Abstract: Anaerobic ammonium oxidation (anammox) by anammox bacteria contributes 21 significantly to the global nitrogen cycle, and plays a major role in sustainable wastewater 22 treatment. Anammox bacteria convert ammonium (NH4 + ) to dinitrogen gas (N2) using nitrite (NO2 -23 ) or nitric oxide (NO) as the electron acceptor. In the absence of NO2or NO, anammox bacteria 24 can couple formate oxidation to the reduction of metal oxides such as Fe(III) or Mn(IV). Their 25 genomes contain homologs of Geobacter and Shewanella cytochromes involved in extracellular 26 electron transfer (EET). However, it is still unknown whether anammox bacteria have EET 27 capability and can couple the oxidation of NH4 + with transfer of electrons to carbon-based 28 insoluble extracellular electron acceptors. Here we show using complementary approaches that in 29 the absence of NO2 -, freshwater and marine anammox bacteria couple the oxidation of NH4 + with 30 transfer of electrons to carbon-based insoluble extracellular electron acceptors such as graphene 31 oxide (GO) or electrodes poised at a certain potential in microbial electrolysis cells (MECs). 32 Metagenomics, fluorescence in-situ hybridization and electrochemical analyses coupled with 33 MEC performance confirmed that anammox electrode biofilms were responsible for current 34 generation through EET-dependent oxidation of NH4 + . 15 N-labelling experiments revealed the 35 molecular mechanism of the EET-dependent anammox process. NH4 + was oxidized to N2 via 36 hydroxylamine (NH2OH) as intermediate when electrode was the terminal electron acceptor. 37 Comparative transcriptomics analysis supported isotope labelling experiments and revealed an 38 alternative pathway for NH4 + oxidation coupled to EET when electrode is used as electron acceptor 39 compared to NO2as electron acceptor. To our knowledge, our results provide the first 40 experimental evidence that marine and freshwater anammox bacteria can couple NH4 + oxidation 41 with EET, which is a significant finding, and challenges our perception of a key player of anaerobic 42 oxidation of NH4 + in natural environments and engineered systems. 43 Main text: Anaerobic ammonium oxidation (anammox) by anammox bacteria contributes up to 44 50% of N2 emitted into Earth's atmosphere from the oceans (1, 2). Also, anammox bacteria has 45 been extensively investigated for energy-efficient removal of NH4 + from wastewater (3). Initially, 46 anammox bacteria were assumed to be restricted to NH4 + as electron donor and NO2or NO as 47 electron acceptor (4, 5). More than a decade ago, preliminary experiments showed that Kuenenia 48 stuttgartiensis and Scalindua could couple the oxidation of formate to the reduction of insoluble 49 extracellular electron acceptors such as Fe(III) or Mn(IV) oxides (6, 7). However, the mechanism 50 of how anammox bacteria reduce insoluble extracellular electron acceptors has remained 51 unexplored to date. Also, growth or electrochemical activity was not quantified in these 52 experiments. Further, these experiments could not discriminate between Fe(III) oxide reduction 53 for nutritional acquisition (i.e., via siderophores) versus respiration through extracellular electron 54 transfer (EET) (8). Therefore, with these preliminary experiments it could not be determined if 55 anammox bacteria have EET capability or not. 56 Although preliminary work showed that K. stuttgartiensis could not reduce Mn(IV) or Fe(III) 57 with NH4 + as electron donor (6), the possibility of anammox bacteria to oxidize NH4 + coupled to 58 EET to other insoluble extracellular electron acceptors cannot be ruled out. In fact EET (and set 59 of genes involved with EET) is not uniformly applied to all insoluble extracellular electron 60 acceptors; some electroactive bacteria are not able to transfer electrons to carbon-based insoluble 61 extracellular electron acceptors such as electrodes in bioelectrochemical systems but could reduce 62 metal oxides and vice versa (9). It is known for more than two decades that carbon-based high-63 molecular-weight organic materials, which are ubiquitous in terrestrial and aquatic environments 64 and that are not involved in microbial metabolism (i.e., humic substances) can be used as external 65 electron acceptor for the anaerobic oxidation of compounds (10). Also, it has been reported that 66 anaerobic NH4 + oxidation linked to microbial reduction of natural organic matter fuels nitrogen 67 loss in marine sediments (11). A literature survey of more than 100 EET-capable species indicated 68 that there are many ecological niches for microorganisms able to perform EET (12) . This resonates 69 with a recent finding where Listeria monocytogenes, a host-associated pathogen and fermentative 70 Gram-positive bacterium, was able to respire through a flavin-based EET process and behaved as 71 an electrochemically active microorganism (i.e., able to transfer electrons from oxidized fuel 72 (substrate) to a working electrode via EET process) (13). Further it was reported that anammox 73 bacteria seem to have homologs of Geobacter and Shewanella multi-heme cytochromes that are 74 responsible for EET (14). These observations stimulated us to investigate whether anammox 75 bacteria can couple NH4 + oxidation with EET to carbon-based insoluble extracellular electron 76 acceptor and can behave as electrochemically active bacteria. (Fig. S1B-G) . Also, a previously enriched K. stuttgartiensis (freshwater anammox 86 species) culture was used (4). The anammox cells were incubated anoxically for 216 hours in the 87 presence of 15 NH4 + (4 mM) and graphene oxide (GO) as a proxy for insoluble electron acceptor. 88 No NO2or NO3were added to the incubations. GO particles are bigger than bacterial cells and 89 cannot be internalized, and thus GO can only be reduced by EET (16). Indeed, GO was reduced 90 by anammox bacteria as shown by the formation of suspended reduced GO (rGO), which is black 91 in color and insoluble (Fig. 1A) (16). In contrast, abiotic controls did not form insoluble black 92 precipitates. Reduction of GO to rGO by anammox bacteria was further confirmed by Raman 93 spectroscopy, where the formation of the characteristic 2D and D+D¢ peaks of rGO (17) were 94 detected in the vials with anammox cells (Fig. 1B) , whereas no peaks were detected in the abiotic 95 control. Further, isotope analysis of the produced N2 gas showed that anammox cells were capable 96 of 30 N2 formation (Fig. 1C ). In contrast, 29 N2 production was not significant in any of the tested 97 anammox species or controls, suggesting that unlabeled NO2or NO3were not involved. The 98 production of 30 N2 indicated that the anammox cultures use a different mechanism for NH4 + 99 oxidation in the presence of an insoluble extracellular electron acceptor (further explained below). 100 Gas production was not observed in the abiotic control (Fig. 1C) . To determine if anammox 101 bacteria are still dominant after incubation with GO, we extracted and sequenced total DNA from 102 the Brocadia and Scalindua vials at the end of the experiment. Differential coverage showed that 103 the metagenomes were dominated by anammox bacteria (Fig. S2A removal were observed in any of the abiotic controls. Subsequently, the Ca. Brocadia culture was 119 inoculated into the MEC and operated under optimal conditions for anammox (i.e., addition of 120 NH4 + and NO2 -). Under this scenario, NH4 + and NO2were completely removed from the medium 121 without any current generation ( Fig. 2A) . Stoichiometric ratios of consumed NO2 − to consumed 122 NH4 + (∆NO2 − /∆NH4 + ) and produced NO3 − to consumed NH4 + (∆NO3 − /∆NH4 + ) were in the range 123 of 1.0-1.3 and 0.12-0.18, respectively, which are close to the theoretical ratios of the anammox 124 reaction (18). These ratios indicated that anammox bacteria were responsible for NH4 + removal in 125 the MEC. Subsequently, NO2was gradually decreased to 0 mM leaving the electrodes as the sole 126 electron acceptor. When the exogenous electron acceptor (i.e., NO2 -) was completely removed 127 from the feed, anammox cells began to form a biofilm on the surface of the electrodes (Fig. S1I ) 128 and current generation coupled to NH4 + oxidation was observed in the absence of NO2 -( Fig. 2A) . 129 Further, NO2and NO3 − were below the detection limit at all time points when the working 130 electrode was used as the sole electron acceptor. The magnitude of current generation was 131 proportional to the NH4 + concentration ( Fig. 2A) and maximum current density was observed at 132 set potential of 0.4 V vs Ag/AgCl. There was no visible biofilm growth and current generation at The mole of electrons transferred to the electrode per mole of NH4 + oxidized to N2 (Table S1) 166 was stoichiometrically close to equation 1 (Eq. 1). Also, electron balance calculations showed that 167 coulombic efficiency (CE) was >80% for all NH4 + concentrations and anammox cultures tested in 168 the experiments with electrodes as the sole electron acceptor (Table S1 ). (Table S1 ). In addition, NH4 + oxidation and current production were not affected by the addition 176 of Penicillin G (Fig. S4) (Table S8) . This observation agrees with the NH4 + removal and 248 oxidation to N2 observed in the MECs and isotope labeling experiments ( Fig. 2A, Fig 3A) . The 249 genes encoding for NO and NO2reductases (nir genes) and their redox couples were significantly 250 downregulated when electrode was used as the electron acceptor (Table S8 ). This is expected as 251 NO2was not added in the electrode-dependent anammox process. Also, this supports the 252 hypothesis that NO is not an intermediate of the electrode-dependent anammox process and that 253 there was no effect of PTIO when NO2was replaced by the electrode as electron acceptor ( suggest an alternative pathway for NH4 + oxidation coupled to EET when working electrode is used 272 as electron acceptor compared to NO2as electron acceptor. 273 In conclusion, our study provides the first experimental evidence that phylogenetically and 