Anaerobic cell suspension experiments with enrichment cultures

All steps of cell suspension experiments were conducted under anaerobic conditions. After incubating overnight with 200 μM unlabeled phenylacetate, 4.5 L of the enrichment culture was harvested anaerobically by centrifugation (13,400× g, 4 °C, 10 min), resuspended in 50 mL of anaerobic 10 mM sodium phosphate buffer (pH 7.5) in the anaerobic glove box, and 17 mL of the cell suspension (16.4 mg protein) was distributed into duplicate 20-mL glass vials sealed with 24-mm diameter polytetrafluoroethylene (PTFE) Mininert screw-cap valves (Sigma-Aldrich). Each vial was amended with 200 μM of labeled phenylacetate (phenylacetic acid-2-¹³C). Gaseous (static headspace) and 150 μL liquid samples were taken at time zero and every 30 min thereafter up to 210 min. Headspace samples (100 μL) were taken with a 500-μL gastight syringe (Sample-Lok Series A-2; Sigma-Aldrich) and analyzed immediately by gas chromatography/mass spectrometry (GC/MS) as described in the Supplementary Material (Text S1). Liquid samples for liquid chromatography/mass spectrometry (LC/MS) were centrifuged to remove whole cells, 100 μL of supernatant was diluted 50% with high-purity methanol, and these samples were stored in sealed glass vials with 250-μL glass inserts at 4 °C until LC/MS analysis (described in the Supplementary Material, Text S1). Analogous experiments were performed with labeled phenylalanine (L-phenylalanine-β-¹³C) substituting for labeled phenylacetate.