2 13c acetate

Optical rotations were measured on a Jasco P2000 digital polarimeter (Jasco, Cremella, Italy). UV spectra were acquired on a Jasco V-650 spectrophotometer, NMR spectra were recorded on a Bruker Avance DRX 600 (Bruker, Milan, Italy) equipped with a cryoprobe operating at 600 MHz for protons. Chemical shift values are reported in ppm (δ) and referenced to internal signals of residual protons (CD₃OD ¹H δ3.34, ¹³C 49.0 ppm). High-resolution mass spectra were acquired on a Q-Exactive Hybrid Quadrupole-Orbitrap mass spectrometer (Thermo Scientific, Milan, Italy); HPLC analyses have been performed on a Jasco system (PU-2089 Plus-quaternary gradient pump equipped with a Jasco MD-2018 Plus photodiode array detector). [1-¹³C]-acetate, [2-¹³C]-acetate and [1,2-¹³C₂]-acetate, [1-¹³C]-glycolate (all sodium salts), and salicylhydroxamic acid (SHAM) were obtained from Sigma Aldrich (Milan, Italy). Solvents were purchased from VWR (Milan, Italy) and were HPLC-grade.

[2-¹³C]-acetate labeling batch experiments were conducted in biological triplicates for each reactor from Experiment 1. Each serum bottle had a total volume of 122 mL, a working volume of 20 mL and was filled with 18.74 mL reactor effluent and 1.26 mL of 1 M [2-¹³C]-acetate (Sigma Aldrich, USA) in an anaerobic chamber (97% N₂, 3% H₂). The bottles were incubated at 37 °C for 20 h. Gas composition was analyzed using the gas chromatograph Clarus 580 (PerkinElmer, Germany) and the ratio of ¹²CO:¹³CO₂ as well as ¹²CH₄:¹³CH₄ was determined using the gas chromatography-mass spectrometer (GC–MS) Clarus 600 (PerkinElmer, Germany) as described previously [50 (link)].