Involvement of Tetrahymena pyriformis and selected fungi in the elimination of anthracene, and toxicity assessment of the biotransformation products.

Anthracene (AC) is a non-mutagenic and non-carcinogenic, low-molecular-weight polycyclic aromatic hydrocarbon present in the environment. Its toxicity can be dramatically increased after solar-light exposure. Biotransformation capacities of AC by Tetrahymena pyriformis and a selection of eight micromycetes were studied, and the ability of these microorganisms to detoxify the polluted ecosystems was assessed. We showed that T. pyriformis was able to accumulate high amounts of AC without any transformation. In contrast, the fungi Cunninghamella elegans, Absidia fusca, Absidia cylindrospora, Rhodotorula glutinis, and Aspergillus terreus were able to transform AC with a high efficiency. Cytotoxicity assays conducted on HeLa cells and T. pyriformis showed that crude extract from A. fusca culture medium obtained after AC biotransformation was not toxic. For A. fusca and A. cylindrospora, 1-4 dihydroxyanthraquinone was shown to be the major product during the biotransformation process. This compound seemed to be a dead-end metabolite at least for the Absidia strains. The cytotoxicity of 1-4 dihydroxyanthraquinone was higher than that of AC to T. pyriformis but lower to HeLa cells. On the whole our results showed that the microorganisms studied were all able to decontaminate an AC-polluted ecosystem, either by accumulating or transforming the compound. A possible detoxification process resulting from AC biotransformation can be considered only using the human cell model.

Fungal bioconversion of 2,4-dichlorophenoxyacetic acid (2,4-D) and 2,4-dichlorophenol (2,4-DCP).

Ninety strains of fungi from the collection of our mycology laboratory were tested in Galzy and Slonimski (GS) synthetic liquid medium for their ability to degrade the herbicide 2,4-dichlorophenoxyacetic acid (2,4-D) and its by-product, 2,4-dichlorophenol (2,4-DCP) at 100 mg l(-1), each. Evolution of the amounts of each chemical in the culture media was monitored by HPLC. After 5 days of cultivation, the best results were obtained with Aspergillus penicilloides and Mortierella isabellina for 2,4-D and with Chrysosporium pannorum and Mucor genevensis for 2,4-DCP. The data collected seemed to prove, on one hand, that the strains responses varied with the taxonomic groups and the chemicals tested, and, on the other hand, that 2,4-D was less accessible to fungal degradation than 2,4-DCP. In each case, kinetics studies with the two most efficient strains revealed that there was a lag phase of 1 day before the onset of 2,4-D degradation, whereas there was none during 2,4-DCP degradation. Moreover, 2,4-DCP was detected transiently during 2,4-D degradation. Finally, M. isabellina improved its degradation potential in Tartaric Acid (TA) medium relative to GS and Malt Extract (ME) media.

Assessment of anthracene toxicity toward environmental eukaryotic microorganisms: Tetrahymena pyriformis and selected micromycetes.

The toxicity of anthracene, a nonmutagenic, noncarcinogenic, low-molecular-weight polycyclic aromatic hydrocarbon present in the environment, was assessed using a ciliated protozoan, Tetrahymena pyriformis, and a selection of 10 micromycetes from different taxonomic groups living in two environmental compartments, namely aquatic and soil ecosystems. With T. pyriformis, a concentration-dependent inhibitory effect was shown on the cell proliferation rate, IC(50) = 33.40+/-4.84 mg/L (kinetic method). Phagocytosis of nonsoluble anthracene was confirmed by the presence of digestive vacuoles with photon microscopy. In fungi, anthracene did not exhibit a fungicide effect but showed a fungistatic action. Except for Absidia fusca and Cladosporium herbarum, the micromycetes showed a concentration-dependent decrease in growth. From IC(50) values determined by endpoint or kinetic methods, Rhodotorula glutinis and all of the Ascomycotina (except for Penicillium chrysogenum) were the most sensitive species, while Phanerochaete chrysosporium, P. chrysogenum, and the two Deuteromycotina were more resistant to anthracene. Our discussion focuses on the evaluation of toxicity by the two methods used for the calculation of the IC(50) values (endpoint and kinetic), the advantages of studying growth by a kinetic method (the dynamic aspect), and a comparison of toxicity to T. pyriformis and the different micromycetes.

Biodegradation capability of Absidia fusca Linnemann towards environmental pollutants.

The purpose of this work was to study the bioremediation capability of Absidia fusca Linnemann (Zygomycete) towards different classes of xenobiotics (lignin-derived compounds, chloroaromatic compounds, polycyclic aromatic hydrocarbons) the presence of which in contaminated soils, water and sediments poses a significant risk to the environment and human health. Two strains from different origins were compared. One was from an official collection and grown in non-inducing conditions, while the other was isolated during the course of the survey of fungal flora in a polluted soil from Annaba (Algeria). All data were analyzed and results validated via a statistical treatment. We showed the effect of the factors studied (origin of the strain, xenobiotic) but also the interactions between these factors. The strain of A. fusca isolated from a polluted soil was able to efficiently degrade most of the xenobiotics tested, particularly: pentachlorophenol, phenol, catechol, guaiacol and ferulic acid. This property also existed in the other strain but at a very low level.

Removal of herbicides from liquid media by fungi isolated from a contaminated soil.

Fungi were isolated from soil samples corresponding to pesticide-contaminated soil (CS) and noncontaminated soil (NCS) in the Annaba vicinity (Algeria) and identified. The number of isolates obtained from CS and NCS were 263 and 288, respectively. The most frequent species (Aspergillus fumigatus, A. niger, A. terreus, Absidia corymbifera, and Rhizopus microsporus var microsporus) were not sensitive to the pesticides. The growth of the genus Trichoderma was inhibited by the pesticides, while genera Absidia and Fusarium were stimulated. The 53 species isolated were assayed for their ability to remove metribuzin from liquid medium. Only Botrytis cinerea from NCS and Sordaria superba and Absidia fusca from CS removed more than 50% of the compound after 5 d. Metamitron was very resistant. Among the 21 species tested, only Alternaria solani (from NCS), Drechslera australiensis (from CS and NCS), and Absidia fusca (from CS) reduced the concentration in the medium more than 10% (10-16%). Twelve species were grown with linuron, seven of them were inefficient in removing this compound. The two strains of Sordaria macrospora yielded 22 to 25% depletion, while Botrytis cinerea depleted linuron almost completely. Among the 31 species assayed for their ability to eliminate metobromuron, Botrytis cinerea (from CS and NCS) depleted almost completely the chemical from the medium. Rhizopus oryzae and Absidia fusca from CS removed 40 and 47% of the compound, respectively. No systematic relationships were observed between the soil contamination and herbicide elimination capacities of soil fungi. Absidia fusca and Botrytis cinerea were particularly interesting for bioremediation purposes because they were able to transform efficiently three of the four compounds assayed.

Biodegradation of anthracene and fluoranthene by fungi isolated from an experimental constructed wetland for wastewater treatment.

Pilot-scale constructed wetlands were used to treat water contaminated by polycyclic aromatic hydrocarbons (PAHs), particularly fluoranthene, and the possible role of fungi present in these ecosystems was investigated. A total of 40 fungal species (24 genera) were isolated and identified from samples (gravel and sediments) from a contaminated wetland and a control wetland. All of them were assayed for their ability to remove anthracene (AC) and fluoranthene (FA) from liquid medium. FA was degraded efficiently by 33 species while only 2 species were able to remove AC over 70%. A selection of 10 strains of micromycetes belonging to various taxonomic groups was further investigated for FA and AC degradation, toxicity assays and phenoloxidases (POx) detection. Interesting and not previously reported species were revealed (Absidia cylindrospora, Cladosporium sphaerospermum, and Ulocladium chartarum). They were all able to highly degrade the PAH-model compounds chosen. An interesting inducibility was noted for Ulocladium chartarum. Degradative ability of fungi was not related to their extracellular POx activity. This study may contribute to the improvement of constructed wetlands for water treatment, which may be enriched in efficient fungi.

Biodegradation of pyrene by sediment fungi.

Micromycetes were isolated from PAHS-contaminated sediment and identified. They were investigated for pyrene degradation (10 mg l-1) in liquid synthetic medium for two days. Among the 41 strains isolated, 10 highly degraded pyrene (> 2.4 mg g-1 dry weight): two Zygomycetes (Mucor racemosus, M. racemosus var. sphaerosporus), 6 Deuteromycetes (Gliocladium virens, Penicillium simplicissimum, P. janthinellum, Phialophora alba, P. hoffmannii, Trichoderma harzianum), a Dematiaceae (Scopulariopsis brumptii) and a Sphaeropsidale (Coniothyrium fuckelii). Zygomycetes appeared as one of the most efficient taxonomic groups, especially with Mucor racemosus. Penicillium crustosum was the only strain that did not degrade pyrene. Among the 10 fungi which were performant for pyrene degradation, nine were not yet reported in the literature and showed a real value for PAH remediation.

Exserohilum israeli, a new species isolated from soil from Timna Park (Israel), and its physiological properties.

Fungi isolated from soil in Timna Park (Israel) were found to belong to a new species of Exserohilum for which the name Erserohilum israeli sp. nov. is proposed. The main physiological properties of members of this species are discussed and the influence of temperature and copper concentrations on the growth and morphology of the fungus were investigated.

Antimicrobial and antitumor activities of mycosporulone.

The conditions for optimal production of mycosporulone (1) are given. Its cytotoxic, antimicrobial, and antitumor activities are described. The biological activities of 1 were compared with those of known antibacterial, antifungal, and antitumor agents. The compound was particularly active against Pseudomonas aeruginosa and Staphylococcus aureus (resistant to penicillin). Compound 1 was not toxic to normal human cells (MRC(5)), although it exhibited cytotoxic activity against the human tumor cell lines MDA-MB 231 and PC(3) and the murine L-1210 leukemia cell line.

Effects of culture parameters on the degradation of 2,4-dichlorophenoxyacetic acid (2,4-D) and 2,4-dichlorophenol (2,4-DCP) by selected fungi. Groupe pour l'Etude du Devenir des Xénobiotiques dans l'Environnement (GEDEXE).

In order to enhance 2,4-D and 2,4-DCP degradation by four selected fungi (Cunninghamella elegans, C. echinulata, Rhizoctonia solani and Verticillium lecanii), three culture parameters (initial chemical concentration, amounts of glucose and nitrogen) were varied. The levels of both xenobiotics in the culture media were monitored by HPLC analysis after five days of cultivation. The best results were obtained at low initial concentration (20 mg.L-1 vs 100) and with low amounts of glucose (5 g.L-1 vs 10) and nitrogen (2.4 mM vs 24). When these two elements were lacking from the culture media, biodegradation was not suppressed, but took place to a lesser extent. Thus, initial chemical concentration and amounts of carbon and nitrogen, in the culture medium, were shown to strongly influence the extent of 2,4-D and 2,4-DCP removal by fungi.

Degradation of three phenylurea herbicides (chlortoluron, isoproturon and diuron) by micromycetes isolated from soil.

As part of a study conducted on the fate of xenobiotics in the environment, a selection of 100 strains of micromycetes (Ascomycetes, Basidiomycetes and Yeasts) have been cultivated in liquid synthetic medium with 3 phenylurea herbicides: chlortoluron and isoproturon (100mg L-1) and diuron (20mg L-1). While 17 strains depleted isoproturon over 50% only 4 depleted diuron and 2 chlortoluron at the same level. The best results were obtained with Bjerkandera adusta and Oxysporus sp which were the most efficient towards the 3 substrates. After 2 weeks Bjerkandera adusta depleted chlortoluron 98%, diuron 92% and isoproturon 88%.

Degradation of phenolic and chloroaromatic compounds by Coprinus spp.

Three species of Coprinus: C. sp, C. cinereus and C. micaceus were compared on solid media for some aspects of their physiological behaviour and cultural requirements (temperature, pH, substrate). Constitutive extracellular enzymatic activities were also determined. The Coprinus spp. exhibited different physiological and cultural features. Cultures of the 3 Coprinus species in synthetic liquid medium showed an efficient degradation of phenolic lignin model compounds (catechol, ferulic acid, guaiacol, phenol, protocatechuic acid syringic acid and vanillic acid) and pentachloronitrobenzene, while pentachlorophenol was not metabolized after 5 days perhaps because of a strong adsorption on mycelial biomass. It was suggested that phenoloxidases were not necessarily required for the metabolization of these compounds. Coprinus species may share a common degrading system for monomeric phenolic and chloroaromatic compounds.

Soil mycoflora from the Dead Sea Oases of Ein Gedi and Einot Zuqim (Israel).

Samples were taken from the top 10 cm of soils from 24 points in the Ein Gedi area. Among 329 isolates, 142 species were identified: 11 genera of ascomycetes, one genus of coelomycetes, 28 genera of hyphomycetes, 7 genera of zygomycetes and one yeast, in addition to some unidentified basidiomycetes. The hyphomycetes were represented by 17 dematiaceous, 9 mucedinaceous and two tuberculariaceous. Melanconiaceous and stilbellaceous genera were not found. Two new varieties of Microascus recently described were reisolated. No strict thermophiles or halophiles were obtained. There is apparently no very characteristic or specific fungal flora of the Dead Sea Oases although it was different from that found in the desert soil surrounding this area.

Fungal microflora biodiversity as a function of pollution in Oued Sebou (Morocco).

An inventory of the fungal microflora present in sediments collected at 11 sites along Oued Sebou has been established. The influence of some climatic parameters (temperature, rainfall) and of pollution on the composition of the fungal communities has been questioned. Emericella nidulans, Eurotium amstelodami, Neosartotya fisheri var. glabrum (Ascomycetes), Dreschslera biseptata (Dematiaceae), Penicillium citrinum (Mucedinaceae) and Phoma sp. (Sphaeropsidale) can be considered as strains sensitive to pollution. Penicillium janthelinum, Trichoderma koningii (Mucedinaceae) and Candida albicans (Yeast) were found only at the most polluted sites; Talaromyces flavus (Ascomycete) Rhodotorula rubra, Saccharomyces cerevisiae (Yeasts) and Fusarium oxysporum (Tuberculariales) were more often found at heavily polluted sites than at lightly polluted ones.

Exserohilum sodomii, a new species isolated from soil near the Dead Sea (Israel).

Exserohilum sodomii sp. nov., is described. This new species was isolated from a soil sample from the Dead Sea surroundings. Its main physiological properties, as well as the influence of temperature and salts concentration in the culture medium on growth and morphology of the fungus were investigated and discussed.

Biodegradation of three substituted phenylurea herbicides (chlortoluron, diuron, and isoproturon) by soil fungi. A comparative study.

As a part of a study conducted on the fate of xenobiotics in the environment, a selection of 90 strains of micromycetes, mostly isolated from soil and belonging to various taxonomic groups, have been cultivated in liquid synthetic medium with chlortoluron (100 mg.L-1), diuron (20 mg.L-1), and isoproturon (100 mg.L-1) for 5 days. Evaluation of the chemicals in the culture media was made by HPLC. Our results show a wide variation not only with taxonomic groups but also with the species, and with the tested chemicals. On the whole, 4, 7, and 11% of the strains depleted respectively chlortoluron, diuron, and isoproturon, at 50% or over. Rhizoctonia solani was the only strain that depleted each of the 3 substituted phenylureas over 70%, nevertheless, the growth of this fungus was slightly inhibited by diuron. The very fast disappearance of the chemicals from the culture media was only due to biotic phenomena, as no adsorption occurred on the fungal biomass. So, depletion translated a real biodegradation of the tested substrates.

Influence of environmental adaptation on the sensitivity of micromycetes to pentachlorophenol toxicity in microcosms.

The search for fungal strains capable of pentachlorophenol degradation led to the isolation and identification of 98 different micromycetes. When these strains were submitted to toxicity tests in aquatic microcosms at concentrations of 10 mgL-1 and 100 mgL-1 PCP, inhibition of growth was 57% and 36% respectively of the total isolated and identified strains. Among the strains inhibited at 10 mgL-1, 6 can serve as bioindicators of PCP pollution while the strains resistant can be regarded as potential PCP biodegraders. It was confirmed that fungal strains isolated from sites contaminated by chemically different products manifest different levels of sensitivity to PCP toxicity and probably different biodegradation potentials.

Evaluation of acrodontiolamide, a chlorinated compound produced by Acrodontium salmoneum de Hoog for cytotoxicity and antimicrobial activity.

A new antifungal compound has been isolated from the culture medium of Acrodontium salmoneum de Hoog. Its structure was previously elucidated and was named acrodontiolamide. However, this compound is not characteristically produced by the genus Acrodontium, it is rather a feature of one isolate of A. Salmoneum coming from the soil of the grotto of La Pierre Saint Martin (France). Production, purification, cytotoxicity and antimicrobial activities of acrodontiolamide are described. Concerning microorganisms, inhibitory activity seems to be specifically restricted to phytopathogenic and entomapathogenic fungi. Acrodontiolamide is not cytotoxic to either normal human cultured cells or tumor cells.

Comparison of the toxicity of various lignin-related phenolic compounds toward selected fungi perfecti and fungi imperfecti.

A selection of 88 micromycetes belonging to various taxonomic groups was grown on malt-agar medium containing different lignin-related phenolic compounds (ferulic, protocatechuic, syringic and vanillic acids, catechol, guaiacol, and phenol) at concentrations ranging from 100 to 1000 mg/liter. Growth and morphological changes were recorded after 15 days of incubation. Behavior of fungi in contact with the different phenolics were compared. Some strains were very sensitive and might constitute specific or general bioindicators of pollution. The relationships between origin of isolates or constitutive phenoloxidase activity and resistance or sensitivity of fungi to phenolics were researched. No evidence of implication of these two parameters was found.

Biological activity of a heptaketide metabolite from Pleiochaeta setosa.

An uncommon heptaketide metabolite, setosol (2,8-dimethyl-4 methoxy-6,10,11-trihydroxy-benzo-oxaonin), was isolated from a liquid culture filtrate of the fungus Pleiochaeta setosa. The biological activity of the molecule was studied by using 12 microbial strains consisting of three bacteria, three yeasts and six fungi. The level of activity was compared with those of known antibiotics and antifungal agents. The metabolite exhibited antifungal and antibiotic activity against Drechslera oryzae, Gerlachia oryzae, Pyricularia oryzae, Cryptococcus neoformans, and Staphylococcus aureus. The acetylated derivative of setosol did not inhibit the growth of any of the target pathogens. Phytotoxicity studies on whole lupine leaves show that setosol is implicated in the pathogenesis of the brown spot disease of lupines since artificial inoculation of the leaves with the metabolite provoked lesions similar to the characteristic brown spots and lesions on lupine leaves infected by the fungus.