Suspect screening of emerging pollutants and their major transformation products in wastewaters treated with fungi by liquid chromatography coupled to a high resolution mass spectrometry.

A new approach for the screening of 33 pharmaceuticals and 113 of their known transformation products in wastewaters was developed. The methodology is based on the analysis of samples by liquid chromatography coupled to high resolution mass spectrometry (HRMS) followed by data processing using specific software and manual confirmation. A home-made library was built with the transformation products reported in literature for the target pharmaceuticals after treatment with various fungi. The method was applied to the search of these contaminants in 67 samples generated along treatment of wastewaters with white-rot fungus Trametes versicolor. The screening methodology allowed the detection of different transformation products (TPs) generated from degradation of parent compounds after fungal treatment. This approach can be a useful tool for the rapid screening and tentative detection of emerging contaminants during water treatment in both full and batch-scale studies when pure standards are not available.

Non conventional biological treatment based on Trametes versicolor for the elimination of recalcitrant anticancer drugs in hospital wastewater.

This work presents a study about the elimination of anticancer drugs, a group of pollutants considered recalcitrant during conventional activated sludge wastewater treatment, using a biological treatment based on the fungus Trametes versicolor. A 10-L fluidized bed bioreactor inoculated with this fungus was set up in order to evaluate the removal of 10 selected anticancer drugs in real hospital wastewater. Almost all the tested anticancer drugs were completely removed from the wastewater at the end of the batch experiment (8 days) with the exception of Ifosfamide and Tamoxifen. These two recalcitrant compounds, together with Cyclophosphamide, were selected for further studies to test their degradability by T. versicolor under optimal growth conditions. Cyclophosphamide and Ifosfamide were inalterable during batch experiments both at high and low concentration, whereas Tamoxifen exhibited a decrease in its concentration along the treatment. Two positional isomers of a hydroxylated form of Tamoxifen were identified during this experiment using a high resolution mass spectrometry based on ultra-high performance chromatography coupled to an Orbitrap detector (LTQ-Velos Orbitrap). Finally the identified transformation products of Tamoxifen were monitored in the bioreactor run with real hospital wastewater.

Hospital wastewater treatment by fungal bioreactor: removal efficiency for pharmaceuticals and endocrine disruptor compounds.

Hospital effluents contribute to the occurrence of emerging contaminants in the environment due to their high load of pharmaceutical active compounds (PhACs) and some endocrine disruptor compounds (EDCs). Nowadays, hospital wastewaters are co-treated with urban wastewater; however, the dilution factor and the inefficiency of wastewater treatment plants in the removal of PhACs and EDCs make inappropriate the co-treatment of both effluents. In this paper, a new alternative to pre-treat hospital wastewater concerning the removal of PhACs and EDCs is presented. The treatment was carried out in a batch fluidized bed bioreactor under sterile and non-sterile conditions with Trametes versicolor pellets. Results on non-sterile experiments pointed out that 46 out of the 51 detected PhACs and EDCs were partially to completely removed. The total initial PhAC amount into the bioreactor was 8185 µg in sterile treatment and 8426 µg in non-sterile treatment, and the overall load elimination was 83.2% and 53.3% in their respective treatments. In addition, the Microtox test showed reduction of wastewater toxicity after the treatment. Hence, the good efficiency of the fungal treatment regarding removal of the wide diversity of PhACs and EDCs detected in hospital effluents is demonstrated.

Biodegradation of the X-ray contrast agent iopromide and the fluoroquinolone antibiotic ofloxacin by the white rot fungus Trametes versicolor in hospital wastewaters and identification of degradation products.

This paper describes the degradation of the X-ray contrast agent iopromide (IOP) and the antibiotic ofloxacin (OFLOX) by the white-rot-fungus Trametes versicolor. Batch studies in synthetic medium revealed that between 60 and 80% of IOP and OFLOX were removed when spiked at approximately 12 mg L(-1) and 10 mg L(-1), respectively. A significant number of transformation products (TPs) were identified for both pharmaceuticals, confirming their degradation. IOP TPs were attributed to two principal reactions: (i) sequential deiodination of the aromatic ring and (ii) N-dealkylation of the amide at the hydroxylated side chain of the molecule. On the other hand, OFLOX transformation products were attributed mainly to the oxidation, hydroxylation and cleavage of the piperazine ring. Experiments in 10 L-bioreactor with fungal biomass fluidized by air pulses operated in batch achieved high percentage of degradation of IOP and OFLOX when load with sterile (87% IOP, 98.5% OFLOX) and unsterile (65.4% IOP, 99% OFLOX) hospital wastewater (HWW) at their real concentration (µg L(-1) level). Some of the most relevant IOP and OFLOX TPs identified in synthetic medium were also detected in bioreactor samples. Acute toxicity tests indicated a reduction of the toxicity in the final culture broth from both experiments in synthetic medium and in batch bioreactor.

Synthesis and characterization of combined cross-linked laccase and tyrosinase aggregates transforming acetaminophen as a model phenolic compound in wastewaters.

Laccase (EC 1.10.3.2) and tyrosinases (EC 1.14.18.1) are ubiquitous enzymes present in nature as they are known to originate from bacteria, fungi, plants, etc. Both laccase and tyrosinase are copper-containing phenoloxidases requiring readily available O2 without auxiliary cofactor for their catalytic transformation of numerous phenolic substrates. In the present study, laccase and tyrosinase have been insolubilized as combined crosslinked enzyme aggregates (combi-CLEA) using chitosan, a renewable and biodegradable polymer, as crosslinker. The combi-CLEA, with specific activity of 12.3 U/g for laccase and 167.4 U/g for tyrosinase, exhibited high enzymatic activity at pH5-8 and temperature at 5-30°C, significant resistance to denaturation and no diffusional restriction to its active site based upon the Michaelis-Menten kinetic parameters. Subsequently, the combi-CLEA was applied to the transformation of acetaminophen as a model phenolic compound in samples of real wastewaters in order to evaluate the potential efficiency of the biocatalyst. In batch mode the combi-CLEA transformed more than 80% to nearly 100% of acetaminophen from the municipal wastewater and more than 90% from the hospital wastewater. UPLC-MS analysis of acetaminophen metabolites showed the formation of its oligomers as dimers, trimers and tetramers due to the laccase and 3-hydroxyacetaminophen due to the tyrosinase.

Degradation of pharmaceuticals in non-sterile urban wastewater by Trametes versicolor in a fluidized bed bioreactor.

The constant detection of pharmaceuticals (PhACs) in the environment demonstrates the inefficiency of conventional wastewater treatment plants to completely remove them from wastewaters. So far, many studies have shown the feasibility of using white rot fungi to remove these contaminants. However, none of them have studied the degradation of several PhACs in real urban wastewater under non-sterile conditions, where mixtures of contaminants presents at low concentrations (ng L(-1) to µg L(-1)) as well as other active microorganisms are present. In this work, a batch fluidized bed bioreactor was used to study, for the first time, the degradation of PhACs present in urban wastewaters at their pre-existent concentrations under non-sterile conditions. Glucose and ammonium tartrate were continuously supplied as carbon and nitrogen source, respectively, and pH was maintained at 4.5. Complete removal of 7 out of the 10 initially detected PhACs was achieved in non-sterile treatment, while only 2 were partially removed and 1 of the PhACs analyzed increased its concentration. In addition, Microtox test showed an important reduction of toxicity in the wastewater after the treatment.

Degradation of carbamazepine by Trametes versicolor in an air pulsed fluidized bed bioreactor and identification of intermediates.

The paper describes the aerobic degradation of carbamazepine (CBZ), an anti-epileptic drug widely found in aquatic environment, from Erlenmeyer flask to bioreactor by the white-rot fungus Trametes versicolor. In Erlenmeyer flask, CBZ at approximately 9 mg L(-1) was almost completely eliminated (94%) after 6 d, while at near environmentally relevant concentrations of 50 µg L(-1), 61% of the contaminant was degraded in 7 d. Acridone, acridine, 10,11-dihydro-10,11-dihydroxy-CBZ, and 10, 11-epoxy-CBZ were identified as major metabolites, confirming the degradation of CBZ. The degradation process was then carried out in an air pulsed fluidized bioreactor operated in batch and continuous mode. Around 96% of CBZ was removed after 2 days in batch mode operation, and 10,11-dihydro-10,11-epoxycarbamazepine was found as unique metabolite. In bioreactor operated in continuous mode with a hydraulic retention time of 3 d, 54% of the inflow concentration (approx. 200 µg L(-1)) was reduced at the steady state (25 d) with a CBZ degradation rate of 11.9 µg CBZ g(-1) dry weight d(-1). No metabolite was detected in the culture broth. Acute toxicity tests (Microtox) indicated that the final culture broth in both batch and continuous mode operation were non toxic, with 15 min EC50 values of 24% and 77%, respectively.

White-rot fungus-mediated degradation of the analgesic ketoprofen and identification of intermediates by HPLC-DAD-MS and NMR.

Ketoprofen is a nonsteroidal anti-inflammatory drug that has been detected in the environment in the range of ng L(-1)-microg L(-1) due to its low degradability in some wastewater treatment plants. In this study, the use of the white-rot fungus Trametes versicolor to effectively degrade ketoprofen in a defined liquid medium was assessed. The fungus eliminated ketoprofen to nondetectable levels in 24h when it was added at 10mgL(-1) whereas at low concentration of 40microgL(-1) it was almost completely removed (95%) after 5h. Low extracellular laccase activity was detected in the T. versicolor cultures but the addition of the laccase-mediator system did not lead to ketoprofen oxidation. The cytochrome P-450 inhibitor 1-aminobenzotriazole reduced ketoprofen oxidation. These data suggest that the first oxidation step is cytochrome P450 mediated. During time-course degradation experiments, three intermediates were structurally elucidated and quantified by HPLC-DAD-MS and NMR: 2-[3-(4-hydroxybenzoyl)phenyl]-propanoic acid, 2-[(3-hydroxy(phenyl)methyl)phenyl]-propanoic acid, and 2-(3-benzoyl-4-hydroxyphenyl)-propanoic acid. The latter was reported for the first time in biological systems. After 7 d of incubation, only small amounts of 2-[(3-hydroxy(phenyl)methyl)phenyl]-propanoic acid (0.08mg) remained in the liquid medium in comparison with the initial ketoprofen dose (1.0mg), suggesting possible mineralization of ketoprofen.

Degradation of the drug sodium diclofenac by Trametes versicolor pellets and identification of some intermediates by NMR.

Degradation of diclofenac sodium, a nonsteroidal anti-inflammatory drug widely found in the aquatic environment, was assessed using the white-rot fungus Trametes versicolor. Almost complete diclofenac removal (> or = 94%) occurred the first hour with T. versicolor pellets when the drug was added at relatively high (10 mg L(-1)) and environmentally relevant low (45 microg L(-1)) concentrations in a defined liquid medium. In vivo and in vitro experiments using the cytochrome P450 inhibitor 1-aminobenzotriazole and purified laccase, respectively, suggested at least two different mechanisms employed by T. versicolor to initiate diclofenac degradation. Two hydroxylated metabolites, 4'-hydroxydiclofenac and 5-hydroxydiclofenac, were structurally elucidated by nuclear magnetic resonance as degradation intermediates in fungal cultures spiked with diclofenac. Both parent compound and intermediates disappeared after 24 h leading to a decrease in ecotoxicity calculated by the Microtox test. Laccase-catalyzed transformation of diclofenac led to the formation of 4-(2,6-dichlorophenylamino)-1,3-benzenedimethanol, which was not detected in in vivo experiments probably due to the low laccase activity levels observed through the first hours of incubation.