Degradation of Polycyclic Aromatic Hydrocarbons by Crude Extracts from Spent Mushroom Substrate and its Possible Mechanisms

Biodegradation of polycyclic aromatic hydrocarbons (PAHs) by pure laccase has been reported, but the high cost limited its application in environmental bioremediation. Here, we reported a study about PAHs degradation by crude extracts (CEs) containing laccase, which were obtained by extracting four spent mushroom (Agaricus bisporus, Pleurotus eryngii, Pleurotus ostreatus, and Coprinus comatus) substrates. The results showed that anthracene, benzo[a]pyrene, and benzo[a]anthracene were top three degradable PAHs by CEs while naphthalene was most recalcitrant. The PAHs oxidation was enhanced in the presence of 2,2-azino-bis-(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS). Laccase included in CE might play a major role in PAHs degradation. The maximum degradation rate of anthracene and benzo[a]pyrene was observed by using crude extracts from P. eryngii while the highest laccase activities were found in crude extracts from A. bisporus, moreover, crude extracts from P. eryngii, which contained less laccase activities, degraded more anthracene and benzo[a]pyrene than pure laccase with higher laccase activities. The lack of correlation between laccase activity and PAHs degradation rate indicated that other factors might also influence the PAHs degradation. Boiled CEs were added to determine the effect on PAHs degradation by laccase. The results showed that all four boiled CEs had improved the PAHs oxidation. The maximum improvement was observed by adding CEs from P. eryngii. It suggested that some mediators indeed existed in CEs and CEs from P. eryngii contained most. As a result, CEs from P. eryngii has the most application potential in PAHs bioremediation.     

天然介体在产漆酶真菌氧化蒽和芘中的重要作用（英文稿）

【目的】研究了氧化还原介体在产漆酶真菌氧化蒽和芘的作用。【方法】通过非变性电泳和酶活力分析。【结果】发现血红密孔菌Z-1和木蹄层孔菌Z-5只产漆酶,其最大酶产量分别为11.90 U/mL和4.83 U/mL,不产木质素过氧化酶和锰过氧化物酶。木蹄层孔菌Z-5的胞外液尽管具有较低的漆酶活性,但是氧化了74.3%的蒽和12.4%的芘,高于血红密孔菌Z-1对蒽和芘的氧化率,提示天然介体可能存在于真菌胞外液中并且影响了漆酶对多环芳烃的氧化。实验进一步表明,木蹄层孔菌Z-5灭活和不灭活的超滤液以及灭活的胞外液对纯漆酶氧化多环芳烃的促进作用均大于血红密孔菌Z-1,说明木蹄层孔菌Z-5的天然介体比血红密孔菌Z-1能够更为有效地促进多环芳烃氧化。【结论】氧化还原结体在产漆酶真菌降解底物过程中发挥了重要作用,这也解释了木蹄层孔菌Z-5胞外液尽管漆酶活性不高,但是具有较大多环芳烃氧化率的原因。     

Natural Mediators in the Oxidation of Polycyclic Aromatic Hydrocarbons by Laccase Mediator Systems

The oxidation of polycyclic aromatic compounds was studied in systems consisting of laccase from Trametes versicolor and so-called mediator compounds. The enzymatic oxidation of acenaphthene, acenaphthylene, anthracene, and fluorene was mediated by various laccase substrates (phenols and aromatic amines) or compounds produced and secreted by white rot fungi. The best natural mediators, such as phenol, aniline, 4-hydroxybenzoic acid, and 4-hydroxybenzyl alcohol were as efficient as the previously described synthetic compounds ABTS [2,2′-azino-bis-(3-ethylbenzothiazoline-6-sulfonic acid)] and 1-hydroxybenzotriazole. The oxidation efficiency increased proportionally with the redox potentials of the phenolic mediators up to a maximum value of 0.9 V and decreased thereafter with redox potentials exceeding this value. Natural compounds such as methionine, cysteine, and reduced glutathione, containing sulfhydryl groups, were also active as mediator compounds.     

Polycyclic Aromatic Hydrocarbon Metabolism by White Rot Fungi and Oxidation by Coriolopsis gallica UAMH 8260 Laccase

We studied the metabolism of polycyclic aromatic hydrocarbons (PAHs) by using white rot fungi previously identified as organisms that metabolize polychlorinated biphenyls. Bran flakes medium, which has been shown to support production of high levels of laccase and manganese peroxidase, was used as the growth medium. Ten fungi grown for 5 days in this medium in the presence of anthracene, pyrene, or phenanthrene, each at a concentration of 5 μg/ml could metabolize these PAHs. We studied the oxidation of 10 PAHs by using laccase purified from Coriolopsis gallica. The reaction mixtures contained 20 μM PAH, 15% acetonitrile in 60 mM phosphate buffer (pH 6), 1 mM 2,2′-azinobis-(3-ethylbenzthiazoline-6-sulfonate) (ABTS), and 5 U of laccase. Laccase exhibited 91% of its maximum activity in the absence of acetonitrile. The following seven PAHs were oxidized by laccase: benzo[a]pyrene, 9-methylanthracene, 2-methylanthracene, anthracene, biphenylene, acenaphthene, and phenanthrene. There was no clear relationship between the ionization potential of the substrate and the first-order rate constant (k) for substrate loss in vitro in the presence of ABTS. The effects of mediating substrates were examined further by using anthracene as the substrate. Hydroxybenzotriazole (HBT) (1 mM) supported approximately one-half the anthracene oxidation rate (k = 2.4 h⁻¹) that ABTS (1 mM) supported (k = 5.2 h⁻¹), but 1 mM HBT plus 1 mM ABTS increased the oxidation rate ninefold compared with the oxidation rate in the presence of ABTS, to 45 h⁻¹. Laccase purified from Pleurotus ostreatus had an activity similar to that of C. gallica laccase with HBT alone, with ABTS alone, and with 1 mM HBT plus 1 mM ABTS. Mass spectra of products obtained from oxidation of anthracene and acenaphthene revealed that the dione derivatives of these compounds were present.     

First evidence of catalytic mediation by phenolic compounds in the laccase-induced oxidation of lignin models.

The sulfonephthalein indicator, phenol red, exhibits an unusually slow rate of oxidation by laccase from Poliporus pinsitus, in spite of the fact that it is a phenol and therefore a natural substrate for this phenoloxidase enzyme. Nevertheless, after prolonged exposure to laccase (24 h) phenol red is oxidized by more than 90%. We found that phenol red, which can be oxidatively converted into a resonance-stabilized phenoxy radical, performs as a mediator in the laccase-catalyzed oxidation of a nonphenolic substrate (4-methoxybenzyl alcohol) and also of a hindered phenol (2,4,6-tri-tert-butylphenol). In particular, phenol red was found to be at least 10 times more efficient than 3-hydroxyanthranilate (a reported natural phenolic mediator of laccase) in the oxidation of 4-methoxybenzyl alcohol. Other phenols, which do not bear structural analogies to phenol red, underwent rapid degradation and did not perform as laccase mediators. On the other hand, several variously substituted sulfonephthaleins, of different pK2 values, mediated the laccase catalysis, the most efficient being dichlorophenol red, which has the lowest pK2 of the series. The mediating efficiency of phenol red and dichlorophenol red was found to be pH dependent, as was their oxidation Ep value (determined by cyclic voltammetry). We argue that the relative abundance of the phenoxy anion, which is easier to oxidize than the protonated phenol, may be one of the factors determining the efficiency of a phenolic mediator, together with its ability to form relatively stable oxidized intermediates that react with the desired substrate before being depleted in undesired routes.     

Natural mediators in the oxidation of polycyclic aromatic hydrocarbons by laccase mediator systems

The oxidation of polycyclic aromatic compounds was studied in systems consisting of laccase from Trametes versicolor and so-called mediator compounds. The enzymatic oxidation of acenaphthene, acenaphthylene, anthracene, and fluorene was mediated by various laccase substrates (phenols and aromatic amines) or compounds produced and secreted by white rot fungi. The best natural mediators, such as phenol, aniline, 4-hydroxybenzoic acid, and 4-hydroxybenzyl alcohol were as efficient as the previously described synthetic compounds ABTS [2,2'-azino-bis-(3-ethylbenzothiazoline-6-sulfonic acid)] and 1-hydroxybenzotriazole. The oxidation efficiency increased proportionally with the redox potentials of the phenolic mediators up to a maximum value of 0.9 V and decreased thereafter with redox potentials exceeding this value. Natural compounds such as methionine, cysteine, and reduced glutathione, containing sulfhydryl groups, were also active as mediator compounds.     

Catalytic Efficiency of some Mediators in Laccase-Catalyzed Alcohol Oxidation

The enzyme laccase oxidises phenolic groups of lignin but not the non-phenolic ones. Redox mediators activate laccase towards the non-phenolic groups, particularly the benzyl alcohols. The oxidation step is performed by the oxidised form of the mediator, generated on its interaction with laccase. The oxidised mediator can follow an electron transfer, a radical hydrogen atom transfer or an ionic mechanism in the oxidation of the non-phenolic subunits. Support for these conclusions is provided by (i) investigating the product pattern with suitable probe substrates, (ii) measuring the intramolecular kinetic isotope effect. Determination of electrochemical properties and bond dissociation energies via semiempirical calculations enabled us to rationalise the origin of the different mechanistic behaviour of the mediators. Finally, a comparison of different laccase-mediator-systems (LMS), when applied to the delignification of wood pulp, indicates violuric acid as the most efficient mediator, in an oxidation that is selectively directed towards lignin only.     

Oxidation of polycyclic aromatic hydrocarbons by laccase of Coriolus hirsutus

The oxidation of five polycyclic aromatic hydrocarbons; anthracene, benzo()pyrene, fluoranthene, phenanthrene and pyrene was catalyzed by laccase from Coriolus hirsutus in the presence of the redox mediators, 2,2-azinobis(3-ethylbenzthiazoline-6-sulfonate) (ABTS) and 1-hydroxybenzotriazole (HBT). In the ABTS-mediated system, benzo()pyrene was the most rapidly oxidized substrate, with anthracene being the most rapidly oxidized in the HBT-mediated system. There was no clear relationship between the ionization potential and the oxidation of the substrates. ABTS increased the oxidation of benzo()pyrene more than HBT but the oxidation of the other PAHs tested were the opposite. The mediators used in conjunction increased the oxidation of benzo()pyrene compared to using the mediators alone.     

Degradation of anthracene by laccase of Trametes versicolor in the presence of different mediator compounds

 Laccase of Trametes versicolor was generally able to oxidize anthracene in vitro. After 72 h incubation about 35% of the anthracene was transformed stoichiometrically to 9,10-anthraquinone. Transformation of anthracene increased rapidly in the presence of different mediators that readily generate stable radicals: 2,2′-azino-bis-(3-ethylbenzthiazoline-6-sulfonic acid) (ABTS) and 1-hydroxybenzotriazole. For the reaction, the presence of both the laccase and the mediator was necessary. In the presence of 0.005 mM 1-hydroxybenzotriazole this conversion had removed 47% of the anthracene after 72 h; 75% of the substrate was oxidized during this period when ABTS (1 mM) was used as mediator. In contrast to reactions without or with only low concentrations of a mediator, there was a discrepancy between the disappearance of anthracene and the formation of 9,10-anthraquinone in mediator-forced reactions. Coupling-products of mediators with anthracene degradation products were found. Anthracene disappeared nearly completely after incubation for 72 h with laccase in a 0.1 mM solution of 1-hydroxybenzotriazole and was transformed to 9,10-anthraquinone in about 80% yield; 90% of the substrate was transformed in the presence of ABTS (2.0 mM) resulting again in 80% quinone. Phenothiazine was not effective in this system. Received: 9 April 1996/Received last revision: 7 June 1996/Accepted: 10 June 1996     

Influence of very low doses of mediators on fungal laccase activity - nonlinearity beyond imagination

Laccase, an enzyme responsible for aerobic transformations of natural phenolics, in industrial applications requires the presence of low-molecular substances known as mediators, which accelerate oxidation processes. However, the use of mediators is limited by their toxicity and the high costs of exploitation. The activation of extracellular laccase in growing fungal culture with highly diluted mediators, ABTS and HBT is described. Two high laccase-producing fungal strains, Trametes versicolor and Cerrena unicolor, were used in this study as a source of enzyme. Selected dilutions of the mediators significantly increased the activity of extracellular laccase during 14 days of cultivation what was distinctly visible in PAGE technique and in colorimetric tests. The same mediator dilutions increased demethylation properties of laccase, which was demonstrated during incubation of enzyme with veratric acid. It was established that the activation effect was assigned to specific dilutions of mediators. Our dose-response dilution process smoothly passes into the range of action of homeopathic dilutions and is of interest for homeopaths.     

Oxidation of polycyclic aromatic hydrocarbons by the bacterial laccase CueO from E. coli

Laccases produced by white rot fungi are capable of rapidly oxidizing benzo[a]pyrene. We hypothesize that the polycyclic aromatic hydrocarbon (PAH)-degrading bacteria producing laccase can enhance the degree of benzo[a]pyrene mineralization. However, fungal laccases are glycoproteins which cannot be glycosylated in bacteria, and there is no evidence to show that bacterial laccases can oxidize benzo[a]pyrene. In this study, the in vitro oxidation of PAHs by crude preparations of the bacterial laccase, CueO, from Escherichia coli was investigated. The results revealed that the crude CueO catalyzed the oxidation of anthracene and benzo[a]pyrene in the same way as the fungal laccase from Trametes versicolor, but showed specific characteristics such as thermostability and copper dependence. In the presence of 2,2'-azino-bis-(3-ethylbenzothiazoline-6-sulfonic acid), high amounts of anthracene and benzo[a]pyrene, 80% and 97%, respectively, were transformed under optimal conditions of 60°C, pH 5, and 5 mmol l(-1) CuCl(2) after a 24-h incubation period. Other PAHs including fluorene, acenaphthylene, phenanthrene, and benzo[a]anthracene were also oxidized by the crude CueO. These findings indicated the potential application of prokaryotic laccases in enhancing the mineralization of benzo[a]pyrene by PAH-degrading bacteria.     

Predicting the Efficacy of Polycyclic Aromatic Hydrocarbon Bioremediation in Creosote-Contaminated Soil Using Bioavailability Assays

Nonexhaustive extraction (propanol, butanol, hydroxypropyl-β-cyclodextrin [HPCD]), persulfate oxidation and biodegradability assays were employed to determine the bioavailability of polycyclic aromatic hydrocarbons (PAHs) in creosote-contaminated soil. After 16 weeks incubation, greater than 89% of three-ring compounds (acenaphthene, anthracene, fluorene, and phenanthrene) and 21% to 79% of four-ring compounds (benz[a]anthracene, chrysene, fluoranthene, and pyrene) were degraded by the indigenous microorganisms under biopile conditions. No significant decrease in five- (benzo[a]pyrene, benzo[b+k]fluoranthene) and six-ring compounds (benz[g,h,i]perylene, indeno[1,2,3-c,d]pyrene) was observed. Desorption of PAHs using propanol or butanol could not predict PAH biodegradability: low-molecular-weight PAH biodegradability was underestimated whereas high-molecular-weight PAH biodegradability was overestimated. Persulfate oxidation and HPCD extraction of creosote-contaminated soil was able to predict three- and four-ring PAH biodegradability; however, the biodegradability of five-ring PAHs was overestimated. These results demonstrate that persulfate oxidation and HPCD extraction are good predictors of PAH biodegradability for compounds with octanol-water partitioning coefficients of < 6.     

Oxidative enzymes possess catalytic activity in systems with ionic liquids

Oxidative enzymes, laccase C from Trametes sp. and horseradish and soybean peroxidases, catalyzed oxidation reactions in systems with ionic liquids whose content varied from several volume percent to almost total non-aqueous ionic liquids. Similar to the effects produced by standard organic solvents used in non-aqueous enzymology, catalytic activity of the enzymes was decreased by adding a water-miscible ionic liquid, 4-methyl-N-butylpyridinium tetrafluoroborate, or by suspending the enzyme in a water-immiscible ionic liquid, 1-butyl-3-methylimdizaolium hexafluorophosphate. For the oxidation of anthracene, catalyzed by laccase C and assisted by a number of mediators, addition of 4-methyl-N-butylpyridinium tetrafluoroborate, instead of tert-butanol, increased the yield of the oxidation product several-fold.     

The degradation of three-ringed polycyclic aromatic hydrocarbons by wood-inhabiting fungus Pleurotus ostreatus and soil-inhabiting fungus Agaricus bisporus

The degradation of two isomeric three-ringed polycyclic aromatic hydrocarbons by the white rot fungus Pleurotus ostreatus D1 and the litter-decomposing fungus Agaricus bisporus F-8 was studied. Despite some differences, the degradation of phenanthrene and anthracene followed the same scheme, forming quinone metabolites at the first stage. The further fate of these metabolites was determined by the composition of the ligninolytic enzyme complexes of the fungi. The quinone metabolites of phenanthrene and anthracene produced in the presence of only laccase were observed to accumulate, whereas those formed in presence of laccase and versatile peroxidase were metabolized further to form products that were further included in basal metabolism (e.g. phthalic acid). Laccase can catalyze the initial attack on the PAH molecule, which leads to the formation of quinones, and that peroxidase ensures their further oxidation, which eventually leads to PAH mineralization.A. bisporus, which produced only laccase, metabolized phenanthrene and anthracene to give the corresponding quinones as the dominant metabolites. No products of further utilization of these compounds were detected. Thus, the fungi's affiliation with different ecophysiological groups and their cultivation conditions affect the composition and dynamics of production of the ligninolytic enzyme complex and the completeness of PAH utilization.     

Purification and characterization of a thermostable laccase with unique oxidative characteristics from <Emphasis Type="Italic">Trametes hirsuta</Emphasis>

A laccase was purified from Trametes hirsuta. This laccase was classified as a “white” or “yellow” laccase. pH 2.4 was optimal for the oxidation of ABTS and pH 2.5 for DMP. DMP oxidation was optimal at 85°C. The half-life of this laccase was 70 min at 75°C, and 5 h at 65°C. Non-phenolic dyes, such as Methyl Red, were oxidized by purified laccase without mediators. The enzyme was not inhibited by Cu²⁺, Mn²⁺, or EDTA. These are atypical laccase characteristics that make it a good candidate for theoretical and applied research.     

Detoxification of polycyclic aromatic hydrocarbons by fungi

Summary The polycyclic aromatic hydrocarbons (PAHs) are a group of hazardous environmental pollutants, many of which are acutely toxic, mutagenic, or carcinogenic. A diverse group of fungi, includingAspergillus ochraceus, Cunninghamella elegans, Phanerochaete chrysosporium, Saccharomyces cerevisiae, andSyncephalastrum racemosum, have the ability to oxidize PAHs. The PAHs anthracene, benz[a]anthracene, benzo[a]pyrene, fluoranthene, fluorene, naphthalene, phenanthrene, and pyrene, as well as several methyl-, nitro-, and fluoro-substituted PAHs, are metabolized by one or more of these fungi. Unsubstituted PAHs are oxidized initially to arene oxides,trans-dihydrodiols, phenols, quinones, and tetralones. Phenols andtrans-dihydrodiols may be further metabolized, and thus detoxified, by conjugation with sulfate, glucuronic acid, glucose, or xylose. Although dihydrodiol epoxides and other mutagenic and carcinogenic compounds have been detected as minor fungal metabolites of a few PAHs, most transformations performed by fungi reduce the mutagenicity and thus detoxify the PAHs.     

Laccase-mediated degradation and reduction of toxicity of the postharvest fungicide imazalil

The degradation of the postharvest fungicide imazalil by the oxidative enzyme laccase was investigated in the presence of a reaction mediator (a laccase/mediator system). Of various natural mediators tested, imazalil was degraded most effectively in the presence of 4-hydroxybenzoic acid while the absence of a mediator resulted in no degradation. In this system, the mediator compound was oxidized by laccase and then the oxidized mediator oxidized imazalil. The study on the cytotoxicity of the degraded-imazalil solution against mouse fibroblast L929 cells revealed toxicity of 4-hydroxybenzoic acid. The cytotoxic study also suggested that instead of 4-hydroxybenzoic acid, the used of methyl 4-hydroxybenzoate as the reaction mediator reduced the intrinsic cytotoxicity of imazalil.     

Mediator facilitated,laccase catalysed oxidation of granular potato starch and the physico-chemical characterisation of the oxidized products

The primary hydroxyl groups in potato starch were selectively oxidized to the corresponding aldehyde and carboxylic acid functionalities by mediators like TEMPO, using laccase from fungi as catalytic oxidant and oxygen as the primary oxidant. Oxidized starch products with degree of substitution (DS_CHO ranging from 0.16 to 16.4/100AGU and DS_COOH from 0.01 to 3.71 carboxyl groups/100AGU) were obtained with mediator facilitated enzymatic oxidation. Maximum conversion of the primary alcohol group was obtained at a pH of 5, with TEMPO as mediator, under oxygen bubbling and two step administration of Trametes versicolor laccase (200 + 200 nkat/g of starch). The oxidized products were characterised by IR spectroscopy, XRD and thermal studies. In the oxidized samples, the larger starch granules exhibited cracks and fractures in comparison to the smaller granules which were relatively unaffected, as observed from the microstructural studies.     

Characterization of fluoranthene- and pyrene-degrading bacteria isolated from PAH-contaminated soils and sediments

Sixteen environmental samples, from the United States, Germany and Norway, with histories of previous exposure to either creosote, diesel fuel or coal tar materials, were screened for bacteria which could degrade high molecular weight (HMW) polycyclic aromatic hydrocarbons (PAHs). A modified version of the spray plate technique was used for the isolations. Using fluoranthene (FLA) and pyrene (PYR) as model HMW PAHs, we isolated 28 strains on FLA and 21 strains on PYR. FLA degraders were defined as able to grow on FLA but not PYR. PYR degraders grew on both PAHs. All PYR degraders were found to be Gram-positive and all FLA degraders were Gram-negative. GC-FAME analysis showed that many of the PYR degraders were Mycobacterium spp and many of the FLA degraders were Sphingomonas spp. Comparison of the metabolic characteristics of the strains using the spray plate technique and direct growth studies revealed that more than half of the FLA degraders (59%) were able to cometabolize PYR (ie, they produced clearing zones or colored metabolites on spray plates but did not grow on the PAH) and the ability of many of these strains to cometabolize fluorene, anthracene, benzo[b]fluorene, benzo[a]anthracene and benzo[a]pyrene was significantly affected by pre-exposure to phenanthrene. Studies on the metabolic products produced from PYR cometabolism by strain EPA 505 suggested the possibility of attack at two different sites on the PYR molecule. However, the inability to derive degradable carbon from initial opening of one of the PYR rings probably accounted for the lack of growth on this PAH by the FLA-degrading strains. The PYR degraders on the other hand, were less able to cometabolize HMW PAHs, even following pre-exposure to PHE. Characterization of the FLA degradation pathway for several of the Sphingomonas isolates indicated oxidation and ring opening through to acenaphthenone as the principle metabolite. Strain CO6, however, also oxidized FLA through fluorenone, suggesting a dual attack on the FLA molecule, similar to that observed by others in Mycobacterium spp. Journal of Industrial Microbiology & Biotechnology (2000) 24, 100–112. Received 01 May 1999/ Accepted in revised form 01 November 1999     

Integrated Approach for Polycyclic Aromatic Hydrocarbon Solubilization from the Soil Matrix to Enhance Bioremediation

Polycyclic aromatic hydrocarbons (PAHs) are known to be toxic to living organisms and have been identified as carcinogenic. In this study, a pathway of surfactant flushing, chemical oxidation, and biological treatment is proposed to remediate the soils polluted with the hydrophobic PAHs. Different surfactants such as Tween 80, Brij 35, sodium dodecyl sulfate (SDS), and polyethylene glycol (PEG) 6000 were tested in order to increase the PAH solubilization from the soil matrix. The maximum desorption efficiency of naphthalene and anthracene were found to be 56.5% and 59%, respectively, when Brij and SDS were used. The soluble PAH in the aqueous phase was amended with sodium thiosulfate (3%) to oxidize the PAH into a more bioavailable form. The chemical oxidation with subsequent biodegradation by Pseudomonas aeruginosa exhibited the relatively high PAH degradation rate (1.24 times higher) when compared with chemical oxidation alone. These results display the efficiency of chemical pretreatment of PAH-contaminated soil for improved bioremediation.