Quantifying Volatile Fatty Acids by GC

Molar proportions of propionate and butyrate used as an input for the correlation analysis of the current study were calculated as follows: (individual VFA mM/ total VFA mM) × 100 and reported in our companion study Bennett et al. (2021) (link). Briefly, samples for VFA analyses were processed according to Ruiz-Moreno et al. (2015) (link) by centrifuging at 10,000 × g for 15 min. Supernatant was mixed with a solution of crotonic acid and metaphosphoric acid to freeze overnight, and then centrifuged again at 10,000 × g for 15 min. Resulting supernatant was mixed with ethyl acetate, vortexed and the top layer transferred to a chromatography injection vial for gas chromatography (Agilent 7820A GC, Agilent Technologies, Palo Alto, CA) with a flame ionization detector and a capillary column (CP-WAX 58 FFAP 25 m 0.53 mm, Varian CP7767, Varian Analytical Instruments, Walnut Creek, CA) maintained at 110 °C, with injector temperature at 200 °C and detector at 220 °C.