Porapak q column

Gasses were sampled with a Pressure-Lok syringe (VICI, Houston, TX, United States); and the concentration of methane and hydrogen was determined using a gas chromatograph (7890A, Agilent, Santa Clara, CA, United States) equipped with an 80/100-mesh Porapak Q column (Supelco; Sigma-Aldrich, St. Louis, MO, United States) and a thermal conductivity detector (Liu et al., 2014 (link)). The unit of CH₄ concentration was converted from partial pressure in headspace to mmol L^–1 in liquid medium by using Avogadro’s law. Liquid samples (0.5 ml) were collected periodically with a sterile syringe, centrifuged, and filtered through 0.22-μm filters. Concentrations of butyrate and acetate in culture medium were determined by high-performance liquid chromatography with a ZORBAX SB-Aq C18 organic acid column (250 by 4.6 mm; particle size 5 μm; Agilent) at a flow rate of 0.8 ml min^–1. The UV absorbance detector was set at 210 nm (Zhang et al., 2018 (link)).

Modified diluted nitrate mineral salt (DNMS) medium [49 (link)] supplemented with 5 μM lanthanum (LaCl₃) was used as a growth medium in 120 ml serum vials (with 20 ml culture volume), with substrate gas as the only source of C and energy. Methane (20%), ethane (10%) and propane (10%), individually or in a mixture at varying concentrations, were injected (percentage v/v in headspace) in sealed serum vials as C substrates. The growth of liquid cultures was monitored by measuring the optical density at 540 nm. Concentrations of substrate gases in the cultures were quantified using a gas chromatograph (GC) using an Agilent 7820A GC equipped with a Porapak Q column (Supelco) coupled to a flame ionisation detector (FID) to measure methane, ethane and propane concentrations as previously described [49 (link)]. Comparison of growth of Methylocella sp. PC4 with and without lanthanum (Fig. 6) was performed in 50 ml polypropylene falcon tubes (with 15 ml culture volume) to avoid any contamination of lanthanides from glassware. As many methanotrophs do not store well frozen [113 ], cultures were maintained on plates or in liquid.

At the end of the incubation, total pressure of each bottle was measured with a Druck DPI 104-IS Pressure Gauge (GE Measurements; Billerica, MA) and a subsample of 10 mL of gas was collected for CH₄ and CO₂ analysis. The concentration of both gases were determined in a Gow Mac thermal conductivity series 580 gas chromatography (Gow Mac Instrument, Bridgewater, NJ) equipped with a Porapak Q column (Supelco, 60 °C, 30 mL/min of helium 99.99% carrier gas). The final GP volumes were corrected by subtracting the final GP of the blank bottles.
The remaining fermented media was used for the determination of in vitro true OM digestibility. The media was dried in a ventilated oven at 55 °C, then NDF was analyzed according to Mertens (2002) (link) and adapted to the Ankom²⁰⁰ Fiber Analyzer (Ankom Technology). The in vitro true OM digestibility was calculated according to Goering and Van Soest (1970) : in vitro true OM digestibility (%) = (iOM – rNDF)/(iOM), in which iOM was the incubated OM and rNDF the residual NDF after 48 h of incubation minus the NDF content in the blank bottles.