Gcms qp2010s system

To determine the quantity of alcohols and acids in the cultures, 1 mL of cell suspension was centrifuged (17,000 ×g, 2 min, room temperature) and 100 µL of the supernatant was mixed with 900 µL methanol containing 2.2 mM 1,3-propanediol as an internal standard (IS). After centrifugation (17,000 ×g, 5 min, room temperature), 750 µL of the supernatant was transferred to glass vials for measurement. GC–MS analysis was carried out by injecting 1 µL with a 1:10 split into a GCMS-QP2010S system (Shimadzu, Kyoto, Japan) fitted with an InertCap FFAP capillary column (0.25 mm × 30 m, 0.25 µm film thickness) from GL Sciences (Eindhoven, Netherlands). The temperature profile started with an initial 3-min hold at 50 °C followed by a gradient of 35 °C min⁻¹ to 220 °C with a final hold at 220 °C for 2 min.

Fermentation products were quantified by GC/MS on a Shimadzu GCMS-QP2010S system (Shimadzu, Kyoto, Japan). The culture supernatant was diluted 1:10 in methanol containing 5.5 mM 1,3-propanediol as an internal standard. The temperature profile of the GC protocol included an initial 1-min step at 60 °C followed by a temperature gradient of 5 °C/min to 70 °C and 35 °C/min to 220 °C, and then a 2-min hold before cooling to 60 °C for the next run. Retention times and the quantification of ethanol, butanol and acetate were established using GC/MS-grade standards.

All organic starting materials and solvents were analytically pure and used without further purification. KOH (99.99%) was obtained from Sigma-Aldrich (St. Louis, MO, USA). Nuclear magnetic resonance (NMR) spectra were recorded on ECA-400 or 600 spectrometers (JEOL, Tokyo, Japan) using CDCl₃ and DMSO-d₆ as a solvent at 298 K. ¹H-NMR (400 MHz, 600 MHz) chemical shifts (δ) were referenced to internal standard TMS (for ¹H, δ = 0.00 ppm). ¹³C-NMR (100 MHz, 125 MHz) chemical shifts were referenced to internal solvent (δ = 77.16 ppm in CDCl₃; 39.52 ppm in DMSO-d₆). Mass spectra (MS) were obtained on a GCMS-QP2010S system (Shimadzu Kyoto, Japan), the high-resolution mass spectra (ESI) were obtained with a micrOTOF-Q 10142 spectrometer (Agilent, California, CA, USA). The melting points are uncorrected.