Lignin demethylation and polysaccharide decomposition in spruce sapwood degraded by brown rot fungi
Introduction
Wood decay fungi are essential for sustained productivity in terrestrial ecosystems. In addition to their role in metal and nutrient cycling in aerobic environments (Jellison et al., 1997, Ostrofsky et al., 1997, Connolly et al., 1999, Hobbie et al., 1999), they are the main biological agents for the conversion of vascular plant tissue to humic materials (e.g. Blanchette et al., 1994, Eriksson et al., 1990) which are ultimately mineralized by soil fungi and bacteria. On a global basis, vascular plant tissue makes up the largest living pool of actively cycling organic matter on earth. Lignin, a primarily ether-linked phenylpropanoid biopolymer found in wood cells and in small amounts in foliar tissue, makes up the second most abundant biopolymer after cellulose. Brown-rot (BR) basidiomycete fungi, the dominant wood decay fungi in coniferous forests, play a unique role in the conversion of wood to coarse debris and soil organic matter, as they are selective for the metabolism of the polysaccharide components of wood over that of lignin (e.g. Blanchette et al., 1994) (see Fig. 1). White-rot fungi, in contrast, can degrade lignin and cellulose simultaneously or selectively degrade lignin (Eriksson et al., 1990). During BR degradation, the methoxyl carbons of lignin are removed, generating an aromatic hydroxyl-rich product abundant in ortho-hydroxy substitution (Ander et al., 1988, Agusin et al., 1989, Enoki et al., 1988, Jin et al., 1990, Filley et al., 2000). Further lignin alteration is thought to be quantitatively minimal but associated with oxidation of the aliphatic side chains and potentially limited lignin depolymerization (e.g. Agosin et al., 1989).
The mechanism of BR decay is not yet determined, but is the focus of active research in mycology, plant pathology and soil sciences. The initial stages of decay are thought to involve the action of Fenton chemistry (Fe2++H2O2) for the production of hydroxyl anions and radicals (Koenigs, 1974). Phenolic compounds produced by the fungi function as ferric iron chelators and sources of electrons for iron reduction (e.g. Chanhoke et al., 1991, Enoki et al., 1997, Goodell et al., 1997, Kerem et al., 1999, Paszczynski et al., 1999). These low molecular weight reactants, unlike enzymes, are small enough to penetrate the sound wood lignocellulose fabric, and have been shown in immunolabeling studies to be present throughout the S2 layer of the BR-degraded cell wall (Jellison et al., 1997). Additionally, many studies have demonstrated that the lignin-rich residues remaining after BR decay can be enriched in metals, particularly calcium, iron, manganese, and magnesium (Ostrofsky et al., 1997, Jellison et al., 1997). BR residues, therefore, are potential sites for enhanced metal concentration and sources of aromatic dihydroxy-rich, altered lignin to forest soils and groundwater.
The extent to which lignin derived carbon is partitioned in soil and water and influences the biogeochemistry of its surroundings is controlled by many factors including its original chemical and structural composition and, in no small part, the nature of the chemical alteration imparted to it during microbial degradation (e.g. Bianchi et al., 1997, Hedges and Oades, 1997). For example, the chemical functionality of degraded plant components (e.g. polyphenolic aromatic acids and alcohols or aliphatic acids) determines the ability of soil and dissolved organic matter to chelate and retain metals such as iron, aluminum, calcium and magnesium (e.g. Tan and Binger, 1986, Schnitzer et al., 1984, Xu and Goodell, 2001). Also, the presence of phenolic compounds, in particular ortho-hydroxy aromatic alcohols (catechols) enables facile redox reactions with ferric iron (e.g. Voelker and Sulzberger, 1996, Paszczynski et al., 1999). The binding capacity of soil and dissolved organic matter for cations and mineral surfaces significantly influences residence time and preservation of organic matter pools with different functional groups (e.g. Miltner and Zech, 1998, Schmidt et al., 2000). The ability of organic matter to effectively bind to metal hydroxy oxides, or clay surfaces bears directly on the efficiency of sorptive protection mechanisms in soils (Henrichs, 1995, Kaiser and Guggenberger, 2000).
We present here the results from a study of spruce sapwood degraded by two common BR basidiomycete fungi, Gloeophyllum trabeum and Postia placenta, in a 32-week laboratory time series inoculation study. The overall goal of this study was to access the chemical alterations imparted to lignin and cellulose during this little understood but ubiquitous process, and address any interdependent relationships in the chemistry of degradation of these two biopolymers by BR fungi. We conducted a molecular biomarker (using 13C-tetramethylammonium hydroxide thermochemolysis) and bulk spectroscopic (using solid-state 13C NMR) assessment of fresh and degraded residues. Herein, we discuss the relationships between lignin demethylation (catechol production), wood mass loss, and polysaccharide (cellulose and hemicellulose) depletion in BR wood and relate the identified chemistry to potential geochemical influence in soils.
Section snippets
Brown-rot inoculation of spruce sapwood
Red spruce (Picea rubens (Sarg.)) sapwood blocks were degraded using a modified ASTM soil block procedure (ASTM, 1994). The soil mix (80 g) was added to 500 ml glass chambers along with 200 ml distilled and deionized water. Two birch (Betula spp.) feeder strips (1×40×40 mm) were placed on the soil surface in each jar and inoculated with four small (2×2 mm) blocks of malt agar supporting the growth of either Postia placenta (Mad-698-R) or Gloeophyllum trabeum (Mad-617-R). Spruce blocks (40×20×20
Weight loss during decay
With increasing time in the degradation experiment the wood blocks changed in color, shape, and weight in a fashion characteristic of BR decay. The blocks progressively turned dark brown taking on a friable texture and shrinking in volume with time. The changes were first visible at 2 weeks time near the base of the blocks in contact with the inoculation strip. By 4 weeks the base of the blocks had shrunk significantly and the originally rectangular blocks took on an inverted trapezoidal shape
Comparison of P. placenta and G. trabeum-induced wood alteration
The lignin demethylation and polysaccharide loss exhibited in G. trabeum and P. placenta degradation (Fig. 5, Fig. 6) is consistent with the known utilization pathways of BR fungi (e.g. Enoki et al., 1988, Jin et al., 1990, Blanchette et al., 1994, Filley et al., 2000). Important insights into BR chemistry and possibly lignin structure are revealed by closer examination of the differences in the relative degree of demethylation among the monomers and in the relative amount of polysaccharide and
Conclusion
The 13C-TMAH thermochemolysis of the residues remaining after a 32 month inoculation experiment using two BR fungi, G. trabeum and P. placenta, showed progressive demethylation of all monomers with a mean demethylation at 22%. G. trabeum degraded wood exhibited a greater degree of demethylation compared to P. Placenta degraded wood. Wood decayed by P. placenta exhibited a greater degree of lignin side chain oxidation as determined by yield of G6. The demethylation of the lignin macromolecule
Acknowledgments
The authors would like to acknowledge helpful comments regarding this manuscript by Rose Filley and Mark Teece and two additional reviewers. We would also like to acknowledge the support of USDA grants 97-34158-502301-35103-09935 and the Carnegie Institution of Washington. This is paper 2520 of the Maine Agricultural and Forestry Experiment Station.
Associate Editor—J. Rice
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