Extracellular electron transfer-dependent anaerobic oxidation of ammonium by anammox bacteria

Abstract

Anaerobic ammonium oxidation (anammox) bacteria contribute significantly to the global nitrogen cycle and play a major role in sustainable wastewater treatment. Anammox bacteria convert ammonium (NH₄⁺) to dinitrogen gas (N₂) using intracellular electron acceptors such as nitrite (NO₂^-) or nitric oxide (NO). However, it is still unknown whether anammox bacteria have extracellular electron transfer (EET) capability with transfer of electrons to insoluble extracellular electron acceptors. Here we show that freshwater and marine anammox bacteria couple the oxidation of NH₄⁺ with transfer of electrons to insoluble extracellular electron acceptors such as graphene oxide or electrodes in microbial electrolysis cells. ¹⁵N-labeling experiments revealed that NH₄⁺ was oxidized to N₂ via hydroxylamine (NH₂OH) as intermediate, and comparative transcriptomics analysis revealed an alternative pathway for NH₄⁺ oxidation with electrode as electron acceptor. Complete NH₄⁺ oxidation to N₂ without accumulation of NO₂^- and NO₃^- was achieved in EET-dependent anammox. These findings are promising in the context of implementing EET-dependent anammox process for energy-efficient treatment of nitrogen.

Fig. 1. NH₄⁺ oxidation coupled with extracellular electron transfer.

a Photographs of serum vials after 216 h of incubation with different species of anammox bacteria, ¹⁵NH₄⁺, and graphene oxide (GO). The presence of black precipitates indicates the formation of reduced GO (rGO). No obvious change in color was observed in the abiotic control vials after the same period of incubation with ¹⁵NH₄⁺ and GO. b Raman spectra of the vials after 216 h of incubation. Peaks in bands of 2D and D + D′ located at ∼2700 and ∼2900 cm⁻¹, respectively, indicate the formation of rGO. c ³⁰N₂ production by different anammox bacteria from ¹⁵NH₄⁺ and GO as the sole electron acceptor. Anammox cells were incubated with 4 mM ¹⁵NH₄⁺ and GO to a final concentration of 200 mg L⁻¹. There was no ²⁹N₂ formation throughout the experiment. NO and N₂O were not detected throughout the experiment. Results from triplicate serum vial experiments are represented as mean ± SD.

Fig. 2. Anammox bacteria are electrochemically active.

a–d Ammonium oxidation coupled to current generation in chronoamperometry experiment conducted in one single-chamber multiple working electrode microbial electrolysis cell (MEC) inoculated with Ca. Brocadia. a MEC was operated initially under different set potentials with the addition of nitrite, which is the preferred electron acceptor for ammonium oxidation by anammox bacteria, followed by operation with working electrodes as sole electron acceptors. The highlighted area in blue refers to the operation of MEC in the presence of nitrite. The black arrow indicates the addition of allylthiourea (ATU), a compound that selectively inhibits nitrifiers. b MEC operated under open circuit voltage (OCV) mode. c MEC operated at different set potentials and with the addition of nitrite. d MEC operated at different set potentials and without the addition of ammonium and then with the addition of ammonium followed by autoclaving. The black arrow in (d) indicates autoclaving followed by re-connecting of the MECs. Red dashed lines in (a–d) represent a change of batch. e Cyclic Voltammogram (1 mV s⁻¹) of anammox biofilm grown on working electrodes (i.e., anodes) operated at different set potentials and growth periods following inoculation in MEC. f Confocal laser scanning microscopy images of a thin cross-section of the graphite rod anodes (0.6 V vs. standard hydrogen electrode (SHE) applied potential). The images are showing the in-situ spatial organization of all bacteria (green), anammox bacteria (red), and the merged micrograph (yellow). Fluorescence in-situ hybridization was performed with EUB I, II, and III probes for all bacteria^, and Alexa647-labeled Amx820 probe for anammox bacteria^,. The dotted outline indicates the graphite rod anode surface. The white arrow indicates the biofilm. The scale bars represent 20 μm in length.

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.