Production and Degradation of Oxalic Acid by Brown Rot Fungi

Our results show that all of the brown rot fungi tested produce oxalic acid in liquid as well as in semisolid cultures. Gloeophyllum trabeum, which accumulates the lowest amount of oxalic acid during decay of pine holocellulose, showed the highest polysaccharide-depolymerizing activity. Semisolid cultures inoculated with this fungus rapidly converted ¹⁴C-labeled oxalic acid to CO₂ during cellulose depolymerization. The other brown rot fungi also oxidized ¹⁴C-labeled oxalic acid, although less rapidly. In contrast, semisolid cultures inoculated with the white rot fungus Coriolus versicolor did not significantly catabolize the acid and did not depolymerize the holocellulose during decay. Semisolid cultures of G. trabeum amended with desferrioxamine, a specific iron-chelating agent, were unable to lower the degree of polymerization of cellulose or to oxidize ¹⁴C-labeled oxalic acid to the extent or at the rate that control cultures did. These results suggest that both iron and oxalic acid are involved in cellulose depolymerization by brown rot fungi.     

Gene cloning and heterologous expression of pyranose 2-oxidase from the brown-rot fungus, <Emphasis Type="Italic">Gloeophyllum</Emphasis><Emphasis Type="Italic">trabeum</Emphasis>

A pyranose 2-oxidase gene from the brown-rot basidiomycete Gloeophyllum trabeum was isolated using homology-based degenerate PCR. The gene structure was determined and compared to that of several pyranose 2-oxidases cloned from white-rot fungi. The G. trabeum pyranose 2-oxidase gene consists of 16 coding exons with canonical promoter CAAT and TATA elements in the 5′UTR. The corresponding G. trabeum cDNA was cloned and contains an ORF of 1,962 base pairs encoding a 653 amino acid polypeptide with a predicted molecular weight of 72 kDa. A Hisx6 tagged recombinant G. trabeum pyranose 2-oxidase was generated and expressed heterologously in Escherichia coli yielding 15 U enzyme activity per ml of induced culture. Structural alignment and phylogenetic analysis were performed and are discussed.     

Wood durability of home-garden teak against brown-rot and white-rot fungi

Natural decay resistance of teak wood grown in home-garden forestry and the factors influencing decay resistance were determined in comparison with that of a typical forest plantation. Accelerated laboratory tests were conducted on 1800 wood samples drawn from 15 trees of three planted sites. Analysis of variance based on a univariate mixed model showed that planted site, fungal species, and their interaction terms were important sources of variation in decay resistance. With increasing decay resistance from centre to periphery of the heartwood, radial position was a critical factor and the interaction effect of fungal species × radial position was significant in influencing the durability. No significant differences were found in decay resistance either between the opposite radii or due to the various possible interaction terms of radii with the site, fungal species and radial position. There were significant differences in decay resistance against brown-rot fungi between wet and dry sites of home-garden teak although differences against white-rot fungi were non-significant among the three planted sites. Polyporus palustris was the more aggressive brown-rot fungus than Gloeophyllum trabeum. The higher susceptibility of wet site home-garden teak to brown-rot decay was associated with a paler colour of the wood and lower extractive content.     

Detection of metal-chelating compounds from wood-rotting fungi Trametes versicolorand Wolfiporia cocos

For many years, the wood decay process by fungi was associated almost exclusively with production of lignocellulolytic enzymes. However, recent studies by electron microscopy have shown that fungal enzymes are too large to penetrate into the cell wall at an early stage of decay. Thus, the hypothesis that low molecular mass agents may initiate the breakdown of both cellulose and lignin was proposed. The purpose of this work was to detect low molecular mass compounds, with metal-chelating capability, from liquid cultures of two wood-rot fungi. The brown-rot fungus Wolfiporia cocos produced the highest chrome azurol S (CAS) reaction, simultaneously reducing the pH of the malt extract medium. In contrast, the white-rot fungus Trametes versicolor did not react with CAS and the pH remained approximately constant during the culture period. The presence of hydroxamate derivatives and oxalic acid was detected in extracts of low molecular mass of both fungi. Moreover, in W. cocos extracts, catecholate derivatives were also detected. Accumulation of oxalic acid was greater in W. cocos than in T. versicolor at the end of the culture period, and this might be responsible for the strong response from W. cocos in the CAS reaction.     

The effect of copper on the growth of wood-rotting fungi and a blue-stain fungus

The effect of copper (II) ions on the growth of three brown-rot fungi, six white-rot fungi and one blue-stain fungus in solid medium was evaluated. The fungi were grown in malt extract agar with different concentrations of copper added, and the radial growth rate was determined. At the end of the incubation period, the mycelial biomass and the media pH were determined. The white-rot and blue-stain fungus grew up to 3 mM and 6 mM copper, respectively and the brown-rot fungi were the only ones that grew up to 10 mM, with higher growth rates than those shown by the other fungi. In general, the brown-rot fungi produced greater acidification in the culture media than the white-rot fungi and blue-stain fungus, and the acidification increased when the amount of copper was increased. The biomass production for the different species, in the absence or presence of copper, was not related to the radial growth rate, and the fungal species that produced the greatest biomass amounts did not correspond to those that presented the highest growth rates. The brown-rot fungi Wolfiporia cocos and Laetiporus sulfureus and blue-stain fungus Ophiostoma sp. demonstrated greater tolerance to high copper concentrations in solid medium than the white-rot fungi, determined as radial growth rate. On the other hand, the highest biomass producers in solid medium with copper added were the white-rot fungi Ganoderma australe and Trametes versicolor and the brown-rot fungus Gloeophyllum trabeum.     

Differences in crystalline cellulose modification due to degradation by brown and white rot fungi

Wood-decaying basidiomycetes are some of the most effective bioconverters of lignocellulose in nature, however the way they alter wood crystalline cellulose on a molecular level is still not well understood. To address this, we examined and compared changes in wood undergoing decay by two species of brown rot fungi, Gloeophyllum trabeum and Meruliporia incrassata, and two species of white rot fungi, Irpex lacteus and Pycnoporus sanguineus, using X-ray diffraction (XRD) and ¹³C solid-state nuclear magnetic resonance (NMR) spectroscopy. The overall percent crystallinity in wood undergoing decay by M. incrassata, G. trabeum, and I. lacteus appeared to decrease according to the stage of decay, while in wood decayed by P. sanguineus the crystallinity was found to increase during some stages of degradation. This result is suggested to be potentially due to the different decay strategies employed by these fungi. The average spacing between the 200 cellulose crystal planes was significantly decreased in wood degraded by brown rot, whereas changes observed in wood degraded by the two white rot fungi examined varied according to the selectivity for lignin. The conclusions were supported by a quantitative analysis of the structural components in the wood before and during decay confirming the distinct differences observed for brown and white rot fungi. The results from this study were consistent with differences in degradation methods previously reported among fungal species, specifically more non-enzymatic degradation in brown rot versus more enzymatic degradation in white rot.     

Fungal accumulation of metals from building materials during brown rot wood decay

This study analyzes the accumulation and translocation of metal ions in wood during the degradation performed by one strain of each of the three brown rot fungi; Serpula lacrymans, Meruliporia incrassata and Coniophora puteana. These fungi species are inhabitants of the built environment where the prevention and understanding of fungal decay is of high priority. This study focuses on the influence of various building materials in relation to fungal growth and metal uptake. Changes in the concentration of iron, manganese, calcium and copper ions in the decayed wood were analyzed by induced coupled plasma spectroscopy and related to wood weight loss and oxalic acid accumulation. Metal transport into the fungal inoculated wood was found to be dependent on the individual strain/species. The S. lacrymans strain caused a significant increase in total iron whereas the concentration of copper ions in the wood appeared decreased after 10 weeks of decay. Wood inoculated with the M. incrassata isolate showed the contrary tendency with high copper accumulation and low iron increase despite similar weight losses for the two strains. However, significantly lower oxalic acid accumulation was recorded in M. incrassata degraded wood. The addition of a building material resulted in increased weight loss in wood degraded by C. puteana in the soil-block test; however, this could not be directly linked specifically to the accumulation of any of the four metals recorded. The accumulation of oxalic acid seemed to influence the iron uptake. The study assessing the influence of the presence of soil and glass in the soil-block test revealed that soil contributed the majority of the metals for uptake by the fungi and contributed to increased weight loss. The varying uptake observed among the three brown rot fungi strains toward the four metals analyzed may be related to the specific non-enzymatic and enzymatic properties including bio-chelators employed by each of the species during wood decay.     

The isolation and immunolocalization of iron-binding compounds produced by Gloeophyllum trabeum

Summary Low molecular weight iron-binding compounds are produced by the brown-rot fungus Gloeophyllum trabeum. These chelators may function in scavenging transition metals for fungal metabolism and extracellular enzyme production. Because of the low molecular mass of the chelate-metal complex (below 1000 Da), and the oxidizing potential of the bound transition metals, certain chelating compounds could also play a role in the early stages of cellulose depolymerization by brown-rot fungi. High-affinity iron-binding compounds were isolated and partially purified from both liquid cultures of the brown-rot Gloeophyllum trabeum and from infected wood. Chelating compounds purified by thin-layer chromatography were used to prepare specific antibodies. These antibodies were shown to detect the chelator in infected wood and liquid fungal cultures by enzyme-linked immunosorbent assay and could be used in immunotransmission electron microscopy to visualize the high-affinity iron-binding compounds in situ. Elucidating the physiological roles of fungal chelate-metal complexes and determining their function in lignocellulose depolymerization will help us to better understand the mechanism of wood biodegradation.Publication no. 1549 Maine Agricultural Experiment Station Offprint requests to: J. Jellison     

Fungal bioleaching of metals in preservative-treated wood

Twenty-four brown-rot and 10 white-rot fungi were screened to evaluate their applicability for detoxification of preservative-treated wood impregnated with copper and chromium (CC) salts. Brown-rot fungi generally showed higher tolerance towards copper inhibition than white-rot fungi. Additionally, brown-rot fungi were found to accumulate considerable quantities of oxalic acid (up to 44.3 mM) in liquid medium, while white-rot fungi generally accumulated only traces of this organic acid. Oxalic acid is a strong organic acid capable of complexing a variety of heavy metals. Four Antrodia vaillantii and two Poria placenta brown-rot strains that displayed both a high copper tolerance and a high oxalic acid production were selected for further study. The brown-rot fungi effectively decayed wood containing up to 4.4% CC causing corrected mass losses of up to 24.3% in 4 weeks. Fungal treatment was also found to promote extensive leaching of chromium (up to 52.4%), but only moderate leaching of copper (15.6% or less). These results indicate the potential of solid-state fermentation with copper-tolerant fungi for the remediation of preservative-treated wood. Improving the solubility of copper will be an important challenge for future research.     

A proposed role of oxalic acid in wood decay systems of wood-rotting basidiomycetes

Abstract: The possible roles of oxalic acid, veratryl alcohol, and manganese were investigated in relation to lignin biodegradation by white-rot basidiomycetes. Oxalate inhibited both lignin peroxidase (LiP) and manganese-peroxidase (MnP). and was decarboxylated by the mediation of veratryl alcohol and Mn. Oxalate was shown to regulate the mineralization of lignin in the in vivo system of Phanerochaete chrysosporium . In the brown-rot wood decay process, oxalic acid may serve as an acid catalyst as well as an electron donor for the Fenton reaction, to breakdown cellulose and hemicellulose. Oxaloacetase and glyoxylate oxidase may play a key role in production of oxalic acid by white-rot and brown-rot basidiomycetes such as Phanerochaete chrysosporium, Coriolus versicolor and Tyromyces palustris . A possible role of oxalate metabolism is discussed in relation to the physiology of wood-rotting fungi.     

Effect of carbon source on the production of oxalic acid and hydrogen peroxide by brown-rot fungus Poria placenta

Oxalic acid and hydrogen peroxide have been suggested to be essential in the degradation of wood carbohydrates by brown-rot fungi. The production of oxalic acid, hydrogen peroxide and endo-β-1,4-glucanase activity by the brown-rot fungus Poria placenta was studied on crystalline cellulose, amorphous cellulose and glucose media. Oxalic acid and hydrogen peroxide by P. placenta were clearly produced on culture media containing either crystalline or amorphous cellulose. Oxalic acid and hydrogen peroxide were formed simultaneously and highest amounts of oxalic acid (1.0 g l⁻¹) and hydrogen peroxide (39.5 μM) were obtained on amorphous cellulose after 3 weeks cultivation. On glucose medium the amounts were low. The endoglucanase activity was observed to increase during the cultivation and was most pronounced on glucose medium and thus indicated the constitutive characteristics of the brown-rot cellulases.     

A synergism between oxalic acid and polygalacturonases in the depolymerization of potato tuber tissue

Rhizoctonia bataticola produced oxalic acid in vitro and in vivo during pathogenesis of patato tuber. Polygalacturonase (PG) was also detected in culture filtrates of the rot-causing organism. Levels of maceration and cell death in tuber tissue were higher when a mixture of oxalic acid and PG was used than when either oxalic acid or PG were used alone.     

Oxalic acid overproduction by copper-tolerant brown-rot basidiomycetes on southern yellow pine treated with copper-based preservatives

Accumulation of oxalic acid (OA) by brown-rot fungi and precipitation of copper oxalate crystals in wood decayed by copper-tolerant decay fungi has implicated OA in the mechanism of copper tolerance. Understanding the role of OA in copper tolerance is important due to an increasing reliance on copper-based wood preservatives. In this study, four copper-tolerant brown-rot fungi were evaluated for decay capacity and OA production in early stages of exposure to four waterborne copper-based wood preservatives (ammonical copper quat type B and D, ammonical copper citrate, and chromated copper arsenate, type C) and one oilborne copper-based wood preservative (oxine copper) in southern yellow pine blocks. Weight losses were less than 14% during the 4-week incubation. The presence of copper in waterborne preservatives uniformly stimulated OA production by the test fungi within 2 weeks of exposure of the treated blocks to test fungi; 66% to 93% more OA was produced in treated blocks than untreated controls. Oxine copper, a nickel-containing oilborne preservative, prevented both weight loss and OA production in all fungi tested.     

An NADH:Quinone Oxidoreductase Active during Biodegradation by the Brown-Rot Basidiomycete Gloeophyllum trabeum

The brown-rot basidiomycete Gloeophyllum trabeum uses a quinone redox cycle to generate extracellular Fenton reagent, a key component of the biodegradative system expressed by this highly destructive wood decay fungus. The hitherto uncharacterized quinone reductase that drives this cycle is a potential target for inhibitors of wood decay. We have identified the major quinone reductase expressed by G. trabeum under conditions that elicit high levels of quinone redox cycling. The enzyme comprises two identical 22-kDa subunits, each with one molecule of flavin mononucleotide. It is specific for NADH as the reductant and uses the quinones produced by G. trabeum (2,5-dimethoxy-1,4-benzoquinone and 4,5-dimethoxy-1,2-benzoquinone) as electron acceptors. The affinity of the reductase for these quinones is so high that precise kinetic parameters were not obtainable, but it is clear that k_cat/K_m for the quinones is greater than 10⁸ M⁻¹ s⁻¹. The reductase is encoded by a gene with substantial similarity to NAD(P)H:quinone reductase genes from other fungi. The G. trabeum quinone reductase may function in quinone detoxification, a role often proposed for these enzymes, but we hypothesize that the fungus has recruited it to drive extracellular oxyradical production.     

Protecting wood from mould,decay, and termites with multi-component biocide systems

Biocides must be developed for controlling mould establishment on cellulose-based building materials. Accordingly, biocides intended for indoor applications must be non-toxic, non-volatile, odourless, hypoallergenic, and able to provide long-term protection under conditions of high humidity. Multi-component biocide systems were tested in American Wood-Preservers’ Association soil block tests for inhibition of brown-rot and white-rot decay fungi and American Society for Testing and Materials standard tests for inhibition of mould fungi and termites. Multi-component systems combining a borate base supplemented with either 0.1% azole or 0.5% thujaplicin, performed well against the two brown-rot fungi Postia placenta and Gloeophyllum trabeum; the white-rot fungus Coriolus versicolor; the three mould fungi Aspergillus niger, Penicillium chrysogenum, and Trichoderma viride; and the subterranean termite Reticulitermes flavipes (Kollar). It was concluded that for interior applications borate-based multi-component biocide systems can protect wood from decay fungi, mould fungi, and termites, and that a system containing thiabendazole provided protection at a lower retention than the other biocides in this study. Synergy was observed between the borate base and voriconazole in inhibition of mould.     

Regulatory aspects of endoglucanase production by the brown-rot fungusGloeophyllum trabeum

Regulation of endoglucanase formation by the brown-rot fungusGloeophyllum trabeum was investigated. This fungus produced endoglucanases in the presence of monosaccharides such as glucose or mannose as the sole carbon source, but the expression of these enzymes was four to five times higher in the presence of cellulose or cellobiose. In a lactose- or glucose-containing medium, endoglucanase production was induced by cellobiose. Glucose and glycerol did not repress enzyme production. We concluded that endoglucanase production by brown-rot fungi is inducible by cellulose and not subject to catabolite repression. Cellobiose is the most effective inducer of the system.     

Lignocellulosic polysaccharides and lignin degradation by wood decay fungi: the relevance of nonenzymatic Fenton-based reactions

The brown rot fungus Wolfiporia cocos and the selective white rot fungus Perenniporia medulla-panis produce peptides and phenolate-derivative compounds as low molecular weight Fe³⁺-reductants. Phenolates were the major compounds with Fe³⁺-reducing activity in both fungi and displayed Fe³⁺-reducing activity at pH 2.0 and 4.5 in the absence and presence of oxalic acid. The chemical structures of these compounds were identified. Together with Fe³⁺ and H₂O₂ (mediated Fenton reaction) they produced oxygen radicals that oxidized lignocellulosic polysaccharides and lignin extensively in vitro under conditions similar to those found in vivo. These results indicate that, in addition to the extensively studied Gloeophyllum trabeum—a model brown rot fungus—other brown rot fungi as well as selective white rot fungi, possess the means to promote Fenton chemistry to degrade cellulose and hemicellulose, and to modify lignin. Moreover, new information is provided, particularly regarding how lignin is attacked, and either repolymerized or solubilized depending on the type of fungal attack, and suggests a new pathway for selective white rot degradation of wood. The importance of Fenton reactions mediated by phenolates operating separately or synergistically with carbohydrate-degrading enzymes in brown rot fungi, and lignin-modifying enzymes in white rot fungi is discussed. This research improves our understanding of natural processes in carbon cycling in the environment, which may enable the exploration of novel methods for bioconversion of lignocellulose in the production of biofuels or polymers, in addition to the development of new and better ways to protect wood from degradation by microorganisms.     

Organic Acid Production by Basidiomycetes: I. Screening of Acid-Producing Strains

Sixty-seven strains belonging to 47 species of Basidiomycetes were examined for their acid-producing abilities in glucose media, in both the presence and absence of CaCO₃, in stationary and shake cultures. Some strains were found to produce large quantities of oxalic acid. The oxalic acid-producing strains could be separated into two groups. Strains of one group (mostly brown-rot fungi) were able to produce oxalic acid, regardless of whether CaCO₃ was present in the medium. Strains of the other group (mostly white-rot fungi) were characterized by their ability to produce oxalic acid only when CaCO₃ was added to the medium. With the latter group, shake-culturing was generally more effective than stationary culturing in respect to acid production. In the CaCO₃-containing media, Schizophyllum commune, Merulius tremellosus, and Porodisculus pendulus were found to produce substantial amounts of L-malic acid as a main metabolic product, along with small quantities of oxalic and other acids in shake cultures. Especially, S. commune and M. tremellosus may be employed as malic acid-producing species.     

Study of the structure of biodegraded wood using the water vapour sorption method

The dynamics of the biodegradation of wood by brown-rot fungi (Coniophora puteana, Poria placenta, and Gloephyllum trabeum) was investigated by the water vapour sorption method. The change in wood microstructure characteristics (specific surface and concentration of surface hydrophilic centres) with increasing exposure time correlated with reduction in mass and change in composition. Two-to-eight-nanometer-wide micropores, whose size and volume depended on the fungal species and exposure time, appeared in the wood. Methodological aspects of the application of sorption methods should be taken into account in the interpretation of the results.     

Biodeterioration of brazilwood Caesalpinia echinata Lam. (Leguminosae—Caesalpinioideae) by rot fungi and termites

The heartwood of Caesalpinia echinata Lam. (Leguminosae) (commonly called brazilwood) is used for violin bow manufacture due to the unique vibrational and physical properties found in the wood. In the present work, the effects of Pycnoporus sanguineus (white-rot fungus), Gloeophyllum trabeum (brown-rot fungus), Chaetomium globosum (soft-rot fungus), and Cryptotermes brevis (dry-wood termite) on weight losses and chemical composition of extractives and cell-wall polysaccharides of C. echinata wood were investigated under laboratory conditions and compared to those obtained for Anadenanthera macrocarpa, Eucalyptus grandis, and Pinus elliottii. The heartwood of C. echinata was found to be as resistant as A. macrocarpa to the decay fungi tested and to the attack of the dry-wood termite. Pinitol and galactopinitol A were the main sugar alcohols found in the extractives of wood of C. echinata, their presence, however, did not appear related to the resistance to fungal decay. Although only incipient stages of decay were found, the modifications in cell-wall polysaccharide composition of heartwood of C. echinata by rot fungi were related to decrease in polymers other than xylans. The high resistance of C. echinata to xylophages is probably due to the presence of toxic extractives in the wood.