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.     

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.     

Changes in Molecular Size Distribution of Cellulose during Attack by White Rot and Brown Rot Fungi

The kinetics of cotton cellulose depolymerization by the brown rot fungus Postia placenta and the white rot fungus Phanerochaete chrysosporium were investigated with solid-state cultures. The degree of polymerization (DP; the average number of glucosyl residues per cellulose molecule) of cellulose removed from soil-block cultures during degradation by P. placenta was first determined viscosimetrically. Changes in molecular size distribution of cellulose attacked by either fungus were then determined by size exclusion chromatography as the tricarbanilate derivative. The first study with P. placenta revealed two phases of depolymerization: a rapid decrease to a DP of approximately 800 and then a slower decrease to a DP of approximately 250. Almost all depolymerization occurred before weight loss. Determination of the molecular size distribution of cellulose during attack by the brown rot fungus revealed single major peaks centered over progressively lower DPs. Cellulose attacked by P. chrysosporium was continuously consumed and showed a different pattern of change in molecular size distribution than cellulose attacked by P. placenta. At first, a broad peak which shifted at a slightly lower average DP appeared, but as attack progressed the peak narrowed and the average DP increased slightly. From these results, it is apparent that the mechanism of cellulose degradation differs fundamentally between brown and white rot fungi, as represented by the species studied here. We conclude that the brown rot fungus cleaved completely through the amorphous regions of the cellulose microfibrils, whereas the white rot fungus attacked the surfaces of the microfibrils, resulting in a progressive erosion.     

Mechanisms of wood degradation by brown-rot fungi: chelator-mediated cellulose degradation and binding of iron by cellulose

Iron, hydrogen peroxide, biochelators and oxalate are believed to play important roles in cellulose degradation by brown-rot fungi. The effect of these compounds in an 'enhanced' Fenton system on alpha-cellulose degradation was investigated specifically in regard to molecular weight distribution and cellulose-iron affinity. This study shows that the degradative ability of an ultrafiltered low molecular weight preparation of chelating compounds isolated from the brown-rot fungus Gloeophyllum trabeum (termed 'Gt chelator') increased with increasing Gt chelator concentration when the FeIII to Gt chelator ratio was greater than about 30:1. When this ratio was less than 30:1, increasing Gt chelator concentration did not accelerate cellulose degradation. In excess hydrogen peroxide, cellulose degradation increased and then decreased with increasing iron concentration when FeIII was present in excess of the Gt chelator. The critical ratio of FeIII to Gt chelator varied depending on the concentration of hydrogen peroxide in the system. Increasing iron concentration above a critical iron:chelator ratio inhibited cellulose degradation. The optimum pH for cellulose degradation mediated by Gt chelator was around 4.0. A comparison of the effects of 2,3-DHBA (a chelator that reduces iron similarly to Gt chelator) and Gt chelator with respect to cellulose degradation demonstrated the same pattern of cellulose degradation. Cellulose-iron affinity studies were conducted at three pH levels (3.6, 3.8, 4.1), and the binding constants for cellulose-FeIII, cellulose-FeII and cellulose-FeIII in the presence of Gt chelator were calculated. The binding constants for cellulose-FeIII at all three pH levels were much higher than those for cellulose-FeII, and the binding constants for cellulose-FeIII in the presence of Gt chelator were very close to those for cellulose-FeII. This is probably the result of FeIII reduction to FeII by Gt chelator and suggests that chelators from the fungus may be able to sequester iron from cellulose and reduce it in near proximity to the cellulose and thereby better promote depolymerization. The free radical generating system described has potential for use in a variety of industrial processing and pollution control applications.     

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.     

Significant levels of extracellular reactive oxygen species produced by brown rot basidiomycetes on cellulose

It is often proposed that brown rot basidiomycetes use extracellular reactive oxygen species (ROS) to accomplish the initial depolymerization of cellulose in wood, but little evidence has been presented to show that the fungi produce these oxidants in physiologically relevant quantities. We used [(14)C]phenethyl polyacrylate as a radical trap to estimate extracellular ROS production by two brown rot fungi, Gloeophyllum trabeum and Postia placenta, that were degrading cellulose. Both fungi oxidized aromatic rings on the trap to give monohydroxylated and more polar products in significant yields. All of the cultures contained 2,5-dimethoxyhydroquinone, a fungal metabolite that has been shown to drive Fenton chemistry in vitro. These results show that extracellular ROS occur at significant levels in cellulose colonized by brown rot fungi, and suggest that hydroquinone-driven ROS production may contribute to decay by diverse brown rot species.     

Effect of pH and oxalic acid on the reduction of Fe3+ by a biomimetic chelator and on Fe3+ desorption/adsorption onto wood: Implications for brown-rot decay

Fenton reaction is thought to play an important role in wood degradation by brown-rot fungi. In this context, the effect of oxalic acid and pH on iron reduction by a biomimetic fungal chelator and on the adsorption/desorption of iron to/from wood was investigated. The results presented in this work indicate that at pH 2.0 and 4.5 and in the presence of oxalic acid, the phenolate chelator 2,3-dihydroxybenzoic acid (2,3-DHBA) is capable of reducing ferric iron only when the iron is complexed with oxalate to form Fe³⁺-mono-oxalate (Fe(C₂O₄)⁺). Within the pH range tested in this work, this complex formation occurs when the oxalate:Fe³⁺ molar ratio is less than 20 (pH 2.0) or less than 10 (pH 4.5). When aqueous ferric iron was passed through a column packed with milled red spruce (Picea rubens) wood equilibrated at pH 2.0 and 4.5, it was observed that ferric iron binds to wood at pH 4.5 but not at pH 2.0, and the bound iron could then be released by application of oxalic acid at pH 4.5. The release of bound iron was dependent on the amount of oxalic acid applied in the column. When the amount of oxalate was at least 20-fold greater than the amount of iron bound to the wood, all bound iron was released. When Fe–oxalate complexes were applied to the milled wood column equilibrated in the pH range of 2–4.5, iron from Fe–oxalate complexes was bound to the wood only when the pH was 3.6 or higher and the oxalate:Fe³⁺ molar ratio was less than 10. When 2,3-DHBA was evaluated for its ability to release iron bound to the milled wood, it was found that 2,3-DHBA possessed a greater affinity for ferric iron than the wood as 2,3-DHBA was capable of releasing the ferric iron bound to the wood in the pH range 3.6–5.5. These results further the understanding of the mechanisms employed by brown-rot fungi in wood biodegradation processes.     

Kinetic and antigenic similarities for cellobiose dehydrogenase from the brown rot fungus Coniophora puteana and the white rot fungus Phanerochaete chrysosporium

Abstract Cellobiose dehydrogenase was purified from the brown rot fungus Coniophora puteana . Strong cross-reaction was observed with antibodies to cellobiose:quinone oxidoreductase from the white rot fungus Phanerochaete chrysosporium . Kinetic measurements were made with cellobiose as electron donor. Ferricyanide and DCPIP both showed a pH optimum close to pH 4, but activity with ferricyanide declined more rapidly when the pH was raised. Dioxygen reduction to hydrogen peroxide was observed, but at a much lower rate than for other acceptors. These properties are similar to those of cellobiose dehydrogenase from P. chrysosporium , despite differences between brown and white rot modes of decay.     

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.     

Three Native Cellulose-Depolymerizing Endoglucanases from Solid-Substrate Cultures of the Brown Rot Fungus Meruliporia (Serpula) incrassata

Three extracellular cellulose-depolymerizing enzymes from cotton undergoing decay by the brown rot fungus Meruliporia (Serpula) incrassata were isolated by anion-exchange and hydrophobic interaction chromatographies. Depolymerization was detected by analyzing the changes in the molecular size distribution of cotton cellulose by high-performance size-exclusion chromatography. The average degree of polymerization (DP; number of glucosyl residues per cellulose chain) was calculated from the size-exclusion chromatography data. The very acidic purified endoglucanases, Cel 25, Cel 49, and Cel 57, were glycosylated and had molecular weights of 25,200, 48,500, and 57,100, respectively. Two, Cel 25 and Cel 49, depolymerized cotton cellulose and were also very active on carboxymethyl cellulose (CMC). Cel 57, by contrast, significantly depolymerized cotton cellulose but did not release reducing sugars from CMC and only very slightly reduced the viscosity of CMC solutions. Molecular size distributions of cotton cellulose attacked by the three endoglucanases revealed single major peaks that shifted to lower DP positions. A second smaller peak (DP, 10 to 20) was also observed in the size-exclusion chromatograms of cotton attacked by Cel 49 and Cel 57. Under the reaction conditions used, Cel 25, the most active of the cellulases, reduced the weight average DP from 3,438 to 315, solubilizing approximately 20% of the cellulose. The weight average DP values of cotton attacked under the same conditions by Cel 49 and Cel 57 were 814 and 534; weight losses were 9 and 11% respectively.     

Ceriporic acid B, an extracellular metabolite of Ceriporiopsis subvermispora, suppresses the depolymerization of cellulose by the Fenton reaction

The white rot fungus, Ceriporiopsis subvermispora, is able to degrade lignin in wood without intensive damage to cellulose. Since lignin biodegradation by white rot fungi proceeds by radical reactions, accompanied by the production of a large amount of Fe3+-reductant phenols and reductive radical species in the presence of iron ions, molecular oxygen, and H2O2, C. subvermispora has been proposed to possess a biological system which suppresses the production of a cellulolytic active oxygen species, *OH, by the Fenton reaction. In the present paper, we demonstrate that 1-nonadecene-2,3-dicarboxylic acid (ceriporic acid B), an extracellular metabolite of C. subvermispora, strongly inhibited *OH production and the depolymerization of cellulose by the Fenton reaction in the presence of iron ions, cellulose, H2O2, and a reductant for Fe3+, hydroquinone (HQ), at the physiological pH of the fungus.     

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.     

Effect of pH and Oxalate on Hydroquinone-Derived Hydroxyl Radical Formation during Brown Rot Wood Degradation

The redox cycle of 2,5-dimethoxybenzoquinone (2,5-DMBQ) is proposed as a source of reducing equivalent for the regeneration of Fe²⁺ and H₂O₂ in brown rot fungal decay of wood. Oxalate has also been proposed to be the physiological iron reductant. We characterized the effect of pH and oxalate on the 2,5-DMBQ-driven Fenton chemistry and on Fe³⁺ reduction and oxidation. Hydroxyl radical formation was assessed by lipid peroxidation. We found that hydroquinone (2,5-DMHQ) is very stable in the absence of iron at pH 2 to 4, the pH of degraded wood. 2,5-DMHQ readily reduces Fe³⁺ at a rate constant of 4.5 × 10³ M⁻¹s⁻¹ at pH 4.0. Fe²⁺ is also very stable at a low pH. H₂O₂ generation results from the autoxidation of the semiquinone radical and was observed only when 2,5-DMHQ was incubated with Fe³⁺. Consistent with this conclusion, lipid peroxidation occurred only in incubation mixtures containing both 2,5-DMHQ and Fe³⁺. Catalase and hydroxyl radical scavengers were effective inhibitors of lipid peroxidation, whereas superoxide dismutase caused no inhibition. At a low concentration of oxalate (50 μM), ferric ion reduction and lipid peroxidation are enhanced. Thus, the enhancement of both ferric ion reduction and lipid peroxidation may be due to oxalate increasing the solubility of the ferric ion. Increasing the oxalate concentration such that the oxalate/ferric ion ratio favored formation of the 2:1 and 3:1 complexes resulted in inhibition of iron reduction and lipid peroxidation. Our results confirm that hydroxyl radical formation occurs via the 2,5-DMBQ redox cycle.     

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.     

Copper catechol-driven Fenton reactions and their potential role in wood degradation

Metals can potentially play a role in the non-enzymatic processes involved in wood biodegradation. Dihydroxybenzenes reduce Cu(II)–Cu(I), which then react with H₂O₂ driving a Fenton reaction. In this work the degradation of veratryl alcohol (VA), the simplest non-phenolic lignin model compound, via a cuprous Fenton reaction mediated by 1,2-dihydroxybenzene (catechol, CAT) was studied. A factorial experimental design was performed to assess the impact of several experimental variables including, pH, and CAT, CuCl₂ and H₂O₂ concentrations on VA degradation. Optimized conditions were determined using a response surface modeling methodology (RSM). The greatest amount of VA degradation occurred at a CAT:CuCl₂:H₂O₂ ratio of 0.287:0.313:4.062, a pH of 3.6. A time-course measurement for VA degradation was performed under these experimental conditions and after an 8 h reaction period, 31% of the VA was degraded. Under the same experimental conditions, VA degradation by an iron CAT-driven Fenton reaction was more effective than the copper CAT-driven Fenton reaction. In a similar experiment, carboxymethyl cellulose (CMC) depolymerization was also determined. Only the iron CAT-driven Fenton reaction was found to depolymerize CMC. We suggest that the greater redox potential of the Fe(III)CAT complex compared to the Cu(II)CAT complex would dictate that under most environmental conditions, degradation of VA would occur by the iron complex only. This research has important implications for the mechanisms of brown rot fungal degradation in wood because it eliminates a pathway that had previously been proposed as a mechanism explaining free radical generation in the oxidative depolymerization of cellulose in the cell wall.     

The influence of pH on OH scavenger inhibition of damage to deoxyribose by Fenton reaction

Summary Hydroxyl radicals (OH') can be formed in aqueous solution by direct reaction of hydrogen peroxide (H₂O₂) with ferrous salt (Fenton reaction). OH' damage to deoxyribose, measured as formation of thiobarbituric acid-reactive material, was evaluated at different pHs to study the mechanism of action of classical OH' scavengers. OH' scavenger effect on Fe²⁺ oxidation was also evaluated in the same experimental conditions. In the absence of OH' scavengers, OH' damage to deoxyribose is higher at acidic compared to neutral and moderately basic pH. At acidic pH deoxiribose is per se able to inhibit Fe²⁺ oxidation by H₂0₂. Most of OH' scavengers tested inhibit deoxyribose damage and Fe²⁺ oxidation in a similar manner: both inhibitions are most relevant at acidic pH and decrease by increasing the pH. These results are not due to OH' scavenger inhibition of Fenton reaction. The influence of pH on the parameters studied appears to be due to the competition of deoxyribose and OH' scavengers for iron. These results suggest the prominent role of iron binding in the degradation of deoxyribose and in the OH' scavenging ability of different compounds. Results obtained with triethylenetetramine, a iron chelator with a low rate constant with OH', confirm that both deoxyribose and the OH' scavengers interact with iron bringing about a site specific Fenton reaction; that the OH' formed at these sites oxidize these molecules to their radical forms which in turn reduce the Fe^3– produced by Fenton reaction. The results presented indicate that most of classical OH' scavengers exert their effect predominantly by preventing the site specific reaction between Fe²⁺ and H₂0₂ on the deoxyribose molecule.     

A Comparison between the Cellulolytic Activity of White and Brown Rot Fungi. I. The Activity on Insoluble Cellulose

About 70 strains of white and brown rot fungi were cultivated on media, containing filter paper cellulose as the main carbon source. The cellulolytic activity of the culture filtrates was measured after different periods of growth by means of the turbidimetric method. The results obtained indicate a difference between the two types of wood decay fungi as to the capacity of attacking the cellulose used in the medium and in the cellulase test. No significant C₁activity was found in any of the brown rot cultures whereas all white rot fungi tested exerted a measurable activity on the test substrate. The effect of various carbohydrates and some proteins as inducers of cellulase activity was studied. Especially cellobiose and lactose were active on white rot fungi in this respect, particularly in the presence of yeast extract. Also some brown rot fungi exerted C₁-activity after incubation on glucose or cellobiose.     

Decay resistance of wood treated with boric acid and tall oil derivates

In this study, the effect of two boric acid concentrations (1% and 2%) and four derivates of tall oil with varying chemical composition were tested separately and in combination. The tall oil derivates were chosen in a way that they consist of different amounts of free fatty, resin acids and neutral compounds. Decay tests using two brown rot fungi (Postia placenta and Coniophora puteana) were performed on both unleached and leached test samples. Boric acid showed a low weight loss in test samples when exposed to fungal decay before leaching, but no effect after leaching. The tall oil derivates gave better efficacy against decay fungi compared to control, but are not within the range of the efficacy needed for a wood preservative. Double impregnation with boric acid and tall oil derivates gave synergistic effects for several of the double treatments both in unleached and leached samples. In the unleached samples the double treatment gave a better efficacy against decay fungi than tall oil alone. In leached samples a better efficacy against brown rot fungi were achieved than in samples with boron alone and a nearly similar or better efficacy than for tall oil alone. Boric acid at 2% concentration combined with the tall oil derivate consisting of 90% free resin acids (TO-III) showed the best performance against the two decay fungi with a weight loss less than 3% after a modified pure culture test.