Carbamazepine (CBZ) is an environmentally recalcitrant compound highly stable in soil and during wastewater treatment. In this study, we examined the mechanisms by which the white-rot fungus Pleurotus ostreatus metabolizes CBZ in liquid culture using a physiological approach. P. ostreatus PC9 was grown in media known to support different levels of a multiplicity of enzyme systems such as cytochrome P450 (CYP450) and manganese peroxidase (MnP). When both CYP450 and MnP systems were active, 99% of the added CBZ was eliminated from the solution and transformed to 10,11-epoxycarbamazepine. High removal of CBZ was also obtained when either MnP or CYP450 was active. When both CYP450 and MnP were inactivated, only 10 to 30% of the added CBZ was removed. In this latter system, removal of CBZ might be partially attributed to the activity of versatile peroxidase. P. ostreatus was able to eliminate CBZ in liquid culture even when CBZ was added at an environmentally relevant concentration (1 μg L(-1)). On the basis of our study, we suggest that two families of enzymes are involved in the oxidation of CBZ in liquid culture: MnP in a Mn(2+)-dependent or independent manner and CYP450. Our study also highlights the potential of using P. ostreatus for bioremediation systems.

The widely used anticonvulsant pharmaceutical carbamazepine is recalcitrant in many environmental niches and thus poses a challenge in wastewater treatment. We followed the decomposition of carbamazepine by the white-rot fungus Pleurotus ostreatus in liquid culture compared to solid-state fermentation on lignocellulosic substrate where different enzymatic systems are active. Carbamazepine metabolites were identified using liquid chromatography-high-resolution mass spectrometry (LC-Q-TOF-MS). In liquid culture, carbamazepine was only transformed to 10,11-epoxy carbamazepine and 10,11-dihydroxy carbamazepine as a dead-end product. During solid-state fermentation, carbamazepine metabolism resulted in the generation of an additional 22 transformation products, some of which are toxic. Under solid-state-fermentation conditions, 10,11-epoxy carbamazepine was further metabolized via acridine and 10,11-dihydroxy carbamazepine pathways. The latter was further metabolized via five subpathways. When (14)C-carbonyl-labeled carbamazepine was used as the substrate, (14)C-CO2 release amounted to 17.4% of the initial radioactivity after 63 days of incubation. The proposed pathways were validated using metabolites (10,11-epoxy carbamazepine, 10,11-dihydroxy carbamazepine, and acridine) as primary substrates and following their fate at different time points. This work highlights the effect of growth conditions on the transformation pathways of xenobiotics. A better understanding of the fate of pollutants during bioremediation treatments is important for establishment of such technologies.

Transformation products (TPs) of environmental pollutants must be identified to understand biodegradation processes and reaction mechanisms and to assess the efficiency of treatment processes. The combination of oxidation by an electrochemical cell (EC) with analysis by liquid chromatography-high-resolution mass spectrometry (LC-HRMS) is a rapid approach for the determination and identification of TPs generated by natural microbial processes. Electrochemically generated TPs of the recalcitrant pharmaceutical carbamazepine (CBZ) were used for a target screening for TPs formed by the white-rot fungus Pleurotus ostreatus. EC with LC-HRMS facilitates detection and identification of TPs because the product spectrum is not superimposed with biogenic metabolites and elevated substrate concentrations can be used. A group of 10 TPs formed in the microbial process were detected by target screening for molecular ions, and another 4 were detected by screening on the basis of characteristic fragment ions. Three of these TPs have never been reported before. For CBZ, EC with LC-HRMS was found to be more effective than software tools in defining targets for the screening and faster than nontarget screening alone in TP identification. EC with LC-HRMS may be used to feed MS databases with spectra of possible TPs of larger numbers of environmental contaminants for an efficient target screening.