Anaerobic degradation of steroid hormones by novel denitrifying bacteria

During the last decade, the endocrine disrupting activity of steroid hormones both of natural and of anthropogenic origin was recognized as a potential risk to the fertility of wildlife populations in various ecosystems and the human population. Concerns about potential negative ecological effects of steroid hormones have resulted in an increased interest with regard to the elimination of these compounds in soil, sediments, and during wastewater treatment. Analysis of steroid hormones showed that denitrification significantly contributes to the overall elimination of estradiol in the sewage sludge process. However, nothing is known about the bacteria responsible for this estradiol degradation. Also no information exists about the degradation of testosterone under anoxic conditions. This work gives first data about the anaerobic degradation of both estradiol or testosterone by novel denitrifying bacteria. These bacteria (strain AcBE2-1 and strain FS) were isolated in this work from activated sludge and anoxic digested sludge of a municipal wastewater treatment plant with estradiol or testosterone as the sole source of carbon and energy and nitrate as the electron acceptor. Strain AcBE2-1 was isolated from activated sludge with estradiol and nitrate. This bacterium oxidized estradiol beyond estrone completely to carbon dioxide and water by reduction of nitrate to a mixture of dinitrogen monoxide and dinitrogen, with the intermediate accumulation of nitrite. Electron recoveries were between 90 and 100%, taking into account assimilated estradiol. With nitrate as the electron acceptor, the bacterium also grew on estrone, fatty acids (C2 to C6), isobutyrate, crotonate, DL-lactate, or several citric acid cycle intermediates. Phylogenetic analysis of its 16S rRNA gene sequence revealed that strain AcBE2-1 represents a separate line of descent within the family Rhodocyclaceae (Betaproteobacteria). Together with several clone sequences obtained from hitherto uncultivated bacteria, strain AcBE2-1 forms a novel cluster distinct from known lineages. The closest relatives are the cholesterol-degrading, denitrifying bacteria Sterolibacterium denitrificans DSM 13999T and strain 72Chol (= DSM 12783), with <93.9% sequence similarity. The G+C content of the DNA was 61.4 mol%. Detection of a quinone system with ubiquinone Q-8 as the predominant compound and a fatty acid profile that is clearly different from Sterolibacterium denitrificans, supported the results of the phylogenetic analysis. On the basis of 16S rRNA gene sequence data in combination with chemotaxonomic and physiological data, strain AcBE2-1 (= DSM 16959 = JCM 12830) is placed in a new genus Denitratisoma gen. nov. as the type strain of the type species Denitratisoma oestradiolicum gen. nov., sp. nov. Strain FS was isolated from anoxic digested sludge on estradiol and testosterone with nitrate as the electron acceptor. Strain FS represents the first bacterium that anaerobically mineralizes both estradiol (C18) or testosterone (C19). The degradation balance measured in testosterone-limited cultures indicated a complete oxidation to carbon dioxide and water. Estradiol oxidation was almost complete even in nitrate-limited cultures. Estrone and 4-androstene-3,17-dione were identified as intermediates of estradiol and testosterone degradation, respectively. Dinitrogen monoxide was the sole end product of denitrification, without intermediate nitrite formation. Beside estradiol and testosterone, only a narrow spectrum of organic substrates were used as electron donors under denitrifying conditions including estrone, 4-androstene-3,17-dione, acetate, propionate, isobutyrate, valerate, caproate, heptanoate, or glutamate. Comparative 16S rRNA gene sequence analysis revealed that strain FS has no known close relatives and represents a separate line of descent within the Gammaproteobacteria positioned among clone sequences of so far uncultivated bacteria. A distant relationship of about 87.1 % 16S rRNA gene sequence similarity exists to the genera Nevskia, Hydrocarboniphaga, Nitrosococcus, and Ectothiorhodospira, all of which exhibit different physiological properties in comparison with strain FS. The G+C content of the DNA was 61.9 mol%. Based on the high 16S rRNA gene sequence divergence and different physiological properties to previously described bacteria in combination with chemotaxonomic data, strain FS (= DSM 18526) was indicated as the type strain of a type species placed in a new genus Steroidobacter gen. nov., for which the name Steroidobacter denitrificans gen. nov., sp. nov. is proposed. A proteomic approach with Denitratisoma oestradiolicum allowed to demonstrate the induction of estradiol-specific proteins and resulted in the identification of several enzymes for example S-adenosylmethionine synthase, S-adenosylmethionine-dependent methyltransferases, 3-octaprenyl-4-hydroxybenzoate decarboxylase, and several hydroxysteroid dehydrogenases that might be involved in anaerobic estradiol degradation. These initial results represent a prerequisite for a detailed characterization and elucidation of the estradiol degradation pathway in this novel denitrifying bacterium. The obtained amino acid sequence data could be used in future studies to construct degenerated oligonucleotides for cloning and sequencing the respective genes coding for these proteins. The complete nucleotide sequence information will then allow to search for related proteins and to make functional and structural comparisons.

Grantor dc:publisher

Publikationsserver der RWTH Aachen University

Year dc:date

2007

Author dc:creator

• Fahrbach, Michael

Contributors dc:contributor

• Hollender, Juliane

• info:eu-repo/classification/ddc/570
• Biowissenschaften, Biologie
• Estradiol
• Testosteron
• Mikrobieller Abbau
• Bakterien
• Denitrifikation
• Anaerober Abbau
• Steroidhormone
• denitrifizierende Bakterien
• Anaerobic degradation
• denitrifying bacteria
• testosterone
• steroid hormones

Statement dc:rights

• info:eu-repo/semantics/openAccess

Language dc:language

eng

Identifier

https://publications.rwth-aachen.de/search?p=id:%22RWTH-CONV-123259%22

OAI identifier oai:identifier

oai:publications.rwth-aachen.de:61616