key: cord-103523-46hn2249
authors: Shaw, Dario R.; Ali, Muhammad; Katuri, Krishna P.; Gralnick, Jeffrey A.; Reimann, Joachim; Mesman, Rob; van Niftrik, Laura; Jetten, Mike S. M.; Saikaly, Pascal E.
title: Extracellular electron transfer-dependent anaerobic oxidation of ammonium by anammox bacteria
date: 2019-11-26
journal: bioRxiv
DOI: 10.1101/855817
sha: 
doc_id: 103523
cord_uid: 46hn2249

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 (NH4+) to dinitrogen gas (N2) using nitrite (NO2−) or nitric oxide (NO) as the electron acceptor. In the absence of NO2− 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 NH4+ with transfer of electrons to carbon-based insoluble extracellular electron acceptors. Here we show using complementary approaches that in the absence of NO2−, freshwater and marine anammox bacteria couple the oxidation of NH4+ 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 NH4+. 15N-labelling experiments revealed the molecular mechanism of the EET-dependent anammox process. NH4+ was oxidized to N2 via hydroxylamine (NH2OH) as intermediate when electrode was the terminal electron acceptor. Comparative transcriptomics analysis supported isotope labelling experiments and revealed an alternative pathway for NH4+ oxidation coupled to EET when electrode is used as electron acceptor compared to NO2−as electron acceptor. To our knowledge, our results provide the first experimental evidence that marine and freshwater anammox bacteria can couple NH4+ oxidation with EET, which is a significant finding, and challenges our perception of a key player of anaerobic oxidation of NH4+ in natural environments and engineered systems.

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 

Microbial Nitrogen Cycling Processes in Oxygen Minimum 292

Molecular mechanism of 298 anaerobic ammonium oxidation

Nitric oxide-dependent 300 anaerobic ammonium oxidation

Deciphering the 307 evolution and metabolism of an anammox bacterium from a community genome

Enrichment and characterization of 311 marine anammox bacteria associated with global nitrogen gas production

Our analysis showed upregulation under electrode-dependent anammox process of the genes in 1235 the Wood-Ljungdahl pathway for CO2 fixation and acetyl-CoA synthesis

GltS catalyzes the binding of the ammonium-1242 nitrogen to 2-oxoglutarate with the oxidation of Fdred (91). The 2-oxoglutarate used for this 1243 reaction can be provided by the key enzyme of the rTCA cycle 2-oxoglutarate:ferredoxin 1244 oxidoreductase (OGOR) (92)

Iron assimilation in anammox bacteria in EET-dependent anammox process

Surprisingly the proteins involved in iron 1253 transport and assimilation are still unknown. Our analysis revealed that in the absence of soluble 1254 electron acceptors (i.e., NO2 -, NO3 -), Ca. Brocadia electricigens expressed two gene clusters 1255 encoding a siderophore-mediated iron uptake system (Fig. S10, Table S3 and 13). The expressed 1256 siderophore-mediated transport system

Fe(III) uptake relies on beta-barrel TonB-dependent receptors in the 1259 outer membrane (95) and an energy-transducing protein complex TonB-ExbB-ExbD that links the 1260 outer with the inner membrane and generate a proton motive force (94). A periplasmic iron-binding 1261 protein and an ATP-dependent ABC

Fe(III) reduction in the cytoplasm 1264 can be carried out by ferric-chelate reductases/rubredoxins, from which multiple genes were found 1265 to be expressed (Fig. S10, Table S3)

This finding 1269 is in agreement with a previous study using the EET-capable model bacteria Geobacter sulfurreducens 1270 (8), in which it was shown that the pathways required for EET and metal oxide reduction are distinct. 1271 1272 1: Rieske/cytochrome b complex; bc-2: Rieske/cytochrome b complex; bc-3: Rieske/cytochrome 1386 b complex; Cyt c (1 heme): Periplasmic mono-heme c-type cytochrome

Membrane-anchored tetraheme 1388 c-type cytochrome; Cyt c (5 hemes): Outer membrane penta-heme c-type cytochrome

Cytochrome c redox partner of the ETM; Cyt Nir: Cytochrome c; ETM: electron transfer module 1390 for hydrazine synthesis; ExbB: Biopolymer transport protein ExbB/TolQ; ExbD: Biopolymer 1391 transport protein ExbD/TolR; FDH: membrane-bound formate dehydrogenase

FocA: Formate/nitrite transporter; GltS: Glutamate 1393 synthase; HAO BROSI_A0501: Hydroxylamine oxidoreductase; HAO BROSI_A3534: 1394 Hydroxylamine oxidoreductase; HAO EX330_11045: Hydroxylamine oxidoreductase; HDH: 1395 hydrazine dehydrogenase; HZS: hydrazine synthase; membNXR: membrane-bound complex of 1396 the nxr gene cluster; MscL: Large mechanosensitive channel; MscS: Pore-forming small 1397 mechanosensitive channel; NADH-DH: NADH dehydrogenase; Nir BROSI_A0131: nitrite 1398 reductase; NorVW: Flavodoxin nitric oxide reductase; NrfA: ammonium-forming nitrite 1399 reductase

heme): Outer membrane lipoprotein mono-heme c-type cytochrome

OmpA: OmpA-like outer membrane protein, porin; PFdO: Pyruvate 1402 ferredoxin oxidoreductase; PFL: Pyruvate formate lyase; PP binding protein: Iron ABC transporter 1403 periplasmic substrate-binding protein; Rnf: RnfABCDGE type electron transport complex

Rubredoxin/ferric-chelate reductase; S-layer: S-layer protein; TonB: Energy transducer TonB

acetoxime (C3H7NO), and the molecular ion peaks were detected at mass to charge (m/z) = 73 and 848 74 for 14 NH2OH and 15 NH2OH, respectively. 25 M of 14 NH2OH and 15 NH2OH were used as 849 standards. To determine the source of the oxygen used in the electrode-dependent NH4 + oxidation 850 to NH2OH, MECs were incubated with 15 NH4Cl (4 mM, Cambridge Isotope Laboratories) in 851 presence of 10% D2O for 144 hours. Stable isotopes of NH2OH were determined by GC/MS 852 analysis after derivatization using acetone as described above. 853 854 Activity and electron balance calculations 855 Activities of specific anammox ( 29 N2) with nitrite as the preferred electron acceptor and electrode-856 dependent anammox ( 30 N2) with working electrode (0.4 V vs Ag/AgCl) as sole electron acceptor 857 were calculated based on the changes in gas concentrations in single-chamber MEC batch 858 incubations. The activity was normalized against protein content of the biofilm on the electrodes. 859 Protein content was measured as described below (See Analytical methods section). 860 The moles of electrons recovered as current per mole of NH4 + oxidized were calculated using: Analytical methods 870 All samples were filtered through a 0.2 µm pore-size syringe filters (Pall corporation) prior to 871 chemical analysis. NH4 + concentration was determined photometrically using the indophenol 872 method (28) (lower detection limit = 5 μM). Absorbance at a wavelength of 600 nm was 873 determined using multi-label plate readers (SpectraMax Plus 384; Molecular Devices, CA, USA). 874 NO2concentration was determined by the naphthylethylenediamine method (28) (lower detection 875 limit = 5 μM). Samples were mixed with 4.9 mM naphthylethylenediamine solution, and the 876 absorbance was measured at a wavelength of 540 nm. NO3concentration was measured by HACH 877 kits (HACH, CO, USA; lower detection limit = 0.01 mg l -1 NO3 --N). User's guide was followed 878 for these kits and concentrations were measured by spectrophotometer (D5000, HACH, CO, 879 USA). Concentrations of NH2OH and hydrazine (N2H4) were determined colorimetrically as 880 previously described (29). For NH2OH, liquid samples were mixed with 8-quinolinol solution 881 (0.48% (w/v) trichloroacetic acid, 0.2% (w/v) 8-hydroxyquinoline and 0.2 M Na2CO3) and heated 882 at 100°C for 1 min. After cooling down for 15 min, absorbance was measured at 705 nm (30). 883 N2H4 was derivatized with 2% (w/v) p-dimethylaminobenzaldehyde and absorbance at 460 nm 884 was measured (31). The concentration of biomass on the working electrodes was determined as 885 protein concentration using DC Protein Assay Kit (Bio-Rad, Tokyo, Japan) according to 886 manufacturer's instructions. Bovine serum albumin was used as the protein standard. Putative EET-dependent anammox pathway 1031 We provided evidence that phylogenetically distant anammox bacteria can perform EET and are 1032 electrochemically active, and we elucidated the molecular mechanism of NH4 + oxidation, which 1033 by itself are significant findings that changes our perception of a key player in the global nitrogen (Table S3 and Table S8 ). This result is consistent with the NH4 + uptake, oxidation and 1066 final conversion to N2 observed in the MECs and isotope labeling experiments ( Fig. 2A, Fig 3A) . 1067 The requirement of more moles of NH4 + when anammox growth is based on EET compared to 1068 NO2as electron acceptor (Eq. 1), increases the demand of NH4 + import into the cell, which can 1069 explain the upregulation of the ammonium transporters. In contrast, the genes encoding for NO 1070 and NO2reductases (nir genes) and their redox couples were significantly downregulated ( Fig.   1071 S10, Table S8 ). This agrees, with the fact that NO2and NO3were below the detection limit in 1072 the MECs (Fig. 2A, Fig. S3A and B) and there was no effect of PTIO when NO2was replaced by 1073 electrode as electron acceptor (Fig. S8) (Fig. 4) . The four low-potential 1117 electrons released from this reaction must be stored until they are transferred to a redox partner (Fig. 2E, Fig. S3C and D) exhibited oxidation/reduction peaks, which suggests that additional 1182 cytochrome(s) that transfer electrons directly to the electrode via solvent exposed hemes may be 1183 involved. Also, no cytochromes for long-range electron transport were detected in the analysis 1184 (Table S6 and 7), suggesting that EET to electrodes by anammox bacteria rely on a direct EET 1185 mechanism. Homology detection and structure prediction by hidden Markov model comparison 1186 (HMM-HMM) of the highly upregulated penta-heme cytochrome EX330_07910 (Fig. S10 , Table   1187 S6) gave high probability hits to proteins associated to the extracellular matrix and outer membrane 1188 iron respiratory proteins such as MtrF, OmcA and MtrC. Also, it is worth mentioning that the gene 1189 cluster EX330_07910-07915 was one of the most upregulated under set-potential conditions. 1190 Therefore, future work should focus on determining the role of EX330_07910-07915 in the EET-1191 dependent anammox process. Likewise, we also found the expression of outer membrane mono-1192 heme c-type cytochromes (OM Cyt c (1 heme) (Fig. S10, Table S7 ) homologs to G.