4 m sodium hydroxide solution

After centrifuging the fecal and slurry samples at 3134× g for 20 min at 20 °C, 4 mL of supernatant was collected. The supernatant was placed in a 20-mL glass vial, and then 4 mL of chloroform (Merck, Darmstadt, Germany) and 60 μL of 4 M sodium hydroxide solution (Sigma-Aldrich, St. Louis, MO, USA) were also added to the glass vial and mixed with supernatant. The mixture in the glass vial was centrifuged at 3134× g for 20 min at 20 °C, and the chloroform layer was collected and placed into a 2.0-mL gas chromatography vial (Agilent, Santa Clara, CA, USA). Phenols and indoles in the chloroform layer were determined using a gas chromatograph (6890N, Agilent, Santa Clara, CA, USA) that was equipped with a DB-1 column (30 m × 0.25 mm × 0.25 μm, Agilent, Santa Clara, CA, USA) and a flame ionization detector. A sample of 2.0 μL was injected at a 5-to-1 split ratio. The gas chromatography oven was initially set at 40 °C for 5 min, increased to 230 °C at a rate of 10 °C per min, and finally held at 230 °C for 2 min. The injection and detection ports of gas chromatography were maintained at 250 °C.

Slurry samples were centrifuged at 3,134 × ɡ for 20 min at 20°C, and then 4 mL of supernatant was mixed with 4 mL of chloroform (Merck, Darmstadt, Germany) and 60 μL of 4M sodium hydroxide solution (Sigma-Aldrich, St. Louis, MO, USA) in a 20 mL glass vial. The mixture was centrifuged at 3,134 × ɡ for 20 min at 20°C, and the chloroform layer was transferred to a 2.0 mL GC vial (Agilent, Santa Clara, CA, USA). Phenols and indoles were analyzed using a GC (6890N, Agilent, Santa Clara, CA, USA) equipped with a DB-1 column (30 m × 0.25 mm × 0.25 μm, Agilent, Santa Clara, CA, USA) and a FID. The sample injection volume was 2.0 μL with a 5:1 split ratio. The oven temperature was initially 40°C for 5 min, increasing to 230°C at 10°C/min, which was then held at 230°C for 2 min. The injection and detection ports were maintained at 250°C.