Holz Roh Werkst (2006) 64: 415–422 DOI 10.1007/s00107-006-0133-9

ORIGINALARBEITEN · ORIGINALS

Characterization of trembling aspen wood (Populus tremuloides L.) degraded with the white rot fungus Ceriporiopsis subvermispora and MWLs isolated thereof Joon-Weon Choi · Don-Ha Choi · Sye-Hee Ahn · Sung-Suk Lee · Myung-Kil Kim · Dietrich Meier · Oskar Faix · Gary M. Scott

Published online: 2 August 2006 © Springer-Verlag 2006

Abstract Trembling aspen wood (Populus tremuloides L.) was treated with white rot fungus Ceriporiopsis subvermispora for 1, 2, 4, and 6 weeks. As fungal decay advanced, lignin contents were decreased gradually up to ca. 27.5% (based on the Klason residues of the control) after 6 weeks. Alkali solubility of cell wall residues was increased until 4 weeks of fungal treatment, but additional treatment did not cause any effects. Milled wood lignins (MWLs) were isolated from the decayed woods by Bj¨orkman’s procedure and subjected to thioacidolysis and analytical pyrolysis to investigate the modification of lignin structures during fungal degradation. Thioacidolysis revealed that the yields of trithioethylated C6 C3 monomers, as a parameter for frequency of β-O-4 linkages in lignin, were substantially reduced (−20%, based on the control) in MWLs isolated from decayed woods. Analytical pyrolysis revealed that the relative amounts of coniferyl alcohol and sinapyl alcohol in the J.-W. Choi · D.-H. Choi (u) · S.-S. Lee · M.-K. Kim Division of Wood Chemistry and Microbiology, Korea Forest Research Institute, 130-712 Seoul, Korea e-mail: [email protected] S.-H. Ahn Division of Life and Environmental Resources, College of Natural Resources, Daegu University, 712-714 Kyungsan, Korea D. Meier · O. Faix Institute of Wood Chemistry and Chemical Technology of Wood, Federal Research Center for Forestry and Forest Products (BFH), 21031 Hamburg, Germany G. M. Scott Faculty of Paper Science and Engineering, College of Environmental Science and Forestry, State University of New York, Syracuse, New York 13210, USA

pyrolysates were lowered dependent on the biodegradation time, whereas an elevation of C6 C1 and C6 C2 pyrolytic phenols was observed. The results from both analytical methods strongly suggested that β-O-4 linkages were cleaved by C. subvermispora. Specially, degradation of syringyl-type lignin seems to be preferred. Untersuchung von durch den Weißf¨aulepilz (Ceriporiopsis subvermispora) abgebautem Espenholz (Populus tremuloides L.) und daraus isolierten Milled Wood Ligninen (MWLs) Zusammenfassung Espenholz (Populus tremuloides L.) wurde f¨ur eine Dauer von 1, 2, 4 und 6 Wochen dem Weißf¨aulepilz Ceriporiopsis subvermispora ausgesetzt. Nach sechsw¨ochigem biologischem Abbau verringerte sich der Ligningehalt der Holzproben allm¨ahlich bis auf ca. 27,5% des urspr¨unglichen Klason-Ligningehaltes, wohingegen die MeOH-Extraktstoffe erheblich zunahmen. Bis zu einer Inkubationszeit von vier Wochen stieg die Alkalil¨oslichkeit der Zellwandsubstanzen an; eine l¨angere Abbaudauer zeigte jedoch keine weiteren Auswirkungen. Aus dem abgebauten Holz wurden nach dem Bj¨orkmanVerfahren Milled Wood Lignine (MWLs) isoliert und mittels Thioacidolyse und analytischer Pyrolyse wurde die Ver¨anderung der Ligninstruktur durch die Pilzeinwirkung untersucht. Die Thioacidolyse zeigte, dass sich die relativen Mengen von C6 C3 Trithioethylat-Monomeren, die als Parameter f¨ur die H¨aufigkeit der β-O-4 Bindungen im Lignin dienen, aufgrund der enzymatischen Aktivit¨at des Pilzes um 20% verringert hatten. Die relativen Mengen von Coniferyl- und Sinapylalkohol in den Pyrolyseprodukten der MWLs sanken mit der Dauer des biologischen Abbaus, wohingegen gleichzeitig

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eine deutliche Zunahme der Phenole des Typs C6 C1 und C6 C2 beobachtet werden konnte. Die Ergebnisse beider Abbaumethoden deuten darauf hin, dass C. subvermispora die β-O-4 Bindungen, insbesondere die des Syringyl-Lignins, spaltet.

1 Introduction Cost-effective and environmentally friendly removal of lignins is, to date, a challenging task imposed to pulp and paper industries. Increased environmental concern is one of the driving forces to apply biotechnological steps during pulping and bleaching. For decades researchers have paid a high attention to white rot basidiomycetes, as these fungi are able to effectively degrade the lignin moiety of biomass, and thus, can help to improve the quality and economics of pulp production (Messner and Srebotnik 1994). The lignolytic enzyme system of Ceriporiopsis subvermispora was intensively investigated for biopulping processes and for biological treatment of black liquors as well (Guerra et al. 2004, Mosai et al. 1999, Nagarathnamma et al. 1999, Souza-Cruz et al. 2004). This fungus has a high selectivity for lignin degradation and does not damage cellulose microfibrils (Akhtar et al. 1998, Breen and Singleton 1999). Furthermore, C. subvermispora shows equal effectivity for softwood and hardwood degradation (Breen and Singleton 1999). Although lignin peroxidase (LiP) is known to be an enzyme for lignin splitting, which occurs in most white rot fungi, activity of LiP is not detected in liquid culture media of C. subvermispora (Srebotnik et al. 1994, Srebotnik et al. 1997) Therefore, MnP seems to be responsible for depolymerization of lignins, probably by splitting both phenolic and nonphenolic β-O-4 linkages (Ferraz et al. 2003, Guerra et al. 2003, Jensen et al. 1996). Despite extensive studies, lignin degradation mechanisms by ligninolytic system of white rot fungi are still far from being completely understood. Degradation mechanisms were mostly suggested on the basis of model experiments using synthesized compounds, either phenolic or nonphenolic lignin substructures (Daina et al. 2002, Hammel et al. 1985, Kirk et al. 1986, Lundell et al. 1993, Srebotnik et al. 1994, Srebotnik et al. 1997, Umezawa and Higuchi 1987). Recently, several studies on structural modification of natural lignins by extracellular enzymes from white-rot fungi are performed (Ferraz et al. 2003, Guerra et al. 2002, Guerra et al. 2003, Guerra et al. 2004). This kind of investigation is, however, timeconsuming, and thus, rapid characterization methods suited to this purpose are sought-after. Analytical pyrolysis was proven to be a rapid and powerful tool, also for the characterization of wood (Fagus sylvatica) degraded by the white-rot fungi Trametes versicolor, Pleurotus ostreatus and Lentinus edodes, respectively (Faix

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et al. 1991). Taking the suggestions in the quoted paper, we applied analytical pyrolysis for the observation of the whiterot process in this study. Trembling aspen wood (Populus tremuloides L.) was incubated with the white-rot C. subvermispora for 1, 2, 4, and 6 weeks. Milled wood lignins (MWLs) were isolated from the decayed woods according to Bj¨orkman’s method and characterized by analytical pyrolysis as well as by thioacidolysis. The decayed woods were also analyzed by determination of the lignin contents and the alkali solubility of the lignin moiety. The results are interpreted in terms of fungal degradation.

2 Materials and methods 2.1 Preparation of fungal mycelium cocktails The stationary culture solution of C. subvermispora strain SS-3 was kindly provided by Forest Product Laboratory, University of Wisconsin. The fungal mat was removed from inoculation agar plugs with a sterile forceps and transferred to a sterile blender cup. The fungal mat was blended with sterile water (100 ml) in five cycles of 15 s. The fungal mycelium cocktails were poured into sterile water (100 ml) and gently mixed for 5 min before using. Before biodegradation experiment, aspen wood (Populus tremuloides L.) chips (1.8 kg, dry basis) were decontaminated with atmospheric steam for 10 min using a laboratory scale bioreactor and then cooled down to room temperature prior to inoculation. The sterile wood chips (4.0 kg on wet basis) were inoculated with fungal mycelium cocktail (2.77 ml, 5 mg of mycelia/kg dry wood) and corn steep liquor (0.5% of dry wood basis, 18 g/200 ml of sterile water) in each bioreactor. The non-inoculated wood chips similarly incubated in the bioreactor served as controls. The fungal incubation was continued for 1, 2, 4 and 6 weeks, respectively. The bioreactors were placed at 27 ◦ C and the humidity in the bioreactor was kept at 55% by supplying humidified air (1.6 l/h) through a 0.2 µm Millipore filter inline throughout incubation periods. Each fungal treated wood chip was stored without removing the fungal mycelia under −40 ◦ C and freeze-dried before further chemical analysis. 2.2 Determination of weight loss, preparation of cell wall residues and purification of milled wood lignin The weight losses were determined on the basis of the initial and final dry weight of wood chips. The freezedried wood chips were ground to 40–60 mesh and extracted twice with MeOH for 24 h at room temperature. The extract-free samples are called in the following “cell wall residues” (CWR). For milled wood lignin (MWL) isolation, extractive free CWR was finely ground for 24 h

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using a zirconium planetary mono mill under dried condition, and then subjected to extraction with 95% aqueous dioxane. The isolation protocol of Bj¨orkman (1956) was followed. 2.3 Determination of lignin contents and alkaline solubility Lignin contents were determined by the sulfuric acid standard method according to Dence (1992). Hydrolysis residues called “insoluble lignin” were filtered and weighed after dryness at 105 ◦ C and acid soluble lignins were determined by UV spectroscopy at 205 nm (Table 1). Alkali solubility was determined by mixing the CWR in 1 M NaOH for 24 h at 37 ◦ C. The insoluble residues were separated from the solution by centrifugation, washed with distilled water, dried thoroughly in vacuum, and weighed. The contents of alkali solubles were calculated as weight loss. The hydrolysates were acidified to pH 2 with diluted HCl (Fig. 1). After standing for 2 h at room temperature, precipitates were separated from the supernatant by centrifugation. 2.4 Characterization of milled wood lignins Thioacidolysis was performed according to Lapierre’s procedure (1986). Each lignin sample (ca. 10 mg) was dispersed with thioacidolysis reagent [10 ml, 0.2 M BF3 etherate in a 8.75:1 (v/v) dioxane : ethanethiol] in a 20 ml glass tube fitted with Teflon-lined screwcap under nitrogen atmosphere. Thioacidolysis was performed by placing each sample in a heating block held at 100 ◦ C for 4 h. The thioacidolysis products obtained were trimethylsilylated with N,O-bis(trimethylsilyl)trifluoroacetamide (BSTFA, 50 µl) and pyridine (50 µl) at 60 ◦ C for 2 h. The resulting derivatives were applied to a HP 6890 Series GC System equipped with a DB-5 capillary column (60 m × 0.250 mm, 0.25 µm film, J & W Scientific). GC con-

Fig. 1 Schematic diagram of sample preparation for chemical analysis Abb. 1 Schematische Darstellung der Probenvorbereitung f¨ur die chemische Analyse

dition: injector temperature 250 ◦ C; detector temperature 280 ◦ C. Temperature program: 160–250 ◦ C with a heating rate of 2 ◦ C min−1 , the final temperature was held for 5 min. All data are the average values from double determinations. For analytical pyrolysis each sample was pyrolysed at 450 ◦ C for 10 s (Choi et al. 2001, Faix et al. 1990a, Faix et al. 1990b). GC conditions: DB1701 capillary column (60 m × 0.32 mm, 0.25 µm film, J & W Scientific); injector temperature 250 ◦ C; detector temperature 280 ◦ C. Temperature program: 45 ◦ C (holding 4 min), heating rate 3 ◦ C · min−1; the temperature maximum (280 ◦ C) was held for 30 min. The peak integrals are presented as relative % (sum of the areas of identified substances equals to 100). All data presented are the average from double determinations.

Table 1 Determination of weight loss during fungal treatment and lignin contents in extractive-free wood powder (CWR, A), in residues after 1 M NaOH treatment (B) and in alkali solubles (C) obtained from fungal treated (using C. subvermispora) aspen wood Tabelle 1 Bestimmung des Masseverlustes, des Methanolextraktstoffgehalts sowie des Ligningehalts in extraktfreiem Holz (CWR, A), in den R¨uckst¨anden nach 1 M NaOH-Behandlung (B) und im alkalil¨oslichen Teil (C) von pilzinfiziertem (C. subvermispora) Espenholz nach unterschiedlicher Befallsdauer A B C D Extractive Lignin content (%) Residues of Lignin content (%) Precipitates Lignin content (%) Lignin free wood Insoluble Acid 100 g CWR Insoluble Acid from NaOH Insoluble Acid dissolved in powder lignin soluble after NaOH lignin soluble in treatment lignin soluble NaOH by (CWR) lignin Treatment lignin B (g) lignin difference∗ (g) (g) (g) Control 0 100 19.6 0.70 80.1 19.3 1.35 16.2 11.1 4.89 3.7 1 1.4 100 18.0 0.77 77.1 15.8 1.36 14.2 13.4 6.60 5.4 2 2.8 100 17.0 0.79 72.7 16.0 1.06 15.3 18.7 7.70 5.4 4 6.2 100 16.8 0.78 70.4 14.9 1.06 14.9 17.0 7.21 6.3 6 7.9 100 15.2 0.77 71.0 15.4 1.02 14.4 15.3 6.04 4.3 ∗ Based on lignin contents listed in columns A and B: lignin yields in column A minus lignin yields in column B related to 100 g CWR. Time of fungal treatment (weeks)

Weight loss (%)

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to 15.2% (sample after 6 weeks of incubation) corresponding to an apparent lignin loss of 27.5%. The extract-free cell wall residues were treated under mild alkaline condition using 1 M NaOH (Fig. 1). As shown in Fig. 2, the alkali solubility of the control was around 20% but the decayed woods revealed a solubility of up to 30% (after 4 weeks of treatment). The alkali solubility of lignins reached a maximum (6.3 g from 100 g of CWR, see column D in Table 1) after fungal incubation of 4 weeks, whereas the control released 3.7 g of alkali soluble lignin. Incubation longer than 4 weeks decreased the alkali solubility, which should be rationalized on the basis of the “accumulation” of condensed structure, since the amount of β-O-4 linkages has decreased. Fig. 2 Determination of alkaline solubility in 1 M NaOH after fungal treatment of aspen wood with C. subvermispora Abb. 2 Bestimmung der Alkalil¨oslichkeit in 1 M NaOH von pilzinfiziertem (C. subvermispora) Espenholz

3 Results and discussion 3.1 Lignin contents and alkali solubility after fungal treatment After 1 week of fungal treatment the color of the chips turned to brown and this hue was maintained without apparent change for 6 weeks of decay. The weight loss of wood chips ranged from 1.4% to 7.9% (dry wood basis) for 6 weeks of incubation. As Table 1 (in column A) indicates, the total lignin contents were reduced from 19.6% (control)

3.2 Characterization of milled wood lignins by thioacidolysis and analytical pyrolysis For in-depth studies about modification of lignin structures during fungal decay treatment, milled wood lignins were isolated according to Bj¨orkman (1956). The yields of MWLs ranged from 23.2% (isolated from decayed wood) to 27.8% (isolated from the control) (Table 2), based on lignin contents in CWR. The metabolic system of the fungus changes the structure of the wood in such a way that the yields of MWLs are slightly decreased after fungal decay. Table 2 illustrates the yields of the main trithioethylated products, GCHSEt-CHSEt-CH(SEt)2 and S-CHSEt-CHSEt-CH(SEt)2 , obtained from MWLs. It is consensus that these compounds arise from β-O-4 linkages under thioacidolysis condition

Table 2 Characterization of milled wood lignins (MWLs) obtained from aspen wood treated with C. subvesmispora by thioacidolysis. The S/G ratios of analytical pyrolysis are derived from raw data listed in Table 3 Tabelle 2 Untersuchung von Milled Wood Ligninen (MWLs) isoliert von C. subvesmispora behandeltem Espenholz durch Thioacidolyse. Die S/G Verh¨altnisse der analytischen Pyrolyse wurden von den Rohdaten abgeleitet, die in Tabelle 3 aufgef¨uhrt sind Time of fungal treatment (weeks) Control

Yield of MWL∗ (%) 27.8

1

23.2

2

23.3

4

23.5

6

25.6

Average % (based on control) after 1 to 6 week treatment Average loss after 1 to 6 week treatment

Yield of monomeric thioacidolysis products (µmol · g−1 MWL)(% data based on control) G1 644.4 ± 16.2 (100%) 561.1 ± 4.1 (87.1%) 546.7 ± 74.9 (84.8%) 557.8 ± 21.7 (86.6%) 559.4 ± 8.4 (86.8%)

S2 721.4 ± 23.4 (100%) 588.1 ± 9.9 (81.5%) 543.3 ± 27.7 (75.3%) 546.7 ± 0.2 (75.8%) 530.5 ± 19.4 (73.5%)

G+S 1365.8 ± 40.2 (100%) 1149.2 ± 6.9 (84.1%) 1090.0 ± 19.3 (79.8%) 1104.5 ± 19.8 (80.9%) 1089.9 ± 28.7 (79.8%)

86.3%

76.5%

81.2%

100 − 86.3 = 13.7%

100 − 76.5 = 23.5%

100 − 81.2 = 18.8%

S/G ratios based on Thioacidolysis Thioacidolysis Analytical pyrolisis non corrected corrected3 corrected3 1.12 0.79 0.88 1.05

0.74

0.88

0.99

0.70

0.81

0.98

0.69

0.86

0.95

0.67

0.79

∗ : Based on total lignin contents of CWR in Table 1. 1 G: G-CHSEt-CHSEt-CH(SEt) , 2 S: S-CHSEt-CHSEt-CH(SEt) 3 S values both of pyrol2 2 ysis and thioacidolysis were corrected by Boettcher’s equation (B¨ottcher 1993). Corrected S (%) = -0.588 + 0.704 × S % (non corrected), where % G non corrected + % S non corrected = 100 %

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H-types

Pyrolytic Products

MWL purified from aspen wood after fungal treatment Control 1 week 2 weeks 4 weeks 6 weeks 4.29 4.86 4.51 3.91 3.47 0.13 0.19 0.24 0.25 0.24 0.08 0.08 0.10 0.10 0.09 0.36 0.34 0.40 0.40 0.41

phenol o-cresol 4-vinyl phenol 4-hydroxy-benzoic acid methylester Sum

S-type monomers

G-type monomers

Table 3 Characterization of milled wood lignins isolated from untreated and fungal treated aspen wood by analytical pyrolysis Tabelle 3 Untersuchung von Milled Wood Ligninen (MWLs) isoliert von unbehandeltem und mit Weißf¨aulepilz (C. subvermispora) behandeltem Espenholz durch analytische Pyrolyse

419

4.86

5.47

5.25

4.65

4.21

guaiacol, 2-Methoxy-phenol 3-methyl guaiacol 4-methyl guaiacol 3-ethyl guaiacol 4-ethyl guaiacol 4-vinyl guaiacol eugenol isoeugenol (cis) poss.: 3-Methoxy-5-methylphenol isoeugenol (trans) vanillin 6-hydroxy-7-methoxy-indene (cis or trans) 6-hydroxy-7-methoxy-indene (cis or trans) homovanillin methoxy catechol acetoguaiacone guaiacyl aceton propioguaiacone isomer of coniferyl alcohol G-CO-CH=CH2 coniferyl alcohol (cis) coniferyl alcohol (trans) coniferylaldehyde

2.22 0.16 1.78 0.00 0.23 2.71 0.47 0.19 0.13 1.93 4.08 1.08 1.00 2.02 0.00 2.29 0.43 0.16 0.46 2.00 1.22 10.59 3.24

3.23 0.25 2.42 0.10 0.31 3.59 0.54 0.26 0.27 2.39 4.01 0.87 0.83 1.83 0.17 2.35 0.62 0.18 0.43 1.48 0.93 8.09 3.14

3.35 0.31 3.86 0.19 0.52 4.01 2.38 0.44 0.39 3.44 4.03 0.63 0.60 1.91 0.18 2.44 0.75 0.25 0.53 1.20 0.76 4.97 3.09

3.24 0.31 3.65 0.18 0.45 3.84 0.58 0.32 0.40 2.91 4.24 0.79 0.74 2.02 0.19 2.64 0.77 0.24 0.49 1.41 0.81 5.48 3.30

3.41 0.33 4.44 0.21 0.64 4.05 2.35 0.43 0.42 3.64 4.20 0.76 0.72 1.96 0.16 2.73 0.81 0.28 0.57 1.39 0.67 3.78 3.23

Sum

38.38

38.28

40.22

39.00

41.19

4.90 3.31 0.51 4.67 1.02 0.26 0.50 2.35 0.99 3.77 8.72 3.28 3.81 0.92 0.34 0.36 2.69 1.09 7.07 6.21

6.63 4.06 0.59 5.42 1.06 0.22 0.55 1.45 0.91 4.11 7.21 2.80 3.49 1.21 0.37 0.79 1.83 0.85 6.40 6.31

6.24 6.00 0.88 5.23 1.24 0.43 0.87 1.15 0.74 5.15 6.75 2.65 3.46 1.24 0.46 0.85 1.52 0.56 3.06 6.05

6.07 5.81 0.78 5.31 1.12 0.34 0.67 1.56 0.88 4.76 7.34 3.06 3.90 1.39 0.45 0.99 1.67 0.53 3.51 6.18

6.16 6.81 1.04 5.11 1.26 0.45 0.90 1.34 0.82 5.32 6.87 2.67 3.61 1.31 0.50 0.85 1.61 0.39 1.89 5.66

Sum

56.76

56.25

54.52

56.35

54.59

Sum of H Sum of G Sum of S

4.86 38.38 56.76

5.47 38.28 56.25

5.26 40.22 54.52

4.65 39.00 56.35

4.22 41.19 54.59

100.00

100.00

100.00

100.00

100.00

syringol 4-methyl syringol 4-ethyl syringol 4-vinyl syringol 4-allyl syringol 4-propyl syringol 4-propenyl syringol (cis) 6-hydroxy-5,7-dimethoxy-indene (cis or trans) 6-hydroxy-5,7-dimethoxy-indene (cis or trans) 4-propenyl syringol (trans) syringaldehyde homosyringaldehyde acetosyringone syringyl acetone propiosyringone S-CO-CO-CH3 S-CO-CH=CH2 + isomer of sinapyl alcohol sinapyl alcohol (cis) sinapyl alcohol (trans) sinapaldehyde

Total

(Lapierre et al. 1986). The yields are presented in Table 2 in absolute amounts (mg · g−1MWL) and as relative percentages (yields of both compounds obtained from the control are set arbitrarily to 100%) in order to illustrate the rela-

tive changes in monomeric composition of thioacidolysis. The sum of both trithioethylated C6 C3 -products amounts to 1366 µmol · g−1MWL in the control. In comparison with this data, ca. 19% decrease of trithioethylated C6 C3 -products iso-

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lated from decayed woods can be observed for all MWLs (average of all degraded samples). The syringyl type C6 C3 trithioethylated substance suffered on an average more loss (23.5%) than that of the G-type (13.7%). A significant reduction of thioacidolysis products (G+S) was already observed in the early stage of incubation (ca. 20.2% after 2 weeks of incubation). With advanced incubation time this value did not change systematically. It is also obvious that the cleavage of β-O-4 linkages containing S-units was slightly preferred from the very beginning of the fungal treatment. There is no systematic change as a function of incubation time. Analytical pyrolysis was applied as an additional degradation technique. The pyrograms were evaluated by calculation of the relative integrals of identified phenols (Table 3). Among 47 essential pyrolytic products the most salient features were the relative abundances of C6 C3 monomers, such as cinnamyl alcohols and cinnamaldehydes. A quantitative comparison of products obtained from decayed aspen wood MWLs with those obtained from the control indicates that the yields of coniferyl and sinapyl alcohol were dropped significantly after the first two weeks of incubation (Fig. 3), along with an increase of C6 C1 - and C6 C2 -type monomers (methyland vinyl-guaiacol and corresponding syringol derivatives). In contrast, coniferyl- and sinapaldehyde maintained their relative proportions without fluctuation irrespective of the incubation time with the fungus (Fig. 3). The rapidly decreased yields of cinnamyl alcohols in an early stage of fungal degradation are in agreement with the results obtained from thioacidolysis where a decrease of trithioethylated products with complete C3 -side chain was already observed after one week of incubation. C6 C3 pyrolytic monomers are mainly formed from β-aryl ether bonds containing OH-groups in α-position by water splitting during pyrolysis. The C6 C1 and C6 C2 phe-

nols are provided from scission between Cα and Cβ and between Cβ and Cγ , respectively. The increasing intensities of C6 C1 and C6 C2 phenols can be interpreted in two ways: 1. As an indirect evidence for enzymatic cleavage of side chains by C. subvermispora. 2. As a modification of the lignin (e. g., promoting condensation reactions) by C. subvermispora in such a way that the abundance of β-O-4 linkages is decreasing.

Fig. 3 Variation of relative abundances of C6 C3 monomers (left) and sum of C6 C1 and C6 C2 phenols, as well as C1 phenols (guaiacol and syringol) (right) determined by analytical pyrolysis of milled wood lignins isolated from aspen wood after fungal treatment with C. subvermispora. C6 C1 is methyl-guaiacol (respectively methyl-syringol) and C6 C2 is the sum of ethyl- and vinyl-guaiacol (respectively the corresponding syringyl type derivatives)

Abb. 3 Ver¨anderung der relativen Menge an C6 C3 -Monomeren (links) und der Summe der C6 C1 - und C6 C2 -Phenole, sowie der C1 Phenole (Guajakol und Syringol) (rechts), bestimmt durch analytische Pyrolyse der Milled Wood Lignine, die aus dem pilzinfiziertem Espenholz isoliert wurden. C6 C1 ist Methylguajakol (beziehungsweise ¨ und VinylguajaMethylsyringol) und C6 C2 ist die Summe von Athylkol (beziehungsweise des entsprechenden Syringylderivates)

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3.3 S/G ratios obtained by thioacidolysis and analytical pyrolysis Before discussing the S/G ratios obtained from thioacidolysis and analytical pyrolysis one should recall that all degradation techniques give easily rise to S-type products because syringyl units have a low condensation degree within the macromolecule. Accordingly, all degradation techniques lead to an elevated amount of S-units and as a consequence the uncorrected S/G-ratios obtained overestimate the “true” S/G ratios in lignin (Sarkanen and Hergert 1971). B¨ottcher (1993) developed a correction equation based on a great number of MWLs that were pyrolyzed, degraded by nitrobenzene oxidation, and characterized by OMe-determination. As a measure for the “true” S/Gratio (for the calibration of the degradation techniques) the OMe/C9 units were taken. In Table 2, where the correction equation is also presented, the uncorrected S/G ratios based on the noncondensed thioacidolysis degradation products are listed. The same values for pyrolysis are compiled in Table 3. To facilitate a comparison of the S/G ratios obtained by thioacidolysis and pyrolysis, the same correction formula of B¨ottcher (1993) was used. The S/G ratios in Table 2 calculated using both techniques are in good agreement.

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Both techniques demonstrate that S/G ratios are continuously decreased as the fungal degradation is advancing. Accordingly, syringyl units seem to be more easily metabolized by C. subvermispora. However, it is also possible that demethoxylation reactions occur in the course of which G units arise from S units. Moreover, one should bear in mind that the data discussed above were obtained from samples (MWLs) representing only 1/4 of the total lignin of P. tremuloides.

4 Conclusion Trembling aspen wood can successfully delignified with C. subvermispora. Comparison of thioacidolytic results from MWLs isolated from the blank and decayed woods suggests that lignins are depolymerized in an early stage of fungal incubation via cleavage of β-O-4 linkages. The degradation of syringyl units seems to be slightly preferred. Analytical pyrolysis gives hints to cleavage of Cα −Cβ and Cβ −Cγ bonds of the propyl side chains. The yields of pyrolytic products observed as a function of fungal treatment time reveal a lowering of C6 C3 monomers and elevation of C6 C1 and C6 C2 monomers. This observation reflects the lignin fragmentation detected by thioacidolysis in another way. S/G ratios derived from both degradation techniques are in good agreement. Increased lignin solubility in alkali is another manifestation of depolymerization. A change in the solubility parameter after 4 weeks of degradation can be interpreted as a sign of increased condensation reactions which occur parallel to the depolymerization. Acknowledgement This research was supported partially by the grant from Korea Science and Engineering Foundation (KOSEF). We would like to express our gratitude to Mrs. Fortmann for the performance of pyrolysis-GC-MS.

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