Zdena Křesinová

Biodegradation of endocrine-disrupting compounds and suppression of estrogenic activity by ligninolytic fungi

Endocrine-disrupting compounds (EDCs) represent a large group of substances of natural and anthropogenic origin. They are widely distributed in the environment and can pose serious risks to aquatic organisms and to public health. In this study, 4-n-nonylphenol, technical 4-nonylphenol, bisphenol A, 17alpha-ethinylestradiol, and triclosan were biodegraded by eight ligninolytic fungal strains (Irpex lacteus 617/93, Bjerkandera adusta 606/93, Phanerochaete chrysosporium ME 446, Phanerochaete magnoliae CCBAS 134/I, Pleurotus ostreatus 3004 CCBAS 278, Trametes versicolor 167/93, Pycnoporus cinnabarinus CCBAS 595, Dichomitus squalens CCBAS 750). The results show that under the used conditions the fungi were able to degrade the EDCs within 14d of cultivation with exception of B. adusta and P. chrysosporium in the case of triclosane and bisphenol A, respectively. I. lacteus and P. ostreatus were found to be most efficient EDC degraders with their degradation efficiency exceeding 90% or 80%, respectively, in 7d. Both fungi degraded technical 4-nonylphenol, bisphenol-A, and 17alpha-ethinylestradiol below the detection limit within first 3d of cultivation. In general, estrogenic activities assayed with a recombinant yeast test decreased with advanced degradation. However, in case of I. lacteus, P. ostreatus, and P. chrysosporium the yeast assay showed a residual estrogenic activity (28-85% of initial) in 17alpha-ethinylestradiol cultures. Estrogenic activity in B. adusta cultures temporally increased during degradation of technical 4-nonylphenol, suggesting a production of endocrine-active intermediates. Attention was paid also to the effects of EDCs on the ligninolytic enzyme activities of the different fungi strains to evaluate their possible stimulation or suppression of activities during the biodegradation processes.

Biodegradation of PCBs by ligninolytic fungi and characterization of the degradation products.

The aim of the present study was to compare the degrading capabilities of eight ligninolytic fungal representatives towards a technical mixture of polychlorinated biphenyls (Delor 103). Axenic cultures of the fungi, either in complex or N-limited liquid media, were spiked with the technical mixture of Delor 103. All of the fungal strains were able to degrade the pollutant significantly after 6weeks of incubation in both media. Outstanding results were achieved by the treatment with Pleurotus ostreatus, which removed 98.4% and 99.6% of the PCB mixture in complex and mineral media, respectively. This fungus was the only one capable of breaking down penta- and hexachlorinated biphenyls in the complex medium. Ecotoxicological assays performed with the luminescent bacterium Vibrio fischeri demonstrated that all of the fungal strains employed in this study were able to remove the toxicity only temporarily (e.g., after 28d of incubation), while P. ostreatus was capable of suppressing the toxicity associated to PCBs along the whole incubation period in both media. We also performed an extensive set of qualitative GC/MS analyses and chlorinated derivatives of hydroxy- and methoxy-biphenyls were detected along with monoaromatic structures, i.e. chlorobenzoic acids, chlorobenzaldehydes and chlorobenzyl alcohols. This results indicate that both intracellular (cytochrome P-450 monooxigenase, aryl-alcohol dehydrogenase and aryl-aldehyde dehydrogenase) and extracellular (ligninolytic enzymes) enzymatic systems could be involved in the biotransformation of PCB by ligninolytic fungi. The data from this work also document that the fungi are able to degrade further the main metabolites on the PCB pathway (i.e. chlorobenzoic acids) simultaneously with PCBs.

Influence of the bioaccessible fraction of polycyclic aromatic hydrocarbons on the ecotoxicity of historically contaminated soils

Sequential supercritical fluid extraction together with a two-site desorption model were employed to estimate the bioaccessible fraction of polycyclic aromatic hydrocarbons (PAHs) in four historically contaminated soils. The ecotoxicity of the soils was assayed by four different contact tests. The same soils were exposed to exhaustive extraction and the extracts were returned to the soils to ensure total 100% bioaccessibility of the pollutants. Then the soils were assayed again. Statistical evaluation revealed that the predicted bioaccessible PAHs generally correlated with the ecotoxicity responses of the tests. The estimated bioaccessible fractions varied from 10 to 98%. This value increased for PAHs with higher lipophilicity and showed no correlation with the organic carbon content in the soils. The ecotoxicity tests in the study indicated different sensitivity toward PAHs and the tests employing Heterocypris incongruens and Eisenia fetida were found to be more suitable than Lemna minor and Vibrio fischeri. Mortality and growth inhibition of ostracods correlated with all the types of PAHs and earthworm growth inhibition and mortality were preferentially sensitive to PAHs with only 3-4 aromatic rings. Determination of the biota-soil accumulation factors indicated that the earthworm growth inhibition corresponded to increased accumulation of PAHs in the earthworm tissue.

Polycyclic aromatic hydrocarbons degradation and microbial community shifts during co-composting of creosote-treated wood.

The feasibility of decontaminating creosote-treated wood (CTW) by co-composting with agricultural wastes was investigated using two bulking agents, grass cuttings (GC) and broiler litter (BL), each employed at a 1:1 ratio with the matrix. The initial concentration of total polycyclic aromatic hydrocarbons (PAHs) in CTW (26,500 mg kg(-1)) was reduced to 3 and 19% after 240 d in GC and BL compost, respectively. PAH degradation exceeded the predicted bioaccesible threshold, estimated through sequential supercritical CO2 extraction, together with significant detoxification, assessed by contact tests using Vibrio fisheri and Hordeum vulgare. GC composting was characterized by high microbial biomass growth in the early phases, as suggested by phospholipid fatty acid analyses. Based on the 454-pyrosequencing results, fungi (mostly Saccharomycetales) constituted an important portion of the microbial community, and bacteria were characterized by rapid shifts (from Firmicutes (Bacilli) and Actinobacteria to Proteobacteria). However, during BL composting, larger amounts of prokaryotic and eukaryotic PLFA markers were observed during the cooling and maturation phases, which were dominated by Proteobacteria and fungi belonging to the Ascomycota and those putatively related to the Glomeromycota. This work reports the first in-depth analysis of the chemical and microbiological processes that occur during the co-composting of a PAH-contaminated matrix.

Mechanistic Study of 17α-Ethinylestradiol Biodegradation by Pleurotus ostreatus: Tracking of Extracelullar and Intracelullar Degradation Mechanisms

The white rot fungus Pleurotus ostreatus is able to completely remove the synthetic hormone 17α-ethinylestradiol (EE2, 200 μg in 20 mL) from a liquid complex or mineral medium in 3 or 14 days, respectively. Its efficiency has also been documented in the removal of estrogenic activity that correlated with the EE2 degradation. A set of in vitro experiments using various cellular and enzyme fractions has been performed and the results showed that EE2 was degraded by isolated laccase (about 90% within 24 h). The degradation was also tested with concentrated extracellular liquid where degradation reached 50% mainly due to the laccase activity; however, after a supplementation with H₂O₂ and Mn²⁺, residual manganese-dependent peroxidase activities (40 times lower than Lac) raised the degradation to 100%. Moreover, the intracellular fraction and also laccase-like activity associated with fungal mycelium were found to be efficient in the degradation too. Isolated microsomal proteins appeared to also be involved in the process. The degradation was completely suppressed in the presence of cytochrome P-450 inhibitors, piperonylbutoxide and carbon monoxide, indicating a role of this monooxygenase in the degradation process. Attention was also paid to monitoring of changes in the estrogenic activity during these particular in vitro experiments when mainly degradations related to ligninolytic enzymes were found to decrease the estrogenic activity with EE2 removal proportionally. Several novel metabolites of EE2 were detected using different chromatographic method with mass spectrometric techniques (LC-MS, GC-MS) including also [¹³C]-labeled substrates. The results document the involvement of various different simultaneous mechanisms in the EE2 degradation by P. ostreatus by both the ligninolytic system and the eukaryotic machinery of cytochromes P-450.

Inoculum carrier and contaminant bioavailability affect fungal degradation performances of PAH-contaminated solid matrices from a wood preservation plant.

The objective of the study was to investigate the impact of chopped wheat straw (CWS), ground corn cobs (GCC) and commercial pellets (CP), as inoculum carriers, on both growth and polycyclic aromatic hydrocarbons (PAH) degradation performances of Dichomitus squalens, Pleurotus ostreatus and Coprinus comatus. A historically-contaminated soil (HCS) and creosote-treated shavings (CTS) from the Sobeslav wood preservation plant, characterized by different relative abundances of the PAH bioavailable fractions, were used to assess the contaminated matrix effect and its interaction with both carrier and fungal strain. In HCS, best results were obtained with CP-immobilized P. ostreatus, which was able to deplete benzo[a]anthracene, chrysene, benzo[b]fluoranthene (BbF), benzo[k]fluoranthene (BkF) and benzo[a]pyrene (BaP) by 69.1%, 29.7%, 39.7%, 32.8% and 85.2%, respectively. Only few high-molecular mass PAHs such as BbF, BkF and BaP were degraded beyond their respective bioavailable fractions and this effect was confined to a limited number of inoculants. In CTS, only phenanthrene degradation exceeded its respective bioavailability from 1.42 to 1.86-fold. Regardless of both inoculum carrier and fungal species, degradation was positively and significantly (P<0.001) correlated with bioavailability in fungal microcosms on HCS and CTS and such correlation was very similar in the two matrices (R(adj)(2) equal to 0.60 and 0.59, respectively). The ability of white-rot fungi to degrade certain PAHs beyond their bioavailability was experimentally proven by this study. Although CTS and HCS considerably differed in their physico-chemical properties, PAH contents and contaminant aging, the relationship between degradation and bioavailability was not significantly affected by the type of matrix.

Chemical and microbiological characterization of an aged PCB-contaminated soil.

This study was aimed at complex characterization of three soil samples (bulk soil, topsoil and rhizosphere soil) from a site historically contaminated with polychlorinated biphenyls (PCB). The bulk soil was the most highly contaminated, with a PCB concentration of 705.95 mg kg(-1), while the rhizosphere soil was the least contaminated (169.36 mg kg(-1)). PCB degradation intermediates, namely chlorobenzoic acids (CBAs), were detected in all the soil samples, suggesting the occurrence of microbial transformation processes over time. The higher content of organic carbon in the topsoil and rhizosphere soil than in the bulk soil could be linked to the reduced bioaccessibility (bioavailability) of these chlorinated pollutants. However, different proportions of the PCB congener contents and different bioaccessibility of the PCB homologues indicate microbial biotransformation of the compounds. The higher content of organic carbon probably also promoted the growth of microorganisms, as revealed by phospholipid fatty acid (PFLA) quantification. Tag-encoded pyrosequencing analysis showed that the bacterial community structure was significantly similar among the three soils and was predominated by Proteobacteria (44-48%) in all cases. Moreover, analysis at lower taxonomic levels pointed to the presence of genera (Sphingomonas, Bulkholderia, Arthrobacter, Bacillus) including members with reported PCB removal abilities. The fungal community was mostly represented by Basidiomycota and Ascomycota, which accounted for >80% of all the sequences detected in the three soils. Fungal taxa with biodegradation potential (Paxillus, Cryptococcus, Phoma, Mortierella) were also found. These results highlight the potential of the indigenous consortia present at the site as a starting point for PCB bioremediation processes.

Biodegradation of chlorobenzoic acids by ligninolytic fungi

We investigated the abilities of several perspective ligninolytic fungal strains to degrade 12 mono-, di- and trichloro representatives of chlorobenzoic acids (CBAs) under model liquid conditions and in contaminated soil. Attention was also paid to toxicity changes during the degradation, estimated using two luminescent assay variations with Vibrio fischeri. The results show that almost all the fungi were able to efficiently degrade CBAs in liquid media, where Irpex lacteus, Pycnoporus cinnabarinus and Dichomitus squalens appeared to be the most effective in the main factors: degradation and toxicity removal. Analysis of the degradation products revealed that methoxy and hydroxy derivatives were produced together with reduced forms of the original acids. The findings suggest that probably more than one mechanism is involved in the process. Generally, the tested fungal strains were able to degrade CBAs in soil in the 85-99% range within 60 days. Analysis of ergosterol showed that active colonization is an important factor for degradation of CBAs by fungi. The most efficient strains in terms of degradation were I. lacteus, Pleurotus ostreatus, Bjerkandera adusta in soil, which were also able to actively colonize the soil. However, in contrast to P. ostreatus and I. lacteus, B. adusta was not able to significantly reduce the measured toxicity.

Method for analysis of psychopharmaceuticals in real industrial wastewater and groundwater with suspended organic particulate matter using solid phase extraction disks extraction and ultra-high performance liquid chromatography/time-of-flight mass spectrometry.

A rapid and reliable analytical method was developed for the quantitative determination of psychopharmaceuticals, their precursors and by-products in real contaminated samples from a pharmaceutical company in Olomouc (Czech Republic), based on SPE disk extraction and detection by ultra performance liquid chromatography, combined with time-of-flight mass spectrometry. The target compounds were quantified in the real whole-water samples (water including suspended particles), both in the presence of suspended particulate matter (SPM) and high concentrations of other organic pollutants. A total of nine compounds were analyzed which consisted of three commonly used antidepressants (tricyclic antidepressants and antipsychotics), one antitussive agent and five by-products or precursors. At first, the SPE disk method was developed for the extraction of water samples (dissolved analytes, recovery 84-104%) and pressurised liquid extraction technique was verified for solid matrices (sludge samples, recovery 81-95%). In order to evaluate the SPE disk technique for whole water samples containing SPM, non contaminated groundwater samples were also loaded with different amounts (100 and 300mgL(-1)) of real contaminated sludge originating from the same locality. The recoveries from the whole-water samples obtained by SPE disk method ranged between 67 and 119% after the addition of the most contaminated sludge. The final method was applied to several real groundwater (whole-water) samples from the industrial area and high concentrations (up to 10(3)μgL(-1)) of the target compounds were detected. The results of this study document and indicate the feasibility of the SPE disk method for analysis of groundwater.

Hydroxylated Anthraquinones Produced by Geosmithia species

Geosmithia fungi are little known symbionts of bark beetles. Secondary metabolites of lilac colored species G. lavendula and other nine Geosmithia species were investigated in order to elucidate their possible role in the interactions of the fungi with environment. Hydroxylated anthraquinones (yellow, orange, and red pigments), were found to be the most abundant compounds produced into the medium during the submerged cultivation. Three main compounds were identified as 1,3,6,8-tetrahydroxyanthraquinone (1), rhodolamprometrin (1-acetyl-2,4,5,7-tetrahydroxyanthraquinone; 2), and 1-acetyl-2,4,5,7,8-pentahydroxyanthraquinone (3). Compounds 2 and 3 (representing the majority of produced metabolites) inhibited the growth of G+-bacteria Staphylococcus aureus and Bacillus subtilis with minimum inhibitory concentration of 64–512 μg/mL. Anti-inflammatory activity detected as inhibition of cyclooxygenase-2 was found only for compound 3 at 1 and 10 μg/mL. Compound 2 interfered with the morphology, compound 3 with cell-cycle dynamics of adherent mammalian cell lines.