Gc 2014 gas

I-screen supernatant samples were centrifuged (~4000× g, 5 min) and sterile-filtered (0.45 µm). A mixture of formic acid (20%), methanol and 2-ethyl butyric acid (internal standard, 2 mg/mL in methanol) was added. A 3 µL sample with a split ratio of 75.0 was injected on a GC-column (ZB-5HT inferno, ID 0.52 mm, film thickness 0.10 µm; Zebron; Phenomenex, Torrance, CA, USA) in a Shimadzu GC-2014 gas chromatograph. SCFA parameters analyzed were: acetic acid, propionic acid, iso-butyric acid, butyric acid, and iso-valeric acid. For the analysis of lactate content in the I-screen samples, samples were centrifuged (~4000× g, 5 min) and the clear supernatant was sterilized by using a 0.45 µm cellulose acetate membrane filter Z746487 (Whatman^®, Cytiva, Little Chalfont, UK). The D- and L-lactate was determined by the Arena 20XL analyzer. The assay is based on the enzyme-catalyzed reaction of lactic acid + NAD ↔ Pyruvate + NADH. The amount of NADH generated is stoichiometrically linked to the amount of lactic acid present and is measured by the amount of light absorbed by NADH at a wavelength of 340 nm. By using both the L-lactate dehydrogenase and D-lactate dehydrogenase enzymes in separate reactions, the individual concentrations of L-lactic acid and D-lactic acid were determined.

FeMo-co synthesis and apo-NifDK reconstitution assays were carried
out in vitro in an anaerobic chamber, as previously described.^{34 (link)} For the in vitro synthesis of FeMo-co, each
100 μL reaction contained 3 μM NifH^Av, 1 μM OsNifB, 1.5 μM apo-NifEN^Av, 0.6 μM apo-NifDK^Av, 17.5 μM Na₂MoO₄, 175 μM R-homocitrate, 9 μM [Fe₄–S₄]cluster-loaded NifU^Ec (holo-NifU^Ec), 125 μM SAM, 1 mg/mL BSA, and the
ATP-regenerating mixture (1.23 mM ATP, 18 mM phosphocreatine disodium
salt, 2.2 mM MgCl₂, 3 mM DTH, 46 μg/mL creatine phosphokinase).
For the positive control FeMo-co synthesis assay, holo-NifU^Ec was omitted, and OsNifB was replaced
with 2.5 μM NifB-co. The reactants were incubated for 60 min
at 30 °C. For the acetylene reduction assays, 500 μL of
the ATP-regenerating mixture and 2.0 μM NifH^Av were added to the reaction tube. The reaction mixture was
then transferred to 9 mL serum vials under an argon/acetylene (94%/6%)
atmosphere. The reaction was incubated for 20 min at 30 °C. To
measure ethylene formation, 50 μL of the gas phase was taken
from the reaction vials and injected in the Shimadzu GC-2014 gas chromatographer
equipped with the Porapak N 80/100 column (Shimadzu, Kyoto, Japan).

The fatty acid compositions and contents were determined using the method previously described by Xu et al. [12 (link)]. The freeze-dried biomass (25 mg) was trans-methylated with 2% H₂SO₄ in a methanol:toluene mixture (90:10, v:v) at 80 °C for 1.5 h. The contents of fatty acid methyl esters (FAMEs) were determined using a GC-2014 gas chromatograph spectrometer with a flame ionization detector (Shimadzu; Kyoto, Japan), equipped with a 30 m fused silica DB-WAX capillary column (Agilent Technologies; Santa Clara, CA, USA). The temperature of the injection port was maintained at 260 °C. The column temperature was programmed from 140 °C to 240 °C at 10 °C min⁻¹ with a hold of 5 min at 240 °C. High-purity argon was used as the carrier gas at a flow rate of 1.2 mL min⁻¹. Individual peaks of FAMEs were identified through comparisons of retention times with 37 fatty acid standards (Nu-Chek-Prep; Elysian, MN, USA). Each measurement was performed in triplicate.