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. 2020 Apr 28;11(1):2058.
doi: 10.1038/s41467-020-16016-y.

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

Affiliations

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

Dario R Shaw et al. Nat Commun. .

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.

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Conflict of interest statement

The authors declare no competing interests.

Figures

Fig. 1
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
Fig. 2. Anammox bacteria are electrochemically active.
ad 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 (ad) 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
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.

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