Agilent 7890a

The methanolic extract of the alga was analyzed using Agilent 7890A Gas Chromatography/Mass Spectrometry (GC/MS) (Agilent 7890A, Agilent Technologies Inc., Santa Clara, CA, USA) and 5975 A mass spectrophotometer using an HP-5 MS capillary column (polydimethylsiloxane with a length of 30 m, an internal diameter of 0.25 mm, and a thickness of 0.25 μm). The initial temperature of the oven was set to increase from 80 to 180 °C at a rate of 8 °C/min. Helium was used as the carrier gas whose speed was 1 mm/min along the column length. The injection valve was set in the split mode at a ratio of 1:500 and an injection temperature of 250 °C. The mass spectrum was 40–500 mass/load and ionization energy of 70 eV. The whole run time was 55 min. The mass libraries Wily 2007 and NIST were employed to identify the compounds. Data were processed in the Windows-based Chemstation software. The relative percentage of the extract constituents was expressed in percent based on the peak level.

Detailed information is provided in the Supplementary Materials and Methods. The first in vitro experiment (“In vitro Experiment 1”) was conducted to compare the fermentative activities, including gases and VFA production of Jersey and Holstein rumen microbiomes by incubating the TMR fed to the animals according to a previously published procedure [94 (link)]. In vitro batch cultures were incubated at 39.5°C for 48 h. CH₄ and H₂ concentrations were determined through gas chromatography (Agilent 7890A, Agilent Inc., Palo Alto, CA) [22 (link)]. Methane production was then calculated by using the equation of Wang et al. [95 (link)]. The second in vitro experiment (“In vitro Experiment 2”) was conducted to compare the activities of Jersey and Holstein rumen microbiomes to use H₂ by adding molecular deuterium (²H₂ or D₂) to the incubation bottle headspace. Samples of each bottle headspace (2 ml) were collected at 1, 4, 8, 12, 24, 36, and 48 h of incubation and used to measure headspace H₂ and CH₄ concentrations through gas chromatography (Agilent 7890A, Agilent Inc., Palo Alto, CA) [22 (link)]. Samples from the liquid phase of the bottle in the two experiments were collected from each bottle after finishing the incubations and snap frozen in liquid N₂, and stored at −80°C for further analysis of fermentation end products.

1,1,2,2-tetrachloroethene (≥99.5%), 1,1,2-trichloroethene (99.5%), trans-1,2-dichloroethene (trans-1,2-DCE; ≥98%), 1,1-dichloroethene (1,1-DCE; ≥99%), cis-1,2-dichloroethene (cis-1,2-DCE; ≥97%), 1,2-dichloroethane (1,2-DCA; ≥99%) and vinyl chloride (VC; 100 μg/mL in methanol) were purchased from Sigma-Aldrich (St. Louis, MO, United States). All other chemicals used to prepare analytical standards or feed solutions were analytical reagent grade.
Two milliliters of liquid samples obtained from the cathode chambers were immediately transferred to sealed 10 mL bottles filled with the high purity N₂ gas (≥99.99%). The bottles were placed in a 25°C shaker for 30 min to reach the equilibrium. Concentrations of volatile organic compounds including PCE, TCE, trans-1,2-DCE, 1,1-DCE, cis-1,2-DCE, 1,2-DCA, and VC in the headspace (8 mL) were determined using a gas chromatograph (Agilent 7890A, Palo Alto, CA, United States) equipped with a 63Ni electron capture detector and DB-1301 column (30 m × 250 μm × 0.25 μm, Agilent). Ethene and methane were determined using a gas chromatograph (Agilent 7890A, Palo Alto, CA, United States) equipped with flame ionization detector (FID) and HP-5 column (30 m × 250 μm × 0.25 μm, Agilent). Headspace concentrations were converted to aqueous-phase using tabulated Henry’s law constants (Chen et al., 2018 (link)).