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 (NukonTM, 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.
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 (NukonTM, 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.