| The concentration profiles of the batch tests performed with the culture operated at Lac/N ratio 2.97. The tested substrate combinations shown are (A) lactate in the absence of an electron acceptor, (B) lactate with nitrate, (C) acetate with nitrate and (D) propionate with nitrate. 

| The concentration profiles of the batch tests performed with the culture operated at Lac/N ratio 2.97. The tested substrate combinations shown are (A) lactate in the absence of an electron acceptor, (B) lactate with nitrate, (C) acetate with nitrate and (D) propionate with nitrate. 

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Denitrification and dissimilatory reduction to ammonium (DNRA) are competing nitrate-reduction processes that entail important biogeochemical consequences for nitrogen retention/removal in natural and man-made ecosystems. The nature of the available carbon source and electron donor have been suggested to play an important role on the outcome of thi...

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... the experiments a slightly positive pressure was maintained in the vials to avoid oxygen leakage into the bottles and to facilitate sampling. Incubation times varied between 3 and 6 h and the sampling interval varied from 45-90 min ( Figures S1, S2). ...
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... sources were always supplied in higher initial concentrations (5 mM) than electron acceptor (4 mM) to assure electron-excessive conditions. A full overview of the batch results can be found in Figures S1, S2 and the resulting conversion rates in Tables S5, S6. ...
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... Figure 1 the concentration profiles of the four most relevant the batch tests performed with the culture operated at Lac/N ratio 2.97 are shown. In the absence of an electron acceptor, one mole lactate was fermented to 0.37 mole of acetate and 0.69 mole of propionate ( Figure 1A), with no measurable production of H 2 . ...
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... Figure 1 the concentration profiles of the four most relevant the batch tests performed with the culture operated at Lac/N ratio 2.97 are shown. In the absence of an electron acceptor, one mole lactate was fermented to 0.37 mole of acetate and 0.69 mole of propionate ( Figure 1A), with no measurable production of H 2 . Batch tests using lactate together with nitrate or nitrite as electron acceptor showed similar rate of propionate production and a transient acetate accumulation (Figure 1B). ...
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... the absence of an electron acceptor, one mole lactate was fermented to 0.37 mole of acetate and 0.69 mole of propionate ( Figure 1A), with no measurable production of H 2 . Batch tests using lactate together with nitrate or nitrite as electron acceptor showed similar rate of propionate production and a transient acetate accumulation (Figure 1B). The acetate accumulation was lower with nitrite as electron acceptor compared to nitrate. ...
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... acetate as electron donor, the conversion appeared to be slower than lactate depletion for the same concentrations of respective electron acceptor ( Figure 1C). Propionate, when used TABLE 5A | Net conversion rates (mmol/h) in the reactor steady states for the different influent Lac/N ratios (mol/mol). ...
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... an electron donor, was only consumed for the conversion of nitrate into nitrite but at an insignificant rate ( Figure 1D). ...

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... A high concentration of lactate facilitated nitrate reduction to ammonium, which was attributable to the presence of more electron donors ( Figure S1). However, given the stoichiometry of lactate oxidation coupled with nitrate reduction, the lactate added in this study should not be fully oxidized to CO 2 , but may form other intermediates, such as propionate, ethanol, and formate [51]. When cultured with 100 µM As(V), this strain transformed 50% of As(V) to As(III) during DNRA (Figure 2). ...
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... Apart from Sulfuricurvum spp., several other lineages detected in our libraries are also known for DNRA, such as Geobacter spp. (van den Berg et al., 2017), members of the Desulfocapsaceae (Arshad et al., 2017;Bell et al., 2020), or Sulfurimonas spp. (Bell et al., 2020). ...
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... Thus, lactate and ammonium/ammonia may be linked by lactic acid producing bacteria (e.g., Streptococcus, Lactobacillus and Actinomyces) which can convert arginine into ammonia 60 . Additionally, lactic acid can be used during, and stimulate, DNRA 240 . We hypothesize that the salivary ammonium/ammonia levels under the in vivo conditions of this study may partly reflect the amount of lactic acid producing bacteria, or that they were metabolically linked to lactate in a different way. ...
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... The genome of Shewanella loihica strain PV-4 encodes the complete set of conventional denitrification and DNRA pathways, and relatively high C/N ratios upregulated their DNRA functional genes (e.g., nrfA) (Yoon et al., 2015). In another study, higher lactate/NO 3 ratio (2.97) also promoted the co-existence of fermentative and respiratory DNRA, and enhanced their activities (Van Den Berg et al., 2017). Meta-analysis also suggested that DNRA rate was positively correlated with C/NO 3 -(Van Den Berg et al., 2016;Pandey et al., 2020). ...
... Type of electron donor also was another important factor. Compared with heterotrophic denitrifiers, fermentative DNRA bacteria prefer to use less oxidative and labile organic carbon sources (Tiedje, 1988;Van Den Berg et al., 2017). L-sorbose or D-cellobiose enriched denitrifiers (e.g., Klebsiella), while D-glucose, D-fructose and citrate enriched DNRA bacteria (e.g., Escherichia, Sulfurospirillum) (Carlson et al., 2020). ...
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... A second potential anti-caries prebiotic is nitrate, but current in vivo evidence is limited (Rosier et al., 2018). It is estimated that humans obtain more than 80% of dietary nitrate from vegetablesa food group unequivocally associated with health benefits (Link et al., 2004;Wang et al., 2014;Turati et al., 2015;Lundberg et al., 2018). The salivary glands contain electrogenic sialin 2NO 3 -/H + transporters that concentrate plasma nitrate into the saliva (Qu et al., 2016), leading to high salivary nitrate concentrations (100-500 mM during fasting, which is~10x higher than in plasma, and 5-8 mM after a nitrate-containing meal) (Lundberg and Govoni, 2004;Hezel and Weitzberg, 2015). ...
... Thus, lactate and ammonia may be linked by lactic acid producing bacteria (e.g., Streptococcus, Lactobacillus and Actinomyces) which can convert arginine into ammonia (Liu et al., 2012). Additionally, lactate can be used during, and stimulate, DNRA (van den Berg et al., 2017). We hypothesize that the salivary ammonia levels under the in vivo conditions of this study may partly reflect the amount of lactic acid producing bacteria, or that they were metabolically linked to lactate in a different way. ...
... Another source of nitrate is drinking water and high nitrate levels in water, resulting from agricultural contamination, have been associated with cancer and other adverse health effects (Ward et al., 2018). However, we obtain over 80% of nitrate from vegetables, which are considered protective against cancer and other diseases (Link and Potter, 2004;Wang et al., 2014;Turati et al., 2015). This includes nitraterich vegetables such as lettuce and spinach, which are considered protective against different types of cancer (mouth, pharynx, larynx, oesophagus and stomach) (Mills et al., 2017). ...
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... However, glucose efficiency as a denitrification C substrate may decline if fermentative bacteria compete with denitrifiers for C (Paul et al., 1989). Given that acetate is generally considered to be a non-fermentable substrate (van den Berg, Elis ario, Kuenen, Kleerebezem, & van Loosdrecht, 2017), the lower N 2 emissions observed on day 14 in the LU and LD soils under glucose may have also resulted from greater microbial competition for glucose between fermentative organisms and denitrifiers. However, the fact that the glucose-treated AD soil had similar N 2 emissions to the acetate-treated soil at day 14, suggests that the microbial community in the AD soil was also responding differently to substrate addition with respect to the LU and LD soils due to potential effects of the lower P and C status on the microbial biomass and community structure as noted above. ...
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Carbon (C) substrates are critical for regulating denitrification, a process that results in nitrous oxide (N2O) and dinitrogen (N2) emissions from soil. However, the impacts of C substrates on concomitant soil emissions of carbon dioxide (CO2) and N2O under varying soil types and soil water contents are not well studied. Three repacked Pallic grassland soils, varying in texture and phosphorus (P) status, containing NO3‐‐¹⁵N were held at three levels of matric potential (ψ, ‐3, ‐5 and ‐7 kPa), while receiving daily substrate additions (acetate, glucose, water control) for 14 days. The CO2 and N2O emissions were measured daily. Additionally, the N2O/(N2+N2O) ratios were determined using ¹⁵N on days 3 and 14. Results showed that N2O emissions increased exponentially as soil gas diffusivity declined, N2O peak emissions were higher with glucose than with acetate addition, with a range (± standard deviation) of 0.1 ± 0.0 to 42.7 ± 2.1 mg N m⁻² h⁻¹. The highest cumulative N2O emission (2.5 ± 0.2 g N m‐2) was measured following glucose addition with a soil ψ of ‐3 kPa. In comparison with added glucose, acetate resulted in a 2‐fold increase in N2 emissions in soils with relatively low gas diffusivities. The N2O:(N2O+N2) emissions ratios varied with substrate (glucose, 0.91; acetate 0.81) on day 3, and had declined by day 14 under substrate addition (≤ 0.10). Cumulative CO2 emissions were enhanced with increasing soil gas diffusivity and were higher for soils amended with glucose (ranging from 22.5 ± 1.3 to 36.6 ± 1.8, g C m‐2) than for those amended with acetate. Collectively, the results demonstrate that the increase of N2O, N2, and CO2 emissions, and changes in the N2O/(N2+N2O) ratio vary over time in response to C substrate type and soil gas diffusivity. This article is protected by copyright. All rights reserved.
... Oyster biodeposits, and/or active nitrification across oxic/anoxic interfaces can provide significant amounts of nitrate in the underlying sediments (Jensen et al., 1994;Newell et al., 2005 and references therein); thus, we consider that fermentative DNRA may not be the primary form of DNRA occurring underneath BC in our study. This is supported by our iTAG data that show low relative abundances (<0.5%) of fermentative bacteria that can perform DNRA and influence the competition between denitrification and DNRA in favor of DNRA (e.g., taxa affiliated to Clostridia, Vibrio, Desulfovibrio, Bacillus, and Pseudomonas sp.; Burgin and Hamilton, 2007;van den Berg et al., 2017). ...
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Coastal ecosystems are impacted by excessive nutrient inputs that cause degradation of water quality and impairments of ecosystem functioning. Regulatory and management efforts to enhance nutrient export from coastal ecosystems include sustainable oyster aquaculture that removes nitrogen in the form of oyster biomass and increases particulate export to underlying sediments where increased organic material may enhance microbial denitrification. To better understand the impacts of oyster aquaculture on nitrogen removal, we examined bacterial processes in sediments underlying three of the most common aquaculture methods that vary in the proximity of oysters to the sediments. Sediment samples underlying sites managed with these different aquaculture methods were examined using the 16S rRNA gene to assess microbial community structure, gene expression analyses to examine nitrogen and sulfur cycling genes, and nitrogen gas flux measurements. All sites were located in the same hydrodynamic setting within Waquoit Bay, MA during 2018 and 2019. Although sediments under the different oyster farming practices showed similar communities, ordination analysis revealed discrete community groups formed along the sampling season. Measured N 2 fluxes and expression of key genes involved in denitrification, anaerobic ammonium oxidation (anammox), and dissimilatory nitrate reduction to ammonium (DNRA) increased during mid-summer and into fall in both years primarily under bottom cages. While all three oyster growing methods enhanced nitrogen removal relative to the control site, gene expression data indicate that the nitrogen retaining process of DNRA is particularly enhanced after end of July under bottom cages, and to a lesser extent, under suspended and floating bags. The choice of gear can also potentially increase processes that induce nitrogen retention in the form of ammonia in the underlying sediments over time, thus causing deviations from predicted nitrogen removal. If nitrogen removal is a primary objective, monitoring for these shifts is essential for making decisions about siting and size of aquaculture sites from year to year.
... However, the NH 4 + concentrations were overall low, suggesting limited importance of this unwanted process. Interestingly, many DNRA bacteria are also fermenting (Muyzer & Stams, 2008;Van Den Berg, Elisário, Kuenen, Kleerebezem, & van Loosdrecht, 2017), and hence the DNRA bacteria may also contribute to fermentation and thereby support denitrification despite being competitors for NO 3 − . ...
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Denitrifying woodchip bioreactors (DWBs) are potential low‐cost technologies for the removal of nitrate (NO3−) in water through denitrification. However, if environmental conditions do not support microbial communities performing complete denitrification, other N transformation processes will occur resulting in the export of nitrite (NO2−), nitrous oxide (N2O), or ammonium (NH4+). In order to identify the factors controlling the relative accumulation of NO2−, N2O, and/or NH4+ in DWBs, porewater samples were collected over two operational years from a DWB designed for removing NO3− from mine water. Woodchip samples were collected at the end of the operational period. Changes in the abundances of functional genes involved in denitrification, N2O reduction, and dissimilatory nitrate reduction to ammonium were correlated with pore water chemistry and temperature. Temporal changes in the abundance of the denitrification gene nirS were significantly correlated with increases in porewater N2O concentrations, and indicated the preferential selection of incomplete denitrifying pathways ending with N2O. Temperature and the TOC/NO3− ratio were strongly correlated with NH4+ concentrations and inversely correlated with the ratio between denitrification genes and the genes indicative of ammonification (∑nir/nrfA), suggesting an environmental control on NO3− transformations. Overall, our results for a DWB operated at hydraulic residence times of 1.0 ‐ 2.6 days demonstrate the temporal development in the microbial community and indicate an increased potential for N2O emissions with time from the DWB.
... Dissimilatory nitrate/nitrite reduction to ammonium is catalyzed by the microorganisms carrying cytochrome c 552 nitrite reductases (encoded by nrfA genes) or NADHdependent nitrite reductases (encoded by nirB genes), often incorrectly generalized as assimilatory nitrite reductases (13). According to the current limited knowledge, NO 2 Ϫto-NH 4 ϩ reduction may serve as the electron acceptor reaction for respiration (respiratory DNRA) or the electron dump for NADH regeneration in fermentation of complex organics (fermentative DNRA) (14)(15)(16). ...
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Dissimilatory nitrate/nitrite reduction to ammonium (DNRA) has recently regained attention as a nitrogen retention pathway that may potentially be harnessed to alleviate nitrogen loss resulting from denitrification. Until recently, the ecophysiology of DNRA bacteria inhabiting agricultural soils has remained largely unexplored, due to the difficulty in targeted enrichment and isolation of DNRA microorganisms. In this study, >100 DNRA bacteria were isolated from NO3−-reducing anoxic enrichment cultures established with rice paddy soils using a newly developed colorimetric screening method. Six of these isolates, each assigned to a different genus, were characterized to improve the understanding of DNRA physiology. All the isolates carried nrfA and/or nirB, and the Bacillus sp. strain possessed a clade II nosZ gene conferring the capacity for N2O reduction. A common prominent physiological feature observed in the isolates was NO2− accumulation before NH4+ production, which was further examined with Citrobacter sp. strain DNRA3 (possessing nrfA and nirB) and Enterobacter sp. strain DNRA5 (possessing only nirB). Both isolates showed inhibition of NO2−-to-NH4+ reduction at submillimolar NO3− concentrations and downregulation of nrfA or nirB transcription when NO3− was being reduced to NO2−. In batch and chemostat experiments, both isolates produced NH4+ from NO3− reduction when incubated with excess organic electron donors, while incubation with excess NO3− resulted in NO2− buildup but no substantial NH4+ production, presumably due to inhibitory NO3− concentrations. This previously overlooked link between NO3− repression of NO2−-to-NH4+ reduction and the C-to-N ratio regulation of DNRA activity may be a key mechanism underpinning denitrification-versus-DNRA competition in soil.
... Geobacter lovleyi has emerged as a model representative of environmental bacteria that drives DNRA in nature (15). This organism has been shown to dominate a microbial community in an enrichment culture grown under limiting nitrate (16)(17)(18), a condition that enhances DNRA over denitrification. The switch from nitrate to nitrite as the electron acceptor in the enrichment culture did not change the dominant ribotype, suggesting that the DNRA pathway of G. lovleyi can efficiently use both substrates (17). ...
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Full-text available
Cytochrome c nitrite reductase (NrfA) catalyzes the reduction of nitrite to ammonium in the dissimilatory nitrate reduction to ammonium (DNRA) pathway, a process that competes with denitrification, conserves nitrogen, and minimizes nutrient loss in soils. The environmental bacterium Geobacter lovleyi has recently been recognized as a key driver of DNRA in nature, but its enzymatic pathway is still uncharacterized. To address this limitation, here we overexpressed, purified, and characterized G. lovleyi NrfA. We observed that the enzyme crystallizes as a dimer, but remains monomeric in solution. Importantly, its crystal structure at 2.55 Å resolution revealed the presence of an arginine residue in the region otherwise occupied by calcium in canonical NrfA enzymes. The presence of EDTA did not affect the activity of G. lovleyi NrfA, and site-directed mutagenesis of this arginine reduced enzymatic activity < 3% of the wild-type levels. Phylogenetic analysis revealed four separate emergences of Arg-containing NrfA enzymes. Thus, the Ca ²⁺ -independent, Arg-containing NrfA from G. lovleyi represents a new subclass of cytochrome c nitrite reductase. Most genera from the exclusive clades of Arg-containing NrfA proteins are also represented in clades containing Ca ²⁺ -dependent enzymes, suggesting convergent evolution.