Testosterone Degradation in Strain GDN1

Strain GDN1 was aerobically grown in a phosphate-buffered shake-flask (300 ml in 1 L-Erlenmeyer flask) containing 1 mM testosterone. 17α-Ethinylestradiol (50 μM; indigestible by strain GDN1) was added as an internal control. Nitrate was omitted from the medium. In 1 L of distilled water, the medium contained the following: 0.29 g testosterone, 2.0 g NH₄Cl, 0.5 g MgSO₄7 H₂O, and 0.1 g CaCl₂2 H₂O. After autoclaving, sterile 50 ml KH₂PO₄-K₂HPO₄ buffer solution (1 M, pH 6.0), vitamins (1 mL/L)^{54 (link)}, EDTA-chelated mixture of trace elements (1 mL/L)^{55 (link)}, and selenite and tungstate solution (1 mL/L)^{56 (link)} were added. The aerobic culture was incubated at 37°C in an orbital shaker (180 rpm). The cultural samples (5 mL) were withdrawn every 6 h (0 ~ 30 h). The testosterone-derived intermediates extracted from the cultural samples were identified and quantified using UPLC – HRMS The androgenic activity of the cultural samples was determined as described later.