Fig. 3. Mechanism of NH₄⁺ oxidation with extracellular electron transfer.

a Time course of the anaerobic oxidation of ¹⁵NH₄⁺ to ²⁹N₂ and ³⁰N₂. The single-chamber microbial electrolysis cells (MECs) with mature biofilm on the working electrodes operated at 0.6 V vs. SHE were fed with 4 mM ¹⁵NH₄⁺ and 1 mM ¹⁴NO₂⁻. Under these conditions, anammox bacteria will consume first the preferred electron acceptor (i.e., ¹⁴NO₂⁻) and form ²⁹N₂ and then the remaining ¹⁵NH₄⁺ will be oxidized to the final product (³⁰N₂) through the electrode-dependent anammox process. NO and N₂O were not detected throughout the experiment. Results from triplicate MEC reactors are presented as mean ± SD. b Determination of NH₂OH as the intermediate of the electrode-dependent anammox process. The MECs with mature biofilm on the working electrodes operated at 0.6 V vs. SHE were fed with 4 mM ¹⁵NH₄⁺ and 2 mM ¹⁴NH₂OH. Under these conditions, anammox bacteria would preferentially consume the unlabeled pool of hydroxylamine (i.e., ¹⁴NH₂OH), leading to the accumulation of ¹⁵NH₂OH due to the oxidation of ¹⁵NH₄⁺. Samples were derivatized using acetone, and isotopic ratios were determined by gas chromatography mass spectrometry (GC/MS). Results from triplicate MEC reactors are presented as mean ± SD. c Ion mass chromatograms of hydroxylamine derivatization with acetone. The MECs with mature biofilm (Ca. Brocadia) on the working electrodes operated at 0.6 V vs. standard hydrogen electrode (SHE) were fed with 4 mM ¹⁵NH₄⁺ and 10% deuterium oxide (D₂O). The mass to charge (m/z) of 73, 74, and 75 corresponds to derivatization products of ¹⁴NH₂OH, ¹⁵NH₂OH, and ¹⁵NH₂OD, respectively, with acetone determined by GC/MS. Twenty microliters of ¹⁴NH₂OH and ¹⁵NH₂OH were used as standards. The 73 m/z (top) at a retention time of 8.6 min arises from the acetone used for derivatization. The 75 m/z (bottom) accumulation over the course of the experiment indicates that the oxygen used in the anaerobic oxidation of ammonium originates from OH⁻ of the water molecule.