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

Abstract Anaerobic ammonium oxidation (anammox) by anammox bacteria contributes significantly to the global nitrogen cycle, and plays a major role in sustainable wastewater treatment. Anammox bacteria convert ammonium (NH 4 + ) to dinitrogen gas (N 2 ) using nitrite (NO 2 − ) or nitric oxide (NO) as the electron acceptor. In the absence of NO 2 − or NO, anammox bacteria can couple formate oxidation to the reduction of metal oxides such as Fe(III) or Mn(IV). Their genomes contain homologs of Geobacter and Shewanella cytochromes involved in extracellular electron transfer (EET). However, it is still unknown whether anammox bacteria have EET capability and can couple the oxidation of NH 4 + with transfer of electrons to carbon-based insoluble extracellular electron acceptors. Here we show using complementary approaches that in the absence of NO 2 − , freshwater and marine anammox bacteria couple the oxidation of NH 4 + with transfer of electrons to carbon-based insoluble extracellular electron acceptors such as graphene oxide (GO) or electrodes poised at a certain potential in microbial electrolysis cells (MECs). Metagenomics, fluorescence in-situ hybridization and electrochemical analyses coupled with MEC performance confirmed that anammox electrode biofilms were responsible for current generation through EET-dependent oxidation of NH 4 + . 15 N-labelling experiments revealed the molecular mechanism of the EET-dependent anammox process. NH 4 + was oxidized to N 2 via hydroxylamine (NH 2 OH) as intermediate when electrode was the terminal electron acceptor. Comparative transcriptomics analysis supported isotope labelling experiments and revealed an alternative pathway for NH 4 + oxidation coupled to EET when electrode is used as electron acceptor compared to NO 2 − as electron acceptor. To our knowledge, our results provide the first experimental evidence that marine and freshwater anammox bacteria can couple NH 4 + oxidation with EET, which is a significant finding, and challenges our perception of a key player of anaerobic oxidation of NH 4 + in natural environments and engineered systems.