Fungal Diversity and Use in Decomposition of Environmental Pollutants

This article presents a critical review of the actual state of fungal activities on environmental pollutants, fungal diversity, the use of fungi in the degradation of chemical pollutants, enzyme degrading systems and perspectives on the use of fungi in bioremediation and unexplored research. The ability of fungi to transform or metabolize chemical pollutants has received much attention due to environmental persistence and chemical toxicity. The fungal degradation of xenobiotics is looked upon as an effective method of removing these pollutants from the environment by a process which is currently known as bioremediation. This review summarizes information from fundamental works that have revealed that a wide variety of fungi are capable of degrading an equally wide range of toxical chemical. The capacity of non-ligninolytic and ligninolytic fungi in the bioremediation of polycyclic aromatic hydrocarbon (PAHs), benzene-toluene-ethylbenzene-xylene (BTEX), chlorophenols, polychlorinated biphenyl, munitions waste and pesticides have been discussed. Besides this, several extracellular enzymes are involved in the metabolism of xenobiotic compounds as well as other factors related to these processes.     

Studies on the role of proteases in the white-rot fungus Trametes versicolor: effect of PMSF and chloroquine on ligninolytic enzymes activity

In the present study we investigated the possibility of proteinases, intracellular and extracellular, being involved in the regulation of ligninolytic activities in cultures of Trametes versicolor during the shift from primary growth (i.e. trophophase) to idiophase triggered by nitrogen or carbon starvation. These studies were performed using specific inhibitors added to the cultures of T. versicolor. Addition of PMSF (irreversible inhibitor of serine proteinases) or chloroquine (the lysosomotropic agent inhibiting intralysosomal degradation of proteins) revealed distinct differences in the activity of ligninolytic enzymes between nutrient-deprived and non-starved cultures. The addition of PMSF during the transfer of mycelia to the nutrient limited media significantly enhanced the activities of laccase (2-7-fold) and of unspecified peroxidases (2-4-fold). The activity of lignin peroxidase decreased with PMSF, both in tropho- and in idiophasic cultures. The enhanced activities of laccase and general peroxidases (horseradish peroxidase-like, HRP-like) were accompanied by markedly altered patterns of both intracellular and extracellular proteolytic activities revealed by electrophoretic analysis with polyacrylamide gels containing the copolymerized substrate (haemoglobin or gelatin, respectively). The experiments with chloroquine added to nutrient-deprived cultures showed that inhibition of vacuolar proteolysis resulted in lowered activities of laccase and peroxidase. Electrophoretic analysis revealed altered patterns of intracellular proteinases upon chloroquine addition to nutrient-starved cultures. Moreover, chloroquine was found to enhance the activity of proteases secreted in carbon-starved cultures. From the results it is concluded that both intracellular (including vacuolar) and extracellular proteases are involved in the regulation of laccase and peroxidase activity in cultures of T. versicolor under nutrient limitation.     

Fungal diversity and use in decomposition of environmental pollutants

This article presents a critical review of the actual state of fungal activities on environmental pollutants, fungal diversity, the use of fungi in the degradation of chemical pollutants, enzyme degrading systems and perspectives on the use of fungi in bioremediation and unexplored research. The ability of fungi to transform or metabolize chemical pollutants has received much attention due to environmental persistence and chemical toxicity. The fungal degradation of xenobiotics is looked upon as an effective method of removing these pollutants from the environment by a process which is currently known as bioremediation. This review summarizes information from fundamental works that have revealed that a wide variety of fungi are capable of degrading an equally wide range of toxical chemical. The capacity of non-ligninolytic and ligninolytic fungi in the bioremediation of polycyclic aromatic hydrocarbon (PAHs), benzene-toluene-ethylbenzene-xylene (BTEX), chlorophenols, polychlorinated biphenyl, munitions waste and pesticides have been discussed. Besides this, several extracellular enzymes are involved in the metabolism of xenobiotic compounds as well as other factors related to these processes.     

Metabolism of PAH by fungi and correlation with extracellular enzymatic activities

The activity to metabolize the polycyclic aromatic hydrocarbons (PAH) phenanthrene, anthracene, pyrene, fluorene and fluoranthene by Trametes versicolor, Pleurotus ostreatus (white rot fungi), Laetiporus sulphureus, Daedaela quercina, Flamulina velutipes (brown rot fungi), Marasmiellus sp. (litter decaying fungus) and Penicillium sp. M 1 (isolated from a PAH contaminated soil sample) were compared. Screening methods for the presence of exoenzymes (peroxidases, polyphenoloxidases, “radical generating” enzymes) were evaluated for their use in screenings for fungi degrading PAH. Laetiporus sulphureus and Penicillium sp. M 1 cometabolize several PAH with rates comparable to white rot fungi. In most of the cases the patterns of extracellular peroxidases indicate the potential of fungi to degrade PAH.     

Treatment of Colored Effluents with Lignin-Degrading Enzymes: An Emerging Role of Marine-Derived Fungi

Some of the industries that discharge highly colored effluents are paper and pulp mills, textiles and dye-making industries, alcohol distilleries, and leather industries. Terrestrial white-rot basidiomycetous fungi and their lignin-degrading enzymes laccase, manganese-peroxidase and lignin peroxidases are useful in the treatment of colored industrial effluents and other xenobiotics. Free mycelia, mycelial pellets, immobilized fungi or their lignin-degrading enzymes from terrestrial fungi have been reported in treatment of several effluents. Marine obligate or facultative (marine-derived) fungi may have unique properties but have not been explored sufficiently for this purpose. This article presents a critical review of bioremediation potential of such fungi and their lignin-degrading enzymes in comparison with the state-of-the-art in terrestrial white-rot fungi.     

Purification of a new manganese peroxidase of the white-rot fungus Irpex lacteus, and degradation of polycyclic aromatic hydrocarbons by the enzyme

The white-rot fungus Irpex lacteus has been reported to be an efficient degrader of polycyclic aromatic hydrocarbons, polychlorinated biphenyls and pentachlorophenol. The fungus produces ligninolytic enzymes laccase, lignin peroxidase and manganese peroxidase (MnP), the latter being the major one produced. MnP was purified using anion exchange and size exclusion chromatography. SDS-PAGE showed the purified MnP to be a monomeric protein of 37 kDa (37.5 kDa using MALDI-TOF) with an isoelectric point at 3.55. The pH optimum was relatively broad, from 4.0 to 7.0 with a peak at pH 5.5. Kinetic constants K(m) were 8 microM for H(2)O(2) and 12 or 31 microM for Mn(2+) depending on the substrate. The enzyme did not perform oxidation in the absence of H(2)O(2) or Mn(2+). MnP was active at 5-70 degrees C with an optimum between 50-60 degrees C. At temperatures above 65 degrees C the enzyme rapidly lost activity. Degradation of four representatives of PAHs (phenanthrene, anthracene, fluoranthene, and pyrene) was tested and the enzyme showed the ability to degrade them in vitro. Major degradation products of anthracene were identified. The results confirm the role of MnP in PAH degradation by I. lacteus, including cleavage of the aromatic ring.     

Enzyme-assisted bioremediation approach for synthetic dyes and polycyclic aromatic hydrocarbons degradation

Environmental protection from emerging pollutants has become a significant challenge for mankind as an increasing number of contaminants, including synthetic dyes and polycyclic aromatic hydrocarbons (PAHs), represent a serious risk to ecological and environmental balance. Most synthetic dyes have complex aromatic structures and are resistant to degrade by classical approaches, such as physical and chemical processes, including adsorption, chemical coagulation, flocculation, ion exchange, membrane separation, froth flotation, and reverse osmosis. Enzymes-assisted catalytic transformation of pollutants has become a potential alternative to classical methods because of their ability to react with complex compounds, a quick degradation rate, and producing less harmful by-products. Plant peroxidases, and microbial laccase and lignin-degrading peroxidases (manganese and lignin peroxidase) have gained significant attention for treating aromatic waste due to their capability of oxidizing and detoxifying a wide range of recalcitrant xenobiotics, including PAHs and synthetic dyes. Peroxidases being efficient biocatalysts detoxify an array of toxic compounds by simple free-radical mechanism resulting in the formation of oxidized and depolymerized products of significantly reduced toxicity. Moreover, it is an ecofriendly and economically favorable approach towards the biodegradation of recalcitrant and toxic industrial waste. Among microbial and plant peroxidases, bacterial enzymes have broad substrate specificity and can transform a wide range of recalcitrant substrates. Ligninolytic enzymes oxidize the aromatic ring into quinones and acids by producing free hydroxyl radicals instead of dihydrodiols and mineralize aromatic hydrocarbon in combination with cytochrome P450, monooxygenases, and epoxide hydrolases. In the review, an attempt has been made to provide detailed knowledge about the availability of inexpensive peroxidases sources, their mechanism of action, and degradation potential. The present review summarizes the exploitation of peroxidases from plants, bacteria, and fungus (manganese peroxidase, lignin peroxidase, and laccases) for detoxification and degradation of textile dyes as well as PAHs. Conclusively, peroxidases have great potential to react with almost all classes of synthetic dyes and most PAHs due to broad substrate specificity and transformed them into less harmful metabolites.     

Biological functioning of PAH-polluted and thermal desorption-treated soils assessed by fauna and microbial bioindicators

A large number of soil bioindicators were used to assess biological diversity and activity in soil polluted with polycyclic aromatic hydrocarbons (PAHs) and the same soil after thermal desorption (TD) treatment. Abundance and biodiversity of bacteria, fungi, protozoa, nematodes and microarthropods, as well as functional parameters such as enzymatic activities and soil respiration, were assessed during a two year period of in situ monitoring. We investigated the influence of vegetation (spontaneous vegetation and Medicago sativa) and TD treatment on biological functioning. Multivariate analysis was performed to analyze the whole data set. A principal response curve (PRC) technique was used to evaluate the different treatments (various vegetation and contaminated vs. TD soil) contrasted with control (bare) soil over time. Our results indicated the value of using a number of complementary bioindicators, describing both diversity and functions, to assess the influence of vegetation on soil and discriminate polluted from thermal desorption (TD)-treated soil. Plants had an influence on the abundance and activity of all organisms examined in our study, favoring the whole trophic chain development. However, although TD-treated soil had a high abundance and diversity of microorganisms and fauna, enzymatic activities were weak because of the strong physical and chemical modifications of this soil.     

Lignin‐modifying enzymes in filamentous basidiomycetes – ecological,functional and phylogenetic review

Filamentous fungi owe powerful abilities for decomposition of the extensive plant material, lignocellulose, and thereby are indispensable for the Earth's carbon cycle, generation of soil humic matter and formation of soil fine structure. The filamentous wood‐decaying fungi belong to the phyla Basidiomycota and Ascomycota, and are unique organisms specified to degradation of the xylem cell wall components (cellulose, hemicelluloses, lignins and extractives). The basidiomycetous wood‐decaying fungi form brackets, caps or resupinaceous (corticioid) fruiting bodies when growing on wood for dissemination of their sexual basidiospores. In particular, the ability to decompose the aromatic lignin polymers in wood is mostly restricted to the white rot basidiomycetes. The white‐rot decay of wood is possible due to secretion of organic acids, secondary metabolites, and oxidoreductive metalloenzymes, heme peroxidases and laccases, encoded by divergent gene families in these fungi. The brown rot basidiomycetes obviously depend more on a non‐enzymatic strategy for decomposition of wood cellulose and modification of lignin. This review gives a current ecological, genomic, and protein functional and phylogenetic perspective of the wood and lignocellulose‐decaying basidiomycetous fungi. (© 2010 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)     

Activities of phenol oxidizing enzymes of ectomycorrhizal fungi in axenic culture and in symbiosis with Scots pine (Pinus sylvestris L.)

The ectomycorrhizal fungi Suillus granulatus and Paxillus involutus were grown in both liquid culture (LC) and in symbiosis with Pinus sylvestris seedlings under sterile conditions. In LC, the activities of tyrosinase, laccase and peroxidase, and also the oxidation rates of litter- and lignin-derived phenolics were determined. LC mycelia of S. granulatus showed intracellular tyrosinase, laccase, and peroxidase activities. In the culture fluid, extracellular laccase and tyrosinase activities were found. P. involutus predominantly produced intracellular laccase. LC mycelia of S. granulatus suspended in buffer oxidized a range of phenolic acids and mono- and diphenolic compounds by intracellular mechanisms more effectively than did mycelia of P. involutus. In addition, LC mycelia of S. granulatus oxidized p-cresol also by extracellular mechanisms. In symbiosis with Scots pine, S. granulatus and P. involutus increased the level of peroxidase in the fungus/root homogenate and in the nutrient solution of the mycorrhizal plants. Polyphenol oxidase activities were only found with S. granulatus as the mycorrhizal symbiont, and were possibly responsible for the high rate of p-cresol oxidation by this mycorrhizal association. The results suggest that fungal phenol oxidizing enzymes of ectomycorrhizas can substantially contribute to humification and detoxification processes in soil.     

Characterization of Rhodococcus opacus R7, a strain able to degrade naphthalene and o-xylene isolated from a polycyclic aromatic hydrocarbon-contaminated soil

Rhodococcus opacus R7 was isolated from a soil contaminated with polycyclic aromatic hydrocarbons for its ability to grow on naphthalene. The strain was also able to degrade o-xylene, the isomer of xylenes most recalcitrant to microbial degradation. The catabolic pathways for naphthalene and o-xylene were investigated by identification of metabolites in R. opacus R7 cultures performed with the two hydrocarbons and by evaluation of some enzymes involved in the metabolism of these compounds. 1,2-Dihydro-1,2-dihydroxynaphthalene, salicylic and gentisic acids were identified as metabolites in cultures exposed to naphthalene. This suggests that the degradation occurs through the dioxygenation of the aromatic ring with the formation of 1,2-dihydro-1,2-dihydroxynaphthalene, dehydrogenated to the corresponding 1,2-dihydroxy derivative which is further oxidized to salicylic acid, a key intermediate of naphthalene metabolism; this compound is converted to gentisic acid cleaved by a gentisate 1,2-dioxygenase. From R. opacus R7 cultures supplied with o-xylene, 2,3-dimethylphenol and 3,4-dimethylcatechol were observed. The pathway of o-xylene involves the monooxygenation of the benzene nucleus leading to dimethylphenol which is further metabolised to 3,4-dimethylcatechol, followed by a meta cleavage reaction, catalyzed by the catechol 2,3-dioxygenase. R. opacus R7 is the first strain thus far described both in Gram-negative and Gram-positive bacteria which has the ability to degrade both a polycyclic aromatic hydrocarbon such as naphthalene and a monocyclic aromatic hydrocarbon such as o-xylene.     

The enigmatic physiological roles of AhpCF,Gpx, Npr and Kat in peroxide stress response of Enterococcus faecium

In this work, the physiological roles of the primary peroxide scavenging activities of Enterococcus faecium AUS0004 strain were analysed. This healthcare-associated pathogen harbours genes encoding putative NADH peroxidase (Npr), alkyl hydroperoxide reductase (AhpCF), glutathione peroxidase (Gpx) and manganese-dependent catalase (Mn-Kat). Gene expression analyses showed that npr and kat genes are especially and significantly induced in cells treated with hydrogen peroxide (H₂O₂) and cumene hydroperoxide (CuOOH), which suggested an important function of these enzymes to protect E. faecium against peroxide stress. Mutants affected in one or several predicted anti-oxidative activities mentioned above showed that neither the peroxidases nor the catalase are implicated in the defence against peroxide challenges. However, our investigations allowed us to show that Npr is responsible for the degradation of approximately 45% of metabolically derived H₂O₂ which avoids accumulation of the peroxide to lethal concentrations.     

Invasion of wood by basidiomycetous fungi VI. Quantitative but not qualitative differences in the pathovirulence of pathogenic and saprophytic decay fungi

Wood dowels sterilely overgrown with test mycelia of pathogenic and saprophytic basidiomycetous wood-decay fungi, respectively, were inserted into drill holes in the stems of 3 hardwood (Fagus sylvatica, Betula verrucosa, Sorbus aucuparia) and 1 softwood tree species (Picea abies). The host trees 40-120 mm in diameter were part of the light-starved understory of a timber forest. Eighteen to 52 months after inoculation the trees were dissected and the colonized stemwood was microbio-logically examined. Extension growth and survival of the test mycelia in trees of known vitality were taken as a basis for fungal pathovirulence rating. Basidiomycetous wood-decay fungi reach at best the status of physiologically facultative pathogens. They only colonized trees of the lowest vitality classes in the understory. For the expected range of pathovirulence, an appropriate host tree to be chosen had a proper range of vitality which expressed itself in a crown volume of 5 to 70% that of the crown of a free-standing tree and a width of the current annual growth ring from 0.5 to 2.5 mm in dependence of the tree species. Increasingly moribund trees with low crown volumes and narrow growth rings favoured an unlimited mycelial expansion and survival, while in vital trees fungal expansion gradually came to a standstill within 18 to 52 months, frequently combined with an active killing of both the pathogenic and saprophytic mycelia. Development of pathogenic test mycelia much more depended on the choice of an appropriate host tree species than did development of saprophytic test mycelia. The stem colonization by several late saprophytes was nearly as extensive and durable as the stem colonization by notorious tree pathogens. Pathosism in wood-decay basidiomycetes appeared thus to be a quantitative rather than a qualitative feature. For the dieback of the basidiomycetous test mycelia in the live stem reasons such as wood substrate depletion or antagonistic microbial activities could be excluded. The dieback rate of the test mycelia however was clearly enhanced in trees of increasing vitality, and it was enhanced with prolonged host-fungus interaction times. In comparison with sound dominant trees, the endophyte incidence in the sapwood of moribund P. abies trees was not increased. At the instant of tree death or crown decapitation however a severe colonization with blue stain and soft rot fungi was observed that exerted considerable antagonism on test mycelia low in kratovirulence. The implication of this antagonistic action for the colonization of dying trees by pathogenic decay fungi are discussed.     

Ligninolytic enzymes from Ganoderma spp: Current status and potential applications

White-rot fungal species belonging to Ganoderma have long been used as medicinal mushrooms in many Asian countries. In recent years, however, attention is not just being paid to their pharmacological properties, but to their other potentially valuable features as well, including their secretion of enzymes which decompose lignin. The current literature regarding lignin-modifying enzymes from the genus Ganoderma, their potential uses, and the components, structures and processes of lignocellulose degradation are discussed. The ligninolytic enzymes from the genus Ganoderma, as well as the number of additional enzymes that participate in lignin degradation, are summarized; further, the potential applications of these enzymes are analyzed and probed in this article. This review will provide insight on the valuable applications of Ganoderma spp. and will serve as a useful reference on the use of lignocellulose degradation as a means of environmental protection.     

Soilborne filamentous fungi in Brazil

The Atlantic Rainforest is a Brazilian ecosystem that is being rapidly being destroyed, along with the abiotic and biotic factors present in it. Among the biotic factors, the fungi are found in the soil which, besides being of major importance in terms of ecological niches, also have broad and significant applications in biotechnology. In order to assess the biodiversity of these microorganisms in this type of ecosystem, the Banhado Grande region was chosen at the Jureia-Itatins Ecology Station, in the state of Sao Paulo, Brazil. Within this region, two areas were delimited for study, one covered with natural (primary) vegetation and the other containing vegetation that regenerated following the planting of rice crops, referred to here as secondary. Collection of compound soil samples were taken (depth 0-15 cm) over a period of two and a half years, with the litter first being removed, during dry/cold and humid/hot periods. After sifting the samples, they were appropriately processed using the serial dilution technique to isolate the fungi from the soil. Six different culture media were used, having pHs of 4.5, 7.0 and 9.0. Altogether, 1,211 strains were isolated, divided into the following groups: Hyphomycetes, the most abundant followed by Ascomycetes, Zygomycetes, Coelomycetes, and Oomycetes. From these, 112 species were identified, 8 down to the genus level, and those that did not produce conidia were grouped as Mycelia sterilia. Among the strains, 67 were cellulolytic, 32 originated solely in soil under natural vegetation, and 26 originated solely in soil under secondary vegetation.     

Spaltung des α-L-Arabinofuranosids, β-D-Glucopyranosids und β-Cellobiosids von 4-Nitrophenol durch Enzyme verschiedener Pilze — ein Beitrag in Verbindung mit Versuchen zur Steigerung der Selektivität der Tumortherapie

To carry out long-term experiments as part of a therapy concept of malignant tumours using inactive transport forms of cancerostatic substances and their specific cleavage in the acidic pH region of the tumours by application of extraneous enzymes, we require enzymes with similar catalytic and pharmacokinetic properties which differ from each other in immunological respect. In the search for such enzymes, the α-L-arabinofuranosidases from 12 different fungi, among them 9 basidiomycetes, were studied. The enzymes mentioned were demonstrable in all fungi. Optimum pH values ranged between 2.5 and 5.5. The Km values for the cleavage of α-L-arabinofuranoside were, in most cases, 0.5 to 1.8 moles · liter^?1 · 10^?3. With regard to pH dependence, the α-L-arabino-furanosidases of most of the fungi investigated proved adequate for the long-term trials envisaged. 4-nitrophenyl-β-D-glucopyranoside and -β-cellobioside were also cleaved by enzyme preparations of all the 11 fungi investigated. The β-D-glucopyranosidases showed a less favourable pH dependence than the α-L-arabinofuranosidases. The cleavage of 4-nitrophenyl-β-cellobioside, on the contrary, showed mostly a comparatively favourable pH dependence. On the basis of the coinciding optimal pH values and the occurrence of 4-nitrophenyl-β-D-glucopyranoside as an intermediate product in the cleavage of the corresponding cellobioside, we assume that both substrates are cleaved by β-glucosidase. Because the occurrence of the glucoside during the cleavage of cellobioside is undesirable for the therapeutic trial, a method is proposed for selection of an appropriate cellobioside splitting enzyme basing on the present studies and the relevant literature.     

Proteolytic activities in cultures of selected white-rot fungi

The presence of multiple intracellular and extracellular proteolytic activities in trophophasic (nutrientrich) and idiophasic (carbon-or nitrogen-starved) cultures of the white-rot fungi Trametes versicolor and Phlebia radiata was demonstrated by electrophoresis on polyacrylamide gels containing denatured haemoglobin as a substrate. In the trophophasic cultures of T. versicolor, seven electrophoretically distinguishable proteases were defined using mycelial extracts and six (three clear and three less intensive) of secreted proteases. For P. radiata eight bands of intracellular and five bands (one distinct and four less active) of extracellular proteolytic activities were detected. Gel electrophoresis revealed changes in patterns of secreted and mycelial proteinases upon carbon or nitrogen deprivation. The changes were seen both as an increase in activity of certain bands and as the appearance of new proteolytic bands. Specific activities of extracellular proteinases, assayed under idiophasic (—C or —N) conditions, increased 2—3 fold as compared to those upon nutrient sufficiency. These changes accompanied a shift to secondary metabolism and onset of ligninolytic activity.