6890 series

Sterols in cell homogenates were extracted with a solvent mixture containing chloroform/methanol 2/1 (v/v) spiked with epicoprostanol as the internal standard. Lipids were partitioned in chloroform after the addition of saline and saponified by methanolic potassium hydroxide (0.5 N, 60 °C, 15 min). The fatty acids released were methylated with BF3-methanol (12%, 60 °C, 15 min) to not interfere with the chromatography of sterols. The sterols were re-extracted in hexane and silylated, as described previously^{61 (link)}. The trimethylsilylether derivatives of the sterols were separated by gas chromatography (GC) (Hewlett–Packard 6890 series) in a medium polarity capillary column RTX-65, (65% diphenyl 35% dimethyl polysiloxane, length 30 m, diameter 0.32 mm, film thickness 0.25 μm (Restesk, Evry, France)). The mass spectrometer (Agilent 5975 inert XL) in series with the GC was set up for the detection of positive ions. Ions were produced in the electron impact mode at 70 eV. Sterols were identified by the fragmentogram in the scanning mode and quantified by selective monitoring of the specific ions after normalization with the internal standard epicoprostanol and calibration with weighed standards.

The FAC was determined using a gas chromatography (GC) (Hewlett-Packard 6890 Series). The GC was set with a BPX70 capillary column (60 m × 0.25 mm, i.d. 0.25 μm film thickness), flame ionization detector (FID), electronic integrator, and data processor (J & W Scientific, Folsom, CA, USA). AOCS Official Method Ce 1i-07 was adapted for fatty acid methyl ester (FAME) preparation [41 ]. Helium was used as the carrier gas at a flowrate of 0.8 mL/min with a pre-column split ratio of 100:1. The sample injection volume was 1 μL. The FID detector and injector port temperatures were set at 240 °C while the column temperature remained isothermal at an oven temperature of 185 °C. Each FAME was quantified and expressed in percentage with the rapeseed oil references standards.

The analyses were carried out using full scan mode, with the following parameters: 5 μL injected via automatic liquid sampler; the oven was temperature programmed with an initial isothermal of 2 min at 40°C followed by an increase to 350°C at 10°C per minute followed by a final isothermal at this temperature of 15 min. Helium was used as carrier gas and held at a constant flow at 2.0 ml per minute throughout the analysis. Identification was performed using a Hewlett Packard 6890 series gas chromatograph–mass spectrometer equipped with a 5% phenyl methyl siloxane column (HP-5, 60m, 0.25mm i.d., film thickness 0.25 μm) Compound identification was achieved by interpretation of characteristic mass spectra fragmentation patterns, gas chromatographic relative retention times, and by comparison with literature.

For cycle 1, total biogas production was determined by using gas meters working based on water displacement. For cycles 2 and 3, the gas pressure in each bottle was regularly measured by a pressure meter (Testo 312, Germany) to quantify the volume of biogas production. Gas samples were collected for headspace methane content analysis by gas chromatography (5880A series, Hewlett Packard, USA). The methane production was normalized to the sludge content of each bottle (i.e., norm. ml g⁻¹ digestate) and reported at standard atmospheric pressure and 0 °C. Before and after each treatment, several parameters of the sludge samples were determined. Dissolved organic carbon (DOC) was analysed by a TOC analyser (Shimadzu TOC-V_CPH, Japan) after the samples were filtered through a 0.45 μm polyethersulfone membrane (USA). VFA concentrations, including acetate, propionate, butyrate, isobutyrate, valerate, and isovalerate, were analysed using a gas chromatograph (6890 Series, Hewlett Packard, USA) according to Jonsson and Borén^{12 (link)}. The pH was determined using a pH electrode (InfoLab pH 7310, Germany). Student t-test and Welch’s ANOVA was performed in R software (version 4.0.2) to analyse the difference of methane production amount and rate across the samples.

Total FA composition was analyzed by GC-flame ionization detection (27 (link)). Briefly, lipids from ∼50 mg of liver were extracted by a modified Folch extraction. The lipids were then saponified, reextracted, and derivatized with trimethylsilane. FAs were separated and detected using a Hewlett Packard 6890 series gas chromatograph. Fatty acids were identified based on the retention times from a purified FA standard mixture (GLC-744 Nu-Chek Prep) and normalized to an internal standard of pentadecanoic acid (C15:0).

The phytometabolites of ethanolic extract of F. vasta were determined by GCMS analysis using Agilent, 6890 series, and Hewlett Packard, 5973 mass selective detector. An HP-5MS column with a length of 30m, a diameter of 250 µL, and a film thickness of 0.25 µL were used to achieve the best possible separation. The volume of 1.0 µL of the extract was diluted with the appropriate solvent and injected at 250 °C in a splitless mode. Helium gas as a carrier was used at a constant flow rate of 1.02 mL/min., the temperature was increased gradually, starting at 50–150 °C and increasing by 3 °C per min, with a 10 min holding time at each temperature. The final temperature was set to 300 °C at 10 °C/min. The components were identified using their retention indices, and the mass spectrum was interpreted using the National Institute of Standards and Technology database (NIST) [85 (link)].