Characterization of Metabolites Formed During the Biotransformation of 17α-Ethinylestradiol by Nitrosomonas europaea in Batch and Continuous Flow Bioreactors

Abstract

The biotransformation of 17alpha-ethinylestradiol (EE2) by an ammonia oxidizing bacteria, Nitrosomonas europaea, grown in batch (ammonia-rich) and continuous flow (chemostat, ammonia-limited) reactors was investigated. Both C-14 labeled EE2 (10 gammag/L) and unlabeled EE2 (1 mg/L) were used to facilitate metabolite identification under environmentally relevant physiological conditions. Whole cell ammonia monooxygenase (AMO) activity was not inhibited at the EE2 concentrations used in this study. Characterization of the primary metabolite formed during batch cultivation by liquid chromatography/ion-trap mass spectrometry (LC-ITMS) and nuclear magnetic resonance (NMR) spectroscopy showed modification at the ethinyl group and addition of a carboxyl group. This metabolite (M386) (revealed by m/z 385 in negative mode electrospray LC/ MS) was not formed in the abiotic control. In contrast, biotransformation of EE2 under continuous flow conditions showed formation of a monohydroxylated EE2 (revealed by m/z 311), but not M386. Furthermore, nitrated EE2 derivatives were formed in both batch and continuous flow cultures, as a result of abiotic transformation of EE2 in the presence of high concentrations of nitrite in the bioreactors. Results from this study underscore the importance of physiological state and growth conditions as critical variables that can dictate the metabolic pathway for EE2 biodegradation and the nature of byproducts formed.

Resource dependent biodegradation of estrogens and the role of ammonia oxidising and heterotrophic bacteria

“…The biodegradation of EE2 in particular was examined by Skotnicka-Pitak et al [44] in continuous-flow experiments. Their EE2 loadings were c. 129.5 mg EE2 −1 kg N. europea tCOD d −1 , significantly higher than those of this study, 0.008-0.74 mg EE2 −1 kg AOB tCOD d −1 (mg EE2 biodegraded per kg of N. europea as tCOD per day) and biotransformation of EE2 was observed.…”

“…Furthermore, this research suggests that environmental concentrations of AOB and EE2 are insufficient to support co-metabolism during activated sludge treatment. Whilst it is postulated that the fortuitous activity of monoxygenase is important in the removal of organic contaminants such as trihalomethanes and chloroform [44,45], to date, evidence indicating that AOB can biotransform these chemicals at ng L −1 concentrations remains limited.…”

Pharmaceutical metabolites in the environment: Analytical challenges and ecological risks

“…Instead, laboratory-scale experiments are first performed at exaggerated concentrations to identify metabolites that form and persist. Although this is an accepted approach, microbial degradation of pharmaceuticals under different physiological conditions has been shown to produce different metabolites and therefore must be conducted with caution [71].…”

Biodegradation of atenolol by an enriched nitrifying sludge: Products and pathways

“…Another study showed that the oxidative removal of trace organic contaminants correlated with the removal of ammonium (NH 4 + -N) [9]. Ammonia oxidizing bacteria (AOB) in the nitrifying activated sludge are able to degrade a range of aromatic compounds due to its nonspecific enzyme ammonium monooxygenase (AMO) [10,11], following cometabolism in the obligatory presence of a growth substrate such as ammonium [12]. AMO was capable of oxidizing a broad range of aromatic substrates [10,13], probably due to the mechanism of reaction with oxygenated form of AMO [14].…”