Use of lignin-degrading fungi in the disposal of pentachlorophenol-treated wood

Summary The lignin-degrading fungiPhanerochaete chrysosporium, P. sordida, Trametes hirsuta, andCeriporiopsis subvermispora were evaluated for their ability to decrease the concentration of pentachlorophenol (PCP) and to cause dry weight loss in PCP-treated wood. Hardwood and softwood materials from PCP-treated ammunition boxes that were chipped to pass a 3.8-cm screen were used. All four fungi caused significant weight losses and decreases in the PCP concentration. The largest PCP decrease (84% in 4 weeks) was caused byT. hirsuta, and the smallest decrease was caused byC. subvermispora (37% in 4 weeks). After 4 weeks, the fate of spiked¹⁴C[PCP] in softwood chips inoculated withT. hirsuta was as follows: 27% was mineralized, 42.5% was non-extractable and bound to the chips, 23.5% was associated with fungal hyphae, and 6% was organic-extractable. Decreases of PCP byP. chrysosporium andP. sordida averaged 59% and 57%, respectively. PCP decreases caused byPhanerochaete spp. were not significantly affected by wood type or sterilization and were primarily due to methylation of PCP that resulted in accumulation of pentachloroanisole. Softwood weight losses caused byT. hirsuta, P. chrysosporium andC. subvermispora were respectively, 24, 6.5, and 17%, after 4 weeks. These weight losses are comparable to reported weight losses by these organisms in non-treated softwood. Nutrient supplementation significantly increased weight loss but not percentage decrease of PCP. The results of this research demonstrate the potential for using lignin-degrading fungi to destroy PCP-treated wood.     

In Situ Depletion of Pentachlorophenol from Contaminated Soil by Phanerochaete spp

The ability of two white rot fungi to deplete pentachlorophenol (PCP) from soil, which was contaminated with a commercial wood preservative, was examined in a field study. Inoculation of soil containing 250 to 400 μg of PCP g⁻¹ with either Phanerochaete chrysosporium or P. sordida resulted in an overall decrease of 88 to 91% of PCP in the soil in 6.5 weeks. This decrease was achieved under suboptimal temperatures for the growth and activity of these fungi, and without the addition of inorganic nutrients. Since the soil had a very low organic matter content, peat was included as a source of organic carbon for fungal growth and activity. A small percentage (8 to 13%) of the decrease in the amount of PCP was a result of fungal methylation to pentachloroanisole. Gas chromatographic analysis of sample extracts did not reveal the presence of extractable transformation products other than pentachloroanisole. Thus, when losses of PCP via mineralization and volatilization were negligible, as they were in laboratory-scale studies (R. T. Lamar, J. A. Glaser, and T. K. Kirk, Soil Biol. Biochem. 22:433-440, 1990), most of the PCP was converted to nonextractable soil-bound products. The nature, stability, and toxicity of soil-bound transformation products, under a variety of conditions, must be elucidated before use of these fungi in soil remediation efforts can be considered a viable treatment method.     

Selective hyperproduction of manganese peroxidases byPhanerochaete chrysosporium l-1512 immobilized on nylon net in a bubble-column reactor

Manganese peroxidases were overproduced byPhanerochaete chrysosporium I-1512 immobilized on nylon net in a bubble-column reactor. This study investigates a new design of bioreactor, a compromise between a pneumatic reactor and an immobilized biofilm reactor. The carrier, a sheet of nylon net, was maintained by a cylindrical stainless-steel frame installed vertically. It was characterized by its hydrophilic nature, its surface morphology and its surface roughness.P. chrysosporium adhesion was highly efficient; mycelial hyphae invaded the tridimensional structure and strengthened the bonding to the network, as shown by electron scanning microscopy. High levels of Mn peroxidases were produced by strain I-1512 under conditions of glycerol and nitrogen sufficiency when the medium was supplemented with phospholipid and veratryl alcohol. Yields of 3600 U/l Mn peroxidase were produced after 95 h of incubation, indicating significant productivity for industrial purposes (900 U day^–1 l^–1).     

Toxicity of Pentachlorophenol to Six Species of White Rot Fungi as a Function of Chemical Dose

The growth of six species of white rot fungi was a function of pentachlorophenol (PCP) dose, expressed as mass of PCP per mass of mycelia, at PCP doses ≤35 μg mg of mycelium^-1, and not concentration. At higher doses, Inonotus dryophilus, Perenniporia medulla-panis, and Ganoderma oregonense removed less PCP than three other species of white rot fungi. Phanerochaete chrysosporium grown under nitrogen-deficient conditions was inactivated at PCP doses that under nitrogen-sufficient conditions resulted in only 2-day lag periods in growth. Trametes versicolor was the fastest-growing species that remained viable at higher PCP doses. Both Trametes versicolor and Phellinus badius were able to degrade PCP at higher PCP doses.     

Evaluation of different fungal strains in the decolourisation of synthetic dyes

Of seven fungal strains tested for their ability to decolourise three structurally diverse synthetic dyes, Phanerochaete sordida, Bjerkandera sp. BOS55, Phlebia radiata, and Phanerochaete chrysosporium had average values of maximum decolourisation rates higher than 0.2 [Absorbance] d^–1. All seven fungi produced manganese peroxidase (MnP) but laccase activity was detected only in Phlebia radiata. No lignin peroxidase (LiP) activity was observed.     

Rapid Degradation of Isolated Lignins by Phanerochaete chrysosporium

Phanerochaete chrysosporium degraded purified Kraft lignin, alkali-extracted and dioxane-extracted straw lignin, and lignosulfonates at a similar rate, producing small-molecular-weight (~1,000) soluble products which comprised 25 to 35% of the original lignins. At concentrations of 1 g of lignin liter⁻¹, 90 to 100% of the acid-insoluble Kraft, alkali straw, and dioxane straw lignins were degraded by 1 g of fungal mycelium liter⁻¹ within an active ligninolytic period of 2 to 3 days. Cultures with biomass concentrations as low as 0.16 g liter⁻¹ could also completely degrade 1 g of lignin liter⁻¹ during an active period of 6 to 8 days. The absorbance at 280 nm of 2 g of lignosulfonate liter⁻¹ increased during the first 3 days of incubation and decreased to 35% of the original value during the next 7 days. The capacity of 1 g of cells to degrade alkali-extracted straw lignin under optimized conditions was estimated to be as high as 1.0 g day⁻¹. This degradation occurred with a simultaneous glucose consumption rate of 1.0 g day⁻¹. When glucose or cellular energy resources were depleted, lignin degradation ceased. The ability of P. chrysosporium to degrade the various lignins in a similar manner and at very low biomass concentrations indicates that the enzymes responsible for lignin degradation are nonspecific.     

Selective Medium for Isolating Phanerochaete chrysosporium from Soil

A selective medium was developed that is capable of isolating Phanerochaete chrysosporium from soil. This medium contains 15 ppm of benomyl (15 μg g⁻¹) and 550 ppm of streptomycin sulfate in 2% malt agar and is held at 39°C after inoculation. P. chrysosporium was isolated from three nonsterile forest soils to which the fungus had been added. These soils contained large microbial populations.     

Enhanced production of manganese peroxidase from immobilized <Emphasis Type="Italic">Phanerochaete chrysosporium</Emphasis> due to the increased autolysis of chlamydospore-like cells

The autolysis of chlamydospore-like cells in Phanerochaete chrysosporium immobilized in polyurethane foam correlated with the production of manganese peroxidase (MnP). The maximum specific activity of MnP was 1055 U g dry mycelium^–1 in the immobilized culture, compared with 260 U g dry mycelium^–1 in the submerged culture. Scattered mycelial pellets were formed in the immobilized culture in which almost all of the chlamydospore-like cells were subject to autolysis. However, highly crowded pellets were formed in the free culture, in which only the chlamydospore-like cells in the exterior were subject to autolysis. We propose that the enhanced production of MnP in immobilized cultures of P. chrysosporium is due to increased autolysis of the chlamydospore-like cells.     

Transformation by complementation of a uracil auxotroph of the hyper lignin-degrading basidiomycete Phanerochaete sordida YK-624

Phanerochaete sordida YK-624 is a hyper lignin-degrading basidiomycete possessing greater ligninolytic selectivity than either P. chrysosporium or Trametes versicolor. To construct a gene transformation system for P. sordida YK-624, uracil auxotrophic mutants were generated using a combination of ultraviolet (UV) radiation and 5-fluoroorotate resistance as a selection scheme. An uracil auxotrophic strain (UV-64) was transformed into a uracil prototroph using the marker plasmid pPsURA5 containing the orotate phosphoribosyltransferase gene from P. sordida YK-624. This system generated approximately 50 stable transformants using 2 × 10⁷ protoplasts. Southern blot analysis demonstrated that the transformed pPsURA5 was ectopically integrated into the chromosomal DNA of all transformants. The enhanced green fluorescent protein (EGFP) gene was also introduced into UV-64. The transformed EGFP was expressed in the co-transformants driven by P. sordida glyceraldehyde-3-phosphate dehydrogenase gene promoter and terminator regions.     

Kinetics of endosulfan degradation by Phanerochaete chrysosporium

The chlorinated pesticide, endosulfan, could be degraded by Phanerochaete chrysosporium under non-ligninolytic conditions, and this did not require direct contact with mycelium. The major metabolites formed were endosulfan sulfate and endosulfan diol. The rate of degradation depended on the initial concentration. With 2.5 mg endosulfan l^–1, degradation was at 0.23 mg l^–1 day^–1. The degradation could be described using a nonlinear rate expression that was similar to the Michaelis–Menten equation.     

Influence of environmental parameters on pentachlorophenol biotransformation in soil by Lentinula edodes and Phanerochaete chrysosporium

 The influences of temperature, soil moisture potential and initial pH on the biotransformation of pentachlorophenol (PCP) by the lignicolous fungi Lentinula edodes and Phanerochaete chrysosporium were examined. At 10^°C, L. edodes was more effective in degrading PCP (P<0.05) than P. chrysosporium. At 15^°C similar results were obtained for the two fungi. The highest levels of degradation occurred for both fungi at 25^°C. With P. chrysosporium, the extent of PCP elimination was directly related to soil moisture content and optimal at approximately 47%. With L. edodes, in contrast, the process was inversely related to moisture content and maximal at 26%. The initial soil pH also had a marked influence, and pH 4.0 was optimal for both fungi. Received: 7 August 1995/Accepted: 22 August 1995     

Remediation of pentachlorophenol-contaminated soil by composting with immobilized Phanerochaete chrysosporium

Summary To reduce and eliminate the hazards of pentachlorophenol (PCP) to the soil, the method of inoculating free and immobilized white rot fungi, Phanerochaete chrysosporium to PCP-polluted soils was investigated. Three parallel beakers A, B, C are adopted with the same components of soil, yard waste, straw and bran for aerated composting to degrade the PCP in soil. A was with no inoculants as control, B was added with the inoculants of immobilized P.␣chrysosporium, C was inoculated with non-immobilized P. chrysosporium, and additionally D contained only PCP-contaminated soils also as control. By contrastive analyses, the feasibility of applying composting to the bioremediation of the PCP-polluted soil was discussed. From the experimental results, it could be seen that the degradation rate of PCP by the immobilized fungi exceeded 50% at day 9, while that of the non-immobilized fungi achieved the same rate at day 16. However, the final degradation rates of PCP for both of them were beyond 90% at day 60 and that the rate of A was much lower than the others. The above data have shown that the degradation effect of inoculating P. chrysosporium was better than that of no inoculation, and that of the immobilized fungi was better than that of non-immobilized ones. Meanwhile, shown by all the indicators the composts of A, B and C were mature and stabilized at the end of the experiment. Therefore, the method of composting with immobilized P.␣chrysosporium is effective for the bioremediation of PCP-contaminated soil.     

Influence of Phanerochaete chrysosporium on microbial communities and lignocellulose degradation during solid-state fermentation of rice straw

To investigate the changes of microbial communities and influence of Phanerochaete chrysosporium during solid-state fermentation (SSF) of rice straw, phospholipid fatty acids (PLFA) and lignocellulose components were measured with periodical sampling. The results showed that the lignocellulose degrading ratios in SSF which was inoculated by P. chrysosporium and soil microorganisms were higher than those degraded by culturing a single species. The total amount of PLFAs, as an indicator of microbial biomass, reached the peak on day 6. Principal component analysis (PCA) of the PLFA compositions revealed that P. chrysosporium was well responsible for the succession of microbial community and showed that fungi were the predominant species at the end of the process. The correlation analysis between lignocellulose degrading ratio and PLFA profile in P. chrysosporium suggested that P. chrysosporium promoted lignin degrading as the main fungi with gram-positive bacteria.     

Use of Phanerochaete chrysosporium biomass for the removal of textile dyes from a synthetic effluent

Summary The use of Phanerochaete chrysosporium biomass for the removal of Reactofix Golden Yellow from aqueous solution and eight textile dyes (four azo and four anthraquinone) from a synthetic effluent (0.6 g/l) at different pH, temperature and biomass concentrations was studied. Adsorption was maximum at pH 2.0 and 40 °C using 2.45 g mycelial biomass. The rate constant of adsorption was 1.95×10⁻¹/min for Reactofix Golden Yellow and 1.64×10⁻¹/min for synthetic effluent. In both cases, the equilibrium data fitted well in the Langmuir but not the Freundlich model of adsorption, and the adsorption was biphasic. Adsorption decreased the COD of Reactofix Golden Yellow and synthetic effluent by 54 and 57%, respectively. Desorption (80–84%) of dyes from P. chrysosporium mycelial surface occurred as the pH increased from 2 to 10.     

Contrasting Effects of Elevated Temperature and Invertebrate Grazing Regulate Multispecies Interactions between Decomposer Fungi

Predicting the influence of biotic and abiotic factors on species interactions and ecosystem processes is among the primary aims of community ecologists. The composition of saprotrophic fungal communities is a consequence of competitive mycelial interactions, and a major determinant of woodland decomposition and nutrient cycling rates. Elevation of atmospheric temperature is predicted to drive changes in fungal community development. Top-down regulation of mycelial growth is an important determinant of, and moderator of temperature-driven changes to, two-species interaction outcomes. This study explores the interactive effects of a 4 °C temperature increase and soil invertebrate (collembola or woodlice) grazing on multispecies interactions between cord-forming basidiomycete fungi emerging from colonised beech (Fagus sylvatica) wood blocks. The fungal dominance hierarchy at ambient temperature (16 °C; Phanerochaete velutina > Resinicium bicolor > Hypholoma fasciculare) was altered by elevated temperature (20 °C; R. bicolor > P. velutina > H. fasciculare) in ungrazed systems. Warming promoted the competitive ability of the fungal species (R. bicolor) that was preferentially grazed by all invertebrate species. As a consequence, grazing prevented the effect of temperature on fungal community development and maintained a multispecies assemblage. Decomposition of fungal-colonised wood was stimulated by warming, with implications for increased CO₂ efflux from woodland soil. Analogous to aboveground plant communities, increasing complexity of biotic and abiotic interactions appears to be important in buffering climate change effects on soil decomposers.     

Cloning of Phanerochaete chrysosporium leu2 by Complementation of Bacterial Auxotrophs and Transformation of Fungal Auxotrophs

A Phanerochaete chrysosporium cDNA library was constructed in an expression vector that allows expression in both Escherichia coli and Saccharomyces cerevisiae. This expression vector, λYES, contains the lacZ promoter for expression in E. coli and the GAL1 promoter for expression in yeast. A number of genes were cloned by complementation of bacterial amino acid auxotrophs. The cDNA encoding the β-isopropylmalate dehydrogenase from P. chrysosporium was characterized further. The genomic clone (gleu2) was subsequently isolated and was used successfully as a selectable marker to transform P. chrysosporium auxotrophs for LEU2. Protoplasts for transformation were prepared with readily obtained conidiospores rather than with basidiospores, which were used in previous P. chrysosporium transformation procedures. The method described here allows other genes to be isolated from P. chrysosporium for use as selectable markers.     

Establishment of the white rot fungus Phanerochaete chrysosporium on unsterile straw in solid substrate fermentation systems intended for degradation of pesticides

The effects of different inoculum-loading rates and pre-treatment of wheat straw with formic acid and hot water (50 °C) on the establishment of Phanerochaete chrysosporium on unsterile straw were studied in laboratory scale and in a 1.5-m³ bioreactor. The establishment of P. chrysosporium on unsterile straw was satisfactory. Phanerochaete chrysosporium and other fungi, which developed simultaneously, were able to produce the activity necessary to degrade two herbicides, bentazon and MCPA (4-chloro-2-methylphenoxyacetic acid) in 20 days (65 and 75%, respectively). The decrease of both herbicides coincided with the presence of the activity of the lignin-degrading enzymes lignin peroxidase and manganese peroxidase/laccase. Extensive growth of P. chrysosporium or other lignin-degrading fungi on unsterile straw would be excellent for inexpensive solid substrate systems intended for degradation of pesticides.     

Ergosterol contents of some wood-rotting basidiomycete fungi grown in liquid and solid culture conditions

Ergosterol contents of six wood-rotting basidiomycetes were analyzed under different cultivation conditions. Four white-rot and two brown-rot fungi were cultivated in liquid synthetic medium with low nutrient nitrogen (2 mM) and 0.1% glucose, and ergosterol in mycelial biomasses were measured weekly for 35 days. The highest ergosterol content per fungal dry mass in the white-rot fungi was found in Phanerochaete chrysosporium being 2100 μg g⁻¹, while in Ceriporiopsis subvermispora it was 1700 μg g⁻¹, Phlebia radiata 700 μg g⁻¹, and Physisporinus rivulosus 560 μg g⁻¹. In brown-rot fungi the ergosterol content was in Poria placenta 2868 μg g⁻¹ and in Gloeophyllum trabeum 3915 μg g⁻¹. On agar media, P. chrysosporium and P. radiata reached the highest ergosterol value in 7 days, while in wood block cultures the ergosterol contents were quite stable. The conversion factors for ergosterol-to-fungal biomass varied from 48 and 243, which were lower than values for ascomycetous soil fungi reported in the literature.     

A Novel Extracellular Multicopper Oxidase from Phanerochaete chrysosporium with Ferroxidase Activity

Lignin degradation by the white rot basidiomycete Phanerochaete chrysosporium involves various extracellular oxidative enzymes, including lignin peroxidase, manganese peroxidase, and a peroxide-generating enzyme, glyoxal oxidase. Recent studies have suggested that laccases also may be produced by this fungus, but these conclusions have been controversial. We identified four sequences related to laccases and ferroxidases (Fet3) in a search of the publicly available P. chrysosporium database. One gene, designated mco1, has a typical eukaryotic secretion signal and is transcribed in defined media and in colonized wood. Structural analysis and multiple alignments identified residues common to laccase and Fet3 sequences. A recombinant MCO1 (rMCO1) protein expressed in Aspergillus nidulans had a molecular mass of 78 kDa, as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and the copper I-type center was confirmed by the UV-visible spectrum. rMCO1 oxidized various compounds, including 2,2′-azino(bis-3-ethylbenzthiazoline-6-sulfonate) (ABTS) and aromatic amines, although phenolic compounds were poor substrates. The best substrate was Fe²⁺, with a K_m close to 2 μM. Collectively, these results suggest that the P. chrysosporium genome does not encode a typical laccase but rather encodes a unique extracellular multicopper oxidase with strong ferroxidase activity.