e. without biomass, controls for each medium were prepared. Aerobic conditions and photolysis prevention were ensured by shaking at 150 rpm on an orbital

SRT2104 supplier shaker in the dark. The setups were sampled once a day for MSM-CN and MSM media and twice a day for R2A-UV, by taking 1 mL supernatant after half an hour of sedimentation that was sufficient to ensure not to withdraw much biomass. 200 μL was used for UV-AM and 800 μL for LC-UV measurements. Analyses of sulfamethoxazole UV-AM 200 μL were taken from the setups and directly used for UV-AM as described elsewhere (Herzog et al., submitted) with the following changes applied. Calibration was performed with 1.0, 5.0, 10.0 and 15.0 mg L-1 SMX in high-purity water and the used media to evaluate SGC-CBP30 purchase measurement reliability and background absorbance. 96 well UV-star plates from Greiner Bio-One (Greiner Bio-One GmbH, Frickenhausen, Germany) filled with 200 μL were used for measurements and analyzed with an automated plate reader (EnSpire® Multimode Plate Reader, Perkin Elmer, Rodgau, Germany). Each measurement included an SMX blank (media with SMX but without organisms) was measured to detect changes over time as well as a blank (media without SMX) to detect

background absorbance. LC-UV analysis 800 μL samples obtained from the setups were centrifuged (10 min, 8000 g, 20°C), filtrated through a 0.45 μm membrane filter to EPZ5676 nmr remove cellular debris and biomass and filled into sterile glass flasks. Flasks were stored at-20°C before analysis. Analysis was performed with a Dionex 3000 series HPLC system (Dionex, Idstein, Germany), equipped with an auto sampler. A DAD scanning from 200 to 600 nm was

applied to detect and quantify SMX. Chromatographic separation was achieved on a Nucleosil 120-3 C18 column (250 mm × 3.0 mm i.d., 3 μm particle size) from Macherey Nagel (Düren, Germany) at a column temperature of 25°C. The applied mobile phases were acetonitrile (AN) and water (pH 2.5 using phosphoric acid). The gradient used for the first 5 min was 7% AN followed by Farnesyltransferase 7-30% AN from 5-18 min, 30% AN for minutes 18-30 and finally 7% AN for minutes 30-35. The solvent flow rate was 0.6 mL min-1. The column was allowed to equilibrate for 5 min between injections. Limit of quantification and limit of detection were 0.1 mg L-1 and 0.03 mg L-1, respectively. Taxonomic and phylogenetic identification of isolated pure cultures by 16S rRNA gene sequence analysis DNA of SMX biodegrading organisms was extracted by a standard phenol/chloroform/CTAB extraction method. 16S rRNA gene was subsequently amplified via standard PCR using universal bacterial primers 27f (5-AGA GTT TGA TCM TGG CTC AG-3) and 1492r (5-TAC GGY TAC CTT GTT ACG ACT T-3) [49]. All cultures were sent to MWG Operon (Ebersberg, Germany) for sequencing using again primers 27f and 1492r and resulting in nearly full length 16S rRNA gene sequences. Sequences were analyzed with and submitted to European Nucleotide Archive (http://​www.​ebi.​ac.