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Production of organically-bound chlorine during degradation of birch wood by common white-rot fungi
SOU Bid. Biochem. Vol. 29, No. 2, pp. 191-197, 1997 0 1997 Elsevier ScienceLtd. All riehts reserved
PII: SOO38-0717(96)0024243
Printed in &eat Britain 0038-0717/97$17.00+ 0.00
PRODUCTION OF ORGANICALLY-BOUND CHLORINE DURING DEGRADATION OF BIRCH WOOD BY COMMON WHITE-ROT FUNGI G. ÖBERG*t,
H. BRUNBERG
and 0. HJELM
Department of Water and Environmental Studies, Linköping University, 58 1 83 Linköping, Sweden (Accepted 18 June 1996) Summary-In vitro net production of organically-bound chlorine by common white-rot fungi during degradation of birch wood was investigated. It was found that eight of the nine strains examined caused a significant increase in the total amount of organically-bound chlorine (TOX) during the first 8 weeks of incubation, and seven of these species caused a further increase when incubated for an additional 22 weeks. Extractable, GC-amenable chlorinated organic compounds were detected only in samples of two of the investigated fungi and corresponded to 3%, or less, of the total amount of organically-bound chlorine. Ptossible underlying mechanisms and ecological role for production of organically-bound chlorine by white-rot fungi are discussed. 0 1997 Elsevier Science Ltd
INTRODUCTION The past few years of research have shown that organically-bound halogens are more widespread in the environment than previously assumed, and that they are actually natura1 constituents of soil organic matter (Asplund and Grimvall, 1991; Asplund, 1995; Hjelm et al., 1995). The major part seems to be bound to humic like, high-molecular-weight compounds ( > 1.000 D), while low-molecular
weight, CC-amenable halogenated compounds are rarely present in detectable amounts (de Lijser et al., 1991; Hjelm and Asplund, 1995). The origin of the organically-bound halogens in soil is not known, but recent investigations show that in situ production occurs during degradation of organic matter, and it is indicated that net-production of organohalogens during decomposition of spruce needle litter is related to degradation of lignin (Hjelm ef al., 1995; Öberg et al., 1996b). Degradation of lignin is primarily accomplished by white-rot and litter degrading fungi, but very little attention has been paid to the role of these organisms in the turn-over of organohalogens in the terenvironment. Nonetheless, in vitro restrial experiments have shown that some white-rot fungi are capable of producing halometabolites, and compounds identical to those detected in laboratory studies of some funga.1 species have also been detected in field samples collected below fruiting bodies of the same or other fungi (de Jong et al., 1992, 1994). However, no studies have been dedicated to investi-
gate the role of white-rot fungi in the halogenation of organic matter. The aim of our study was to determine whether net production of organically-bound chlorine occurs during degradation of wood by white-rot fungi and to elucidate if it can be attributed to production of CC-amenable compounds. This was achieved by cultivating nine common species of white-rot fungi on blocks of birch (Betula alba) sapwood under controlled conditions and detecting the total amount of organically-bound chlorine after 8 and 30 weeks. Extractable, CC-amenable chlorinated organic compounds in the samples were measured after 30 weeks of incubation.
MATERIALAND METHODS Terminology
The method we used to determine the total amount of organically-bound chlorine (TOX) is a sum parameter and does not distinguish between the halogens chlorine, bromine and iodine. In our study the only halide added to the cultures was chloride which implies that no other organohaloorganochlorine may be formed. gens than Therefore, we find it more adequate to use the term chlorine rather than halogen when referring to the results of our study. However, when referring to the genera1 discussion on organohalogen formation in soil we use the term halogen. Organisms
*Fermer name: Asphmd. TAuthor for correspondence.
The following white-rot fungal strains were used: Armillaria mellea (Vahl ex Fr.); Bjerkandera adusta 191
192
G. Öberg et al.
(Willd. ex. Fr.) Kamt. (= Polyporus adustus (Willd. ex Fr.)) 73213; Trametes (Coriolus) versicolor (L. ex Fr.) Quél., c.$ Polystrictus versicolor A 361; Ganoderma applanatum (Pers. ex Wallr.) 74505; Heterobasidium annosum Bref., c.f. Phomes annosum L 39-1; Merulius tremellosus (Schrad. ex. Fr.) H 94-1; Phellinus isabellinus (Fr.) Bourd. and Galz. SP 22-1; Phlebia radiata (Fr.) L 12-41 and Poria cinerescens Bres. 70236. Al1 of these isolates were a generous gift from the Department of Forest Products, The Swedish University of Agricultural Sciences. Experìmental procedure
The fungi were first cultivated on malt agar plates, and then inoculated in decay chambers as described by Ander and Eriksson (1975). These chambers were prepared by plating three blocks (4 x 4 x 1 cm) of birch (Betula alba) sapwood in 150 ml Erlenmeyer-flasks containing vermiculite (15 g). Malt extract (75 ml 2% Bacto malt extract, “Difco” certified containing 50 pg Cl ml-‘, NaCl, pH 4.75) was then added to the flasks, and the preparations were subsequently autoclaved and inoculated with smal1 pieces of fungal-laden agar. For each fungus, five flasks containing three sapwood blocks were inoculated and incubated for 8 weeks, and one additional flask with two sapwood blocks was inoculated and then incubated for 30 weeks. Reference samples were prepared by incubating five non-inoculated flasks for 8 weeks. Fungal growth was terminated by freezing the flasks and their contents at -20°C for 24 h. The sapwood blocks with adhered mycelia were removed from the vermiculite containing flasks, and oven-dried at 70°C for 24 h. The sapwood blocks were then individually milled (Cyclotec 1093 sample mi11 Tecator; performed at Jäderas experimental station, Sweden), sifted twice through a 0.05 mm sieve and then oven-dried again at 70°C for 24 h. Chemical analyses
The total amount of organically-bound chlorine (TOX) was determined by using the method for soil samples described by Asplund et al. (1994). Three replicates of each sample were analyzed (20 mg milled wood powder), with an AOX-analyser (Euroglas, model 84/85). The total amount of chlorine (TX), or the sum of inorganic and organic chlorine, was determined by adding 20 mg of ground sapwood directly to the AOX-incinerator. Thereafter, the analysis followed the procedure described for TOX determinations (Asplund et al., 1994). Each analysis was performed with three replicates. Chlorinated compounds amenable for gas chromatography were analyzed after 30 weeks of incubation by extractions of the milled wood-powder; one sample from each fungal culture was examined.
The wood-powder (0.5 g) was first extracted with methylene chloride (25 ml). After decantation, the methylene chloride was dried by deep freezing (-20°C) and evaporated to approximately 1 ml. The extract was passed through a smal1 column of silica (Merck Silicagel 60, 500 mg) to remove compounds which might interfere with the GC analysis. After careful washing of the column (5 x 1.5 ml methylene chloride), the extract was again evaporated to a smal1 volume (100 ~1) and an internal standard was added (1-chlorotetradecane). The extract was analyzed by GC-AED (gas chromatography-atomic emission detection) and GC-MS (gas chromatography-mass spectrometry). One sample of wood from each of the decay chambers inoculated with the fungi causing the largest increases in TOX content, i.e. B. adusta, M. tremellosus, P. cinerescens and P. radiata, was also examined for low-molecular weight organic acids. Milled wood powder (0.5 g) was extracted with NaOH (100 ml, 0.1 M) under an atmosphere of nitrogen. After neutralization, the suspension was centrifuged (5000 rev min-‘, 15 min) to remove particles, and, after decantation, the water phase was acidified with HNOs to pH 2. This acidified water phase was extracted with distilled diethyl ether (2 x 25 ml). The resulting organic phase was dried by deep-freezing (-20°C) and by adding anhydrous MgS04. Thereafter the ether solution was evaporated to approximately 1 ml and was treated in the same manner as the methylene chloride extracts to remove compounds interfering with the GC analysis. After a final evaporation to approximately 200 ~1, the acids in the extract were derivatized with BSTFA (bis(trimethylsilyl)trifluoroacetamide) to make them amenable for gas chromatography. The extract was then analyzed by GC-AED and GCMS. GC-AED analyses were performed on a HP 5890 GC. equipped with a HP 5921 microwaveinduced plasma atomic emission detector and a fused silica column (HP Ultra-1, 50 m x 0.32 mm, phase thickness 0.17 Pm). The carrier gas (He) velocity was 39 cm s-‘. The temperature program, which was identical in the GC-AED and GC-MS analyses, was 40°C for 5 min, then raised to 250°C at a rate of 5°C min-‘, and finally kept at 250°C for 10 min. Aliquots (1.1 ~1) were injected using splitless mode (30 s delay). The responses to carbon and chlorine were measured at 496 and 479 nm, respectively. GC-MS analyses were performed using a Shimadzu QP-2000 mass spectrometer equipped with a fused silica column (J and W DB-1, 60 m x 0.32 mm, 0.25 Pm phase thickness). The carrier gas (He) velocity was 29 cm s-’ and a 60 s delay during injection was used. Al1 other variables were identical to the GC-AED analyses.
Chlorine organically-bound by white-rot fungi Al1 chemicals used were of analytical grade and al1 statistical tests were conducted at a 95% significance level.
RESULTS
Total amount of organically bound chlorine A non-parametric test of the results after 8 weeks of decay (Wilcoxon Rank Sum Test) showed that, compared with the reference, there was a significant increase in organically-bound chlorine in al1 but one (H. annosum), of the investigated species. A pronounced increase in relation to the reference was detected for two of the species, i.e. B. adusta and M. tremellosus, (> 10 ,ug Cl g-’ d.w.), whereas growth of six of the species, i.e. Armillaria mellea. Trametes versicolor, Ganoderma applanatum, Phellinus isabellinus, Phlebia radiata and Poria cinerescens, resulted in a smaller (< 5 pg Cl g-’ d.w.) increase (Fig. 1). After 30 weeks, the concentration of organochlorine in the sapwood blocks was more than 30 pg Cl g-’ d.w. for four of the species, i.e. B. adusta, P. cinerescen:;, M. tremeIIosus and P. radiata. At that time, the concentration in the sapwood blocks inoculated with the other five species (C. versicolor, A. mellea, P. isabellinus, G. applanatum and H. annosum) was less than 20 pg-’ g d.w. The laraccumulated increase in organochlorine gest throughout the incubation was detected for B. adusta (60-70 pg-’ g Lw.), whereas the largest increase during the latter part of the incubation, i.e. weeks 8-30, was observed for P. cinerescens (ca. 50 pg-’ g d.w.).
f
i
193
As no blank and only one E-flask with two sapwood blocks was further incubated for each fungus, the reliability of statistical analysis of concentration values after 30 weeks can be questioned. However, a non-parametric test (Kruskal-Wallis 1-way Anova) of the organochlorine concentrations after 8 weeks of incubation showed that within-sample and between-sample variability differed significantly only for one of the nine fungi. Consequently, there is no reason to use a multivariate model, and the two 30-week sapwood blocks of each fungus can be treated as replicates. Analyses (Wilcoxon Rank Sum W-test) of the means of each sapwood black incubated for 8 weeks (n = 15) and for 30 weeks (n = 2) showed that al1 but two of the fungi (i.e. H. annosum and G. applanatum) caused a significant increase in organically-bound chlorine when further incubated (Fig. 2.) Total amount of chlorine (TX) The total amount of chlorine (TX), i.e. the sum of inorganic and organic chlorine, varied from 180 pg Cl-’ g d.w. in M. tremellosus to 540 pg Cl-’ gd.w. in B. adusta. NO obvious relationship could be seen between the total amount of chlorine (TX) and the total amount of organic chlorine (TOX; Fig. 3). CC-amenable compounds The GC analyses showed that a large number of neutral organic compounds could be extracted from the wood powder. Between 50 and several hundred
t
Fig. 1. Average concentrations and standard deviations (P = 0.05) of organically bound chlorine detected in birch wood (Berula alba) inoculated with nine common species of white-rot fungi and incubated for 8 weeks (n = 5).
Fig. 2. Change in amount of organically-bound chlorine during incubation from week 8 to 30 in birch wood (Betula alba) inoculated with nine common species of white-rot fungi. Wilcoxon Rank Sum W-test of the means of each sapwood black incubated for 8 weeks (n = 15) and for 30 weeks (n = 2) showed that al1 but two of the fungi (i.e. H. annosumand G. applanatum)caused a signifìcant increase in organically-bound chlorine during this period.
194
G.
Öberg et al. cinerescens contained only one GC-amenable compound, most likely a chloro-4-methoxycinnamic acid methyl ester. NO chlorinated acids were found in the samples of P. cinerescens, M. tremellosus or P. radiata. However, in the sample of B. adusta one chlorinated acid, i.e. 2,4-dichlorobenzoic acid, was detected; the chlorine bound in this acid represented 0.2% of the TOX, and, together with the chlorinated aldehydes and alcohols, 3.2% of the TOX could be explained by GC-amenable compounds. The identification of these compounds was based on interpretation of mass spectra and, with the exception of the chloro-methoxycinnamatic acid methyl ester from P. cinerescens, on analyses of model compounds. A detailed description wil1 be published elsewhere (0. Hjelm, H. Borén and G. Öberg, unpubl. data).
DISCUSSION
Fig. 3. Total amount of chlorine (TX) and amount of organically-bound chlorine after growth for 30 weeks by the fungi investigated. The fungi are arranged in order of increasing amount of organically-bound chlorine detected after 30 weeks. peaks were visible in the carbon Channel of the atomic emission detector for each of the samples analyzed. The GC-MS technique was comparably less powerful in determining the chemical structures of the detected compounds: the number of identified compounds varied from zero in A. mellea to around compounds 20 in B. adusta. The non-chlorinated were identified as aliphatic and aromatic aldehydes, aliphatic alcohols, terpenes, alkanes, fatty acids and acetate esters thereof and naphthalene derivates. Chlorinated low-molecular weight compounds were only detected in samples of B. adusta and P. cinerescens; none of the organic compounds detected in the extracts from the other seven fungi were chlorinated. It should be noted that only a smal1 proportion of the detected compounds could be structurally identified, probably because most of the compounds were rather complex and had relatively high molecular-weights. This was, however, of no consequente for the determination of the fraction of TOX that could be ascribed to GCamenable compounds since the atomic-emission detector specifically analyses organically-bound chlorine. For B. adusta and P. cinerescens, the amounts of chlorinated compounds detected by AED corresponded to 3 and O.l%, respectively, of the total amount of organochlorine. Five different compounds could be distinguished in the chlorine Channel of the AED chromatograms of the extract of B. adusta; these compounds were 3-chloro-4methoxybenzaldehyde, 3,5-dichloro-4-methoxybenzaldehyde, 3-chloro-4-methoxybenzylic alcohol, 3,5dichloro-4-methoxybenzylic alcohol and 5-chloro3,4_dimethoxybenzaldehyde. The extract from P.
In our study, all, but one, of the common whiterot fungi investigated caused a significant increase in the organochlorine content after 8 weeks of growth on birch wood. This strongly indicates that the ability to produce organically-bound chlorine during wood decay is widespread among white-rot fungi. Evaluation of the TOX method
The determination of the total amount of organically-bound chlorine is crucial for other conclusions we make in the paper, hence, we begin our discussion by evaluating the method used to determine this variable. The method, which was developed for determination of organohalogens in soil samples (Asplund et al., 1994) is based on microcoulometric titration of halide ions formed during incineration of the sample. The most critical step in the procedure is therefore removal of inorganic halides prior to incineration; this is accomplished by washing with nitrate to induce ion-exchange. Several studies have been devoted to ascertaining whether inorganic halides interfere with the procedure for determination of the total amount organohalogens in soil (Asplund et al., 1994; Asplund, 1995; Hjelm et al., 1995). NO evidente of such interference has been recorded, although it could not be completely ruled out that inorganic halides trapped within microbial cells obstruct the analysis. The samples we used contained a large proportion of microbial cells, hence halides retained in such cells may have caused an overestimation of organically-bound chlorine. If this did occur, a correlation should exist between the sum of inorganic and organic chlorine (TX) and TOX, that is, large amounts of TX should have resulted in substantial amounts of TOX. However, in our experiments, no correlation was seen between the amount of organically-bound
Chlorine organically-bound by white-rot fungi
chlorine and the total amount of chlorine (Fig. 3). For example, grsowth by H. unnosum did not cause an increase in the organochlorine content, but the milled wood co:ntained large amounts of chlorine (TX), in other words, these samples contained large amounts of inor8anic chloride. Fairly large amounts of organically-bound chlorine were detected in M. tremellosus, P. radiata and P. cinerescens, but these three species contained smaller amounts of chloride than H. annosum. The largest amounts of both organically-bound chlorine and inorganic chloride were detected in B. adusta. Al1 of the species investigated belong to the basidiomycetes, and to our knowledge there are no indications that membranes, cel1 walls or other factors within this group of fungi vary with respect to their capacity to comentrate or exclude chloride ions from the cytopla.sma. It is therefore quite unlikely that inorganic halides would interfere in the analysis of organochlorine in wood bearing some fungal species but not ethers. We conclude that it is quite unlikely that inorganic chloride interfered significantly with the analysis of organically-bound chlorine in our investigation. Production of organically bound chlorine by white-rot fungi
Eight of the nine white-rot fungal strains considered in our study caused a significant increase in the amount of organically-bound chlorine during the first 8 weeks of incubation, and seven of these species caused a further increase when incubated for an additional 22 weeks. This suggests that production of organically-bound chlorine is common among white-rot fungi. As of yet, very few other studies have addressed this possibility, but in vitro experiments have shown that cultivated strains of B. udusta produce certain low-molecular weight aromatic chlorinated compounds as secondary metabolites (de Jong et al., 1992), and the same compounds have been detected in field samples collected below fruiting bodies of this fungus (de Jong et al., 1994). In ‘our study, these, as wel1 as some additional chlorinated aromatic compounds, were detected after growth by B. adusta, but they only corresponded to about 3% of the total amount of organochlorine. Nevertheless, it is possible that lowmolecular weight organochlorine compounds were continuously polymerized or incorporated into lignin or similar su’bstances during decomposition of the wood, as has been noted in experiments with chlorinated pollutants such as chlorophenols and chloroarisols (Bollag and Loll, 1983) and the concentration of the metabolites would then be dependent on the interrelationships between the rates of production, polymerization and incorporation. It may thus be hypothesized that, even though the production rate may be rather high, the concentration of certain low-molecular weight compounds
195
wil1 remain fairly constant and rather low under steady-state circumstances. This hypothesis is strengthened by the fact that, after 30 weeks of growth, the largest increase in total amount of organochlorine was observed in the samples that contained low-molecular weight chlororganic compounds, i.e. in the wood bearing B. adusta and P. cinerescens. The production of chlorometabolites by fungi is thus a possible source for the increase in the amount of organochlorine detected in our study. However, a significant increase in the organochlorine content was also recorded in decaying wood that did not contain detectable amounts of chlorinated and GC-amenable low-molecular weight organic compounds. This was observed for six of the eight species that caused a significant increase in the organochlorine content, i.e. A. mehea, G. applanatum, M. tremellosus, P. radiata, P. isabellinus and C. versicolor. Although B. adusta and P. cinerescens were responsible for the largest increase after 30 weeks, after the first 8 weeks M. tremellosus caused an increase that was even larger than that caused by P. cinerescens and almost as large as that caused by B. adusta (17.0 f 2.9, 8.5 + 1.9 and 21.9*3.2pgg-‘, respectively; Fig. 1). This strongly indicates that the increase in organochlorine concentration caused by M. tremellosus and the other five fungi was the result of some other process than chlorometabolite production. However, it is possible, that the above-mentioned fungi actually produced chlorometabolites, but that polymerization or incorporation of these compounds was so extensive that the concentrations remained below the detection limit. In addition to production of halometabolites as discussed above, the increase in organochlorine content of the decaying wood may result from enzymatically-catalyzed production of reactive halogen species, as outlined below. Haloperoxidases are known to catalyze halogenation of an organic substrate in the presence of hydrogen peroxide and halide ions (e.g. Neidleman and Geigert, 1986). In the absente of a suitable organic substrate, haloperoxidases catalyze formation of HOC1 (Geigert et al., 1983), which, in turn, may oxidize and chlorinate almost any organic compound. In vitro experiments with chloroperoxidase (CPO, EC 1.11.1.10) from the fungus Caldariomyces fumago verify that such chlorination results in randomly-chlorinated compounds (Asplund et al., organic 1991). Furthermore, it has been shown that spruce forest soils exhibit chloroperoxidase activity (Asplund et ai., 1991, 1993). Very few studies have addressed haloperoxidase activity in white-rot fungi, although it has been shown that lignin peroxidase from Phanerochaete chrysosporium exhibits bromoperoxidase activity, i.e. it is capable of oxidizing iodide and bromide, but not chloride (Renangathan et al.,
G. Öbelrg el al.
196
1987) indicating that white-rot fungi may be capable of producing exo-enzymes with haloperoxidase activity. Accordingly, the organochlorine found in our study may largely originate from hypochlorous acid formed by the action of exoenzymes possessing chloroperoxidase activity. What
is the ecological
role
of organohalogen
pro-
duction?
Previous studies show that organohalogens are natura1 constituents of organic matter and that a production takes place during degradation of organic matter (Hjelm et al., 1995; Öberg et al., 1996a,b). The ecological role of this production is so far unknown, but some of our recent findings i.e. that the amount of organically bound halogens in soil increases with decreasing pH @berg et al., 1996a); that the production is hampered by conventional forest fertilization (NPK) and seems to be related to lignin degradation (Öberg et al., 1996b), in combination with the results of the present study suggest that production of organochlorine is a common feature among white-rot fungi, and makes it tempting to suggest a causa1 relationship between lignin degradation and production of organohalogens. Such a relation may result from an exo enzymatically catalyzed formation of reactive halogen species as outlined below. Firstly, investigations of microbial degradation of lignin strongly indicate that both the fungal hyphae and the enzymes involved are too large to penetrate the wood pores (Harvey et al., 1986; Flournoy et al., 1991). It has therefore been suggested that water-soluble compounds of low-molecular weight initiate decay by diffusing into cel1 wal1 structures of the wood, thereby enlarging the pore size and allowing enzymes to penetrate and continue the degradative process. The existente of such mediators has not yet been conclusively proven, although several candidate compounds have been suggested (Evans et al., 1993). Secondly, it is wel1 known from the pulp and paper industry that addition of reactive chlorine to pulp causes degradation of lignin, and it is also known that this process is accompanied with an undesired increase in the organohalogen content. Thus, a reactive halogen compound such as hypochlorous acid fits the theoretical description of the sought mediator of lignin degradation. In summation, the suggested relationship between natura1 production of organically-bound halogens and lignin degradation could be the result of active participation of enzymatically-produced hypohalous acid in lignin degradation followed by the production of organicallybound halogens. Nonetheless, it remains to be determined whether white-rot fungi actually produce exo-enzymes capable of catalyzing the formation of reactive halogen species and if such reactive species are involved in lignin degradation.
Acknowledgements-We are indebted to Dr Per Sandén, Dr Eva Enquist and Dr Per Stalnacke for statistical advice and fruitful discussions. This study was supported by the Swedish Council for Forestry and Agricultural Research.
REFERENCES
Ander P. and Eriksson K.-E. (1975) Influence of carbohydrates on lignin degradation by the white-rot fungus Sporotrichum pulverulentum. Svensk Papperstidning 78, 1 643-652. Asplund G. (1995) Origin and occurrence of halogenated organic matter in soil. In Naturally-Produced Organohalogens (A. Grimvall and E. W. B. de Leer, Eds) Kluwer Academie Publishers, Dordrecht: 35-48’ Asplund G., Borén H., Carlsson U. and Grimvall A. (1991) Soil peroxidase-mediated chlorination of fulvic acid. In Humic Substances in the Aquatic and Terrestrial Environment (B. Allard, H. Borén and A. Grimvall, Eds), pp. 475-484. Springer, Berlin. Asplund G., Christiansen J. V. and Grimvall A. (1993) A chloroperoxidase-like catalyst in soil: detection and characterization of some properties. Soil Biotogy & Biochemistry 25, 41-46. Asplund G. and Grimvall A. (1991) Organohalogens in nature, more widespread than previously assumed. Environmental Science and Technology 25, 1346-1350. Asplund G., Grimvall A. and Jonsson S. (1994) Determination of total and leachable amounts of organohalogens in soil. Chemosphere 28, 1467-1475. Bollag J.-M. and Lol1 M. J. (1983) Incorporation of xenobiotics into soil humus. Experientia 39, 1221-1231. Evans C. S., Dutton M. V., Guillen F. and Veness R. G. (1993) Enzymes and smal1 molecular weight agents involved with hgnocellulose degradation. In Lignin Biodegradation and Transformation. Biotechnological Applications (J. C. Duarte, M. C. Ferreira and P. Ander, Eds), pp. 77-79. Forbitee Editions, Lisbon. Flournoy D. S., Kirk T. K. and Highley T. L. (1991) Wood decay by brown-rot fungi; changes in pore structure and cel1 wal1 volume. Holzforschung 45, 383-388. Geigert J., Neidleman S. L. and Dalietos D. J. (1983) Navel haloperoxidase substrates. Journal of Biological Chemistry 258, 2273-2277. Harvey P. J., Schoemaker H. E. and Palmer J. M. (1986) Veratryl alcohol as a mediator and the role of radical cations in lignin biodegradation by Phanerochaete chrysosporium. FEBS Letters 195, 242-246. Hjelm 0. and Asplund G. (1995) Chemical characterization of organohalogens in a coniferous forest soil. In Naturally-Produced Örganohalogens (A. Grimvall and E. W. B. de Leer. Eds), DD. 105-111. Kluwer Academie. Dordrecht. Hjelm O., Johansson M. B. and Öberg-Asplund G. (1995) Organically bound halogens in coniferous forest soil Distribution pattern and evidente of in situ production. Chemosphere 30, 2353-2364. de Jong E., Field J. A., Dings J. A. F. M., Wijnberg J. B. P. A. and de Bont J. A. M. (1992) De novo biosynthesis of chlorinated aromatics by the white-rot fungus Bjerkandera sp. BOS55. FEBS Letters 305, 220-224. de Jong E., Field J. A., Spinnler H.-A., Wijnberg J. B. P. A. and de Bont J. A. M. (1994) Significant biogenesis of chlorinated aromatics by fungi in natura1 environments. Applied Environmental Microbiology 60, 264-270. .L
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Chlorine organically-bound by white-rot fungi Neidleman S. L. and Geigert J. (1986) Biohalogenation: Principles. Basic Roles and Applications. Elhs Horwood, Chichester. Öberg G., Börjesson 1. and Samuelsson B. (1996a) Net change in organically bound halogens in relation to soil pH. Water, Air and Soil Pollution, 89, 351-361. Öberg G., Nordhmd E. and Berg B. (1996b) In situ formation of organically bound halogens during decompo-
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Biochemistry 26,
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PRODUCTION OF ORGANICALLY-BOUND CHLORINE DURING DEGRADATION OF BIRCH WOOD BY COMMON WHITE-ROT FUNGI G. ÖBERG*t,
H. BRUNBERG
and 0. HJELM
Department of Water and Environmental Studies, Linköping University, 58 1 83 Linköping, Sweden (Accepted 18 June 1996) Summary-In vitro net production of organically-bound chlorine by common white-rot fungi during degradation of birch wood was investigated. It was found that eight of the nine strains examined caused a significant increase in the total amount of organically-bound chlorine (TOX) during the first 8 weeks of incubation, and seven of these species caused a further increase when incubated for an additional 22 weeks. Extractable, GC-amenable chlorinated organic compounds were detected only in samples of two of the investigated fungi and corresponded to 3%, or less, of the total amount of organically-bound chlorine. Ptossible underlying mechanisms and ecological role for production of organically-bound chlorine by white-rot fungi are discussed. 0 1997 Elsevier Science Ltd
INTRODUCTION The past few years of research have shown that organically-bound halogens are more widespread in the environment than previously assumed, and that they are actually natura1 constituents of soil organic matter (Asplund and Grimvall, 1991; Asplund, 1995; Hjelm et al., 1995). The major part seems to be bound to humic like, high-molecular-weight compounds ( > 1.000 D), while low-molecular
weight, CC-amenable halogenated compounds are rarely present in detectable amounts (de Lijser et al., 1991; Hjelm and Asplund, 1995). The origin of the organically-bound halogens in soil is not known, but recent investigations show that in situ production occurs during degradation of organic matter, and it is indicated that net-production of organohalogens during decomposition of spruce needle litter is related to degradation of lignin (Hjelm ef al., 1995; Öberg et al., 1996b). Degradation of lignin is primarily accomplished by white-rot and litter degrading fungi, but very little attention has been paid to the role of these organisms in the turn-over of organohalogens in the terenvironment. Nonetheless, in vitro restrial experiments have shown that some white-rot fungi are capable of producing halometabolites, and compounds identical to those detected in laboratory studies of some funga.1 species have also been detected in field samples collected below fruiting bodies of the same or other fungi (de Jong et al., 1992, 1994). However, no studies have been dedicated to investi-
gate the role of white-rot fungi in the halogenation of organic matter. The aim of our study was to determine whether net production of organically-bound chlorine occurs during degradation of wood by white-rot fungi and to elucidate if it can be attributed to production of CC-amenable compounds. This was achieved by cultivating nine common species of white-rot fungi on blocks of birch (Betula alba) sapwood under controlled conditions and detecting the total amount of organically-bound chlorine after 8 and 30 weeks. Extractable, CC-amenable chlorinated organic compounds in the samples were measured after 30 weeks of incubation.
MATERIALAND METHODS Terminology
The method we used to determine the total amount of organically-bound chlorine (TOX) is a sum parameter and does not distinguish between the halogens chlorine, bromine and iodine. In our study the only halide added to the cultures was chloride which implies that no other organohaloorganochlorine may be formed. gens than Therefore, we find it more adequate to use the term chlorine rather than halogen when referring to the results of our study. However, when referring to the genera1 discussion on organohalogen formation in soil we use the term halogen. Organisms
*Fermer name: Asphmd. TAuthor for correspondence.
The following white-rot fungal strains were used: Armillaria mellea (Vahl ex Fr.); Bjerkandera adusta 191
192
G. Öberg et al.
(Willd. ex. Fr.) Kamt. (= Polyporus adustus (Willd. ex Fr.)) 73213; Trametes (Coriolus) versicolor (L. ex Fr.) Quél., c.$ Polystrictus versicolor A 361; Ganoderma applanatum (Pers. ex Wallr.) 74505; Heterobasidium annosum Bref., c.f. Phomes annosum L 39-1; Merulius tremellosus (Schrad. ex. Fr.) H 94-1; Phellinus isabellinus (Fr.) Bourd. and Galz. SP 22-1; Phlebia radiata (Fr.) L 12-41 and Poria cinerescens Bres. 70236. Al1 of these isolates were a generous gift from the Department of Forest Products, The Swedish University of Agricultural Sciences. Experìmental procedure
The fungi were first cultivated on malt agar plates, and then inoculated in decay chambers as described by Ander and Eriksson (1975). These chambers were prepared by plating three blocks (4 x 4 x 1 cm) of birch (Betula alba) sapwood in 150 ml Erlenmeyer-flasks containing vermiculite (15 g). Malt extract (75 ml 2% Bacto malt extract, “Difco” certified containing 50 pg Cl ml-‘, NaCl, pH 4.75) was then added to the flasks, and the preparations were subsequently autoclaved and inoculated with smal1 pieces of fungal-laden agar. For each fungus, five flasks containing three sapwood blocks were inoculated and incubated for 8 weeks, and one additional flask with two sapwood blocks was inoculated and then incubated for 30 weeks. Reference samples were prepared by incubating five non-inoculated flasks for 8 weeks. Fungal growth was terminated by freezing the flasks and their contents at -20°C for 24 h. The sapwood blocks with adhered mycelia were removed from the vermiculite containing flasks, and oven-dried at 70°C for 24 h. The sapwood blocks were then individually milled (Cyclotec 1093 sample mi11 Tecator; performed at Jäderas experimental station, Sweden), sifted twice through a 0.05 mm sieve and then oven-dried again at 70°C for 24 h. Chemical analyses
The total amount of organically-bound chlorine (TOX) was determined by using the method for soil samples described by Asplund et al. (1994). Three replicates of each sample were analyzed (20 mg milled wood powder), with an AOX-analyser (Euroglas, model 84/85). The total amount of chlorine (TX), or the sum of inorganic and organic chlorine, was determined by adding 20 mg of ground sapwood directly to the AOX-incinerator. Thereafter, the analysis followed the procedure described for TOX determinations (Asplund et al., 1994). Each analysis was performed with three replicates. Chlorinated compounds amenable for gas chromatography were analyzed after 30 weeks of incubation by extractions of the milled wood-powder; one sample from each fungal culture was examined.
The wood-powder (0.5 g) was first extracted with methylene chloride (25 ml). After decantation, the methylene chloride was dried by deep freezing (-20°C) and evaporated to approximately 1 ml. The extract was passed through a smal1 column of silica (Merck Silicagel 60, 500 mg) to remove compounds which might interfere with the GC analysis. After careful washing of the column (5 x 1.5 ml methylene chloride), the extract was again evaporated to a smal1 volume (100 ~1) and an internal standard was added (1-chlorotetradecane). The extract was analyzed by GC-AED (gas chromatography-atomic emission detection) and GC-MS (gas chromatography-mass spectrometry). One sample of wood from each of the decay chambers inoculated with the fungi causing the largest increases in TOX content, i.e. B. adusta, M. tremellosus, P. cinerescens and P. radiata, was also examined for low-molecular weight organic acids. Milled wood powder (0.5 g) was extracted with NaOH (100 ml, 0.1 M) under an atmosphere of nitrogen. After neutralization, the suspension was centrifuged (5000 rev min-‘, 15 min) to remove particles, and, after decantation, the water phase was acidified with HNOs to pH 2. This acidified water phase was extracted with distilled diethyl ether (2 x 25 ml). The resulting organic phase was dried by deep-freezing (-20°C) and by adding anhydrous MgS04. Thereafter the ether solution was evaporated to approximately 1 ml and was treated in the same manner as the methylene chloride extracts to remove compounds interfering with the GC analysis. After a final evaporation to approximately 200 ~1, the acids in the extract were derivatized with BSTFA (bis(trimethylsilyl)trifluoroacetamide) to make them amenable for gas chromatography. The extract was then analyzed by GC-AED and GCMS. GC-AED analyses were performed on a HP 5890 GC. equipped with a HP 5921 microwaveinduced plasma atomic emission detector and a fused silica column (HP Ultra-1, 50 m x 0.32 mm, phase thickness 0.17 Pm). The carrier gas (He) velocity was 39 cm s-‘. The temperature program, which was identical in the GC-AED and GC-MS analyses, was 40°C for 5 min, then raised to 250°C at a rate of 5°C min-‘, and finally kept at 250°C for 10 min. Aliquots (1.1 ~1) were injected using splitless mode (30 s delay). The responses to carbon and chlorine were measured at 496 and 479 nm, respectively. GC-MS analyses were performed using a Shimadzu QP-2000 mass spectrometer equipped with a fused silica column (J and W DB-1, 60 m x 0.32 mm, 0.25 Pm phase thickness). The carrier gas (He) velocity was 29 cm s-’ and a 60 s delay during injection was used. Al1 other variables were identical to the GC-AED analyses.
Chlorine organically-bound by white-rot fungi Al1 chemicals used were of analytical grade and al1 statistical tests were conducted at a 95% significance level.
RESULTS
Total amount of organically bound chlorine A non-parametric test of the results after 8 weeks of decay (Wilcoxon Rank Sum Test) showed that, compared with the reference, there was a significant increase in organically-bound chlorine in al1 but one (H. annosum), of the investigated species. A pronounced increase in relation to the reference was detected for two of the species, i.e. B. adusta and M. tremellosus, (> 10 ,ug Cl g-’ d.w.), whereas growth of six of the species, i.e. Armillaria mellea. Trametes versicolor, Ganoderma applanatum, Phellinus isabellinus, Phlebia radiata and Poria cinerescens, resulted in a smaller (< 5 pg Cl g-’ d.w.) increase (Fig. 1). After 30 weeks, the concentration of organochlorine in the sapwood blocks was more than 30 pg Cl g-’ d.w. for four of the species, i.e. B. adusta, P. cinerescen:;, M. tremeIIosus and P. radiata. At that time, the concentration in the sapwood blocks inoculated with the other five species (C. versicolor, A. mellea, P. isabellinus, G. applanatum and H. annosum) was less than 20 pg-’ g d.w. The laraccumulated increase in organochlorine gest throughout the incubation was detected for B. adusta (60-70 pg-’ g Lw.), whereas the largest increase during the latter part of the incubation, i.e. weeks 8-30, was observed for P. cinerescens (ca. 50 pg-’ g d.w.).
f
i
193
As no blank and only one E-flask with two sapwood blocks was further incubated for each fungus, the reliability of statistical analysis of concentration values after 30 weeks can be questioned. However, a non-parametric test (Kruskal-Wallis 1-way Anova) of the organochlorine concentrations after 8 weeks of incubation showed that within-sample and between-sample variability differed significantly only for one of the nine fungi. Consequently, there is no reason to use a multivariate model, and the two 30-week sapwood blocks of each fungus can be treated as replicates. Analyses (Wilcoxon Rank Sum W-test) of the means of each sapwood black incubated for 8 weeks (n = 15) and for 30 weeks (n = 2) showed that al1 but two of the fungi (i.e. H. annosum and G. applanatum) caused a significant increase in organically-bound chlorine when further incubated (Fig. 2.) Total amount of chlorine (TX) The total amount of chlorine (TX), i.e. the sum of inorganic and organic chlorine, varied from 180 pg Cl-’ g d.w. in M. tremellosus to 540 pg Cl-’ gd.w. in B. adusta. NO obvious relationship could be seen between the total amount of chlorine (TX) and the total amount of organic chlorine (TOX; Fig. 3). CC-amenable compounds The GC analyses showed that a large number of neutral organic compounds could be extracted from the wood powder. Between 50 and several hundred
t
Fig. 1. Average concentrations and standard deviations (P = 0.05) of organically bound chlorine detected in birch wood (Berula alba) inoculated with nine common species of white-rot fungi and incubated for 8 weeks (n = 5).
Fig. 2. Change in amount of organically-bound chlorine during incubation from week 8 to 30 in birch wood (Betula alba) inoculated with nine common species of white-rot fungi. Wilcoxon Rank Sum W-test of the means of each sapwood black incubated for 8 weeks (n = 15) and for 30 weeks (n = 2) showed that al1 but two of the fungi (i.e. H. annosumand G. applanatum)caused a signifìcant increase in organically-bound chlorine during this period.
194
G.
Öberg et al. cinerescens contained only one GC-amenable compound, most likely a chloro-4-methoxycinnamic acid methyl ester. NO chlorinated acids were found in the samples of P. cinerescens, M. tremellosus or P. radiata. However, in the sample of B. adusta one chlorinated acid, i.e. 2,4-dichlorobenzoic acid, was detected; the chlorine bound in this acid represented 0.2% of the TOX, and, together with the chlorinated aldehydes and alcohols, 3.2% of the TOX could be explained by GC-amenable compounds. The identification of these compounds was based on interpretation of mass spectra and, with the exception of the chloro-methoxycinnamatic acid methyl ester from P. cinerescens, on analyses of model compounds. A detailed description wil1 be published elsewhere (0. Hjelm, H. Borén and G. Öberg, unpubl. data).
DISCUSSION
Fig. 3. Total amount of chlorine (TX) and amount of organically-bound chlorine after growth for 30 weeks by the fungi investigated. The fungi are arranged in order of increasing amount of organically-bound chlorine detected after 30 weeks. peaks were visible in the carbon Channel of the atomic emission detector for each of the samples analyzed. The GC-MS technique was comparably less powerful in determining the chemical structures of the detected compounds: the number of identified compounds varied from zero in A. mellea to around compounds 20 in B. adusta. The non-chlorinated were identified as aliphatic and aromatic aldehydes, aliphatic alcohols, terpenes, alkanes, fatty acids and acetate esters thereof and naphthalene derivates. Chlorinated low-molecular weight compounds were only detected in samples of B. adusta and P. cinerescens; none of the organic compounds detected in the extracts from the other seven fungi were chlorinated. It should be noted that only a smal1 proportion of the detected compounds could be structurally identified, probably because most of the compounds were rather complex and had relatively high molecular-weights. This was, however, of no consequente for the determination of the fraction of TOX that could be ascribed to GCamenable compounds since the atomic-emission detector specifically analyses organically-bound chlorine. For B. adusta and P. cinerescens, the amounts of chlorinated compounds detected by AED corresponded to 3 and O.l%, respectively, of the total amount of organochlorine. Five different compounds could be distinguished in the chlorine Channel of the AED chromatograms of the extract of B. adusta; these compounds were 3-chloro-4methoxybenzaldehyde, 3,5-dichloro-4-methoxybenzaldehyde, 3-chloro-4-methoxybenzylic alcohol, 3,5dichloro-4-methoxybenzylic alcohol and 5-chloro3,4_dimethoxybenzaldehyde. The extract from P.
In our study, all, but one, of the common whiterot fungi investigated caused a significant increase in the organochlorine content after 8 weeks of growth on birch wood. This strongly indicates that the ability to produce organically-bound chlorine during wood decay is widespread among white-rot fungi. Evaluation of the TOX method
The determination of the total amount of organically-bound chlorine is crucial for other conclusions we make in the paper, hence, we begin our discussion by evaluating the method used to determine this variable. The method, which was developed for determination of organohalogens in soil samples (Asplund et al., 1994) is based on microcoulometric titration of halide ions formed during incineration of the sample. The most critical step in the procedure is therefore removal of inorganic halides prior to incineration; this is accomplished by washing with nitrate to induce ion-exchange. Several studies have been devoted to ascertaining whether inorganic halides interfere with the procedure for determination of the total amount organohalogens in soil (Asplund et al., 1994; Asplund, 1995; Hjelm et al., 1995). NO evidente of such interference has been recorded, although it could not be completely ruled out that inorganic halides trapped within microbial cells obstruct the analysis. The samples we used contained a large proportion of microbial cells, hence halides retained in such cells may have caused an overestimation of organically-bound chlorine. If this did occur, a correlation should exist between the sum of inorganic and organic chlorine (TX) and TOX, that is, large amounts of TX should have resulted in substantial amounts of TOX. However, in our experiments, no correlation was seen between the amount of organically-bound
Chlorine organically-bound by white-rot fungi
chlorine and the total amount of chlorine (Fig. 3). For example, grsowth by H. unnosum did not cause an increase in the organochlorine content, but the milled wood co:ntained large amounts of chlorine (TX), in other words, these samples contained large amounts of inor8anic chloride. Fairly large amounts of organically-bound chlorine were detected in M. tremellosus, P. radiata and P. cinerescens, but these three species contained smaller amounts of chloride than H. annosum. The largest amounts of both organically-bound chlorine and inorganic chloride were detected in B. adusta. Al1 of the species investigated belong to the basidiomycetes, and to our knowledge there are no indications that membranes, cel1 walls or other factors within this group of fungi vary with respect to their capacity to comentrate or exclude chloride ions from the cytopla.sma. It is therefore quite unlikely that inorganic halides would interfere in the analysis of organochlorine in wood bearing some fungal species but not ethers. We conclude that it is quite unlikely that inorganic chloride interfered significantly with the analysis of organically-bound chlorine in our investigation. Production of organically bound chlorine by white-rot fungi
Eight of the nine white-rot fungal strains considered in our study caused a significant increase in the amount of organically-bound chlorine during the first 8 weeks of incubation, and seven of these species caused a further increase when incubated for an additional 22 weeks. This suggests that production of organically-bound chlorine is common among white-rot fungi. As of yet, very few other studies have addressed this possibility, but in vitro experiments have shown that cultivated strains of B. udusta produce certain low-molecular weight aromatic chlorinated compounds as secondary metabolites (de Jong et al., 1992), and the same compounds have been detected in field samples collected below fruiting bodies of this fungus (de Jong et al., 1994). In ‘our study, these, as wel1 as some additional chlorinated aromatic compounds, were detected after growth by B. adusta, but they only corresponded to about 3% of the total amount of organochlorine. Nevertheless, it is possible that lowmolecular weight organochlorine compounds were continuously polymerized or incorporated into lignin or similar su’bstances during decomposition of the wood, as has been noted in experiments with chlorinated pollutants such as chlorophenols and chloroarisols (Bollag and Loll, 1983) and the concentration of the metabolites would then be dependent on the interrelationships between the rates of production, polymerization and incorporation. It may thus be hypothesized that, even though the production rate may be rather high, the concentration of certain low-molecular weight compounds
195
wil1 remain fairly constant and rather low under steady-state circumstances. This hypothesis is strengthened by the fact that, after 30 weeks of growth, the largest increase in total amount of organochlorine was observed in the samples that contained low-molecular weight chlororganic compounds, i.e. in the wood bearing B. adusta and P. cinerescens. The production of chlorometabolites by fungi is thus a possible source for the increase in the amount of organochlorine detected in our study. However, a significant increase in the organochlorine content was also recorded in decaying wood that did not contain detectable amounts of chlorinated and GC-amenable low-molecular weight organic compounds. This was observed for six of the eight species that caused a significant increase in the organochlorine content, i.e. A. mehea, G. applanatum, M. tremellosus, P. radiata, P. isabellinus and C. versicolor. Although B. adusta and P. cinerescens were responsible for the largest increase after 30 weeks, after the first 8 weeks M. tremellosus caused an increase that was even larger than that caused by P. cinerescens and almost as large as that caused by B. adusta (17.0 f 2.9, 8.5 + 1.9 and 21.9*3.2pgg-‘, respectively; Fig. 1). This strongly indicates that the increase in organochlorine concentration caused by M. tremellosus and the other five fungi was the result of some other process than chlorometabolite production. However, it is possible, that the above-mentioned fungi actually produced chlorometabolites, but that polymerization or incorporation of these compounds was so extensive that the concentrations remained below the detection limit. In addition to production of halometabolites as discussed above, the increase in organochlorine content of the decaying wood may result from enzymatically-catalyzed production of reactive halogen species, as outlined below. Haloperoxidases are known to catalyze halogenation of an organic substrate in the presence of hydrogen peroxide and halide ions (e.g. Neidleman and Geigert, 1986). In the absente of a suitable organic substrate, haloperoxidases catalyze formation of HOC1 (Geigert et al., 1983), which, in turn, may oxidize and chlorinate almost any organic compound. In vitro experiments with chloroperoxidase (CPO, EC 1.11.1.10) from the fungus Caldariomyces fumago verify that such chlorination results in randomly-chlorinated compounds (Asplund et al., organic 1991). Furthermore, it has been shown that spruce forest soils exhibit chloroperoxidase activity (Asplund et ai., 1991, 1993). Very few studies have addressed haloperoxidase activity in white-rot fungi, although it has been shown that lignin peroxidase from Phanerochaete chrysosporium exhibits bromoperoxidase activity, i.e. it is capable of oxidizing iodide and bromide, but not chloride (Renangathan et al.,
G. Öbelrg el al.
196
1987) indicating that white-rot fungi may be capable of producing exo-enzymes with haloperoxidase activity. Accordingly, the organochlorine found in our study may largely originate from hypochlorous acid formed by the action of exoenzymes possessing chloroperoxidase activity. What
is the ecological
role
of organohalogen
pro-
duction?
Previous studies show that organohalogens are natura1 constituents of organic matter and that a production takes place during degradation of organic matter (Hjelm et al., 1995; Öberg et al., 1996a,b). The ecological role of this production is so far unknown, but some of our recent findings i.e. that the amount of organically bound halogens in soil increases with decreasing pH @berg et al., 1996a); that the production is hampered by conventional forest fertilization (NPK) and seems to be related to lignin degradation (Öberg et al., 1996b), in combination with the results of the present study suggest that production of organochlorine is a common feature among white-rot fungi, and makes it tempting to suggest a causa1 relationship between lignin degradation and production of organohalogens. Such a relation may result from an exo enzymatically catalyzed formation of reactive halogen species as outlined below. Firstly, investigations of microbial degradation of lignin strongly indicate that both the fungal hyphae and the enzymes involved are too large to penetrate the wood pores (Harvey et al., 1986; Flournoy et al., 1991). It has therefore been suggested that water-soluble compounds of low-molecular weight initiate decay by diffusing into cel1 wal1 structures of the wood, thereby enlarging the pore size and allowing enzymes to penetrate and continue the degradative process. The existente of such mediators has not yet been conclusively proven, although several candidate compounds have been suggested (Evans et al., 1993). Secondly, it is wel1 known from the pulp and paper industry that addition of reactive chlorine to pulp causes degradation of lignin, and it is also known that this process is accompanied with an undesired increase in the organohalogen content. Thus, a reactive halogen compound such as hypochlorous acid fits the theoretical description of the sought mediator of lignin degradation. In summation, the suggested relationship between natura1 production of organically-bound halogens and lignin degradation could be the result of active participation of enzymatically-produced hypohalous acid in lignin degradation followed by the production of organicallybound halogens. Nonetheless, it remains to be determined whether white-rot fungi actually produce exo-enzymes capable of catalyzing the formation of reactive halogen species and if such reactive species are involved in lignin degradation.
Acknowledgements-We are indebted to Dr Per Sandén, Dr Eva Enquist and Dr Per Stalnacke for statistical advice and fruitful discussions. This study was supported by the Swedish Council for Forestry and Agricultural Research.
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