7890b gc system

Batch incubations were performed in 120 mL serum bottles containing 10 mL cells (OD₆₀₀∼0.9 or 1.0) and air. The bottles were closed with a rubber stopper and incubated at 55°C and 350 rpm with different concentrations of methanethiol, methane, or hydrogen sulfide. Methanethiol and methane were obtained from pure stocks, whereas hydrogen sulfide was prepared by mixing sodium sulfide with hydrochloric acid in a closed bottle to create hydrogen sulfide in the gas phase. To quantify methanethiol and hydrogen sulfide, 100 μl from the headspace of the bottles was injected with a glass Hamilton syringe into a gas chromatograph (7890B GC systems Agilent technologies, Santa Clara, CA, United States) equipped with a Carbopack BHT100 glass column (2 m, ID 2 mm) and a flame photometric detector (FPD) (Pol et al., 2018 (link)). Methane was measured as described before (Mohammadi et al., 2019 (link)). The areas obtained through GC injections were used to calculate the methanethiol, hydrogen sulfide and methane concentrations using standard curves. It was experimentally determined that the methanethiol concentration in the liquid is about 1.5 times higher than the methanethiol concentration in the gas phase at 55°C. Dry weight of the cells was determined as described by Picone et al. (2020) (link).

Chromatographic analysis using GC-MS was carried out (Agilent Technologies 7890B GC Systems combined with 5977A Mass Selective Detector). Capillary column (HP-5MS Capillary; 30.0m×0.25mm ID×0.25μm film) and helium as carrier gas were used at a rate of flow of 1.9 ml/min with 1μl injection. The sample was analyzed with the column held initially for 3 min at 40°C after injection, then the temperature was increased to 300°C with a 20°C/min heating ramp, with a 4.0 min hold. Injection was carried out in split-less mode at 300°C. MS scan range was (m/z): 50–550 atomic mass units (AMU) under electron impact (EI) ionization (70 eV).

To determine kinetic parameters of H₂S oxidation by sulfide-adapted cells, batch incubations were performed in 120 mL serum bottles containing 10 mL medium without any trace elements. Trace elements were omitted to minimize the effect of abiotic sulfide oxidation. The bottles were closed with butyl rubber stoppers. Incubations were performed at 55 °C and 350 rpm. H₂S was prepared by mixing Na₂S with HCl in a closed bottle. A volume headspace was taken and injected into 120 mL serum bottles and equilibrated for 30 min before initiating the assay by addition of cells. H₂S was measured by injecting 100 μL of the headspace of the bottles with a Hamilton glass syringe into a GC (7890B GC systems Agilent technologies, Santa Clara, USA) equipped with a Carbopack BHT100 glass column (2 m, ID 2 mm) and a flame photometric detector (FPD). The areas obtained were used to calculate H₂S amounts using calibration standard curves with H₂S. Briefly, 400 μL of a 25 mM Na₂S stock (sodium sulfide nonahydrate, purity >98%, Sigma-Aldrich) was acidified with 2 mL 0.5 M HCl in a 574 mL bottle creating a headspace concentration of 17.4 nmol · mL⁻¹. Small volumes of the headspace were subsequently added to a 1162 mL bottle to create various H₂S concentrations to be injected (0.1 mL) into the GC for calibration. The calibration curve ranged from ~1 nmol · L⁻¹ to 1 μmol · L⁻¹ H₂S.