Quantification of Short-Chain Fatty Acids

Volatile fatty acids and lactic acid, referred to in combination as short-chain fatty acids (SCFA), were analyzed in six replicate fermentation vessels per treatment at the 4-, 10-, and 24-h time-points. The SCFA were analyzed as free acids, using pivalic acid (Sigma-Aldrich, St. Louis, MO, USA) as an internal standard. For this, 400 μL of fermentation fluid and 2.4 mL of 1.0 mM pivalic acid solution were mixed, vigorously shaken for 5 min, and then centrifuged at 3,000 × g for 10 min. Then 800 μL of the supernatant and 400 μL of saturated oxalic acid solution were mixed, incubated at 4°C for 60 min, and centrifuged at 18,000 × g for 10 min. The supernatant was analyzed by gas chromatography (Agilent Technologies, Santa Clara, CA, USA) using a glass column packed with 80/120 Carbopack B-DA/4% Carbowax stationary phase, helium as a carrier gas, and a flame ionization detector. The acids quantified were acetic, propionic, butyric, valeric, isobutyric, 2-methylbutyric, isovaleric, and lactic acid.