The biological fate of 17α-ethinylestradiol (EE2; 500 ng/L to 1 mg/L) and trimethoprim (TMP; 1 μg/L to 1 mg/L) was evaluated with flow through reactors containing an ammonia oxidizing bacterial (AOB) culture, two enriched heterotrophic cultures devoid of nitrifier activity, and nitrifying activated sludge (NAS) cultures. AOBs biotransformed EE2 but not TMP, whereas heterotrophs mineralized EE2, biotransformed TMP, and mineralized EE2-derived metabolites generated by AOBs. Kinetic bioassays showed that AOBs biotransformed EE2 five times faster than heterotrophs. The basal expression of heterotrophic dioxygenase enzymes was sufficient to achieve the high degree of transformation observed at EE2 and TMP concentrations ≤ 1 mg/L, and enhanced enzyme expression was not necessary. The importance of AOBs in removing EE2 and TMP was evaluated further by performing NAS experiments at lower feed concentrations (500-1000 ng/L). EE2 removal slowed markedly after AOBs were inhibited, while TMP removal was not affected by AOB inhibition. Two key EE2 metabolites formed by AOB and heterotrophic laboratory-scale chemostats were also found in independent laboratory-scale mixed culture bioreactors; one of these, sulfo-EE2, was largely resistant to further biodegradation. AOBs and heterotrophs may cooperatively enhance the reliability of treatment systems where efficient removal of EE2 is desired.

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.

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