Acquity 1 class

Lipids were analyzed with a Waters Acquity i-Class coupled to a Waters Synapt G2Si adapted from Isaac et al.⁴⁹ . Pellets were dissolved in 100 µl isopropanol:acetonitrile:water 2:1:1 and kept at 12 °C. 2 µl were injected onto a Waters BEH C18 (2.1 mm × 100 mm, 1.7 µm) column run with the following program: 60% A (A: 10 mM NH₄CHOO, 0.1% formic acid, 60% acetonitrile, 40% water) hold for 2 min, to 35% A until 6.5 min, to 12% A until 12 min, to 1% A until 13 min, hold for 15 min, to 60% A until 15.5 min and hold to a final time of 20 min. B was 10 mM NH₄CHOO, 0.1% formic acid, 10% acetonitrile, and 90% isopropanol. The column temperature was set to 55 °C with a flow rate of 300 µl/min. The source was operated at −1.5 kV, 150 °C, sampling cone and source offset were set to 30 and 80, respectively. Desolvation temperature was 500 °C with 600 l/h desolvation gas flow and 50 l/h cone gas flow as well as 6.5 bar nebulizer pressure. For data analysis, ProgenesisQI was used and searched against HMDB database 3.6 with a minimal score of 45 which corresponds relatively to the cut-off used in the GC/MS profiling following the same statistical analysis.

Liquid chromatography-mass spectrometry (LC-MS) analyses were performed with a UPLC system (Acquity I class; Waters) and a BEH C₁₈ column (2.1 mm × 5 cm) (flow, 0.25 ml/min). Samples were loaded onto the column in 5% solvent B (acetonitrile). After 2 min, a gradient from 5% to 90% solvent B was performed over 6 min. The UPLC system was coupled to an ESI Q-Orbitrap mass spectrometer (Q Exactive; Thermo Scientific), operating either in the positive or negative ion mode.

Pit mud moisture levels were established by drying samples for 3 h at 115 °C. Pit mud pH values were established with a Mettler Toledo pH meter after diluting sample 1:4 (w/v) with dH₂O for 3 h. Pit mud ammonium (NH₄⁺-N) levels were established via extraction in 10% (w/v) NaCl at a 1:10 (w/v) ratio, after which concentrations were measured using a UV spectrophotometer. Acetic acid, butyric acid, and caproic acid were extracted using 15% methanol and quantified via gas chromatography (Agilent 7890, Santa Clara, CA, USA) as described previously [12 (link)]. Lactic acid (LA) levels were quantified via ultra-high-performance liquid chromatography (UPLC, Acquity I-class, Waters, Milford, MA, USA) as previously reported [1 ]. Levels of K⁺, PO₄³⁻, soluble Mg²⁺, and soluble Ca²⁺ in air-dried pit mud were measured via extracting samples with ddH₂O at a 1:10 (w/v) ratio, after which concentrations were measured as reported previously by Zhang et al. using an ion chromatograph (ICS5000⁺, Thermo Fisher, Waltham, MA, USA) equipped with a conductivity detector (ICS-5000⁺-DC) and a CS12 column (Ion Pac, Thermo Fisher, 4 mm × 250 mm) [4 (link)]. The utilized injection volume was 25 μL, with methane sulfonic acid (20 mM) as a carrier fluid at a 1 mL/min flow rate, with a column temperature of 30 °C. Humus levels were determined as detailed previously by Shen [13 ].