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Fungal, ligninolytic enzymes have attracted a great attention for their bioremediation capabilities. A deficient knowledge of regulation of enzyme production, however, hinders the use of ligninolytic fungi in bioremediation applications. In this work, a transcriptional analyses of laccase and manganese peroxidase ( MnP) production by two white rots was combined with determination of pI of the enzymes and the evaluation of 17α-ethinyloestradiol ( EE2) degradation to study regulation mechanisms used by fungi during EE2 degradation. In the cultures of Trametes versicolor the addition of EE2 caused an increase in laccase activity with a maximum of 34.2 ± 6.7 U g−1 of dry mycelia that was observed after 2 days of cultivation. It corresponded to a 4.9 times higher transcription levels of a laccase-encoding gene ( lacB) that were detected in the cultures at the same time. Simultaneously, pI values of the fungal laccases were altered in response to the EE2 treatment. Like T. versicolor, Irpex lacteus was also able to remove 10 mg l−1 EE2 within 3 days of cultivation. While an increase to I. lacteus MnP activity and MnP gene transcription levels was observed at the later phase of the cultivation. It suggests another metabolic role of MnP but EE2 degradation. [ABSTRACT FROM AUTHOR]

The white rot fungus Pleurotus ostreatus HK 35, which is also an edible industrial mushroom commonly cultivated in farms, was tested in the degradation of typical representatives of endocrine disrupters (EDCs; bisphenol A, estrone, 17β-estradiol, estriol, 17α-ethinylestradiol, triclosan and 4-n-nonylphenol); its degradation efficiency under model laboratory conditions was greater than 90% within 12 days and better than that of another published strain P. ostreatus 3004. A spent mushroom substrate from a local farm was tested for its applicability in various batch and trickle-bed reactors in degrading EDCs in model fortified and real communal wastewater. The reactors were tested under various regimes including a pilot-scale trickle-bed reactor, which was finally tested at a wastewater treatment plant. The result revealed that the spent substrate is an efficient biodegradation agent, where the fungus was usually able to remove about 95% of EDCs together with suppression of the estrogenic activity of the sample. The results showed the fungus was able to operate in the presence of bacterial microflora in wastewater without any substantial negative effects on the degradation abilities. Finally, a pilot-scale trickle-bed reactor was installed in a wastewater treatment plant and successfully operated for 10days, where the bioreactor was able to remove more than 76% of EDCs present in the wastewater., (Copyright © 2017 Elsevier B.V. All rights reserved.)

The present study describes a pilot remediation test of a co-mingled plume containing BTEX, chlorinated pollutants and pharmaceuticals. Remediation was attempted using a combination of various approaches, including a pump and treat system applying an advanced oxidation process and targeted direct push injections of calcium peroxide. The remediation process was monitored intensively and extensively throughout the pilot test using various conventional and passive sampling methods, including next-generation amplicon sequencing. The results showed that the injection of oxygen-saturated treated water with residual hydrogen peroxide and elevated temperature enhanced the in situ removal of monoaromatics and chlorinated pollutants. In particular, in combination with the injection of calcium peroxide, the conditions facilitated the in situ bacterial biodegradation of the pollutants. The mean groundwater concentration of benzene decreased from 1349μg·L -1 prior to the test to 3μg·L -1 within 3months after the calcium peroxide injections; additionally, monochlorobenzene decreased from 1545μg·L -1 to 36μg·L -1 , and toluene decreased from 143μg·L -1 to 2μg·L -1 . Furthermore, significant degradation of the contaminants bound to the soil matrix in less permeable zones was observed. Based on a developed 3D model, 90% of toluene and 88% of chlorobenzene bound to the soil were removed during the pilot test, and benzene was removed almost completely. On the other hand, the psychopharmaceuticals were effectively removed by the employed advanced oxidation process only from the treated water, and their concentration in groundwater remained stagnant due to inflow from the surroundings and their absence of in situ degradation. The employment of passive sampling techniques, including passive diffusion bags (PDB) for volatile organic pollutants and their respective transformation products, polar organic compound integrative samplers (POCIS) for the pharmaceuticals and in situ soil microcosms for microbial community analysis, was proven to be suitable for monitoring remediation in saturated zones., (Copyright © 2017 Elsevier B.V. All rights reserved.)

This study was performed to test the feasibility of several decontamination methods for remediating heavily contaminated groundwater in a real contaminated locality in the Czech Republic, where a pharmaceuticals plant has been in operation for more than 80 years. The site is polluted mainly by recalcitrant psychopharmaceuticals and monoaromatic hydrocarbons, such as benzene, toluene and chlorobenzene. For this purpose, an advanced oxidation technique employing UV radiation with hydrogen peroxide dosing was employed, in combination with simple aeration pretreatment. The results showed that UV/H 2 O 2 was an efficient and necessary step for degradation of the pharmaceuticals; however, the monoaromatics were already removed during the aeration step. Characterization of the removal mechanisms participating in the aeration revealed that volatilization, co-precipitation and biodegradation contributed to the process. These findings were supported by bacterial metabolite analyses, phospholipid fatty acid analysis, qPCR of representatives of the degradative genes and detailed characterization of the formed precipitate using Mössbauer spectroscopy and scanning electron microscopy. Further tests were carried out in a continuous arrangement directly connected to the wells already present in the locality. The results documented the feasibility of combination of the photo-reactor employing UV/H 2 O 2 together with aeration pretreatment for 4 months, where the overall decontamination efficiency ranged from 72% to 99% of the pharmaceuticals. We recorded even better results for the monoaromatics decontamination except for one month, when we encountered some technical problems with the aeration pump. This demonstrated the necessity of using the aeration step., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

A considerable amount of scientific evidence indicates that a number of pharmaceuticals that could be detected in the environment can contribute towards the development of problems associated with human reproductive systems, as well as those of wildlife. We investigated the estrogenic and androgenic effects of select pharmaceuticals with high production volume and environmental relevance. We examined the receptor-binding activities of these pharmaceuticals in the T47D human cell line using altered secretion of cytokine CXCL12. Functional yeast-luciferase reporter gene assays were also employed to confirm the mechanism of receptor binding by estrogen and androgen. Non-steroidal anti-inflammatory drugs, namely ibuprofen, diclofenac and antiarrhythmic agent amiodarone showed strong anti-estrogenic effects in the T47D cell line. In the yeast-luciferase assay, these anti-inflammatory drugs also demonstrated anti-estrogenic potency and inhibited the E2 response in a concentration-dependent manner. Amiodarone did not exhibit any response in the yeast-luciferase assay; therefore, the endocrine disruption presumably occurred at a different level without directly involving the receptor. All the anti-inflammatory drugs considered in this study, including ketoprofen, naproxen and clofibrate, exhibited a dose-dependent antagonism towards the androgen receptor in the yeast-luciferase assays. Several other drugs, including the stimulant caffeine, did not show any response in the tests that were employed. A risk assessment analysis using 'Hazard Quotient' suggested a potential risk, especially in the cases of ibuprofen, ketoprofen, diclofenac and clofibrate. The results reveal the intrinsic endocrine disrupting nature of several pharmaceuticals and thus could contribute towards explaining a number of adverse health effects on humans and wildlife., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

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., (Copyright © 2016 Elsevier B.V. All rights reserved.)

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., (Copyright © 2015 Elsevier B.V. All rights reserved.)

Recent studies documented that several processes in filamentous fungi are connected with microsomal enzyme activities. In this work, microsomal subproteomes of Pleurotus ostreatus were analyzed by two-dimensional (2-D) polyacrylamide gel electrophoresis and matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometry analysis. To assess proteome dynamics, microsomal proteins were isolated from fungal cultures after 7 and 12 days of cultivation. Additionally, 10 mg/L of 17α-ethinylestradiol (EE2) was treated with the cultures during 2 days. Despite the EE2 degradation by the fungus reached 97 and 76.3 % in 7- and 12-day-old cultures, respectively, only a minor effect on the composition of microsomal proteins was observed. The changes in protein maps related to ageing prevailed over those induced by EE2. Epoxide hydrolase, known to metabolize EE2, was detected in 12-day-old cultures only which suggests differences in EE2 degradation pathways utilized by fungal cultures of different age. The majority (32 %) of identified microsomal proteins were parts of mitochondrial energy metabolism.

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., (Copyright © 2013 Elsevier B.V. All rights reserved.)

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.

Plasmalogens are a group of lipids with potentially important, and not yet fully known, functions in organisms from bacteria to protozoans, invertebrates, and mammals. They can protect cells against the damaging effects of reactive oxygen species, protect other phospholipids or lipoprotein particles against oxidative stress, and have been implicated as signaling molecules and modulators of membrane dynamics. They have been found in many anaerobic bacterial species, and their biosynthetic pathways differ in aerobic and anaerobic organisms. The use of advanced techniques permits the identification of not only plasmalogen classes but also their positional isomers and often also individual molecular species. This paper describes direct analyses of plasmalogens from natural sources, frequently very unusual, using electrospray ionization mass spectrometry in combination with high-performance liquid chromatography and/or shotgun lipidomics.

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., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

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., (Copyright © 2011 Elsevier B.V. All rights reserved.)

A study was conducted to elaborate a fast, simple and efficient method for determination of 15 isomers chlorobenzoic acids (CBAs) in soil using HPLC-UV. Artificially contaminated soil samples were extracted using accelerated solvent extraction (ASE) with 1% acetic acid in a mixture of hexane and acetone (1:1, V/V) under a pressure of 10.34 MPa and temperature of 150°C. The recovery of the ASE method was above 82%. The extracts were concentrated; dimethyl sulfoxide was used to prevent CBA volatilization and the final analysis was performed with a C18 XBridge HPLC column employing a mobile phase consisting of acetonitrile and 0.1% trifluoracetic acid in water. A HPLC procedure with gradient elution and UV detection was developed and validated. The method exhibited a linear range for 2-CBA; 2,6-CBA; 3-CBA; 4-CBA; 2,3-CBA; 2,3,6-CBA; 2,5-CBA; and 2,4-CBA from 5 to 120 μg/mL with a limit of quantification (LOQ) of 5 μg/mL, RSD from 2.42 to 9.42% and accuracy from 82 ± 2 to 103 ± 3%. The linear range of determination of 2,4,6-CBA, 3,4-CBA, 2,3,5,6-CBA, 3,5-CBA, 2,3,5-CBA, 2,3,4,5,6-CBA and 2,3,4,5-CBA was 10-120 μg/mL with LOQ 10 μg/mL, RSD from 0.74 to 5.84% and accuracy from 94 ± 1 to 114 ± 1%. The optimized analytical procedure was finally applied on two historically PCB contaminated soils and 9 CBAs were quantified in the samples., (Copyright © 2011 Elsevier B.V. All rights reserved.)