4.14. Short Chain Fatty Acid Quantification by GC-MS

Samples were harvested from each bioreactor at 4, 24, and 48 h post inoculation. Samples were centrifuged (Sorvall Legend XTR, ThermoFisher, Waltham, MA, USA) at 5000 rpm for 10 min. The supernatant was drawn into a syringe and sterile filtered using a 0.22 PES (Corning) syringe filter and transferred new 5 mL vials. The fluid was then stored in −80 °C freezer until analysis.

To begin SCFA quantification, the samples were thawed at 40 °C for 30 min. The total SCFAs were extracted from the media with diethyl ether for GCMS (QP2010 Ultra, Shimadzu Scientific, Columbia, MD, USA) analysis. The GC-MS method involved injecting 1 µL of sample into the 260 °C injection port. Using a split ratio of 1:20 and a flow rate of 1.00 mL/min of helium, the sample was deposited on the Stabilwax-DA 30 m × 0.25 mm column (Restek Corporation, Bellefonte, PA, USA) which was held at 125 °C for 1 min and then ramped to 170 °C at 30 °C/min, then to 220 °C at 10 °C/min and then to 250 °C at 50 °C/min where the temperature was held for 3 min. The interface temperature between the GC and MS was held at 250 °C and the ion source temperature was 220 °C.

For each time point, a 2-tailed, unpaired homoscedastic Student’s t-test was run to determine if the concentration difference between the control and the experimental groups was statistically significant, p < 0.05.