15N tracer batch experiments in MECs

To elucidate the molecular mechanism of electrode-dependent anaerobic ammonium oxidation by different anammox bacteria, isotopic labeling experiments were conducted in single-chamber MECs operated at set potential of 0.6 V vs. SHE. All batch incubation experiments were performed in triplicate MECs. MEC incubations without biomass for the ¹⁵N tracer batch experiments were also prepared to exclude any possibility of an abiotic reaction. Standard anaerobic techniques were employed in the batch incubation experiments. All the procedures were performed in the anaerobic chamber (Coy Laboratory Products; Grass Lake Charter Township, MI, USA). Anoxic buffers and solutions were prepared by repeatedly vacuuming and purging helium gas (>99.99%) before the experiments. The purity of ¹⁵N-labeled compounds was greater than 99%. The headspace of the MECs was replaced by repeatedly vacuuming and purging with pure (>99.99%) helium gas. Positive pressure (50–75 kPa) was added to the headspace to prevent unintentional contamination with ambient air during the incubation and gas sampling. Oxidation of NH₄⁺ to N₂ was demonstrated by incubating the MECs with ¹⁵NH₄Cl (Cambridge Isotope Laboratories, 4 mM) and ¹⁴NO₂⁻ (1 mM). The MECs were incubated for 144 h at 30 °C for Ca. Brocadia and K. stuttgartiensis cultures, and at room temperature (~25 °C) for Ca. Scalindua. The concentrations of ²⁸N₂, ²⁹N₂, ³⁰N₂, ¹⁴NO, ¹⁵NO, ²⁸N₂O, ²⁹N₂O, and ³⁰N₂O gas were determined by GC/MS³⁷. Fifty microliters of headspace gas was collected using a gas-tight syringe (VICI; Baton Rouge, LA, USA) and immediately injected into a GC (Agilent 7890A system equipped with a CP-7348 PoraBond Q column) combined with 5975C quadrupole inert MS (Agilent Technologies; Santa Clara, CA, USA). Standard calibration curve of N₂ gas was prepared with ³⁰N₂ standard gas (>98% purity) (Cambridge Isotope Laboratories; Tewksbury, MA, USA).

To investigate whether hydroxylamine (NH₂OH) could be produced directly from NH₄⁺ in electrode-dependent anaerobic ammonium oxidation by anammox bacteria, single-chamber MECs were incubated with ¹⁵NH₄Cl (4 mM, Cambridge Isotope Laboratories) and an unlabeled pool of ¹⁴NH₂OH (2 mM) for 144 h. Liquid samples were taken every day and filtered using a 0.2 μm filter and subjected to determination of ¹⁵NH₂OH and ¹⁴NH₂OH. NH₂OH was determined by GC/MS analysis after derivatization using acetone³⁸. Briefly, 100 µl of liquid sample was mixed with 4 µl of acetone, and 2 µl of the derivatized sample was injected to a GC (Agilent 7890 A system equipped with a CP-7348 PoraBond Q column) combined with 5975 C quadrupole inert MS (Agilent Technologies; Santa Clara, CA, USA) in splitless mode. NH₂OH was derivatized to acetoxime (C₃H₇NO), and the molecular ion peaks were detected at mass to charge (m/z) = 73 and 74 for ¹⁴NH₂OH and ¹⁵NH₂OH, respectively. Twenty-five micrometers of ¹⁴NH₂OH and ¹⁵NH₂OH were used as standards. To determine the source of the oxygen used in the electrode-dependent NH₄⁺ oxidation to NH₂OH, MECs were incubated with ¹⁵NH₄Cl (4 mM, Cambridge Isotope Laboratories) in the presence of 10% D₂O for 144 h. Stable isotopes of NH₂OH were determined by GC/MS analysis after derivatization using acetone as described above.