6890 plus

The amounts of SCFAs in cecal and fecal contents were determined using gas chromatography (Agilent 6890 Plus, Santa Clara, CA, USA) following method described by Zhao et al. [51 (link)].

Plasma butyrate was analyzed using Agilent 5973 mass spectrometry with 6890 Plus gas chromatography system (Agilent Technologies, Santa Clara, CA, USA). In brief, 1 mL of plasma was added to 3 mL of 5% formic acid and 2.5 mL of ethyl acetate. The suspension was homogenized for 30 minutes and centrifuged at 15000 rpm for 5 minutes. Six mL of supernatant was isolated, and 0.1 g sodium sulfate anhydrous was added. Then, the solution was injected into the gas chromatography–mass spectrometry machine.

Biogas production was monitored online using an automated gas metering system. The biogas CH₄ content was determined using a portable analyzer (Biogas 5000, Geotech, Canada). Wet samples were collected daily from a port located at 6 m in height of the vessel. The pH, COD, total solids (TS), VS, total suspended solids (TSS), and volatile suspended solids (VSS) were determined according to the procedures in Standard Methods [16 ]. VFAs (C₂–C₆) and ethanol were measured using a gas chromatograph (6890 plus, Agilent, USA) equipped with an Innowax capillary column and a flame ionization detector. Total nitrogen and total phosphorus were measured using colorimetric test kits (TN-H and TP-H, C-Mac, Korea). Whole DNA was extracted and a real-time polymerase chain reaction (PCR) targeting the 16S rRNA gene of methanogen populations was performed as previously described [17 (link)].

After collection, cecal and fecal contents were stored at −80 °C until analysis of SCFAs using gas chromatography (Agilent 6890 Plus gas chromatograph, Santa Clara, CA, USA) and expressed as µmol/g of cecal/fecal material [29 (link)]. One gram of cecal/fecal sample was thawed and suspended in 5 mL of distilled water followed by homogenization (UltraTurrax T 25, Staufen, Germany) for 3 min, resulting in a 20% (w/v) cecal/fecal suspension. The HCl (5 M) was used to adjust the pH of suspension to 2–3 and was placed on shaker for 10 min at room temperature followed by centrifugation (5000 rpm) for 20 min and the clear supernatant was separated. 2-ethylbutyric acid solution was added in supernatant as internal standard having final concentration of 1 mM and this prepared supernatant was used for the quantification of acetic, propionic, and butyric acids using standards of these fatty acids.

A Hewlett Packard 6890 Plus gas chromatograph with an Agilent 7683 series injector and a 5973 mass selective detector (Hewlett Packard, Palo Alto, CA, USA) were used for qualitative analyses. The compounds were separated on an HP-5MS capillary column (5%-phenyl-methylpolysiloxane, 30 m × 0.25 mm i.d., 0.25 μm film thickness). Helium was used as a carrier gas at a constant flow of 1 mL/min. The GC inlet temperature was changed from 110 °C to 250 °C by increments of 20 °C and finally to 275 °C, which is the temperature used in our routine general unknown screening. The inlet liner was Agilent splitless single taper, deactivated liner with glass wool (Agilent, Palo Alto, CA, USA, P/N: 5062-3587). The initial oven temperature of 60 °C was held for 2 min and then ramped at 20 °C/min to 300 °C with a final hold time of 15 min. The temperature of the transfer line was 280 °C. The ion source and quadrupole temperatures were 230 °C and 150 °C, respectively. The mass selective detector was used in EI scan mode, the ionization energy was 70 eV, and the electron multiplier voltage was set 200 V above autotune value. Data were collected for a splitless injection of 1.0 µL of each tropane in methanol, ethyl acetate and n-hexane in the range from 50 to 550 m/z at a rate of 2.9 scans/s. The software used was MSD Chemstation D.01.02.16.

Individual apple samples (at least six replicates) were placed in a hermetically sealed container equipped with septa, and then incubated for 1 h in the darkness at 25 °C. After incubation, 1 mL was withdrawn from the internal atmosphere of the container and injected into a gas chromatographer (Hewlett-Packard 6890 Plus, Agilent, Santa Clara, CA, USA), equipped with a flame ionization detector with electronically controlled pneumatics and a capillary injector for operations with and without splitting (0–100 psi), following a method modified from de Dios et al. [60 (link)]. The ethylene levels released by each apple were expressed in nanoliters of ethylene per gram of apple per hour (nL·g⁻¹·h⁻¹).