Ernest Marco-Urrea

Ability of white-rot fungi to remove selected pharmaceuticals and identification of degradation products of ibuprofen by Trametes versicolor

A screening using four white-rot fungi (Trametes versicolor, Irpex lacteus, Ganoderma lucidum and Phanerochaete chrysosporium) was performed on the degradation of 10 mg L(-1) of ibuprofen (IBU, anti-inflammatory), clofibric acid (CLOFI, lipid regulator) and carbamazepine (CARBA, antiepileptic/analgetic) after 7 d of incubation. Whereas IBU was extensively degraded by all the fungi tested, T. versicolor was the only strain able to degrade either CLOFI (approximately 91%) and CARBA (approximately 58%), although the latter was also degraded by G. lucidum (approximately 47%). In vitro experiments using manganese peroxidase and laccase-mediator system showed that extracellular fungal enzyme systems did not appear to play a role in the first step of degradation. However, our in vivo studies using the cytochrome P450 inhibitors 1-aminobenzotriazole and piperonyl butoxide suggested that the cytochrome P450 system may be involved in the first step of CLOFI and CARBA oxidation by T. versicolor. During the very early stages of IBU degradation by T. versicolor, two hydroxylated metabolites were detected: 1-hydroxy ibuprofen and 2-hydroxy ibuprofen. These byproducts were subsequently degraded by the fungus to 1,2-dihydroxy ibuprofen, that was not reported in biological systems to date. Furthermore, these results are of particular interest because CLOFI and CARBA are highly persistent in the aquatic environment and they pass unchanged or poorly transformed in wastewater treatment plants.

Biodegradation of the analgesic naproxen by Trametes versicolor and identification of intermediates using HPLC-DAD-MS and NMR

The white-rot fungus Trametes vesicolor degraded naproxen (10 mg L(-1)) in a liquid medium to non-detectable levels after 6h. When naproxen was added in the range of concentrations typically found in the environment (55 microg L(-1)), it was almost completely degraded (95%) after 5h. In vitro degradation experiments with purified laccase and purified laccase plus mediator 1-hydroxybenzotriazol showed slight and almost complete naproxen degradation, respectively. A noticeable inhibition on naproxen degradation was also observed when the cytochrome P450 inhibitor 1-aminobenzotriazole was added to the fungal cultures. These data suggest that both enzymatic systems could play a role in naproxen degradation. 2-(6-hydroxynaphthalen-2-yl)propanoic acid and 1-(6-methoxynaphthalen-2-yl)ethanone were structurally elucidated by HPLC-DAD-MS and NMR as degradation intermediates of naproxen. After 6h of incubation, both parent compound and intermediates disappeared from the medium. The non-toxicity of the treated medium was confirmed by Microtox test.

Degradation of the antibiotics norfloxacin and ciprofloxacin by a white-rot fungus and identification of degradation products.

More than 90% of the antibiotics ciprofloxacin (CIPRO) and norfloxacin (NOR) at 2 mg L(-1) were degraded by Trametes versicolor after 7 days of incubation in malt extract liquid medium. In in vitro assays with purified laccase (16.7 nkat mL(-1)), an extracellular enzyme excreted constitutively by this fungus, 16% of CIPRO was removed after 20 h. The addition of the laccase mediator 2,2-azino-bis-(3-ethylbenzthiazoline-6-sulfonic acid) diammonium salt led to 97.7% and 33.7% degradation of CIPRO and NOR, respectively. Inhibition of CIPRO and NOR degradation by the cytochrome P450 inhibitor 1-aminobenzotriazole suggests that the P450 system also plays a role in the degradation of the two antibiotics. Transformation products of CIPRO and NOR were monitored at different incubation times by triple-quadrupole and quadrupole time-of-flight mass spectrometry, and can be assigned to three different reaction pathways: (i) oxidation of the piperazinyl substituent, (ii) monohydroxylation, and (iii) formation of dimeric products.

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.

Oxidation of atenolol, propranolol, carbamazepine and clofibric acid by a biological Fenton-like system mediated by the white-rot fungus Trametes versicolor

Biological advanced oxidation of the pharmaceuticals clofibric acid (CA), carbamazepine (CBZP), atenolol (ATL) and propranolol (PPL) is reported for the first time. Extracellular oxidizing species were produced through a quinone redox cycling mechanism catalyzed by an intracellular quinone reductase and any of the ligninolytic enzymes of Trametes versicolor after addition of the lignin-derived quinone 2,6-dimethoxy-1,4-benzoquinone (DBQ) and Fe(3+)-oxalate in the medium. Time-course experiments with approximately 10mg L(-1) of initial pharmaceutical concentration resulted in percent degradations above 80% after 6h of incubation. Oxidation of pharmaceuticals was only observed under DBQ redox cycling conditions. A similar degradation pattern was observed when CBZP was added at the environmentally relevant concentration of 50 microg L(-1). Depletion of DBQ due to the attack of oxidizing agents was assumed to be the main limiting factor of pharmaceutical degradation. The main degradation products, that resulted to be pharmaceutical hydroxylated derivatives, were structurally elucidated. The detected 4- and 7-hydroxycarbamazepine intermediates of CBZP degradation were not reported to date. Total disappearance of intermediates was observed in all the experiments at the end of the incubation period.

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.

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 naproxen and carbamazepine in spiked sludge by slurry and solid-phase Trametes versicolor systems

Growth and activity of the white-rot fungus Trametes versicolor on sewage sludge were assessed in bioslurry and solid-phase systems. Bioslurry cultures with different loads of sludge (10%, 25% and 38%, w/v) were performed. A lag phase of at least 2 d appeared in the 25 and 38%-cultures, however, the total fungal biomass was higher for the latter and lower for the 10%-culture after 30 d, as revealed by ergosterol determination. Detectable laccase activity levels were found in the 10 and 25%-cultures (up to 1308 and 2588 AUL(-1), respectively) while it was negligible in the 38%-culture. Important levels of ergosterol and laccase were obtained over a 60 d period in sludge solid-phase cultures amended with different concentrations of wheat straw pellets as lignocellulosic bulking material. Degradation experiments in 25%-bioslurry cultures spiked with naproxene (NAP, analgesic) and carbamazepine (CBZ, antiepileptic) showed depletion of around 47% and 57% within 24h, respectively. Complete depletion of NAP and around 48% for CBZ were achieved within 72 h in sludge solid cultures with 38% bulking material. CBZ degradation is especially remarkable due to its high persistence in wastewater treatment plants. Results showed that T. versicolor may be an interesting bioremediation agent for elimination of emerging pollutants in sewage sludge.

Transformation and carbon isotope fractionation of tetra- and trichloroethene to trans-dichloroethene by Dehalococcoides sp. strain CBDB1.

Dehalococcoides sp. strain CBDB1 reductively dechlorinated perchloroethene (PCE) and trichloroethene (TCE) to predominantly trans-1,2-dichloroethene (trans-DCE). Cell counting by direct microscopy showed that strain CBDB1 used PCE and TCE as electron acceptors for respiratory growth obtaining a growth yield of 3.9 × 10(12) cells per mol of chloride released in both cases. PCE and TCE were dechlorinated to trans- and cis-DCE at an average constant ratio of 3.4 (±0.2):1, which is consistent with the ratios found in several trans-DCE-producing sediments and soils containing uncultured Dehalococcoides-like species. Significant carbon isotope fractionation was observed during PCE and TCE reductive dehalogenation. The enrichment factor of TCE (εC = -11.2) was within the range of previously reported values for TCE dechlorination by other Dehalococcoides species although the tceA gene responsible for ethene generation in the latter cultures was absent in strain CBDB1. On the contrary, the enrichment factor of PCE (εC = -1.6) was 3.8-times lower than that obtained for Dehalococcoides sp. strain 195 although both strains shared a high similarity in the pceA gene responsible for PCE dechlorination in strain 195. In addition, the product-related enrichment factors for TCE dehalogenation were calculated based on product isotope signature of the two accumulated products cis-DCE (εC TCE→cis-DCE = -11.0) and trans-DCE (εC TCE→trans-DCE = -15.9). These results are of particular interest since strain CBDB1 constitutes, together with the recent isolated strain MB, the unique Dehalococcoides species unable to dechlorinate PCE and TCE beyond DCE.

Potential of non-ligninolytic fungi in bioremediation of chlorinated and polycyclic aromatic hydrocarbons.

In previous decades, white-rot fungi as bioremediation agents have been the subjects of scientific research due to the potential use of their unspecific oxidative enzymes. However, some non-white-rot fungi, mainly belonging to the Ascomycota and Zygomycota phylum, have demonstrated their potential in the enzymatic transformation of environmental pollutants, thus overcoming some of the limitations observed in white-rot fungi with respect to growth in neutral pH, resistance to adverse conditions and the capacity to surpass autochthonous microorganisms. Despite their presence in so many soil and water environments, little information exists on the enzymatic mechanisms and degradation pathways involved in the transformation of hydrocarbons by these fungi. This review describes the bioremediation potential of non-ligninolytic fungi with respect to chlorinated hydrocarbons and polycyclic aromatic hydrocarbons (PAHs) and also shows known conversion pathways and the prospects for future research.