Minisart 0.2 μm syringe filter

Exocellular metabolites were identified and quantified by nuclear magnetic resonance (NMR). Broth samples were collected at different times and filtered (Minisart 0.2-μm syringe filter; Sartorius, Göttingen, Germany). The supernatants, consisting of the culture medium, were mixed with 100 μl of D₂O with 2.35 g/liter of TSP-d4 (deuterated trimethylsilylpropanoic acid) as an internal reference. Proton NMR spectra were recorded on an Avance III 800-MHz spectrometer equipped with a 5 mM QCI-P cryoprobe (Bruker, Rheinstatten, Germany). Quantitative ¹H NMR was performed at 280 K, using a 30° pulse and a relaxation delay of 10 s. Two-dimensional ¹H-¹³C heteronuclear single quantum coherence (HSQC) spectra were recorded at 280 K to quantify SHX in the supernatant. Sixteen scans were acquired with 4,096 by 128 datapoints and 13.35- by 60-ppm spectral widths. The spectra were processed and the metabolites were quantified using Topspin 3.1 (Bruker, Rheinstatten, Germany). Extracellular metabolites from three independent biological replicates were analyzed.