title: Identification of triclosan-degrading bacteria using stable isotope probing, fluorescence in situ hybridization and microautoradiography.
abstract: Triclosan is considered a ubiquitous pollutant and can be detected in a wide range of environmental samples. Triclosan removal by wastewater treatment plants has been largely attributed to biodegradation processes; however, very little is known about the micro-organisms involved. In this study, DNA-based stable isotope probing (DNA-SIP) combined with microautoradiography-fluorescence in situ hybridization (MAR-FISH) was applied to identify active triclosan degraders in an enrichment culture inoculated with activated sludge. Clone library sequences of 16S rRNA genes derived from the heavy DNA fractions of enrichment culture incubated with (13)C-labelled triclosan showed a predominant enrichment of a single bacterial clade most closely related to the betaproteobacterial genus Methylobacillus. To verify that members of the genus Methylobacillus were actively utilizing triclosan, a specific probe targeting the Methylobacillus group was designed and applied to the enrichment culture incubated with (14)C-labelled triclosan for MAR-FISH. The MAR-FISH results confirmed a positive uptake of carbon from (14)C-labelled triclosan by the Methylobacillus. The high representation of Methylobacillus in the (13)C-labelled DNA clone library and its observed utilization of (14)C-labelled triclosan by MAR-FISH reveal that these micro-organisms are the primary consumers of triclosan in the enrichment culture. The results from this study show that the combination of SIP and MAR-FISH can shed light on the networks of uncultured micro-organisms involved in degradation of organic micro-pollutants.
authors.affiliation: Department of Biotechnology, Chemistry and Environmental Engineering, Aalborg University, Sohngaardsholmsvej 49, DK-9000 Aalborg, Denmark.
authors.forename: Ihab Bishara
authors.initials: IB
authors.lastname: Lolas
authors.affiliation: Department of Environmental Science, Aarhus University, Frederiksborgsvej 399, 4000 Roskilde, Denmark.|Department of Biotechnology, Chemistry and Environmental Engineering, Aalborg University, Sohngaardsholmsvej 49, DK-9000 Aalborg, Denmark.
authors.forename: Xijuan
authors.initials: X
authors.lastname: Chen
authors.affiliation: Department of Environmental Science, Aarhus University, Frederiksborgsvej 399, 4000 Roskilde, Denmark.
authors.forename: Kai
authors.initials: K
authors.lastname: Bester
authors.affiliation: Department of Biotechnology, Chemistry and Environmental Engineering, Aalborg University, Sohngaardsholmsvej 49, DK-9000 Aalborg, Denmark.
authors.forename: Jeppe Lund
authors.initials: JL
authors.lastname: Nielsen
mesh_terms: D001345:Autoradiography / Q000379:methods*; D001419:Bacteria / Q000145:classification / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism*; D001673:Biodegradation, Environmental; D017404:In Situ Hybridization, Fluorescence / Q000379:methods*; D008969:Molecular Sequence Data; D010802:Phylogeny; D012722:Sewage / Q000382:microbiology*; D014260:Triclosan / Q000378:metabolism*
chemical_list: D012722:Sewage; D014260:Triclosan