Degradation of lignite (low-rank coal) by ligninolytic basidiomycetes and their manganese peroxidase system

Ligninolytic basidiomycetes (wood and leaf-litter-decaying fungi) have the ability to degrade low-rank coal (lignite). Extracellular manganese peroxidase is the crucial enzyme in the depolymerization process of both coal-derived humic substances and native coal. The depolymerization of coal by Mn peroxidase is catalysed via chelated Mn(III) acting as a diffusible mediator with a high redox potential and can be enhanced in the presence of additional mediating agents (e.g. glutathione). The depolymerization process results in the formation of a complex mixture of lower-molecular-mass fulvic-acid-like compounds. Experiments using a synthetic 14C-labeled humic acid demonstrated that the Mn peroxidase-catalyzed depolymerization of humic substances was accompanied by a substantial release of carbon dioxide (17%-50% of the initially added radio-activity was released as 14CO2). Mn peroxidase was found to be a highly stable enzyme that remained active for several weeks under reaction conditions in a liquid reaction mixture and even persisted in sterile and native soil from an opencast mining area for some days.

Formation of bound residues during microbial degradation of [14C]anthracene in soil.

Carbon partitioning and residue formation during microbial degradation of polycyclic aromatic hydrocarbons (PAH) in soil and soil-compost mixtures were examined by using [14C]anthracenes labeled at different positions. In native soil 43.8% of [9-14C]anthracene was mineralized by the autochthonous microflora and 45.4% was transformed into bound residues within 176 days. Addition of compost increased the metabolism (67.2% of the anthracene was mineralized) and decreased the residue formation (20. 7% of the anthracene was transformed). Thus, the higher organic carbon content after compost was added did not increase the level of residue formation. [14C]anthracene labeled at position 1,2,3,4,4a,5a was metabolized more rapidly and resulted in formation of higher levels of residues (28.5%) by the soil-compost mixture than [14C]anthracene radiolabeled at position C-9 (20.7%). Two phases of residue formation were observed in the experiments. In the first phase the original compound was sequestered in the soil, as indicated by its limited extractability. In the second phase metabolites were incorporated into humic substances after microbial degradation of the PAH (biogenic residue formation). PAH metabolites undergo oxidative coupling to phenolic compounds to form nonhydrolyzable humic substance-like macromolecules. We found indications that monomeric educts are coupled by C-C- or either bonds. Hydrolyzable ester bonds or sorption of the parent compounds plays a minor role in residue formation. Moreover, experiments performed with 14CO2 revealed that residues may arise from CO2 in the soil in amounts typical for anthracene biodegradation. The extent of residue formation depends on the metabolic capacity of the soil microflora and the characteristics of the soil. The position of the 14C label is another important factor which controls mineralization and residue formation from metabolized compounds.

Production of manganese peroxidase and organic acids and mineralization of 14C-labelled lignin (14C-DHP) during solid-state fermentation of wheat straw with the white rot fungus nematoloma frowardii

The basidiomycetous fungus Nematoloma frowardii produced manganese peroxidase (MnP) as the predominant ligninolytic enzyme during solid-state fermentation (SSF) of wheat straw. The purified enzyme had a molecular mass of 50 kDa and an isoelectric point of 3.2. In addition to MnP, low levels of laccase and lignin peroxidase were detected. Synthetic 14C-ring-labelled lignin (14C-DHP) was efficiently degraded during SSF. Approximately 75% of the initial radioactivity was released as 14CO2, while only 6% was associated with the residual straw material, including the well-developed fungal biomass. On the basis of this finding we concluded that at least partial extracellular mineralization of lignin may have occurred. This conclusion was supported by the fact that we detected high levels of organic acids in the fermented straw (the maximum concentrations in the water phases of the straw cultures were 45 mM malate, 3.5 mM fumarate, and 10 mM oxalate), which rendered MnP effective and therefore made partial direct mineralization of lignin possible. Experiments performed in a cell-free system, which simulated the conditions in the straw cultures, revealed that MnP in fact converted part of the 14C-DHP to 14CO2 (which accounted for up to 8% of the initial radioactivity added) and 14C-labelled water-soluble products (which accounted for 43% of the initial radioactivity) in the presence of natural levels of organic acids (30 mM malate, 5 mM fumarate).

Oxidative decomposition of malonic acid as basis for the action of manganese peroxidase in the absence of hydrogen peroxide.

Manganese peroxidase (MnP) from the ligninolytic basidiomycetes Phlebia radiata and Nematoloma frowardii was found to decompose malonate oxidatively in the absence of H2O2 in a reaction system consisting of the enzyme, sodium malonate and MnCl2. The enzymatic oxidation resulted in a substantial decrease in malonate concentration and the formation of CO2, oxalate, glyoxylate and formate. Simultaneously with the decomposition of malonate, Mn(II) was oxidized to Mn(III) leading to high transient concentrations of the latter. MnP action in the absence of H2O2 started slowly after a lag period of 3 h. The lag period was considerably shortened after a single addition of Mn(III). Superoxide dismutase and catalase inhibited the enzymatic reaction partly, ascorbate completely. ESR studies demonstrated the formation of a carbon-centered radical during the course of the reaction. We propose that the latter generates peroxides that can be used by MnP to oxidize Mn(II) to Mn(III).

Enzymatic Combustion of Aromatic and Aliphatic Compounds by Manganese Peroxidase from Nematoloma frowardii.

The direct involvement of manganese peroxidase (MnP) in the mineralization of natural and xenobiotic compounds was evaluated. A broad spectrum of aromatic substances were partially mineralized by the MnP system of the white rot fungus Nematoloma frowardii. The cell-free MnP system partially converted several aromatic compounds, including [U-C]pentachlorophenol ([U-C]PCP), [U-C]catechol, [U-C]tyrosine, [U-C]tryptophan, [4,5,9,10-C]pyrene, and [ring U-C]2-amino-4,6-dinitrotoluene ([C]2-AmDNT), to CO(2). Mineralization was dependent on the ratio of MnP activity to concentration of reduced glutathione (thiol-mediated oxidation), a finding which was demonstrated by using [C]2-AmDNT as an example. At [C]2-AmDNT concentrations ranging from 2 to 120 muM, the amount of released CO(2) was directly proportional to the concentration of [C]2-AmDNT. The formation of highly polar products was also observed with [C]2-AmDNT and [U-C]PCP; these products were probably low-molecular-weight carboxylic acids. Among the aliphatic compounds tested, glyoxalate was mineralized to the greatest extent. Eighty-six percent of the COOH-glyoxalate and 9% of the CHO-glyoxalate were converted to CO(2), indicating that decarboxylation reactions may be the final step in MnP-catalyzed mineralization. The extracellular enzymatic combustion catalyzed by MnP could represent an important pathway for the formation of carbon dioxide from recalcitrant xenobiotic compounds and may also have general significance in the overall biodegradation of resistant natural macromolecules, such as lignins and humic substances.

Comparison of phenanthrene and pyrene degradation by different wood-decaying fungi.

The degradation of phenanthrene and pyrene was investigated by using five different wood-decaying fungi. After 63 days of incubation in liquid culture, 13.8 and 4.3% of the [ring U-14C]phenantherene and 2.4 and 1.4% of the [4,5,9,10-14C]pyrene were mineralized by Trametes versicolor and Kuehneromyces mutabilis, respectively. No 14CO2 evolution was detected in either [14C]phenanthrene or [14C]pyrene liquid cultures of Flammulina velutipes, Laetiporus sulphureus, and Agrocybe aegerita. Cultivation in straw cultures demonstrated that, in addition to T. versicolor (15.5%) and K. mutabilis (5.0%), L. sulphureus (10.7%) and A. aegerita (3.7%) were also capable of mineralizing phenanthrene in a period of 63 days. Additionally, K. mutabilis (6.7%), L. sulphureus (4.3%), and A. aegerita (3.3%) mineralized [14C]pyrene in straw cultures. The highest mineralization of [14C] pyrene was detected in straw cultures of T. versicolor (34.1%), which suggested that mineralization of both compounds by fungi may be independent of the number of aromatic rings. Phenanthrene and pyrene metabolites were purified by high-performance liquid chromatography and identified by UV absorption, mass, and 1H nuclear magnetic resonance spectrometry. Fungi capable of mineralizing phenanthrene and pyrene in liquid culture produced enriched metabolites substituted in the K region (C-9,10 position of phenanthrene and C-4,5 position of pyrene), whereas all other fungi investigated produced metabolites substituted in the C-1,2, C-3,4, and C-9,10 positions of phenanthrene and the C-1 position of pyrene.

Novel metabolites in phenanthrene and pyrene transformation by Aspergillus niger.

Aspergillus niger, isolated from hydrocarbon-contaminated soil, was examined for its potential to degrade phenanthrene and pyrene. Two novel metabolites, 1-methoxyphenanthrene and 1-methoxypyrene, were identified by conventional chemical techniques. Minor metabolites identified were 1- and 2-phenanthrol and 1-pyrenol. No 14CO2 evolution was observed in either [14C]phenanthrene or [14C]pyrene cultures.

Degradation of polycyclic aromatic hydrocarbons by manganese peroxidase of Nematoloma frowardii.

The degradation of polycyclic aromatic hydrocarbons by a manganese peroxidase crude preparation of Nematoloma forwardii was demonstrated for a mixture of eight different polycyclic aromatic hydrocarbons, and the five individual polycyclic aromatic hydrocarbons phenanthrene, anthracene, pyrene, fluoranthene, and benzo[alpha]pyrene. Oxidation of polycyclic aromatic hydrocarbons was enhanced by the addition of glutathione, a mediator substance, able to form reactive thiyl radicals. Glutathione-mediated manganese peroxidase (1.96 U ml(-1)) was capable of mineralizing [14C]pyrene (7.3%),[14C]anthracene (4.7%), [14C]benzo[alpha]pyrene (4.0%), [14C]benz(alpha)anthracene 2.9%), and [14C]phenanthrene (2.5%) in a period of 168 h. This is the first description of direct enzymatic mineralization of polycyclic aromatic hydrocarbons by manganese peroxidase, and indicates their important role in the oxidation of polycyclic aromatic hydrocarbons by wood-decaying fungi.

Structure of a laccase-mediated product of coupling of 2,4-diamino-6-nitrotoluene to guaiacol, a model for coupling of 2,4,6-trinitrotoluene metabolites to a humic organic soil matrix.

This work presents laccase-mediated model reactions for coupling of reduced 2,4,6-trinitrotoluene (TNT) metabolites to an organic soil matrix. The structure of an isolated coupling product of 2,4-diamino-6-nitrotoluene (2,4-DANT) to guaiacol as humic constituent was determined. Among several structures, the compound was identified conclusively to be the trinuclear coupling product 5-(2-amino-3-methyl-4-nitroanilino)-3,3(prm1)-dimethoxy-4,4(prm1)-diphenoqu inone. The compound has a weight of 409 g mol(sup-1) and may serve as a model reaction for the biogenic formation of bound residues in soil from TNT by coupling aminotoluenes (reduced TNT metabolites) to humic constituents. A linear correlation of the substrate consumption to the enzyme activity was detected. Based on this observation, the described reaction of 2,4-DANT coupling to guaiacol may be used for determination of laccase activity since the reaction was not inhibited by other compounds of culture supernatants. We propose a two-step mechanism for the coupling reaction because 2,4-DANT was not transformed by laccases in the absence of guaiacol and guaiacol oxidation was independent of the presence of 2,4-DANT. The first reaction step is a laccase-mediated dimerization of two guaiacol monomers with subsequent oxidation to a diphenoquinone. The second step is the nucleophilic addition of 2,4-DANT to the ortho position of the carbonyl group of the diphenoquinone structure.

Screening for fungi intensively mineralizing 2,4,6-trinitrotoluene.

Within a screening program, 91 fungal strains belonging to 32 genera of different ecological and taxonomic groups (wood- and litter-decaying basidiomycetes, saprophytic micromycetes) were tested for their ability to metabolize and mineralize 2,4,6-trinitrotoluene (TNT). All these strains metabolized TNT rapidly by forming monoaminodinitrotoluenes (AmDNT). Micromycetes produced higher amounts of AmDNT than did wood- and litter-decaying basidiomycetes. A significant mineralization of [14C]TNT was only observed for certain wood- and litter-decaying basidiomycetes. The most active strains, Clitocybula dusenii TMb12 and Stropharia rugosa-annulata DSM11372 mineralized 42% and 36% respectively of the initial added [14C]TNT (100 microM corresponding to 4.75 microCi/l) to 14CO2 within 64 days. Micromycetes (deuteromycetes, ascomycetes, zygomycetes) proved to be unable to mineralize [14C]TNT significantly.

Involvement of cytochrome P-450 in the 15alpha-hydroxylation of 13-ethyl-gon-4-ene-3,17-dione by Penicillium raistrickii.

The 15alpha-hydroxylation of 13-ethyl-gon-4-ene-3,17-dione (GD) with different subcellular fractions of Penicillium raistrickii i 477 was investigated. Cytochrome P-450 was shown to be involved in this reaction. The steroid transformation was inhibited by carbon monoxide, metyrapone, p-CMB, iodoacetamide, N-methylmaleimide and several metal ions. The 15alpha-hydroxylase was observed to be dependent on nicotinamide-adenine dinucleotide phosphate (NADPH) replaceable by NaIO4, and the activity was enhanced by a NADPH-regenerating system, indicating the involvement of the NADPH-cytochrome c (P-450) reductase. This was further confirmed by the inhibition of the hydroxylase activity in the presence of cytochrome c. No effect was observed in the presence of azide and antimycin A. Solubilized microsomes gave an absorption maximum at 453 nm in carbon monoxide difference spectrum, and showed a Type-I GD-binding spectrum typically for cytochrome P-450 interaction with substrate. First results about the inducibility of the enzymes involved in the 15alpha-hydroxylation of GD are shown.

Transformation of difluorinated phenols by Penicillium frequentans Bi 7/2.

The Penicillium frequentans strain Bi 7/2, using phenol as a sole source of carbon and energy, transformed the fluorinated phenols 2,3-, 2,4-, 2,5- and 3,4-difluorophenol rapidly. After growth on phenol, resting mycelia of the fungus converted the difluorophenols completely at an initial concentration of 0.5 mM within 6 hours. The corresponding difluorinated catechols were found to be intermediates of all difluorophenols investigated. A relatively unspecific phenol hydroxylase catalyzed this hydroxylation step and showed activities towards all difluorophenols tested. One difluorocatechol was formed from each difluorophenol substituted with fluorine in the ortho-position, whereas two catechols were formed from 3,4-difluorophenol, due to its two vacant ortho-positions. A partial defluorination (50-77%) was observed in all cases.

Effects of ryegrass on biodegradation of hydrocarbons in soil.

The effects of growing ryegrass (Lolium perenne L.) on the biodegradation of hydrocarbons was studied in laboratory scale soil columns. Degradation of hydrocarbons as well as bacterial numbers, soil respiration rates and soil dehydrogenase activities were determined. In the rhizosphere soil system, aliphatic hydrocarbons disappeared faster than in unvegetated columns. Abiotic loss by evaporation was of minor significance. Elimination of pollutants was accompanied by an increase in microbial numbers and activities. The microbial plate counts and soil respiration rates were substantially higher in the rhizosphere than in the bulk soil. The results indicate that biodegradation of hydrocarbons in the rhizosphere is stimulated by plant roots.

Phenol and cresol metabolism in Bacillus pumilus isolated from contaminated groundwater.

From an aquifier contaminated with phenolic compounds seven bacterial strains able to grow on phenol and several mono- and disubstituted alkylphenols as sole source of carbon and energy were isolated. Five isolates belong to the genus Pseudomonas, two to the genus Bacillus. The isolate most active in utilization of the applied xenobiotics was identified as Bacillus pumilus and used for the investigation of the degradation pathways in liquid cultures. Cells of this strain precultured on phenol were able to utilize para-cresol as sole carbon source via the oxidation of the methylsubstituent and intradiol ring cleavage of the resulting protocatechuic acid, whereas an intradiol ring fission of the intermediate 4-methylcatechol led to 4-methylmuconolactone as dead end-product. Cells precultured on meta- and ortho-cresol were able to utilize the respective compounds as sole carbon sources via 3-methylcatechol, which induced the following extradiol ring fission pathway. Cells precultured on phenol were able to cooxidize meta- as well as ortho-cresol to 3-methylcatechol, which was cleaved via an intradiol ring fission, finally leading to the dead end-product 2-methylmuconolactone.

Cometabolic degradation of o-cresol and 2,6-dimethylphenol by Penicillium frequentans Bi 7/2.

o-Cresol induced glucose-grown resting mycelia of Penicillium frequentans Bi 7/2 (ATCC-number: 96048) immediately oxidized o-cresol and other phenols. After precultivation on glucose and phenol degradation started after a lag-phase of 24 hours. Metabolites of o-cresol metabolism were methylhydroquinone, methyl-p-benzoquinone, 2-methyl-5-hydroxyhydroquinone and 2-methyl-5-hydroxy-p-benzoquinone. The initial reaction is probably catalyzed by a NADPH dependent hydroxylase which is specific for o-cresol. The metabolism of 2,6-dimethylphenol (2,6-xylenol) occurred via 2,6-dimethylhydroquinone, 2,6-dimethyl-p-benzoquinone, 2,6-dimethyl-3-hydroxyhydroquinone, 2,6-dimethyl-3-hydroxy-p-benzoquinone and 3-methyl-2-hydroxybenzoic acid.

Unspecific degradation of halogenated phenols by the soil fungus Penicillium frequentans Bi 7/2.

Resting phenol-grown mycelia of the fungus Penicillium frequentans strain Bi 7/2 were shown to be capable of metabolizing various monohalogenated phenols as well as 3,4-dichlorophenol. 2,4.dichlorophenol could be metabolized in the presence of phenol as cosubstrate. In the first degradation step the halogenated phenols were oxidized to the corresponding halocatechols. Halocatechols substituted in para-position (4-halocatechols) were further degraded under formation of 4-carboxymethylenbut-2-en-4-olide. A partial dehalogenation took place splitting the ring system. 3-Halocatechols were cleaved to 2-halomuconic acids as dead end metabolites without a dehalogenation step. Dichlorophenols were only transformed to the corresponding catechols. In addition 3,5-dichloro-catechol was O-methylated to give two isomers of dichloroguiacol. The halogenated catechols with the exception of 4-fluorocatechol partly polymerized oxidatively in the culture fluid to form insoluble dark-brown products. The degradation of halophenols are due to the action of unspecific intracellular enzymes responsible for phenol catabolism (phenol hydroxylase, catechol-1,2-dioxygenase, muconate cycloisomerase I).

Molecular characterization of field isolates of Pseudomonas syringae pv. glycinea differing in coronatine production.

Coronatine-producing and non-producing strains of Pseudomonas syringae pv. glycinea have been examined. We found a connection between copper resistance and synthesis of coronatine. Published data implied that these properties may be encoded on different plasmids. Production of coronatine and copper resistance were also found to be correlated for pv. glycinea in 19 field-isolates from leaf spots of plants in a soybean field and in 28 strains of a bacterial culture collection. Genomic diversity within pv. glycinea was investigated by plasmid profiling, DNA hybridization studies and PCR analysis. All strains unable to produce coronatine (cor-) were sensitive to copper ions and showed no homology to DNA from plasmid pSAY1, which carries a gene cluster for steps in coronatine production. In addition, cor- strains could be distinguished from coronatine-producing strains by a single unique band when amplified by random primer PCR. Plasmid profiles of strains isolated from field-populations during 1983, 1985 and 1990 showed that coronatine-producing and non-producing strains were present. The plasmid patterns also varied in 28 strains examined from a culture collection. No correlation between plasmid patterns and race specificity was observed. Cosmid pSAY1 proved to be an effective probe for detection of the coronatine synthesis genes and also revealed polymorphisms in coronatine producing strains of pv. glycinea.