re¢ner with one or two rotating disks operating typically at 180³C and 8 atm. Hence, the re¢ned material is named thermo-mechanical pulp (TMP). 2.2. Enzyme A fungal laccase, SP504 (EC 1.10.3.2), was supplied by Novo Nordisk A/S, Bagsv×rd, Denmark. Activity was measured as laccase units (LACU), where 1 LACU is de¢ned as the amount of enzyme which under standard conditions (T = 30³C, pH = 5.5) oxidizes 1 mol syringaldazine per minute (Novo Nordisk, 1992). The enzyme was contained in a carbohydrate solution with a speci¢c activity of 1840 LACU/ml. 2.3. Enzyme treatment After the wood ¢bers had reached an equilibrium moisture content (V10% by weight at relative humidity V30^50%) following thawing, the moisture content was determined by heating a portion of ¢bers (afterwards disposed o¡) at 105³C until constant weight was achieved. Deionized water previously heated to T = 40³C was then added to the ¢bers such that a consistency of 4% (by dry ¢ber mass) was obtained whereupon the suspension was left for 30 min. The temperature was kept at 40 þ 2³C during all steps in the treatment procedure. The pH was then adjusted (NaOH) to pH = 4.5 and the suspension left for 30 min and readjusted if necessary. Enzyme was then added to the suspension at a dosage D LACU/g ¢ber dry mass and the suspension stirred for a few minutes. Residence time for the enzyme treatment was 1 h whereupon the water was drained from the ¢bers which were then rinsed with 2 l deionized water and dried for 24 h at 40³C. Treatment with no addition of enzyme served as comparison (control). It has been established (results not shown) that the treatment induced modi¢cations are not due to passive e¡ects of the enzyme, i.e., there were no detectable di¡erences between a treatment where no enzyme was added and a treatment where heat inactivated enzyme was added. For each ¢ber type six di¡erent treatments were made: one control, denoted `S0/B0', and ¢ve with

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varying enzyme doses: 0.3, 3, 10, 30 and 300 LACU/ g, denoted `S1/B1', `S2/B2', T, `S5/B5', respectively, where `Sx' represents spruce, `Bx' beech and `x' identi¢es the treatment. Thus, beech ¢bers (B) obtained from the most intensive enzyme treatment (D = 300 LACU/g) has also been assigned the highest number `5' and is thus denoted `B5'. Beech ¢bers obtained from the least intensive treatment (i.e., the control treatment) have accordingly been assigned the lowest number `0' and are thus denoted `B0'. For transmission infrared (IR) measurements beech ¢bers were ground into a powder using a standard model V.O.S. mill equipped with a 1 mm steel sieve. The powder was then subjected to treatments 0 and 4. It should be mentioned that only relatively small quantitative (not qualitative) di¡erences in the achieved ¢ber modi¢cation arise due to grinding (results not shown). 2.4. Ascorbic acid treatment Dried beech ¢bers obtained from treatments 0 and 4 were subjected to ascorbic acid treatment in order to examine whether radicals could be the only cause of the ¢ber modi¢cation induced by the enzyme treatment. The ¢bers were ¢rst suspended at a consistency of 4% in deionized water for 30 min whereupon ascorbic acid was added in an amount of 0.01 mol/g (dry ¢ber mass). A control treatment without ascorbic acid addition was made as well and pH adjusted (HCl) to that of the ascorbic acid suspension (pH = 2.35 þ 0.05). The suspension was stirred and left in the dark for 3 h (after 3 h: pH = 2.35 þ 0.10). Finally the ¢bers were drained and rinsed with 2 l deionized water. They were then dried for 24 h at 40³C. Four types of ¢bers were thus obtained: (1) previously actively treated ¢bers (treatment 4) subjected to ascorbic acid ^ this type is denoted `B4a'; (2) previously actively treated ¢bers (treatment 4) subjected to deionized water (control) ^ denoted `B40'; (3) ¢bers previously subjected to enzyme control treatment subjected to ascorbic acid ^ denoted `B0a'; and (4) ¢bers previously subjected to enzyme control treatment subjected to deionized water (control) ^ denoted `B00'. Note that the ascorbic acid treatment is subsequent to the enzyme treatment, and the symbol (`0' or `a') added to the symbol of the enzyme treatment re£ects this, e.g., `B4' (beech

¢bers obtained from treatment 4) +`a' (subsequent ascorbic acid treatment of these ¢bers) = `B4a'. 2.5. Spectroscopic measurements All spectroscopic measurements took place on ¢bers conditioned to the ambient environment (T = 20 þ 2³C, relative humidity 40 þ 10%). In presenting the results of these measurements, a result referring to a speci¢c treatment i is obtained as a simple average of ¢ve independent samples obtained from this treatment and the uncertainty as þ the sample standard deviation (unless a di¡erent procedure is explicitly mentioned or results of individual samples are preferred). ESR measurements were performed using a Bruker EMS 104 ESR spectrometer operating in the Xband. The obtained signals were corrected using the signal obtained every V30 min from a highly stable paramagnetic alanine sample, the absorption peak height of which showed less than 1% drift during a day. Approximately 400 mg dry ¢ber mass was contained in the bottom 20 mm of a sample glass tube. These 20 mm of the glass tube were centered in the cavity before each measurement. UV-Vis di¡use re£ectance measurements were performed using a Cintra 40 UV-VIS spectrometer equipped with an integrating sphere and photomultiplier for re£ectance measurements. The ¢bers were contained in a sample cup the bottom of which consists of a quartz window, and the baseline was obtained with the same sample cup ¢lled with barium sulfate. The resolution was 2 nm. Fluorescence emission spectra (non-corrected) were obtained with a Perkin-Elmer LS50B equipped with a powder sample cup and holder and a redsensitive photomultiplier tube. Both slit widths (excitation/emission) were 5 nm. Seven excitation wavelengths were chosen for all measurements ranging from 250 nm to 550 nm in steps of 50 nm and the scan speed was 1500 nm/min. Transmission FT-IR spectra were obtained with a Perkin-Elmer System 2000 equipped with a shuttle accessory. A single transmission spectrum was obtained from eight cycles: each cycle consisted of 64 sample scans preceded and succeeded by 64 background scans (KBr) such that the background spectrum was the average of these 128 scans. This cyclic

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data acquisition minimizes the in£uence of nonsteady temperature and instrumental drift on the spectral quality. Each scan was taken with a resolution of 8 cm31 and data interval of 0.1 cm31 . The detector was a N2 cooled NB1MCT detector.

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3. Data treatment

which is integrated over a semi-sphere (i.e., the equation of radiative transfer does not contain any source term [18]). The absorption coe¤cient K(V) is a sum of the di¡erent types of absorption centers, each type i contributing with a concentration ni and an extinction coe¤cient Oi (V): X K …V † ˆ ni O i …V † …5†

3.1. ESR data

3.3. Emission spectra

All ESR spectra consist of a broad structureless absorption band exhibiting no discernible ¢ne structure. This re£ects the chemical heterogeneity of the ¢ber cell wall giving rise to a distribution of di¡erent radical types with di¡erent substitutions and degrees of delocalization (resonance structures) and therefore a distribution of properties such as g values, ¢ne and hyper¢ne couplings. The in£uence of inter-sample heterogeneity on absorption band shape was found to exceed any systematic e¡ect due to the treatments (results not shown). Therefore, only the ESR signal strengths (integrated absorption) are reported. These are approximated by the absolute peak-to-peak value of the ¢rst derivative spectrum per unit dry mass of the ¢bers contained in the speci¢c sample glass tube and per unit of the alanine sample reference signal.

The fact that the ¢bers are highly e¤cient light scatterers complicates the interpretation of emission spectra. By the use of optical ¢lters placed in the optical path between the excitation side of the spectrometer and the sample cup, and between the emission side and the sample cup, respectively, it becomes apparent (results not shown) that the signal S(V) detected by the spectrometer is composed of three parts:

i

3.2. UV-Vis spectra Each UV-Vis di¡use re£ectance spectrum was transformed to obtain the Kubelka-Munk remission function F. This function is identical to K(V)/s(V), provided the sample is a homogeneous mixture of absorption centers and that the light penetration depth is much smaller than the particle (i.e., ¢ber) dimensions [18]: F …R† ˆ K …V †=s…V † ˆ …13R†2 =2R

…4†

where K(V) is the absorption coe¤cient and s(V) the internal scattering coe¤cient describing the scattering of the £ow of photons by inhomogeneities (cracks, density £uctuations, voids, etc.) in the lignin matrix, which covers most of the ¢ber surfaces. In the following it will be assumed that s(V) is the same for all treatments of beech and spruce, respectively. By using F(R) we implicitly assume that luminescence emission does not contribute to the signal,

S…V † ˆ E…V † ‡ Lem …V † ‡ Lex …V †

…6†

where E is the (uncorrected) sample £uorescence emission, Lem a (roughly) constant background and Lex a `leak' term. The background Lem is caused by the fact that a minor amount of excitation light is able to leak through to the detector, even though the emission side monochromator should (ideally) prevent this when the emission slit position corresponds to a grossly di¡erent wavelength. An analogous leakage phenomenon is found for the excitation side where minor amounts of light with wavelengths different from the excitation wavelength leak through: this is represented by the term Lex , which has maxima re£ecting the spectral emission characteristics of the source xenon lamp. In the analysis of the sample signals the term Lex is ignored since for all emission wavelengths Lex (V)HS(V). Assuming the change of ¢ber emission properties due to various treatments to be characterized solely in terms of emission quenching, two dimension-less quantities, the fractional quenching and the relative fractional quenching, which depend on the excitation wavelength and the ¢ber sample (i.e., the speci¢c treatment), characterize the quenching behavior and are obtained as follows. First, for each excitation wavelength Vex , a mean control emission spectrum GE0 (V)f is obtained from

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the ¢ve independent control emission spectra. The fractional emission quenching Qi (Vex ) at this excitation wavelength, relative to the control mean, for a given ¢ber sample i is then obtained as Qi …V ex † ˆ …GE 0 …V †f3E i …V ††=GE 0 …V †f

…7†

which does not depend on the emission wavelength V (by assumption). The emission spectrum Ei (V) of a given ¢ber sample i is thus related to that of the control (mean) sample by Ei (V) = (13Qi (Vex ))GE0 (V)f and it is seen that the fractional quenching Qi (Vex ) equals 0 in case of no quenching and 1 in case of full quenching (no sample emission) relative to the mean control emission. For each enzyme treated ¢ber sample i, the total emission quenching behavior, relative to the control (mean), may now be expressed in terms of Qi (Vex ) which is invariant to whether or not the emission spectra are corrected. S(V) was, however, used directly for F whereby the results in terms of Q are not corrected for the Lem (V) contribution. But since this contribution varies slowly with Vex , as can be seen from the S(V) level at low emission energies (to the red side of the real emission E) where S(V)WLem (V), and since it is relatively small compared to E(V) (where it is maximal), it has little in£uence on the Vex dependence of Q. The same applies to the D dependence of Q since Lem (V) does not depend on D (as is seen in Fig. 9). In the following we express the emission behavior of a given ¢ber sample in terms of (1) the maximum value of Q as function of Vex , Qi (Vmax ex ), which is again a function of the ¢ber type (beech/spruce) and treatment dose D, and (2) the relative fractional quenching qi (Vex ) de¢ned as qi …V ex † ˆ Qi …V ex †=Qi …V max ex †

The parameters q and Q introduced above are well suited to describe the detectable e¡ect of the enzyme treatment since this does not introduce new £uorescent species but rather non-emitting species which quench £uorescence (relative to control ¢bers) as is demonstrated in Fig. 9. An equivalent approach is found in previous research dealing with conjugated polymer ¢lms where irradiation treatments result in the photogeneration of non-emitting carbonyl defects which are able to quench polymer £uorescence [22]. 3.4. Principal components analysis (PCA) The FT-IR spectra on enzyme treated samples and the UV-Vis and £uorescence data obtained on samples from the ascorbic acid treatment were subjected to a PCA [23] where the B0 and B4 samples were also included for comparison. The matrix for PCA of emission spectra was arranged so that all seven emission spectra from one sample were concatenated and put in one row. 4. Results 4.1. ESR measurements From Fig. 2 it can be seen that the enzyme treatment induces an excess amount of free radicals in the ¢bers which correlates with the applied enzyme dose D. The fact that the signal strength as a function of dose is not a monotonically increasing function may

…8†

Data analysis of emission spectra in terms of deconvolution and subsequent identi¢cation of emission components with speci¢c chromophores [19] is, strictly speaking, meaningless due to the band nature of electronic transitions in this material (and in wood ¢bers in general). However, three-way analysis and deconvolution by use of Parafac [20] and Tucker3 [21] were attempted. The outcome of these attempts did not add anything new to the interpretation of the £uorescence landscapes, and will not be discussed further.

Fig. 2. ESR signal strength (in arbitrary units) as a function of applied enzyme dose D (LACU/g). The control values are 3.87 þ 0.35 for beech (top curve) and 2.93 þ 0.58 for spruce1 .

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Fig. 3. ESR signal strength values (units as in Fig. 2) of enzyme treated beech ¢bers subjected to ascorbic acid treatment.

be caused by limitations in the quantitative reproducibility of the treatment ^ a problem which is most evident for the spruce control value(s)1 . Previous attempts to quantify this excess amount of radicals (in beech ¢bers) resulted in typical spin concentrations of 1 spin per 105 lignin monomeric units (20 LACU/g treatment) [1,2]. The e¡ect of the ascorbic acid treatment on the ESR signal strength is seen in Fig. 3. The control treatment on enzyme-control beech ¢bers is seen to induce radicals. These are possibly generated either as a result of acid catalyzed reactions (pH = 2.35) or due to the swelling of the ¢ber cell wall which may cause rupture of the cell wall polymers. The presence of ascorbic acid has no e¡ect on these radicals. It is

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noteworthy that a simple soaking in mildly acidic solution produces roughly the same excess amount of radicals (or rather ESR signal strength) as several of the enzyme treatments. The control treatment of enzyme treated beech, B40, is seen to e¡ect the decay of more than half of the excess ESR signal (comparing the di¡erences B4-B0, B40-B00) and the presence of ascorbic acid clearly catalyzes this decay since it completely removes the excess ESR signal (B4aWB0a). These observations are supported by an ANOVA (analysis of variance) on the possible sets of two treatments which gave signi¢cant di¡erence (at the 5% level) for {B00, B40} and {B40, B4a}. 4.2. Ground state UV-Vis di¡use re£ectance spectra A main qualitative feature of all spectra for both beech and spruce is the onset of a strong absorption band at V350 nm extending down in wavelength to the UV spectral region as is seen in Fig. 4. The lowest energy transitions (at V350 nm) of this band are generally suggested to be caused by a minority of lignin moieties, namely the most conjugated ones. These are structures (see Fig. 1) such as stilbene or biphenyl moieties or aromatic units containing carbonyls in conjugation to the ring [4,5,24,25]. The majority of lignin moieties have transitions at higher energy (B300 nm) corresponding to their smaller degree of conjugation with small energy variations due to chemical structure variation.

1

The spruce control ESR signal strength is not that obtained for the control ¢bers used as reference for the UV-Vis, emission and IR spectra, but for control ¢bres which may have su¡ered enzyme contamination from the enzyme treated ¢bers during the rinsing/drying step of the enzyme treatment. A new control treatment was produced and used as reference for all spectroscopic techniques except ESR. The ESR signal value of these ¢bers were 3.92 þ 0.25 (as opposed to 2.93 þ 0.26) even though great care was taken to obtain the same treatment conditions. This discrepancy is possibly caused by the di¤culty of controlling the drying conditions to su¤cient accuracy. These conditions may a¡ect the buildup or relaxation of stresses in the cell wall polymers which in turn a¡ects the occurrence of radical producing ruptures of these. Previous independent enzyme treatments on both species have, however, shown the e¡ect of the treatment on the other characteristica (UV-Vis, £uorescence and IR) to be highly reproducible.

Fig. 4. UV-Vis spectra of control (B0) and enzyme treated (B5) ¢bers. The enzyme treated ¢bers show additional absorption, most prominently at 500 nm.

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spectrum to the average control spectrum are depicted for the ¢ve spectra of ¢bers subjected to treatment 2, together with those of the ¢ve single control spectra. These control di¡erence spectra give qualitative information regarding which features can be ascribed to particle scattering e¡ects and to chemical inhomogeneities already present in such ¢ber samples. Four of the ¢ve di¡erence spectra of enzyme treated ¢bers do exhibit ground state depletion since such a depletion is not present in any of the ¢ve control di¡erence spectra, whereas the last (top) difference spectrum probably re£ects inhomogeneous ¢ber modi¢cation due to the treatment, the modi¢cation being stronger for the ¢bers which in this case are probed by the source beam of the spectrometer. If similar plots are made for the other treatments (doses), each plot exhibits this modi¢cation inhomogeneity (results not shown). In Fig. 7 the results of the ascorbic acid treated

Fig. 5. UV-Vis di¡erence spectra. Ordering after decreasing vF(500 nm): B5, B4, B2, B3, B1 (top part of ¢gure) and S5, S2, S3, S4, S1.

In Fig. 5 the di¡erence vF = F i 3F 0 is shown for all enzyme treatments of beech and spruce, where `i' denotes treatment i, `0' the control. It is seen that the enzyme treatment modi¢es the ¢bers to cause a major additional absorption, with peaks at 508 nm (B) and 514 nm (S), and that the shape changes with enzyme dose, i.e., a shoulder develops at V399 nm (B) and V414 nm (S), respectively, as the dose is increased. In the following the prominent absorption at 508/514 nm is denoted absorption A whereas the shoulder is denoted absorption B. Depletion of the ground state absorption is apparent around 350^400 nm for the small doses (0.3 and 3 LACU/g), whereas absorption B,, which begins to dominate for the higher doses, possibly extends below 350 nm, thus obscuring the ground state depletion for higher doses. This depletion is demonstrated for beech in Fig. 6, where the relative di¡erences of each single

Fig. 6. UV-Vis di¡erence spectra, single samples of B2 relative to B0 mean (top part of ¢gure) and single samples of B0 relative to B0 mean.

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Fig. 9. Emission spectra (Vex = 500 nm) as a function of dose D. The small narrow peak at 600 nm is an instrumental artifact due to a `leak' as discussed in Section 3.

Fig. 7. PCA plots of UV-Vis and £uorescence data for ascorbic acid treated beech. Enzyme treated beech (B0, B4) is included for comparison.

beech ¢bers are presented in a PCA plot representing the two components covering most of the inter-sample variation. The control treatment causes a signi¢cant decrease of absorption A and B (caused by treatment 4), correlating with the ESR data, as revealed both by the fact that the separation of the B00 and B40 groups is smaller than that between the B0

Fig. 8. The fractional quenching as a function of dose D (equivalent with i).

and B4 groups, and by the K-M spectral representation (results not shown). The ascorbic acid treatment causes these to disappear completely since no separation of B0a and B4a is found on the PCA plot, which again correlates with the ESR data. 4.3. Emission spectra ^ enzyme treated ¢bers The e¡ect of the enzyme treatment on the £uorescence properties of beech and spruce is the quenching of emission for all excitation energies, the degree of which depends on excitation energy, enzyme dose D and wood species. The gross e¡ect is re£ected in the dependence of Qi (Vmax ex ) on D as depicted in Fig. 8. It is evident that the higher the dose the more pronounced is the £uorescence quenching until it saturates at the highest doses. That the main e¡ect of the enzyme treatment is indeed quenching is exempli¢ed in Fig. 9 which shows the average emission spectra of all treatments for Vex = 500 nm. The same is the case for any other choice of excitation wavelength: no additional emission appears due to absorption A or B. In Fig. 10 is shown the relative fractional quenching q depicted for the lowest and highest enzyme dose. It is seen that q has maxima at approximately 350 nm and 450 nm for both beech and spruce. For max beech Qi (Vmax ex ) is obtained for Vex = 350 nm regardless of enzyme treatment i and for spruce Qi (Vmax ex ) is obtained for Vmax ex = 450 nm at high doses D. At lower doses spruce has two approximately equal Qi (Vmax ex )

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Fig. 10. The relative fractional quenching. Single samples relative to the control mean.

max values at Vmax ex = 350 nm and Vex = 450 nm. In the expression for q we adopt the Qi (Vmax ex ) value for Vmax = 450 nm. A continuous development of qi (Vex ) ex as a function of D (i.e., i) is clearly evident for both beech and spruce but for clarity only the lowest and highest doses are depicted. For beech a development is seen towards a relatively larger maximum at 450 nm if the dose increases. The same is seen for spruce where the maximum at 450 nm is equal to that at 350 nm at low doses, such that higher doses imply the dominance of the 450 nm maximum.

4.4. Emission spectra ^ ascorbic acid treatment The most interesting e¡ect of this treatment on the enzyme treated beech ¢bers is that the removal of the additional UV-Vis absorption, partial for the control but complete for the ascorbic acid treatment, is not re£ected in the emission quenching. The quenching behavior is largely invariant to the ascorbic acid treatment. It can therefore be concluded that the species causing additional absorption in the UV-Vis

are not responsible for the £uorescence quenching. This observation can be derived from Fig. 7 which shows a PCA plot of the ¢rst two principal components separating the emission behavior of ascorbic acid treated ¢bers. The separation corresponds mainly to the Qi (Vmax ex ) values, and the qi (Vex ) behavior found for the (just) enzyme treated ¢bers is preserved (results not shown). 4.5. FT-IR spectra Perhaps the most puzzling result of the spectroscopic measurements is in the IR region of the spectrum. In our studies of the ¢ber modi¢cation several IR techniques with varying surface/bulk sensitivities have been utilized such as photoacoustic FT-IR, diffuse re£ectance FT-IR and FT-IR transmission revealing no detectable e¡ect in the IR. Of these techniques we believe FT-IR transmission with a shuttle accessory to provide a good example since it is probably the most sensitive because of the high number of scans, the cyclic background correction and the

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large number of independent samples/spectrum, namely 10 from each treatment (4 and 0 on previously ground beech ¢bers). These 20 spectra were subjected to a PCA revealing no separation between the two groups (results not shown). 5. Discussion The enzyme treatment results in the generation of at least two types of electronic transitions in the lignin matrix, and even though this happens in a chemically heterogeneous environment, the transition energy as well as the spectral shape (caused by a combination of vibronic and band structure) of at least the low energy transition, absorption A, seems independent of the enzyme dose D. The possibility that absorption A and B express the vibronic structure of a single electronic transition is unlikely due to their separation in energy which is V5400 cm31 (B) and V4700 cm31 (S). We therefore suggest that the modi¢cation (or the part of it) causing these prominent changes in the UV-Vis is characterized by the dose dependent contributions of at least two di¡erent types of transitions. The question is whether these are caused by the same type of species or by two di¡erent types of species. The observed change in absorption is modelled by the expression: vF ˆ F i …V †3F 0 …V † ˆ …1=s†…nl …D†…O l …V †3O l0 …V ††‡ nh …D†…O h …V †3O h0 …V †††

…9†

In case of two species, nl (D) is the concentration of species A, Ol (V) the extinction coe¤cient of this species and Ol0 (V) the extinction coe¤cient of the region (on which the species resides) before the treatment generated the species. Equivalent de¢nitions apply to the second term, where nh (D) is the concentration of species B. If only one type of species is involved, only one (say l) of the two l/h terms in the expression for vF is su¤cient to describe the additional absorption, and most notably the shape should be invariant to D and proportional to Ol (V)3Ol0 (V). The development of the additional absorption with D does, however, favor a two species model since the shape of this is not invariant with D but exhibits the before mentioned ground state depletion at low D and no apparent depletion, but strong absorption,

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at high D. The depletion is thus most evident if D is small enough to make the nl (D) term dominate over the nh (D) term, but large enough to make the nl (D) term exceed experimental uncertainty. In e¡ect we thus assume that the individual properties of the new species do not di¡er much from one of two di¡erent average properties, as expressed by the extinction coe¤cients {Ol (V), Oh (V)} which are average quantities characterizing a population of electronic transitions. The depletion at 350 nm and possibly towards lower energy indicates the involvement of the most conjugated lignin moieties (mentioned above) with average extinction coe¤cients {Ol0 (V),Oh0 (V)} in the formation of these new species, since these moieties are known to be responsible for absorption in this region of the UV-Vis spectrum as previously mentioned. The ascorbic acid treatment showed, however, the presence of additional species generated by the enzyme treatment, which absorb relatively weakly in the UV-Vis but which, contrary to the above mentioned species, A and B, quench emission from the ¢bers. The quenching behavior in terms of q(Vex ) depends in a complex way on the electronic transition energies of the quenching species and on their concentration and location within the lignin of the ¢bers. In works on the emission quenching of conjugated polymers due to carbonyl defects, changes in q(Vex ) as a function of defect concentration are interpreted in terms of the reduction in conjugation lengths due to defect interruption, where a relatively larger amount of short conjugation segments (excited at higher energies) are close to a quenching defect: q thus increases at these higher excitation energies when the defect concentration increases. Maximal defect quenching occurs when longer conjugation segments are excited, at the excitation energy of which q equals 1 by de¢nition [22]2 .

2

Note that the q and Q values do not depend on the relative absorption coe¤cient of the ¢bers from treatment i versus those from the control 0, i.e., Ki (Vex )/K0 (Vex ). Putting this quotient equal to one (in e¡ect) amounts to only considering the quenching e¡ect of the treatment on those chromophores which are not induced by the enzyme treatment.

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Lignin, on the other hand, does not consist of the long conjugation segments characterizing the electronic states of non-natural conjugated polymers but of `segments' consisting of a single or possibly two aromatic units (e.g., a stilbene moiety). It is therefore more likely that the q(Vex ) values found for the enzyme treated ¢bers in this study reveal information on the electronic transition energies of the quenching species generated by the treatment. Assuming a Fo«rster mechanism for energy transfer [26], the quenching species should absorb predominantly at the maximum emission wavelengths corresponding to excitation at 350 and 450 nm, which are approximately 400 and 500 nm, respectively, such that excitation at these wavelengths (350 and 450 nm) leads to the relatively large quenching as is seen on the q(Vex ) plots. The gradual change of these plots as well as the general increase of the degree of quenching with increasing enzyme dose may re£ect a corresponding increase in the concentration of quenching species and change of their localization sites in the lignin. The results of the spectroscopic measurements on enzyme treated ¢bers, and on these ¢bers (beech) subjected to a subsequent ascorbic acid treatment, thus suggest that the species generated by the enzyme treatment divide into two fundamentally di¡erent groups: a. Radical species that absorb strongly in the UV-Vis but which do not quench ¢ber £uorescence to any signi¢cant degree. These cause the detection of two additional electronic transitions: absorption A and B. a. Oxidation products (or other spin-less species) which quench ¢ber £uorescence signi¢cantly but which are not directly detectable in the UV-Vis.

None of the species gives rise to any detectable absorption in the IR. Below we put forward suggestions for the identity of the group (a) and (b) species by comparisons with known properties of likely candidates and attempt to isolate the most likely ones. These suggestions are followed by an attempt to explain the mechanisms to which these species are subjected, during and after the enzymatically catalyzed modi¢cation.

a1. Radical species: neutral phenoxy radicals The enzyme laccase is known to catalyze (among a rich variety of substrates) the oxidation of phenols. A necessary condition for any detectable laccase catalyzed modi¢cation of both beech and spruce is the presence of water soluble extractives (i.e. low molecular weight compounds adsorbed to the dry ¢bers before a treatment) during a treatment [1,2,17]. Accordingly some phenolic extractives may play the role of mediators and extend the laccase catalytic range to the ¢ber surfaces where phenoxy radicals may be created. These may then migrate to interior parts of the lignin matrix by H atom transfer between the phenolic parts of the lignin, thus regenerating the surface exposed phenolic parts. Assuming that such a population of treatment induced phenoxy radicals is stabilized by the lignin matrix rigidity, such radicals (assuming them to establish an equilibrium by the postulated migration) should be predominantly localized at phenolic moieties with the lowest possible H atom abstraction energy (of the phenol-OH), ensuring minimum free energy of the entire system of phenolic radicals. The spectroscopic properties of methoxy-substituted phenoxy radicals (lignin monomers) in solution and on paper have been studied by laser £ash photolysis [27]. These lignin monomer models provide an estimate of in situ lignin matrix phenoxy radical UVVis spectroscopic properties, provided that the spin delocalization in the lignin matrix does not extend over (much) more than single lignin monomeric units. Firstly, of all phenoxy radicals studied, none absorbs only at V508/514 nm (absorption A). The ground state absorption spectra are all dominated by highest extinction coe¤cients in the region 350^400 nm and a somewhat lower (by a factor of 1.2^4) secondary maximum in the region 600^700 nm with solvent polarity shifts of approximately 10^20 nm. This indicates that only absorption B can be ascribed to phenoxy radical species. But considering the decrease of the scattering coe¤cient (sV1/V) with increasing wavelength the (possible) phenoxy radical low energy absorption should be relatively enhanced in the F(V) transformation compared to the high energy absorption in the region 350^400 nm. Therefore the absence of any absorption shoulders in the region 600^700 nm does not support a phenoxy radical as-

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signment of absorption B. The highest secondary maximum extinction coe¤cient at V500 nm was found for the 4-methoxyphenacyl radical generated by photoinduced cleavage of (2,6-dimethoxyphenoxy)-4-methoxyacetophenone. The decay of this radical was, however, found to be relatively much more sensitive to oxygen, which makes a radical of this type an unlikely candidate for the (long lived) A/B species. No information on the absolute value of extinction coe¤cients of the various phenoxy radicals is available. a2. Radical species: electronic hole states/cation radicals Among the known mediators for laccase the most e¤cient ones are not phenols but non-phenolics which presumably only lack an electron in their oxidized form (which is the case for, e.g., ABTS), contrary to the phenols which lack an H atom in their oxidized form. The e¤ciency of a mediator is tied to the rates of pathways which disable the mediator. Among such pathways are mediator-mediator radical couplings and mediator unimolecular decay (e.g., deprotonation) [28]. The compound(s) which mediates between laccase and the ¢ber surface is not known, but it could reasonably well be a non-phenolic low oxidation potential aromatic/conjugated compound, which, subsequent to its laccase catalyzed oxidation to a cation radical, is reduced by electron transfer from the surface exposed lignin matrix. Such an electron transfer would leave behind an electron vacancy (an electronic hole state), i.e., the surface exposed lignin moiety would have a half ¢lled HOMO carrying spin = 1/2, thus similar in this respect to a cation radical. To the best of our knowledge, there are to date no studies available on the UV-Vis spectroscopic properties of an appropriate range of lignin model compound cation radicals. Closest to modeling lignin cation radicals are the radical cations of veratryl alcohol (VA), VA methyl ether and VA tert-butyl [28]. Information is also available [29] for compounds such as methoxy substituted benzene radical cations (methoxybenzene, 1,2-di-, 1,3-di-, 1,4-di- and 1,3,5trimethoxybenzene), some stilbenes (stilbene, 4-methyl- and 4,4P-dimethylstilbene) and 4,4P-dimethoxybiphenyl, all of which, however, lack either the aro-

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matic ring substitution corresponding to the lignin monomer propane chain or a methoxy or hydroxyl substitution analogous to lignin. VA‡ will therefore be adopted as a likely model of lignin hole states. Reassuring for this approach is the fact that the UV-Vis spectra of the cation radicals of VA, VA methyl ether and VA tert-butyl are all similar [28] whereas qualitative di¡erences exist between VA‡ (and its derivatives) and the other compounds mentioned above. The aqueous solution spectrum of VA‡ shows absorption peaks at 300, 430 and 530 nm with maximum extinction coe¤cients of 6000, 1800 and 600 M31 cm31 , respectively [28]: Thus the VA‡ transition at 430 nm is close to the absorption B band position. Assuming then that the localized transitions of possible hole states/cation radicals in lignin can be described (qualitatively) by those of VA‡ , the hole state lowest transitions should be broad and weak and positioned close to the absorption A band position. It is likely then that the absorption caused by these transitions is indistinguishable from that of the stronger absorption A. Absorption A, however, can hardly be reconciled with a localized transition of a hole state (accepting the VA‡ model). We suggest that it can be interpreted as due to hole transfer (i.e., charge transfer) transitions, where such a transition optically couples two hole state localization sites in the lignin. This hypothesis is also supported by the fact that vibrational relaxation to the ground vibrational states of the transferred state should compete e¤ciently with £uorescence from the non-relaxed excited state (the transferred state, which is the relaxed excited state, should be near degenerate in energy to the initial state). Thus this type of charge transfer transition should be non-£uorescent, which is in accordance with the ¢ndings of the emission spectra. It may then be the case that the OCT (V)max of such hole transfer transitions is situated around V508/514 nm (B/S) and that these transitions dominate transitions OLOC (V)max to locally excited states of hole states, i.e., OCT (V)max DOLOC (V)max . Spectroscopic studies of electrochemical doping of, e.g., polypyrrole ¢lms show interesting qualitative similarities to the results found for the enzyme induced modi¢cation. It was found that depending on the state of doping of the ¢lms (i.e., equivalent to enzyme dose D) a population of polarons (hole

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states) and bipolarons (two coupled hole states with no spin) existed with characteristic UV-Vis transitions for each type of species but with only the polarons being detectable by ESR [30^33]. Polarons in such material are relatively long lived with experimental (ESR) half-lives of V1 min, their decay being caused by bipolaron formation [34]. However, if hole states do exist in lignin their decay mechanisms are probably much more complicated (and di¡erent) than those of conjugated aromatic polymers and the relatively high disorder of lignin may prevent both the formation of bound pairs of cation radicals/hole states, i.e., bipolarons, and the attainment of an appropriate geometry of the lignin molecular moiety on which these two unpaired electrons reside for an e¡ective spin-spin pairing into a spin-less singlet state. a3. Radical species: peroxy radicals and other radical species The above mentioned radical species may lead to the formation of peroxy radicals (RO2 ) via the addition of molecular oxygen to phenoxy radicals which are generated either directly or indirectly (i.e., through deprotonation of a cation radical). These may subsequently participate in chain propagation leading to various neutral radical species (R ) in the lignin and hydroperoxide (ROOH) moieties [35]. Hydroperoxide moieties in wood ¢bers are known to decompose rapidly under ambient conditions to create oxidation products containing carbonyl and carboxyl groups [5]. Thus in principle a wide variety of di¡erent types of radical species may be generated by the enzyme treatment. It seems, however, unlikely that any peroxy radical, once created in the lignin, would have a lifetime of several weeks, which is the lifetime of the treatment induced excess UV-Vis absorption and ESR signal strength, since a H atom would always be nearby (sorbed water molecule or surrounding lignin matrix `cage') to e¡ectuate chain propagation and since the hydroperoxide is highly unstable. Neutral non-phenoxy radicals created via chain propagation may thus be generated due to the treatment. Such radicals are also known to be created due to the ¢bration process producing the ¢ber raw material and due to swelling and drying of ¢bers (i.e., control

treatment). Since the excess UV-Vis absorption shape found for the enzyme treated ¢bers is not found if control treatments are compared (results not shown), it suggests that these radicals are not causing the (enzyme treatment induced) additional absorption. b1. Oxidation products: carbonyl structures As explained above we interpret the change in £uorescence behavior due to the enzyme treatment as caused by the formation of non-£uorescent oxidation products which absorb at 400 and 500 nm. Candidates for products absorbing at these wavelengths are quinoid structures which are among the typical oxidation products of lignin models oxidized in solution. In order to elucidate the sensitivity of emission towards such (possible) products an analogous example is found in the defect quenching of the luminescence of polyparaphenylenevinylene (PPV). PPV is a compact aromatic polymer with disorder induced localized electronic states, and is thus similar to lignin in this respect although the conjugation lengths of PPV extend over several monomer units. In PPV a single (photo induced) carbonyl defect per 400 vinyls is su¤cient to reduce the quantum yield of £uorescence by a factor 2 [36]. The change in IR absorbance of the carbonyl vibrational band, corresponding to this defect concentration, was in the order of 1034 . Thus carbonyl containing oxidation products may be produced by the enzyme treatment in low concentrations with no detectable e¡ect on the IR spectrum of the ¢bers, since changes as small as those reported for, e.g., PPV are not likely to be detectable for the heterogeneous ¢bers, but with considerable e¡ect on the ¢ber £uorescence properties. b2. Oxidation products: strongly coupled spins Since the reduction in both the ESR signal and the additional UV-Vis absorption due to the ascorbic acid treatment does not correlate with the behavior of the £uorescence quenching, it is not likely that uncoupled radicals are causing the quenching. It may, however, be possible that pairwise coupled radicals (i.e., bipolarons, mentioned above) exist, which escape both ESR detection (as they are spin-less) and UV-Vis detection. These new electronic states may then be less £uorescent and the increase in quenching

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magnitude with dose then re£ects the fact that more and more chromophores are transformed into bipolarons which are able to accept excitation energy from surrounding chromophores. These bipolarons should then absorb at 400 and 500 nm. More extended strong spin-spin coupling is known to occur in some non-natural conjugated polymers such as polyanaline and its derivatives [37] in which the coupling depends on the occurrence of highly ordered polymer regions (`metallic islands'). Since lignin does not show such degrees of structural order as found in these polymers, these spin coupling effects are not likely to arise. 5.1. Proposal regarding key elements in the ¢ber modi¢cation mechanism The results of the UV-Vis measurements are consistent with assigning local transitions of cation radical/hole state parts of the lignin to absorption B, whereas phenoxy radicals were found to be poor candidates for any of these additional absorptions. Due to the compactness [6] of the lignin matrix of the surface layers of the ¢bers (as opposed to Kraft pulp lignin) an electronic hole state may be liable to migrate e¤ciently to other lignin regions by short range electron transfer, since typical distances between neighboring lignin monomeric units (V4^6 î ) are favorable for high transfer rates between A these. It can be suggested that spatial extension of oxidation deep into the ¢ber cell wall layers can be e¡ectuated by such a hole state `hopping' mechanism between neighboring localization sites, equivalent with electron transfer in the opposite direction, the hole states being initially created on ¢ber surface lignin. Such a mechanism may be related to absorption A which has been found to be inconsistent with proposals of possible local electronic transitions/radical species. Absorption A may correspond to a hole transfer transition [38] where one localization site is optically coupled with a neighboring one. The fact that no (additional) emission is observed due to this dominant absorption is consistent with such an assignment: the excited state is a vibrational unrelaxed ground state of the transferred hole and relaxation then competes e¤ciently with emission. Since the shape of absorption A is also scattering dependent

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through s(V), the absorption coe¤cient K(V) cannot be extracted from the observations. Ignoring the s(V) dependence, the shape does seem Gaussian (with energy), consistent with classical expressions for the extinction coe¤cient of radiative electron transfer [38]. In future work we will focus on obtaining K(V) as well as seeking experimental evidence of this proposed assignment of absorption A. Apart from `hopping' transfer pathways, other reaction pathways will be accessible from this initial state or from a state resulting from a series of transfer steps. Oxidation product formation, which is likely to be preceded by deprotonation, is the outcome of other pathways, as well as, e.g., radical coupling reactions. From studies of photo yellowing of mechanical pulps it is known that photoinduced phenoxy radical formation is an initial reaction step towards the formation of colored products [25,39]. Among these are quinone structures, formed via oxygen attack, which have absorption maxima between 400 nm and 500 nm [25,40] (see footnote 2). The observed £uorescence quenching is thus consistent with the proposal that a fraction of the initially formed cation radicals on the ¢ber surface lignin structure deprotonate and form phenoxy radicals, which subsequently decay into a low concentration of quinone structures, whereas the remaining fraction of cation radicals is transferred by a site-to-site `hopping' mechanism into regions of the lignin where these gain a substantial stability towards deprotonation. We suggest that it is these lignin matrix stabilized cation radicals/hole states that cause the additional absorption in the UV-Vis. These stabilizing regions may impart unfavorable activation energies to deprotonation upon these radicals and they may have poor proton acceptor sites and low oxygen accessibility. The observed ground state depletion in the UV-Vis suggests these hole states to be localized on moieties with low lying electronic transitions (see Fig. 1). If it is assumed that a possible migration mechanism will seek to minimize the free energy of the hole state, hole states should be exposed to a driving force towards low ionization energy lignin moieties. These are typically the condensed stilbene or biphenyl moieties (see Fig. 1). Such a driving force is thus consistent with the observed ground state depletion which should be caused by hole state localization on condensed lignin units.

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The apparently very low level of oxidation products induced by the treatment (as found by IR spectroscopy where no changes in, e.g., the carbonyl band were found) may follow from the fact that the site-to-site transfer rate of the radical species competes e¤ciently with rates of oxidation product formation. The compactness of the lignin matrix has an analogue in organic molecular crystals, in which hole transfer is highly e¤cient with rate constants in the range 1011 ^1014 s31 [41]. Comparing the magnitude of these rate constants with that of VA cation radical deprotonation in solution (measured under similar experimental conditions as those of the enzyme treatment), k = 17 þ 1 s31 [28], as well as with that of the combined average C-C bond cleavage and deprotonation pathway of VA tert-butyl, k = 24 þ 1 s31 [28], it is evident that if hole transfer occurs it is likely to compete e¤ciently with uni-molecular decay pathways of cation radicals generated on ¢ber surface lignin. Taking other reactions with, e.g., radicals in the solution into account, the total decay rate of a surface situated cation radical may still be orders of magnitude below hole transfer rates. The relevance of the above mentioned mechanisms may still apply for the initial formation of a ¢ber surface lignin phenoxy radical since a reversible protonation/deprotonation pathway is known to exist between phenoxy radicals and their corresponding cation radical [28]. 6. Conclusion An enzymatically induced modi¢cation of both beech and spruce TMP ¢bers has been studied using di¡erent spectroscopic techniques. The spectroscopic modi¢cations for both wood species follow the same qualitative trend and are caused by several di¡erent types of species. It has been argued that these divide into primary oxidation products, presumably cation radicals/hole states situated in the lignin matrix (or on extractives), and ¢nal oxidation products, presumably quinone type structures, which are produced by decay pathways of the primary products involving oxygen. It has been postulated that a mechanism, which has hitherto not been acknowledged possible in (natural) polymer oxidative reactions, enables a fraction of primary products to obtain extremely

long lifetimes whereby these primary products can be observed in the UV-Vis. The mechanism is thought to be a combination of the facts (1) that the primary products may migrate between sites in the lignin (and sites on extractives) by electron transfer which competes e¤ciently with pathways which e¡ectuate their decay, and (2) that the migration mechanism eventually, by free energy minimization, carries some of these products to regions of the lignin matrix (or to extractive components) where they are stabilized against decay due to low proton a¤nity sites and limited oxygen permeability. An interesting question pointing to possible future work is whether the primary products observed in this study can also be observed for enzymatic (or other oxidative) treatments of lignin preparations, such as milled wood lignin or dehydrogenative polymerized lignin, or of non-natural aromatic polymers. Suggestions as to the identity of the primary products observed in this research are, however, still hypothetical and work is continuing in our laboratory in order to obtain further clarity on the identity of these products. Acknowledgements We would like to thank the following people for helpful discussions: Dr. D.R. Worrall, Department of Chemistry, University of Loughborough, UK; Prof. J. Ulstrup, Department of Chemistry, The Technical University of Denmark (DTU), Denmark; Prof. J.M. Lawther, The Plant Fibre Laboratory, The Royal Agricultural and Veterinary University, Denmark.

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