Hp 5890

At the end of each incubation time, the fermentation vessels were placed in an ice bath to measure the headspace gas pressure in each vessel using a digital readout voltmeter (Laurel Electronics, Inc., Costa Mesa, CA, USA). Gas samples for CH₄ and CO₂ analysis were transferred into a vacuum test tube (Vacutainer, Becton Dickinson, Franklin Lakes, NJ, USA) and analyzed by gas chromatography (Agilent Technologies HP 5890; Santa Clara, CA) using a TCD detector with a Column Carboxen 1006PLOT capillary column 30 m × 0.53 mm (Supelco), following the procedure described in Kim et al. [15 (link)]. A standard mixture of CH₄ and CO₂ (RIGAS, Daejeon, Korea) was used to determine the CH₄ concentrations of samples. Vacuum tubes containing gas samples under refrigeration were warmed prior to gas analysis by allowing the tubes to equilibrate in a gas chromotograph (Agilent Technologies HP 5890; Santa Clara, CA, USA) for at least 30 min.
The amount of microbial growth rate was determined as optical density (OD) value at 550 nm with a spectrophotometer (Model 680, Bio-Rad Laboratories, Hercules, CA, USA). For measuring glucose, 200 μL of supernatant was mixed with 600 μL of DNS solution and incubated for 5 min in a boiling water bath. Glucose concentration was the OD at 595 nm, determined with a microplate reader (Model 680, Bio-Rad Laboratories, Irvine, CA, USA) [24 (link)].

The chemical components of OS8P were analyzed using a gas chromatograph with mass spectrometry (GC-MS), following the modified method of Chit-aree et al. (19 (link)). Sample preparation involved mixing 0.1 mg of OS8P with 1 ml of anhydrous ether (Merck, Germany). GC-MS analysis was conducted using an Agilent HP 5890 gas chromatography instrument coupled with an Agilent MSD HP 5970 mass selective detector and HP 59970 MS Chemstation with a 59973 NBS mass spectral library (NBS_REVF). A fused silica column of 25 m × 0.20 mm × 0.25 µm film thickness (Varian Inc., Agilent Technologies, USA) was used for separation, and each extracted sample of 1 µl was injected in an injector operation. High-purity helium gas (99.999%) was used as a carrier gas at a 1.0 ml/min flow rate. The ion source and quadrupole mass analyzer were kept at 230 and 150°C, respectively, and the analysis was conducted for 60 min per sample. The MSD Chemstation software (Agilent Technologies) and commercial libraries (NIST02.L, NIST05.L, and Wiley7Nist05.L) were used to identify the chemical compounds of OS8P.

In current study, VFAs include acetic acid (C2:0), propionic acid (C3:0), butyric acid (C4:0), isobutyric acid (isoC4:0), valeric acid (C5:0), and isovaleric acid (isoC5:0). Every 2 mL of incubation fluid from each fermentation bottle on a time scale t₂ were centrifuged at 10,000 × g and 4°C for 15 min. 1.5 mL of supernatant solution was collected, which was immediately mixed and homogenized with 0.15 mL 25% metaphosphoric acid. The mixture solution was centrifuged again at 10,000 × g at 4°C for 15 min, and the supernatant solution was collected to determine each VFA content by gas chromatography (HP5890, Agilent 5890; Agilent Technologies Co. Ltd, USA). The DB-FFAP column (Agilent, No.: 122-3232, 30 m in length with a 0.25 mm i.d. and 0.25 um thickness) was used. The parameters of this column were set as the attenuation in a nitrogen split ratio of 1:50, hydrogen flow 30 mL/min, airflow 365 mL/min, injector temperature 250°C, column temperature 150°C and detector temperature 220°C with N₂ as carrier gas at a flow rate of 0.8 mL/min. The relative response factor, represented as the peak of each VFA, was calculated against a standard VFA mixture analyzed following every 10 measurements.