Propionate

At 6, 12, 24, 36, and 48 h time points after inoculation, we measured total gas production from fermentation as overpressure using a graduated syringe and passing a needle through the rubber stopper prior to unsealing the tubes. For pH measurements, the supernatant was transferred to a separate 15 ml Falcon tube and measured using a pH meter. We collected two aliquots from each tube, one for SCFA (0.4 ml) and other for DNA extraction (1 ml) and stored the aliquots at −80°C. An internal standard (157.5 μl of 4-methyl valeric acid, 1.47 ml of 85% phosphoric acid, 39 mg of copper sulfate pentahydrate in a total volume of 25 ml) was immediately added to the SCFA aliquots samples before vortexing.
We measured SCFAs as previously described (Tuncil et al., 2018b (link)). Briefly, 4 μl of the supernatants were analyzed on a fused-silica capillary column (Nukon^TM, SUPELCO No: 40369-03A, Bellefonte, PA, United States) using a gas chromatograph (GC-FID 7890A, Agilent Technologies, Inc.) (Tuncil et al., 2017 (link)). We used 4- methylvaleric acid (Fisher Scientific) as an internal standard and acetate, propionate, and butyrate (Fisher Scientific, Hampton, NH, United States) to generate standard curves.

SCFA analyses were performed as previously described^{35 (link)}. Briefly, the samples were thawed at room temperature and centrifuged at 13,000 rpm for 10 mins. Supernatants (4 µl) were analyzed using a gas chromatography (GC-FID 7890 A; Agilent Technologies Inc.) on a fused silica capillary column (Nukon^TM SUPELCO No: 40369-03A, Bellefonte, PA) under the following conditions: Injector temperature at 230 °C; initial oven temperature at 100 °C; temperature increase of 8 °C/min to 200 °C with a hold for 3 min at final temperature. Helium was used as a carrier gas at 0.75 ml/min. Acetate (catalog number: A38S), propionate (catalog number: A258), and butyrate (catalog number: AC108111000) purchased from Fisher Scientific (Hampton, NH) were used as external standards. 4-methylvaleric acid (catalog number: AAA1540506, Fisher Scientific) was used as an internal standard for quantification.