Large-scale phenotyping of 1,000 fungal strains for the degradation of non-natural, industrial compounds.

Fungal biotechnology is set to play a keystone role in the emerging bioeconomy, notably to address pollution issues arising from human activities. Because they preserve biological diversity, Biological Resource Centres are considered as critical infrastructures to support the development of biotechnological solutions. Here, we report the first large-scale phenotyping of more than 1,000 fungal strains with evaluation of their growth and degradation potential towards five industrial, human-designed and recalcitrant compounds, including two synthetic dyes, two lignocellulose-derived compounds and a synthetic plastic polymer. We draw a functional map over the phylogenetic diversity of Basidiomycota and Ascomycota, to guide the selection of fungal taxa to be tested for dedicated biotechnological applications. We evidence a functional diversity at all taxonomic ranks, including between strains of a same species. Beyond demonstrating the tremendous potential of filamentous fungi, our results pave the avenue for further functional exploration to solve the ever-growing issue of ecosystems pollution.

Use of soil fungi in the biosorption of three trace metals (Cd, Cu, Pb): promising candidates for treatment technology?

Trace metal contamination is a widespread and complex environmental problem. Because fungi are capable of growing in adverse environments, several fungal species could have an interesting potential in remediation technologies for metal contaminated environments. This study proposes to test the ability to tolerate and biosorb three trace metals (Cd, Cu and Pb) of 28 fungal isolates collected from different soils. First, a tolerance assay in agar medium was performed. Each isolate was grown in the presence of Cd, Cu, and Pb at different concentrations. Then, we exposed each soil fungus to 50 mg L-1 of Cd, Cu, or Pb during 3 days in liquid medium. Parameters such as biomass production, pH, and biosorption were evaluated. The results showed that responses to metal exposure are very diverse even with fungi isolated from the same soil sample, or belonging to the same genera. Several isolates could be considered as good metal biosorbents and could be used in future mycoremediation studies. Among the 28 fungi tested, Absidia cylindrospora biosorbed more than 45% of Cd and Pb, Chaetomium atrobrunneum biosorbed more than 45% of Cd, Cu, Pb, and Coprinellus micaceus biosorbed 100% of Pb.

Comparison of tolerance and biosorption of three trace metals (Cd, Cu, Pb) by the soil fungus Absidia cylindrospora.

Trace metals cause deterioration of the soil and constitute a major concern for the environment and human health. Bioremediation could be an effective solution for the rectification of contaminated soils. Fungi could play an important role in biodegradation because of the morphology of their mycelium (highly reactive and extensive biological surface) and its physiology (high tolerance to many stresses, production of enzymes and secondary metabolites). Fungi can effectively biosequestrate, or biotransform many organic and inorganic contaminants into a non-bioavailable form. This experiment was designed to evaluate the tolerance and the biosorption abilities of the fungus Absidia cylindrospora against three trace metals: Cadmium (Cd), Copper (Cu), and Lead (Pb). Firstly, the tolerance of the strain was evaluated on metal-enriched malt extract agar (MEA). Secondly, the strain was exposed to trace metals, in a liquid malt extract medium. After 3 or 7 days of exposure, the quantities of absorbed and adsorbed metals were measured with Inductively Coupled Plasma-Optical Emission Spectrometry (ICP-OES). Biomass production and pH evolution were also evaluated during the test. Our experiment revealed differences between the three metals. In agar medium, Cd and Pb were better tolerated than Cu. In liquid medium, Cd and Pb were mostly absorbed whereas Cu was mostly adsorbed. A. cylindrospora biosorbed 14% of Cu, 59% of Pb and 68% of Cd when exposed for 3 days at 50 mg L-1.

Molecular screening of xerophilic Aspergillus strains producing mycophenolic acid.

Mycophenolic acid (MPA) is the fungal secondary metabolite displaying several biological properties. Up to now, screening of fungal strains producing MPA has mainly been the result of the search of this molecule in their culture medium by chemical methods. Here we developed a molecular approach by targeting the expression level of the MpaC gene encoding the polyketide synthase, one of the key enzymes involved in the MPA synthesis. Thirty xerophilic Aspergillus strains were identified using the RNA polymerase II subunit and the ß-tubulin genes. Seven Aspergillus species were evidenced. The expression level of the MpaC gene was quantified and compared to the MPA production rate. Only Aspergillus pseudoglaucus and all the eight strains of this species produced MPA. While the MpaC gene was not expressed or weakly expressed in the MPA non-producing strains, all the A. pseudoglaucus strains presented a high level of expression of this gene. The highest expression level of the MpaC gene among the MPA non-producing strains was significantly lower than the lowest expression level of this gene in the MPA producing strains. To our knowledge, this is the first study that demonstrates the effectiveness of molecular approach for the screening of MPA-producing species.

Autochthonous ascomycetes in depollution of polychlorinated biphenyls contaminated soil and sediment.

We investigated the capacity of a consortium of ascomycetous strains, Doratomyces nanus, Doratomyces purpureofuscus, Doratomyces verrucisporus, Myceliophthora thermophila, Phoma eupyrena and Thermoascus crustaceus in the mycoremediation of historically contaminated soil and sediment by polychlorinated biphenyls (PCBs). Analyses of 15 PCB concentrations in three mesocosms containing soil from which the fungal strains had previously been isolated, revealed significant PCB depletions of 16.9% for the 6 indicator PCBs (i-PCBs) and 18.7% for the total 15 PCBs analyzed after 6months treatment. The degradation rate did not statistically vary whether the soil had been treated with non-inoculated straw or colonized straw or without straw and inoculated with the consortium of the six strains. Concerning the sediment, we evidenced significant depletions of 31.8% for the 6 i-PCBs and 33.3% for the 15 PCB congeners. The PCB depletions affected most of the 15 PCBs analyzed without preference for lower chlorinated congeners. Bioaugmented strains were evidenced in different mesocosms, but their reintroduction, after six months treatment, did not improve the rate of PCB degradation, suggesting that the biodegradation could affect the bioavailable PCB fraction. Our results demonstrate that the ascomycetous strains potentially adapted to PCBs may be propitious to the remediation of PCB contaminated sites.

Potential of autochthonous fungal strains isolated from contaminated soils for degradation of polychlorinated biphenyls.

Up to now, most studies on polychlorinated biphenyl (PCB) bioremediation have examined the ability of model fungal strains to biodegrade PCBs. Yet, there is limited information concerning the potential of autochthonous filamentous fungal strains in the biodegradation of PCBs and their possible use in the environmental technologies. In this study, we investigated the capacity of autochthonous fungal strains in the biodegradation of PCBs by isolating 24 taxa from former industrial sites highly contaminated by PCBs. Microscopic and molecular analyses using the internal transcribed spacer (ITS) region revealed that the fungal strains belonged to the phyla Ascomycota (19 strains) and Zygomycota (five strains). The chromatography gas analysis revealed evidence of degradation of seven PCB congeners. With the exception of Circinella muscae which presented no degradation potential, the other fungal strains exhibited a rate of biodegradation ranging from 29 to 85 % after 7 d of incubation in liquid medium. Among these strains, Doratomyces nanus, Doratomyces purpureofuscus, Doratomyces verrucisporus, Myceliophthora thermophila, Phoma eupyrena, and Thermoascus crustaceus showed remarkable degradation ability (>70 %) regardless of the number of chlorine substituents on the biphenyl nucleus and a high tolerance towards PCBs. To our knowledge, this is the first study that demonstrates the ability of PCB degradation by these species and indicates the potential effectiveness of some autochthonous fungal strains in bioremediation systems.

Potentiality of the cox1 gene in the taxonomic resolution of soil fungi.

We explored the potential of the cox1 gene in the species resolution of soil fungi and compared it with the nuclear internal transcribed spacer (ITS) and small subunit (SSU)-rDNA. Conserved primers allowing the amplification of the fungal cox1 gene were designed, and a total of 47 isolates of Zygomycota and Ascomycota were investigated. The analysis revealed a lack of introns in >90% of the isolates. Comparison of the species of each of the six studied genera showed high interspecific sequence polymorphisms. Indeed, the average of nucleotide variations (4.2-11%) according to the genus, due mainly to the nucleotide substitutions, led to the taxonomic resolution of all the species studied regarding both ITS and SSU-rDNA, in which <88% were discriminated. The phylogenetic analysis performed after alignment of the cox1 gene across distant fungal species was in accordance with the well-known taxonomic position of the species studied and no overlap was observed between intra- and interspecific variations. These results clearly demonstrated that the cox1 sequences could provide good molecular markers for the determination of the species composition of environmental samples and constitute an important advance to study soil fungal biodiversity.

CE-SSCP and CE-FLA, simple and high-throughput alternatives for fungal diversity studies.

Fungal communities are key components of soil, but the study of their ecological significance is limited by a lack of appropriated methods. For instance, the assessment of fungi occurrence and spatio-temporal variation in soil requires the analysis of a large number of samples. The molecular signature methods provide a useful tool to monitor these microbial communities and can be easily adapted to capillary electrophoresis (CE) allowing high-throughput studies. Here we assess the suitability of CE-FLA (Fragment Length Polymorphism, denaturing conditions) and CE-SSCP (Single-Stranded Conformation Polymorphism, native conditions) applied to environmental studies since they require a short molecular marker and no post-PCR treatments. We amplified the ITS1 region from 22 fungal strains isolated from an alpine ecosystem and from total genomic DNA of alpine and infiltration basin soils. The CE-FLA and CE-SSCP separated 17 and 15 peaks respectively from a mixture of 19 strains. For the alpine soil-metagenomic DNA, the FLA displayed more peaks than the SSCP and the converse result was found for infiltration basin sediments. We concluded that CE-FLA and CE-SSCP of ITS1 region provided complementary information. In order to improve CE-SSCP sensitivity, we tested its resolution according to migration temperature and found 32 degrees C to be optimal. Because of their simplicity, quickness and reproducibility, we found that these two methods were promising for high-throughput studies of soil fungal communities.

Phylogenetic analysis of the Aspergillus niger aggregate in relation to feruloyl esterase activity.

Species of the Aspergillus niger aggregate are known to produce feruloyl esterases, enzymes involved in the degradation of cell wall polymers. However, species delineation is difficult in these fungi. We combined AFLP analysis with ITS rDNA and beta-tubulin sequencing to characterize the isolates of this aggregate in terms of feruloyl esterase production. A preliminary re-examination of isolates based on comparison of ITS rDNA and beta-tubulin sequences with those of typical taxa deposited in international collections led us to re-identify the isolates as members of the species A. niger, A. foetidus and A. tubingensis. Molecular clustering based on beta-tubulin data and AFLP analysis showed that the strains of A. niger formed a homogenous phylogenetic group distinguished by either zero or type A feruloyl esterase activity, while strains A. foetidus and A. tubingensis exhibited type B feruloyl esterase activity when grown on sugar beet pulp.

Mycoflora and multimycotoxin detection in corn silage: experimental study.

Agricultural activities involve the use of crop preservation such as "trench-type" silo, which can sometimes be contaminated by fungi. To investigate the exposure of livestock and farm workers to fungal spores and mycotoxins, a multimycotoxin analysis method has been developed. Six mycotoxins (aflatoxin B1, citrinin, deoxynivalenol, gliotoxin, ochratoxin A, and zearalenone) were quantified by high-performance liquid chromatography coupled to mass spectrometry after solid-phase extraction. An experimental study of fungal species and mycotoxins was conducted in corn silage (Normandy, France) during 9 months of monitoring. The results indicated the recurrence of around 20 different species, with some of them being potentially toxigenic fungi such as Aspergillus fumigatus, Aspergillus parasiticus, Fusarium verticillioides, and Monascus ruber, and the detection of aflatoxin B1 (4-34 ppb), citrinin (4-25 ppb), zearalenone (23-41 ppb), and deoxynivalenol (100-213 ppb). This suggested a possible chronic exposure to low levels of mycotoxins.

Fungal microflora and ochratoxin a risk in French vineyards.

To evaluate the ochratoxin A risk in French vineyards, five winemaking regions were investigated. An exhaustive survey of the fungal microflora of 60 grape samples was carried out at two development stages of the berries: end of veraison and harvest time. Potentially toxinogenic fungi isolated from grapes were assessed in vitro for ochratoxin A production. Ochratoxin A was also quantified in musts by high-performance liquid chromatography after cleanup on immunoaffinity columns. Among the 90 species identified, almost half are listed as mycotoxin producers, but only 2 are potentially ochratoxinogenic: Aspergillus carbonarius and Aspergillus niger. Among these strains, only A. carbonarius, isolated from the Languedoc region at harvest time, was found to produce ochratoxin A. These results were in accordance with the presence of ochratoxin A in French southern region musts (0.01-0.43 microg/L) and confirmed the major implication of A. carbonarius in ochratoxin A contamination.

Effects of fungal bioaugmentation and cyclodextrin amendment on fluorene degradation in soil slurry.

This study assesses the potential of fungal bioaugmentation and the effect of maltosyl-cyclodextrin amendment, as an approach to accelerate fluorene biodegradation in soil slurries. 47 fungal strains isolated from a contaminated site were tested in the biodegradation of fluorene. Results showed the greater efficiency of "adaptated" fungi isolated from contaminated soil vs. reference strains belonging to the collection of the laboratory. These assays allowed us to select the most efficient strain, Absidia cylindrospora, which was used in a bioaugmentation process. In the presence of Absidia cylindrospora, more than 90% of the fluorene was removed in 288 h while 576 h were necessary in the absence of fungal bioaugmentation. Maltosyl-cyclodextrin, a branched-cyclodextrin was chosen in order to optimize fluorene bioavailability and biodegradation in soil slurries. The results of this study indicate that Absidia cylindrospora and maltosyl-cyclodextrin could be used successfully in bioremediation systems.

Fungal flora and ochratoxin a production in grapes and musts from france.

Eleven samples of grapes and musts used in red table wines were investigated for the occurrence of potential ochratoxin A (OTA)-producing molds. From these samples, 59 filamentous fungi and 2 yeasts were isolated. Among the 30 genera isolated, Deuteromycetes were the most frequent (70%) followed by Ascomycetes (10%). Six of the eleven grapes samples were contaminated by potentially ochratoxinogenic strains (Penicillium chrysogenum and Aspergillus carbonarius). When cultivated in vitro on solid complex media, the 14 strains of A. carbonarius produced OTA. No other species produced OTA under the same conditions. Among must samples, eight of eleven were found to be contaminated by OTA (concentrations from <10 to 461 ng/L). There is a strong correlation between the presence of ochratoxin-producing strains on grapes and OTA in musts. These findings should be connected with the OTA contamination of human blood in these areas and in France.