Quantification of glycerol consumption during fermentations using GC-MS

Samples were taken for glycerol analysis by aseptically removing 1000 μl culture from the fermentation vessel. Cells were removed by centrifugation for 2 min at 10,500×g, and the cell supernatant was carefully transferred to a clean, sterile 1.5 ml Eppendorf tube and stored at − 20 °C for further analysis. Prior to GC-MS analysis, glycerol was first derivatised to glycerol triacetate and separated from the culture supernatant (Wu, et al. 2011). This was done by adding 10 μl NMIM (N-methylmidazole) to 10 μl culture supernatant in a 1.5-ml Eppendorf tube. A volume of 75 μl acetic anhydride was then added, and samples were incubated at room temperature for 5 min. After incubation, 100 μl dH2O was added and the sample was vortexed for 10 s. Dichloromethane was then added at a volume of 100 μl, and 10 μl hexadecane was also added as an internal standard. The sample was vortexed briefly and left to separate at room temperature. The organic phase was then collected, and 0.1 g Na₂SO4 (anhydrous) was added to remove any residual aqueous phase from the sample. Samples were then filtered using grade 1 filter paper (Whatman® qualitative filter paper, grade 1) and added to clean, labelled HPLC vials (Sigma-Aldrich).

GC analysis was performed on an Agilent system equipped with an MS detector (Agilent Technologies, CA, USA). An HP-5 silica-based capillary column (30 m × 0.25 mm × 0.25 μm) was used for the separation of the glycerol derivative with a split ratio of 50:1. Compressed helium was used as the carrier gas at a flow rate of 1 ml min⁻¹ in constant-flow mode. The initial column temperature was 140 °C for 2 min which then increased to 250 °C at a rate of 20 °C min⁻¹ and maintained for 2 min. The inlet temperature was set at 210 °C and the detector temperature was 250 °C.