0.22 μm polyvinylidene fluoride syringe filters

Four Paenarthrobacter spp. were selected for degradation experiments based on their different sad gene arrangements. The Paenarthrobacter spp. were grown in fresh LB medium at room temperature with shaking at 180 rpm to exponential phase (OD₅₉₅ of 1.0–1.5). The biomass was harvested by centrifugation at 3725 g for 20 min (Beckman Coulter Avant J-15R) and washed with mineral salt medium (MSM, Table S1) trice. The biodegradation experiment was carried out in triplicate in 250-ml Erlenmeyer flasks containing 150 ml of MSM amended with 100 mg/L sulfonamide as the sole carbon source. Inoculum of Paenarthrobacter sp. was added in a set of three flasks to a target initial OD₅₉₅ of 0.1. The control treatment was inoculated with mixed sterilized cultures to monitor the abiotic loss of sulfonamides. All the flasks were sealed with sterile breathable sealing film and incubated at room temperature and 180 rpm. A 2-ml suspension of Paenarthrobacter sp. was withdrawn at designed intervals from three independent flasks and centrifuged at 20000 g for 2 min in a 4°C pre-chilled centrifuge. The supernatant was filtered with 0.22-μm polyvinylidene fluoride syringe filters (Millipore, Germany) and stored at 4°C until analysis. The modified Gompertz model was applied to fit the degradation data.

Clostridium enriched conditioned media were prepared from all four individual isolates following a previously described method with some modifications [19 ]. Briefly, each Clostridium isolate grown on SBA plates was used to inoculate cooked meat glucose starch medium (45 mL) supplemented with yeast extract (0.0005 %), hemin (0.1 %), and vitamin K (1 %) and incubated in a 35 ^oC anaerobic chamber (Don Whitley Scientific, United Kingdom) for 48 h. After the incubation, enriched media were centrifuged at 10,000 × g for 40 min at 4 ^oC and supernatants were filter-sterilized using 0.22 μm polyvinylidene fluoride syringe filters (Millipore, Ireland). Sterile conditioned media were aliquoted and stored frozen at -20 ^oC until use. Conditioned media prepared from FS01, FS2.2, FS03, and FS04 isolates were denoted as FS01CM, FS2.2CM, FS03CM, and FS04CM respectively.