Elsevier

Environmental Pollution

Volume 158, Issue 8, August 2010, Pages 2572-2581
Environmental Pollution

Anaerobic testosterone degradation in Steroidobacter denitrificans – Identification of transformation products

https://doi.org/10.1016/j.envpol.2010.05.017Get rights and content

Abstract

The transformation of the androgenic steroid testosterone by gammaproteobacterium Steroidobacter denitrificans was studied under denitrifying conditions. For the first time, growth experiments showed that testosterone was mineralized under consumption of nitrate and concurrent biomass production. Experiments with cell suspensions using [4-14C]-testosterone revealed the intermediate production of several transformation products (TPs). Characterisation of ten TPs was carried out by means of HPLC coupled to high resolution mass spectrometry with atmospheric pressure chemical ionization as well as 1H and 13C NMR spectroscopy. 3β-hydroxy-5α-androstan-17-one (trans-androsterone) was formed in the highest amount followed by 5α-androstan-3,17-dione. The data suggests that several dehydrogenation and hydrogenation processes take place concurrently in ring A and D because no consistent time-resolved pattern of TP peaks was observed and assays using 2 TPs as substrates resulted in essentially the same TPs. The further transformation of testosterone in S. denitrificans seems to be very efficient and fast without formation of detectable intermediates.

Introduction

Steroid hormones have come into focus as environmental pollutants because they are able to act as endocrine disruptors in aquatic environments and are widely excreted by animals and humans. Testosterone, produced in the testis of males and in the ovaries of females, derives biosynthetically from cholesterol and regulates metabolism, growth, and reproduction in mammals. The most likely sources of testosterone and of its known urinary excreted human metabolites (4-androstene-3,17-dione, 3-hydroxy-androstane-17-one, 17-hydroxy-androstane-3-one, androstane-3,17-dione, 3,17-dihydroxy-androstane, 3,17-dihydroxy-5-androstene, 3-hydroxy-5-androstene-17-one, 17-hydroxy-androstene-3-one) for aquatic environments are municipal sewage sludge, paper mill effluents, and runoff from agricultural production sites (Jacobsen et al., 2005, Jenkins et al., 2003, Liu et al., 2009, Thomas et al., 2002). Thomas et al. (2002) have shown that wild fish living downstream of exposed waters were negatively affected by androgenic steroids causing endocrine disruption.

Microbial transformation of testosterone was observed in several environmental matrices such as soils (Casey et al., 2004, Lorenzen et al., 2005), soils amended with swine manure, soil-water slurries (Lee et al., 2003), stream sediments (Bradley et al., 2009), and within the wastewater treatment process (Esperanza et al., 2007). In most cases transformation under aerobic conditions was investigated. Yang et al. (2009) showed recently for manure-borne bacteria that the half-life under anaerobic conditions was significantly increased.

Testosterone, 4-androstene-3,17-dione, and other steroids, such as estradiol and cholesterol, are natural substances that can be completely degraded by several bacterial genera under aerobic conditions (Talalay et al., 1952, Kieslich, 1985, Fujii et al., 2003). Strains of the obligate aerobic betaproteobacterium Comamonas testosteroni (Talalay et al., 1952, Tamaoka et al., 1987) mineralize testosterone to carbon dioxide and water through the well-studied and oxygen-dependent 9,10-seco pathway (Coulter and Talalay, 1968, Sih and Whitlock, 1968, Horinouchi et al., 2004). However, in anoxic environments, key reactions of the 9,10-seco pathway, such as activation of the growth substrate by oxygen-dependent hydroxylation and its subsequent oxygenolytic cleavage, cannot take place. Recently, we enriched and isolated the denitrifying gammaproteobacterium S. denitrificans strain FST with estradiol as the sole carbon and energy source from digested sewage sludge. Strain FST is the first characterized bacterium able to grow on testosterone as the sole source of carbon and energy and with nitrate as the electron acceptor (Fahrbach et al., 2008). Therefore, it must be able to utilize metabolic strategies that do not depend on oxygen for degrading the stable steroid sub-structure.

During wastewater treatment, in freshwater sediments or carbon-rich soils oxygen is rapidly consumed resulting in anoxic conditions. In such environmental compartments microbial transformation of androgenic steroids under denitrifying conditions might be an important process. As there is currently no information available about the anaerobic transformation pathway of androgenic steroids such as testosterone, our aim was to study the transformation of testosterone under denitrifying conditions by strain FST. Our specific goals were to (i) determine quantitatively the consumption of testosterone including an electron balance under nitrate reducing conditions and (ii) to characterize the initial degradation pathway by means of the detection of intermediate TPs. To allow identifying TPs testosterone concentrations which are orders of magnitude higher than in environmental systems were applied. Only this strategy allowed the clear identification of TPs without reference standards using a complementary approach consisting of liquid chromatography combined with high resolution mass spectrometry and nuclear magnetic resonance spectroscopy.

Section snippets

Chemicals

[4-14C]-testosterone was obtained from Hartmann Analytic (Braunschweig, Germany). Unlabeled testosterone, the TPs androsta-1,4-diene-3,17-dione (M2), 3β-hydroxy-5 α-androstane-17-one (M5) (all Fluka, Germany) (Table 2) and all other chemicals were of the highest purity available. DMSO-D6 (99 96 atom%) and CDCl3 (99 8 atom%) were purchased from Deutero (Kastellaun, Germany).

Bacteria, growth media, and culture conditions

The gammaproteobacterium S. denitrificans strain FST (= DSM 18526T) was isolated and enriched from anoxic digested sludge

Complete degradation of testosterone with nitrate as the electron acceptor

Growth experiments clearly showed that testosterone was degraded by S. denitrificans under consumption of nitrate and concurrent biomass and dinitrogen monooxide production (Fig. 1). When incubated with different amounts of steroid but equal initial amounts of nitrate (Table 1), cell dry mass increased with higher amounts of substrate dissimilated. At the time of harvesting of the assays, consumed nitrate was converted nearly stoichiometrically to N2O (average 105.8 ± 4.3%). In the control

Acknowledgements

This work was funded by a grant from the Swiss National Science Foundation (Project no. 113482). We would like to thank Manfred Elend for conducting the NMR analyses as well as Heinz Singer and Marc Suter for discussion of MS data.

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