Meta phosphoric acid solution

Five milliliters of slurry were mixed with 1 mL of 25% meta-phosphoric acid solution (Sigma-Aldrich, St. Louis, MO, USA) and 0.05 mL of saturated mercury (II) chloride solution (Sigma-Aldrich, St. Louis, MO, USA) in a 15 mL plastic tube. The mixed solution was then centrifuged at 3,134 × ɡ for 20 min at 20°C. One milliliter of supernatant was subsequently centrifuged for 10 min at 13,800 × ɡ and filtered through a 0.2 μm filter (Whatman, Uppsala, Sweden). Filtrates were transferred to 2.0 mL GC vials (Agilent, Santa Clara, CA, USA). The concentration of VFAs was analyzed using a GC (6890N, Agilent, Santa Clara, CA, USA) equipped with a HP-INNOWax column (30 m × 0.25 mm × 0.25 μm; Agilent, Santa Clara, CA, USA) and a flame ionization detector (FID). The sample injection volume was 0.2 μL with a 10:1 split ratio. The oven temperature was initially temperature of 80°C for 2 min, increasing to 120°C at 20°C/min, then to 205°C at 10°C/min, and finally held at 205°C for 2 min. The injection and detection ports were maintained at 250°C.

For the VFA analysis, 1 mL of a 25% meta-phosphoric acid solution (Sigma-Aldrich, St. Louis, MO, USA) was added to each of 5 g of feces and 5 mL of slurry in a 15 mL plastic tube, and 0.05 mL of a saturated mercury (II) chloride solution (Sigma-Aldrich, St. Louis, MO, USA) was also added to the plastic tube. Then, the solution was centrifuged at 3134× g for 20 min at 20 °C, and then 1 mL of supernatant was collected. The supernatant was also centrifuged for 10 min at 13,800× g and filtered through a 0.2-μm Whatman filter (Whatman, Uppsala, Sweden). The filtrates were transferred to 2 mL gas chromatography vials (Agilent, Santa Clara, CA, USA). The concentration of VFA was determined using a gas chromatograph (6890N, Agilent, Santa Clara, CA, USA) that was equipped with an HP-INNOWax column (30 m × 0.25 mm × 0.25 μm; Agilent, Santa Clara, CA, USA) and a flame ionization detector. A sample of 0.2 μL was injected at a 10-to-1 split ratio. The gas chromatography oven was initially set at 80 °C for 2 min and increased to 120 °C at a rate of 20 °C per min. The oven temperature was then increased to 205 °C at 10 °C per min, and finally held at 205 °C for 2 min. The injection and detection ports of gas chromatography were maintained at 250 °C.