Degradation of benzo[a]pyrene by the litter-decomposing basidiomycete Stropharia coronilla: role of manganese peroxidase

The litter-decomposing basidiomycete Stropharia coronilla, which preferably colonizes grasslands, was found to be capable of metabolizing and mineralizing benzo[a]pyrene (BaP) in liquid culture. Manganese(II) ions (Mn(2+)) supplied at a concentration of 200 micro M stimulated considerably both the conversion and the mineralization of BaP; the fungus metabolized and mineralized about four and twelve times, respectively, more of the BaP in the presence of supplemental Mn(2+) than in the basal medium. This stimulating effect could be attributed to the ligninolytic enzyme manganese peroxidase (MnP), whose activity increased after the addition of Mn(2+). Crude and purified MnP from S. coronilla oxidized BaP efficiently in a cell-free reaction mixture (in vitro), a process which was enhanced by the surfactant Tween 80. Thus, 100 mg of BaP liter(-1) was converted in an in vitro reaction solution containing 1 U of MnP ml(-1) within 24 h. A clear indication was found that BaP-1,6-quinone was formed as a transient metabolite, which disappeared over the further course of the reaction. The treatment of a mixture of 16 different polycyclic aromatic hydrocarbons (PAHs) selected by the U.S. Environmental Protection Agency as model standards for PAH analysis (total concentration, 320 mg liter(-1)) with MnP resulted in concentration decreases of 10 to 100% for the individual compounds, and again the stimulating effect of Tween 80 was observed. Probably due to their lower ionization potentials, poorly bioavailable, high-molecular-mass PAHs such as BaP, benzo(g,h,i)perylene, and indeno(1,2,3-c,d)pyrene were converted to larger extents than low-molecular-mass ones (e.g., phenanthrene and fluoranthene).     

Degradation of Benzo[a]pyrene by the Litter-Decomposing Basidiomycete Stropharia coronilla: Role of Manganese Peroxidase

The litter-decomposing basidiomycete Stropharia coronilla, which preferably colonizes grasslands, was found to be capable of metabolizing and mineralizing benzo[a]pyrene (BaP) in liquid culture. Manganese(II) ions (Mn²⁺) supplied at a concentration of 200 μM stimulated considerably both the conversion and the mineralization of BaP; the fungus metabolized and mineralized about four and twelve times, respectively, more of the BaP in the presence of supplemental Mn²⁺ than in the basal medium. This stimulating effect could be attributed to the ligninolytic enzyme manganese peroxidase (MnP), whose activity increased after the addition of Mn²⁺. Crude and purified MnP from S. coronilla oxidized BaP efficiently in a cell-free reaction mixture (in vitro), a process which was enhanced by the surfactant Tween 80. Thus, 100 mg of BaP liter⁻¹ was converted in an in vitro reaction solution containing 1 U of MnP ml⁻¹ within 24 h. A clear indication was found that BaP-1,6-quinone was formed as a transient metabolite, which disappeared over the further course of the reaction. The treatment of a mixture of 16 different polycyclic aromatic hydrocarbons (PAHs) selected by the U.S. Environmental Protection Agency as model standards for PAH analysis (total concentration, 320 mg liter⁻¹) with MnP resulted in concentration decreases of 10 to 100% for the individual compounds, and again the stimulating effect of Tween 80 was observed. Probably due to their lower ionization potentials, poorly bioavailable, high-molecular-mass PAHs such as BaP, benzo(g,h,i)perylene, and indeno(1,2,3-c,d)pyrene were converted to larger extents than low-molecular-mass ones (e.g., phenanthrene and fluoranthene).     

Mn(II) Regulation of Lignin Peroxidases and Manganese-Dependent Peroxidases from Lignin-Degrading White Rot Fungi

Two families of peroxidases—lignin peroxidase (LiP) and manganese-dependent lignin peroxidase (MnP)—are formed by the lignin-degrading white rot basidiomycete Phanerochaete chrysosporium and other white rot fungi. Isoenzymes of these enzyme families carry out reactions important to the biodegradation of lignin. This research investigated the regulation of LiP and MnP production by Mn(II). In liquid culture, LiP titers varied as an inverse function of and MnP titers varied as a direct function of the Mn(II) concentration. The extracellular isoenzyme profiles differed radically at low and high Mn(II) levels, whereas other fermentation parameters, including extracellular protein concentrations, the glucose consumption rate, and the accumulation of cell dry weight, did not change significantly with the Mn(II) concentration. In the absence of Mn(II), extracellular LiP isoenzymes predominated, whereas in the presence of Mn(II), MnP isoenzymes were dominant. The release of ¹⁴CO₂ from ¹⁴C-labeled dehydrogenative polymerizate lignin was likewise affected by Mn(II). The rate of ¹⁴CO₂ release increased at low Mn(II) and decreased at high Mn(II) concentrations. This regulatory effect of Mn(II) occurred with five strains of P. chrysosporium, two other species of Phanerochaete, three species of Phlebia, Lentinula edodes, and Phellinus pini.     

Roles of manganese and organic acid chelators in regulating lignin degradation and biosynthesis of peroxidases by Phanerochaete chrysosporium.

We studied the effect of manganese and various organic chelators on the distribution, depolymerization, and mineralization of synthetic 14C-labeled lignins (DHP) in cultures of Phanerochaete chrysosporium. In the presence of high levels of manganese [Mn(II) or Mn(III)], along with a suitable chelator, lignin peroxidase (LiP) production was repressed and manganese peroxidase (MnP) production was stimulated. Even though partial lignin depolymerization was observed under these conditions, further depolymerization of the polymer to smaller compounds was more efficient when low levels of manganese were present. LiPs were prevalent under these latter conditions, but MnPs were also present. Mineralization was more efficient with low manganese. These studies indicate that MnP performs the initial steps of DHP depolymerization but that LiP is necessary for further degradation of the polymer to lower-molecular-weight products and mineralization. We also conclude that a soluble Mn(II)-Mn(III) organic acid complex is necessary to repress LiP.     

Production of ligninolytic exoenzymes and14C-Pyrene mineralization byPleurotus sp. in lignocellulose substrate

Pleurotus sp. was grown in liquid medium and on a solid straw substrate, and activities of laccase and manganese-dependent peroxidase (MnP) were recorded. The activities were the highest in a rich, glucose corn-steep liquid medium. In straw cultures, laccase activity was about ten times lower. Under solid state conditions, MnP production was the highest during days 20–40, when laccase activity already had declined. In straw cultures, mineralization of¹⁴C-pyrene was measured as release of¹⁴CO₂. The highest rates of pyrene mineralization occurred during days 20–45,i.e. the period of high MnP activities, suggesting a role of this enzyme in PAH degradation. Within 60d, 24% of pyrene was mineralized.     

Roles of manganese and organic acid chelators in regulating lignin degradation and biosynthesis of peroxidases by Phanerochaete chrysosporium.

We studied the effect of manganese and various organic chelators on the distribution, depolymerization, and mineralization of synthetic 14C-labeled lignins (DHP) in cultures of Phanerochaete chrysosporium. In the presence of high levels of manganese [Mn(II) or Mn(III)], along with a suitable chelator, lignin peroxidase (LiP) production was repressed and manganese peroxidase (MnP) production was stimulated. Even though partial lignin depolymerization was observed under these conditions, further depolymerization of the polymer to smaller compounds was more efficient when low levels of manganese were present. LiPs were prevalent under these latter conditions, but MnPs were also present. Mineralization was more efficient with low manganese. These studies indicate that MnP performs the initial steps of DHP depolymerization but that LiP is necessary for further degradation of the polymer to lower-molecular-weight products and mineralization. We also conclude that a soluble Mn(II)-Mn(III) organic acid complex is necessary to repress LiP.     

Degradation of humic acids by the litter-decomposing basidiomycete Collybia dryophila

The basidiomycete Collybia dryophila K209, which colonizes forest soil, was found to decompose a natural humic acid isolated from pine-forest litter (LHA) and a synthetic (14)C-labeled humic acid ((14)C-HA) prepared from [U-(14)C]catechol in liquid culture. Degradation resulted in the formation of polar, lower-molecular-mass fulvic acid (FA) and carbon dioxide. HA decomposition was considerably enhanced in the presence of Mn(2+) (200 microM), leading to 75% conversion of LHA and 50% mineralization of (14)C-HA (compared to 60% and 20%, respectively, in the absence of Mn(2+)). There was a strong indication that manganese peroxidase (MnP), the production of which was noticeably increased in Mn(2+)-supplemented cultures, was responsible for this effect. The enzyme was produced as a single protein with a pI of 4.7 and a molecular mass of 44 kDa. During solid-state cultivation, C. dryophila released substantial amounts of water-soluble FA (predominantly of 0.9 kDa molecular mass) from insoluble litter material. The results indicate that basidiomycetes such as C. dryophila which colonize forest litter and soil are involved in humus turnover by their recycling of high-molecular-mass humic substances. Extracellular MnP seems to be a key enzyme in the conversion process.     

Manganese-Dependent Cleavage of Nonphenolic Lignin Structures by Ceriporiopsis subvermispora in the Absence of Lignin Peroxidase

Many ligninolytic fungi appear to lack lignin peroxidase (LiP), the enzyme generally thought to cleave the major, recalcitrant, nonphenolic structures in lignin. At least one such fungus, Ceriporiopsis subvermispora, is nevertheless able to degrade these nonphenolic structures. Experiments showed that wood block cultures and defined liquid medium cultures of C. subvermispora rapidly depolymerized and mineralized a (sup14)C-labeled, polyethylene glycol-linked, high-molecular-weight (beta)-O-4 lignin model compound (model I) that represents the major nonphenolic structure of lignin. The fungus cleaved model I between C(inf(alpha)) and C(inf(beta)) to release benzylic fragments, which were shown in isotope trapping experiments to be major products of model I metabolism. The C(inf(alpha))-C(inf(beta)) cleavage of (beta)-O-4 lignin structures to release benzylic fragments is characteristic of LiP catalysis, but assays of C. subvermispora liquid cultures that were metabolizing model I confirmed that the fungus produced no detectable LiP activity. Three results pointed, instead, to the participation of a different enzyme, manganese peroxidase (MnP), in the degradation of nonphenolic lignin structures by C. subvermispora. (i) The degradation of model I and of exhaustively methylated (nonphenolic), (sup14)C-labeled, synthetic lignin by the fungus in liquid cultures was almost completely inhibited when the Mn concentration of the medium was decreased from 35 (mu)M to approximately 5 (mu)M. (ii) The fungus degraded model I and methylated lignin significantly faster in the presence of Tween 80, a source of unsaturated fatty acids, than it did in the presence of Tween 20, which contains only saturated fatty acids. Previous work has shown that nonphenolic lignin structures are degraded during the MnP-mediated peroxidation of unsaturated lipids. (iii) In experiments with MnP, Mn(II), and unsaturated lipid in vitro, this system mimicked intact C. subvermispora cultures in that it cleaved nonphenolic (beta)-O-4 lignin model compounds between C(inf(alpha)) and C(inf(beta)) to release a benzylic fragment.     

Extracellular Enzyme Production and Synthetic Lignin Mineralization by Ceriporiopsis subvermispora

The ability of the white rot fungus Ceriporiopsis subvermispora to mineralize ¹⁴C-synthetic lignin was studied under different culture conditions, and the levels of two extracellular enzymes were monitored. The highest mineralization rates (28% after 28 days) were obtained in cultures containing a growth-limiting amount of nitrogen source (1.0 mM ammonium tartrate); under this condition, the levels of manganese peroxidase (MnP) and laccase present in the culture supernatant solutions were very low compared with cultures containing 10 mM of the nitrogen source. In contrast, cultures containing a limiting concentration of the carbon source (0.1% glucose) showed low levels of both enzymes and also very low mineralization rates compared with cultures containing 1% glucose. Cultures containing 11 ppm of Mn(II) showed a higher rate of mineralization than those containing 0.3 or 40 ppm of this cation. Levels of MnP and laccase were higher when 40 ppm of Mn(II) was used. Mineralization rates were slightly higher in cultures flushed daily with oxygen, whereas laccase levels were lower and MnP levels were approximately the same as in cultures maintained under an air atmosphere. The presence of 0.4 mM veratryl alcohol reduced both mineralization rates and MnP levels, without affecting laccase levels. Lignin peroxidase activity was not detected under any condition. Addition of purified lignin peroxidase to the cultures in the presence or absence of veratryl alcohol did not enhance mineralization rates significantly.     

Degradation and mineralization of high-molecular-weight polycyclic aromatic hydrocarbons by defined fungal-bacterial cocultures

This study investigated the biodegradation of high-molecular-weight polycyclic aromatic hydrocarbons (PAHs) in liquid media and soil by bacteria (Stenotrophomonas maltophilia VUN 10,010 and bacterial consortium VUN 10,009) and a fungus (Penicillium janthinellum VUO 10, 201) that were isolated from separate creosote- and manufactured-gas plant-contaminated soils. The bacteria could use pyrene as their sole carbon and energy source in a basal salts medium (BSM) and mineralized significant amounts of benzo[a]pyrene cometabolically when pyrene was also present in BSM. P. janthinellum VUO 10,201 could not utilize any high-molecular-weight PAH as sole carbon and energy source but could partially degrade these if cultured in a nutrient broth. Although small amounts of chrysene, benz[a]anthracene, benzo[a]pyrene, and dibenz[a,h]anthracene were degraded by axenic cultures of these isolates in BSM containing a single PAH, such conditions did not support significant microbial growth or PAH mineralization. However, significant degradation of, and microbial growth on, pyrene, chrysene, benz[a]anthracene, benzo[a]pyrene, and dibenz[a,h]anthracene, each as a single PAH in BSM, occurred when P. janthinellum VUO 10,201 and either bacterial consortium VUN 10,009 or S. maltophilia VUN 10,010 were combined in the one culture, i.e., fungal-bacterial cocultures: 25% of the benzo[a]pyrene was mineralized to CO(2) by these cocultures over 49 days, accompanied by transient accumulation and disappearance of intermediates detected by high-pressure liquid chromatography. Inoculation of fungal-bacterial cocultures into PAH-contaminated soil resulted in significantly improved degradation of high-molecular-weight PAHs, benzo[a]pyrene mineralization (53% of added [(14)C]benzo[a]pyrene was recovered as (14)CO(2) in 100 days), and reduction in the mutagenicity of organic soil extracts, compared with the indigenous microbes and soil amended with only axenic inocula.     

Laccase-catalyzed oxidation of Mn(2+) in the presence of natural Mn(3+) chelators as a novel source of extracellular H(2)O(2) production and its impact on manganese peroxidase

A purified and electrophoretically homogeneous blue laccase from the litter-decaying basidiomycete Stropharia rugosoannulata with a molecular mass of approximately 66 kDa oxidized Mn(2+) to Mn(3+), as assessed in the presence of the Mn chelators oxalate, malonate, and pyrophosphate. At rate-saturating concentrations (100 mM) of these chelators and at pH 5.0, Mn(3+) complexes were produced at 0.15, 0.05, and 0.10 micromol/min/mg of protein, respectively. Concomitantly, application of oxalate and malonate, but not pyrophosphate, led to H(2)O(2) formation and tetranitromethane (TNM) reduction indicative for the presence of superoxide anion radical. Employing oxalate, H(2)O(2) production, and TNM reduction significantly exceeded those found for malonate. Evidence is provided that, in the presence of oxalate or malonate, laccase reactions involve enzyme-catalyzed Mn(2+) oxidation and abiotic decomposition of these organic chelators by the resulting Mn(3+), which leads to formation of superoxide and its subsequent reduction to H(2)O(2). A partially purified manganese peroxidase (MnP) from the same organism did not produce Mn(3+) complexes in assays containing 1 mM Mn(2+) and 100 mM oxalate or malonate, but omitting an additional H(2)O(2) source. However, addition of laccase initiated MnP reactions. The results are in support of a physiological role of laccase-catalyzed Mn(2+) oxidation in providing H(2)O(2) for extracellular oxidation reactions and demonstrate a novel type of laccase-MnP cooperation relevant to biodegradation of lignin and xenobiotics.     

Transformation and mineralization of benzo[<Emphasis Type="Italic">a</Emphasis>]pyrene by microbial cultures enriched on mixtures of three- and four-ring polycyclic aromatic hydrocarbons

Microorganisms originating from a soil contaminated by low levels of polycyclic aromatic hydrocarbons (PAHs) were enriched with three- and four-ring PAHs as primary substrates in the presence of benzo[a]pyrene (BaP). Most enrichment cultures, isolated in the presence or absence of a sorptive matrix, significantly transformed BaP. Evidence of BaP mineralization was obtained with cultures enriched on phenanthrene and anthracene. Our findings supplement literature data suggesting the wide occurrence of microbial activity against BaP. Journal of Industrial Microbiology & Biotechnology (2002) 28, 70–73 DOI: 10.1038/sj/jim/7000211 Received 11 December 2000/ Accepted in revised form 04 September 2001     

Polycyclic aromatic hydrocarbon-degrading capabilities of Phanerochaete laevis HHB-1625 and its extracellular ligninolytic enzymes.

The ability of Phanerochaete laevis HHB-1625 to transform polycyclic aromatic hydrocarbons (PAHs) in liquid culture was studied in relation to its complement of extracellular ligninolytic enzymes. In nitrogen-limited liquid medium, P. laevis produced high levels of manganese peroxidase (MnP). MnP activity was strongly regulated by the amount of Mn2+ in the culture medium, as has been previously shown for several other white rot species. Low levels of laccase were also detected. No lignin peroxidase (LiP) was found in the culture medium, either by spectrophotometric assay or by Western blotting (immunoblotting). Despite the apparent reliance of the strain primarily on MnP, liquid cultures of P. laevis were capable of extensive transformation of anthracene, phenanthrene, benz[a]anthracene, and benzo[a]pyrene. Crude extracellular peroxidases from P. laevis transformed all of the above PAHs, either in MnP-Mn2+ reactions or in MnP-based lipid peroxidation systems. In contrast to previously published studies with Phanerochaete chrysosporium, metabolism of each of the four PAHs yielded predominantly polar products, with no significant accumulation of quinones. Further studies with benz[a]anthracene and its 7,12-dione indicated that only small amounts of quinone products were ever present in P. laevis cultures and that quinone intermediates of PAH metabolism were degraded faster and more extensively by P. laevis than by P. chrysosporium.     

Glutathione-mediated mineralization of 14C-labeled 2-amino-4,6-dinitrotoluene by manganese-dependent peroxidase H5 from the white-rot fungus Phanerochaete chrysosporium

Manganese-dependent peroxidase (MnP) H5 from the white-rot fungus Phanerochaete chrysosporium, in the presence of either Mn(II) (10 mM) or GSH (10 mM), was able to mineralize ¹⁴C-U-ring-labeled 2-amino-4,6-dinitrotoluene (2-A-4,6-DNT) up to 29% in 12 days. When both Mn(II) and GSH were present, the mineralization extent reached 82%. On the other hand, no significant mineralization was observed in the absence of both Mn(II) and GSH, suggesting the requirement of a mediator [either Mn(II) or GSH] for the degradation of 2-A-4,6-DNT by MnP. Using electron spin resonance (ESR) techniques, it was found that the glutathionyl free radical (GS^•) was produced through the oxidation of GSH by MnP in the presence as well as in the absence of Mn(II). GS^• was also generated through the direct oxidation of GSH by Mn(III). Our results strongly suggest the involvement of GS^• in the GSH-mediated mineralization of 2-A-4,6-DNT by MnP. Received: 18 February 2000 / Received revision: 24 May 2000 / Accepted: 26 May 2000     

Transformation and mineralization of 2,4,6-trinitrotoluene (TNT) by manganese peroxidase from the white-rot basidiomycete Phlebia radiata

The degradation of the nitroaromatic pollutant 2,4,6-trinitrotoluene (TNT) by the manganese-dependent peroxidase (MnP) of the white-rot fungus Phlebia radiata and the main reduction products formed were investigated. In the presence of small amounts of reduced glutathione (10 mM), a concentrated cell-free preparation of MnP from P. radiata exhibiting an activity of 36 nkat/ml (36 nmol Mn(II) oxidized per sec and per ml) transformed 10 mg/l of TNT within three days. The same preparation was capable of completely transforming the reduced derivatives of TNT. When present at 10 mg/l, the aminodinitrotoluenes were transformed in less than two days and the diaminonitrotoluenes in less than three hours. Experiments with ¹⁴C-U-ring labeled TNT and 2-amino-4,6-dinitrotoluene showed that these compounds were mineralized by 22% and 76%, respectively, within 5 days. Higher concentrations of reduced glutathione (50 mM) led to a severe inhibition of the degradation process. It is concluded that Phlebia radiata is a good candidate for the biodegradation of TNT as well as its reduction metabolites.     

Acanthocytes of Stropharia rugosoannulata function as a nematode-attacking device

Efficient killing of nematodes by Stropharia rugosoannulata Farlow ex Murrill cultures was observed. This fungus showed the ability to immobilize the free-living nematode Panagrellus redivivus Goodey within minutes and to immobilize the pine wilt nematode Bursaphelenchus xylophilus (Steiner & Buhrer) Nickle within hours on agar plates. Moreover, P. redivivus worms were completely degraded by the fungus within 24 to 48 h. The cultures of S. rugosoannulata studied shared the characteristic of abundantly producing cells with finger-like projections called acanthocytes. We showed that the nematode-attacking activity of this fungus is carried out by these spiny acanthocytes and that mechanical force is an important factor in the process. Furthermore, the growth and nematode-attacking activity of the fungus in soil were also determined, and our results suggest that acanthocytes are functional in soil.     

Effect of tobacco smoke components on organogenesis in plant tissues

Callus tissue derived from haploid plants of Nicotiana tabacumdifferentiated when inoculated on media fortified with differentconcentrations of tobacco smoke components: benzo(a)pyrene (BaP),benzo(e)pyrene (BeP), dibenz(a, h)anthracene (DBA), pyrene andchrysene. Neither any auxins nor cytokinins were present inthe medium. BaP was found to be the most potent morphogeneticagent. However, these treatments did not induce any vegetativebuds on the callus obtained from diploid plants of tobacco. ¹ This study was carried out under Contract No. 12-14-100-10341(73)with the Agricultural Research Service, U. S. Department ofAgriculture, administered by the Eastern Marketing and NutritionResearch Division, 600 East Mermaid Lane, Philadelphia, Pennsylvania19118, U.S.A. (Received February 4, 1971; )     

Manganese Peroxidase-Dependent Oxidation of Glyoxylic and Oxalic Acids Synthesized by Ceriporiopsis subvermispora Produces Extracellular Hydrogen Peroxide

The ligninolytic system of the basidiomycete Ceriporiopsis subvermispora is composed of manganese peroxidase (MnP) and laccase. In this work, the source of extracellular hydrogen peroxide required for MnP activity was investigated. Our attention was focused on the possibility that hydrogen peroxide might be generated by MnP itself through the oxidation of organic acids secreted by the fungus. Both oxalate and glyoxylate were found in the extracellular fluid of C. subvermispora cultures grown in chemically defined media, where MnP is also secreted. The in vivo oxidation of oxalate was measured; ¹⁴CO₂ evolution was monitored after addition of exogenous [¹⁴C]oxalate to cultures at constant specific activity. In standard cultures, evolution of CO₂ from oxalate was maximal at day 6, although the MnP titers were highest at day 12, the oxalate concentration was maximal (2.5 mM) at day 10, and the glyoxylate concentration was maximal (0.24 mM) at day 5. However, in cultures containing low nitrogen levels, in which the pH is more stable, a better correlation between MnP titers and mineralization of oxalate was observed. Both MnP activity and oxidation of [¹⁴C]oxalate were negligible in cultures lacking Mn(II). In vitro assays confirmed that Mn(II)-dependent oxidation of [¹⁴C]oxalate by MnP occurs and that this reaction is stimulated by glyoxylate at the concentrations found in cultures. In addition, both organic acids supported phenol red oxidation by MnP without added hydrogen peroxide, and glyoxylate was more reactive than oxalate in this reaction. Based on these results, a model is proposed for the extracellular production of hydrogen peroxide by C. subvermispora.