Dissimilatory Nitrate Reduction To Ammonium Activity Research Articles

Long-term operation and dynamic response of dissimilatory nitrate reduction to ammonium process under low-frequency infrared electromagnetic field

Dissimilatory nitrate reduction to ammonium (DNRA) received more attention for its ability to recover ammonium. This study investigated the possibility of low-frequency infrared electromagnetic field (IR-EMF) to improve DNRA. The optimal IR-EMF intensity of 0.04 μT could effectively improve DNRA activity of nonwoven fabric membrane bioreactors. In the long-term operation, the average ammonium conversion efficiency was enhanced by 117.7% and 62.5% under 0.04 μT and 0.06 μT IR-EMF, respectively. The highest nrfA-gene abundance and potential DNRA rate were obtained under 0.04 μT IR-EMF exposure. Bacteroidetes fragilis, Shewanelle oneidensis MR-1, and Thauera sp. RT1901 were selected to investigate the dynamic response of nitrogen transformation and energy metabolism to IR-EMF. The transcriptome sequencing and RT-qPCR results suggested that IR-EMF could enhance both denitrification and DNRA process, mainly by improving ATP synthesis to boost metabolic activity. This study provided an efficient method for the nitrogen recovery via DNRA process by applying IR-EMF.

Study on the effectiveness and mechanism of a sustainable dual slow-release model to improve N utilization efficiency and reduce N pollution in black soil

Long-term intensive cultivation has led to serious N loss and low N fertilizer utilization efficiency (NUE) in black soil areas. The lost N is not only a waste of resources but also a serious pollution threat to the environment, leading to the decline in water quality and food safety and the greenhouse effect. In the present study, a stable dual slow-release model, CPCS-Urea, was prepared by in situ polymerization using nitrapyrin, urea and melamine-formaldehyde resin as raw materials. The effect of the dual slow-release model was systematically evaluated using two consecutive years of field experiments. Five treatments were established in the field experiment: no N fertilizer (N0), urea (N180), 1 % CPEC-Urea, 0.5 % CPCS-Urea, and 1 % CPCS-Urea. The results showed that the new dual slow-release CPCS-Urea model outperformed both the use of urea and the traditional slow-release CPEC-Urea model in reducing N losses and improving NUE. The application of CPCS-Urea reduced nitrate (NO3−) leaching by 28.2 %–47.2 % and N2O emissions by 36.5 %–42.4 % and increased NUE by 20.7 %–28.5 % compared to urea application. The CPCS-Urea model modulated the activity of ammonia-oxidizing bacteria (AOB) and dissimilatory nitrate reduction to ammonium (DNRA) bacteria in soil, showing a significant decrease in AOB activity and an increase in DNRA activity. This results in a lower soil NO3−-N yield and a 53.1 %–72.0 % increase in NH4+-N content, providing sufficient N for the entire growth and development cycle of maize. In short, the dual slow-release CPCS-Urea model has great application prospects for promoting agricultural development in black soil areas.

Sinking krill carcasses as hotspots of microbial carbon and nitrogen cycling in the Arctic

Krill represent a major link between primary producers and higher trophic levels in polar marine food webs. Potential links to lower trophic levels, such as heterotrophic microorganisms, are less well documented. Here, we studied the kinetics of microbial degradation of sinking carcasses of two dominant krill species Thysanoessa raschii and Meganyctiphanes norvegica from Southwest Greenland. Degradation experiments under oxic conditions showed that 6.0-9.1% of carbon and 6.4-7.1% of nitrogen were lost from the carcasses after one week. Aerobic microbial respiration and the release of dissolved organic carbon were the main pathways of carbon loss from the carcasses. Ammonium release generally contributed the most to carcass nitrogen loss. Oxygen micro profiling revealed anoxic conditions inside krill carcasses/specimens, allowing anaerobic nitrogen cycling through denitrification and dissimilatory nitrate reduction to ammonium (DNRA). Denitrification rates were up to 5.3 and 127.7 nmol N carcass-1 d-1 for T. raschii and M. norvegica, respectively, making krill carcasses hotspots of nitrogen loss in the oxygenated water column of the fjord. Carcass-associated DNRA rates were up to 4-fold higher than denitrification rates, but the combined activity of these two anaerobic respiration processes did not contribute significantly to carbon loss from the carcasses. Living krill specimens did not harbor any significant denitrification and DNRA activity despite having an anoxic gut as revealed by micro profiling. The investigated krill carcasses sink fast (1500-3000 m d-1) and our data show that only a small fraction of the associated carbon is lost during descent. Based on data on krill distribution, our findings are used to discuss the potential importance of sinking krill carcasses for sustaining benthic food webs in the Arctic.

Quantifying the effect of the nitrogen biogeochemical processes on the distribution of ammonium in the riverbank filtration system

Ammonium (NH4+) enrichment of riverbank filtration (RBF) systems is gaining popularity. However, most previous research has concentrated on NO3− removal efficiencies, while the mechanisms of NH4+ enrichment remain unknown. A nitrogen biogeochemical process model was developed for the quantitative analysis of NH4+ enrichment in the Kaladian well field in northwest Songyuan City, NE China. Data from laboratory experiments and in-situ monitoring were used to determine initial values and calibrate the thermodynamic/kinetic parameters representing nitrogen (N) biogeochemical reactions. (1) The NO3− from river was subjected to denitrification (DNF) and dissimilatory nitrate reduction to ammonium (DNRA) within 10–14 m of the shore, whereas the NH4+ in groundwater was caused by DNRA, organic nitrogen mineralization (MIN), and mixing with laterally recharged high NH4+ groundwater. (2) DNF and DNRA were regulated by hydrodynamic processes, with the ranges of these processes being more significant in the wet season due to a higher hydraulic gradient. MIN occurred widely throughout the water flow path, with temperature primarily controlling the rates of the three reactions. (3) DNRA activity was relatively higher in the wet season when the water temperature was higher within 10–14 m of the shore. In the wet season, DNRA contributed 25%–30% to NO3− reduction, which was higher than in the dry season (5%–10%). DNRA contributed at least 40% and 15% to NH4+ enrichment in the wet and dry seasons, respectively. (4). Organic N in media gradually released NH4+ into groundwater via MIN and desorption across the entire flow path, with contributions to NH4+ enrichment reaching 75% and 85%, respectively, in the wet and dry seasons.

Identification of dissimilatory nitrate reduction to ammonium (DNRA) and denitrification in the dynamic cake layer of a full-scale anoixc dynamic membrane bioreactor for treating hotel laundry wastewater

Identification of dissimilatory nitrate reduction to ammonium (DNRA) and denitrification in the dynamic cake layer of a full-scale anoixc dynamic membrane bioreactor (AnDMBR) for treating hotel laundry wastewater was studied. A series of experiments were conducted to understand the contributions of DNRA and canonical denitrification activities in the dynamic cake layer of the AnDMBR. The dynamic cake layer developed included two phases - a steady transmembrane pressure (TMP) increase at 0.24 kPa/day followed by a sharp TMP jump at 1.26 kPa/day four to five days after the AnDMBR start-up. The nitrogen mass balance results showed that canonical denitrification was predominant during the development of the dynamic cake layer. However, DNRA activity and accumulation of bacteria equipped with a complete DNRA pathway showed a positive correlation to the development of the dynamic cake layer. Our metagenomic analysis identified an approximately 18% of the dynamic cake layer bacterial community has a complete DNRA pathway. Pannonibacter (1%), Thauera (0.8%) and Pseudomonas (3%) contained all genes encoding for funcional enzymes of both DNRA (nitrate reductase and DNRA nitrite reductase) and denitrification (nitrate reductase, nitrous oxide reductase and nitric oxide reductase). No other metagenome-assembled genomes (MAGs) possessed a complete cononical denitrification pathway, indicating food-chain-like interactions of denitrifiers in the dynamic cake layer. We found that COD loading rate could be used to control DNRA and canonical denitrification activities during the dynamic cake layer formation.

Microbially facilitated nitrogen cycling in tropical corals

Tropical scleractinian corals support a diverse assemblage of microbial symbionts. This ‘microbiome’ possesses the requisite functional diversity to conduct a range of nitrogen (N) transformations including denitrification, nitrification, nitrogen fixation and dissimilatory nitrate reduction to ammonium (DNRA). Very little direct evidence has been presented to date verifying that these processes are active within tropical corals. Here we use a combination of stable isotope techniques, nutrient uptake calculations and captured metagenomics to quantify rates of nitrogen cycling processes in a selection of tropical scleractinian corals. Denitrification activity was detected in all species, albeit with very low rates, signifying limited importance in holobiont N removal. Relatively greater nitrogen fixation activity confirms that corals are net N importers to reef systems. Low net nitrification activity suggests limited N regeneration capacity; however substantial gross nitrification activity may be concealed through nitrate consumption. Based on nrfA gene abundance and measured inorganic N fluxes, we calculated significant DNRA activity in the studied corals, which has important implications for coral reef N cycling and warrants more targeted investigation. Through the quantification and characterisation of all relevant N-cycling processes, this study provides clarity on the subject of tropical coral-associated biogeochemical N-cycling.

Metagenomic insights into nitrogen and phosphorus cycling at the soil aggregate scale driven by organic material amendments

The soil microbiome, existing as interconnected communities closely associated with soil aggregates, is the key driver in nutrient cycling. However, the underlying genomic information encoding the machinery of the soil microbiome involved in nutrient cycling at the soil aggregate scale is barely known. Here comparative metagenomics and genome binning were applied to investigate microbial functional profiles at the soil aggregate scale under different organic material amendments in a long-term field experiment. Soil samples were sieved into large macroaggregates (>2 mm), macroaggregates (0.25–2 mm) and microaggregates (<0.25 mm). Microbial taxonomic and functional alpha diversity were significantly correlated to soil NO3− and SOC. The highest abundance of nasB, nirK, and amoA genes, which are responsible for denitrification and ammonia oxidizers driving nitrification, was observed in microaggregates. Both manure and peat treatments significantly decreased the abundance of napA and nrfA that encode enzymes involved in dissimilatory nitrate reduction to ammonium (DNRA). As a biomarker for soil inorganic P solubilization, the relative abundance of gcd was significantly increased in macroaggregates and large macroaggregates. Three nearly complete genomes of Nitrososphaeraceae (AOA) and seven bacterial genomes were shown to harbor a series of genes involved in nitrification and P solubilization, respectively. Our study provides comprehensive insights into the microbial genetic potential for DNRA and P-solubilizing activity across different soil aggregate fractions and fertilization regimes.

Measurement of the Potential Rates of Dissimilatory Nitrate Reduction to Ammonium Based on 14NH4 +/15NH4 + Analyses via Sequential Conversion to N2O.

The importance of understanding the fate of nitrate (NO3-), which is the dominant N species transferred from terrestrial to aquatic ecosystems, has been increasing because global nitrogen loads have dramatically increased following industrialization. Dissimilatory nitrate reduction to ammonium (DNRA) and denitrification are both microbial processes that use NO3- for respiration. Compared to denitrification, quantitative determinations of the DNRA activity have been carried out only to a limited extent. This has led to an insufficient understanding of the importance of DNRA in NO3- transformations and the regulating factors of this process. The objective of this paper is to provide a detailed procedure for the measurement of the potential DNRA rate in environmental samples. In brief, the potential DNRA rate can be calculated from the 15N-labeled ammonium (15NH4+) accumulation rate in 15NO3- added incubation. The determination of the 14NH4+ and 15NH4+ concentrations described in this paper is comprised of the following steps. First, the NH4+ in the sample is extracted and trapped on an acidified glass filter as ammonium salt. Second, the trapped ammonium is eluted and oxidized to NO3- via persulfate oxidation. Third, the NO3- is converted to N2O via an N2O reductase deficient denitrifier. Finally, the converted N2O is analyzed using a previously developed quadrupole gas chromatography-mass spectrometry system. We applied this method to salt marsh sediments and calculated their potential DNRA rates, demonstrating that the proposed procedures allow a simple and more rapid determination compared to previously described methods.

Dissimilatory nitrate ammonification and N2 fixation helps maintain nitrogen nutrition in resource-limited rice paddies

Un-fertilized rice paddies have shown maintained soil nitrogen (N) status, stable N supply to the rice plant and sustained rice yields at moderate levels for hundreds of years. Microbial N2 fixation is known to contribute N to un-fertilized paddies, but it cannot fully explain the maintained N nutrition, where favourable conditions exist for N loss by denitrification. We used 15N tracer, 15N2 uptake, acetylene reduction assay and qPCR to simultaneously investigate N2 fixation, dissimilatory nitrate reduction to ammonium (DNRA), denitrification and related microbial gene abundances in long-term low (or no) and high N input rice paddies of Myanmar. We also determined how varying soil organic carbon-to-nitrate (SOC/NO3−) ratios affect nitrate partitioning between DNRA and denitrification by manipulating these ratios through labile organic carbon addition. We observed more than 2.5 times higher N2 fixation (1.49–2.08 μg N g−1 soil day−1) and significantly higher N2 fixing gene (nifH) abundance in low compared with high N input paddies. Up to 60% of the soil nitrate (1.51–2.67 μg NO3−-N g−1 soil day−1) was ammonified through DNRA, and only 15% was lost as N2 through denitrification in low N input paddies, whereas denitrification exceeded DNRA in high N input paddies. The microbial gene related to DNRA activity (nrfA) was also higher in low input than in high input rice paddies. We found that nitrate retention can be improved in high N input rice paddies by maintaining a higher soil organic carbon-to-nitrate ratio. Our findings highlight the unique microbial N-cycling strategies in resource-limited paddies which support maintained N nutrition of the paddy system.

Dissimilatory nitrate reduction to ammonium dominates soil nitrate retention capacity in subtropical forests

The abundance, community composition and activity of key nitrogen (N)-cycling functional guilds were monitored at a site where coniferous Cunninghamia lanceolate and broadleaved Mytilaria laosensis were planted in 1993. Leaf cellulose, litter C/N ratio, leaf dry matter content, soil inorganic N content were significantly higher under C. lanceolata, while specific leaf area, litter production, litter magnesium, soil C and soil C/NO3− ratio were higher under M. laosensis. The 15N tracing experiment together with quantitative PCR revealed that autotrophic nitrification rates, as well as the abundances of ammonia-oxidizing archaea and comammox Nitrospira were significantly higher under C. lanceolate than M. laosensis. However, M. laosensis exhibited substantially higher nitrate retention capacity via dissimilatory nitrate reduction to ammonium (DNRA), accompanied by a significantly higher abundance of nrfA gene than C. lanceolate. The Illumina sequencing indicated that tree species markedly affected soil bacterial community composition regardless of the soil layers. Redundancy analysis suggested that litter C/N ratio was the most influential factor explaining functional gene abundances and bacterial communities. Taken together, our findings showed that M. laosensis improved soil N retention capacity mainly through inhibiting autotrophic nitrification while enhancing DNRA activity. This study highlights the importance of tree species identity in influencing the microbially-mediated N cycling and bacterial community composition.

Soil substrates rather than gene abundance dominate DNRA capacity in the Spartina alterniflora ecotones of estuarine and intertidal wetlands

Dissimilatory nitrate reduction to ammonium (DNRA) plays an important role in keeping nitrate retention as a more bioavailable form (ammonium) in estuarine and intertidal environments. However, the effects of soil abiotic and biotic characteristics on DNRA in Spartina alterniflora ecotones of estuarine and intertidal wetlands remain unclear. In this study, we used nitrogen isotope tracing and molecular approaches to investigate DNRA activity, and abiotic and biotic factors of both rhizosphere and non-rhizosphere soils in Spartina alterniflora ecotones of the Yangtze estuarine and intertidal wetlands. DNRA varied significantly throughout the sampling sites, with potential rates of 0.53–3.57 nmol N g−1 h−1. The rates of DNRA were significantly higher in rhizosphere than non-rhizosphere soils at the oligohaline sites. Salinity had more influence on DNRA activity than Spartina alterniflora at the brackish sites. Total organic carbon, nitrate, Fe (II) and sulfide were significantly correlated with DNRA rates and nrfA gene abundance. Soil substrates strongly affected DNRA activity in rhizosphere soil, while nrfA gene abundance was the predominant factor mediating DNRA activity in non-rhizosphere soil. DNRA contributed more to the total nitrate reduction at the brackish than oligohaline sites, suggesting that DNRA plays an important role in nitrate reduction in estuarine and intertidal wetlands. This study suggests that soil substrates rather than nrfA gene dominate DNRA activity in estuarine and intertidal wetlands after Spartina alterniflora invasion. Our results are helpful to understand the importance of soil characteristics changes induced by the exotic plant invasion to nitrogen cycling in estuarine and intertidal wetlands.

Nitrogen Addition Decreases Dissimilatory Nitrate Reduction to Ammonium in Rice Paddies.

Dissimilatory nitrate reduction to ammonium (DNRA), denitrification, anaerobic ammonium oxidation (anammox), and biological N2 fixation (BNF) can influence the nitrogen (N) use efficiency of rice production. While the effect of N application on BNF is known, little is known about its effect on NO3- partitioning between DNRA, denitrification, and anammox. Here, we investigated the effect of N application on DNRA, denitrification, anammox, and BNF and on the abundance of relevant genes in three paddy soils in Australia. Rice was grown in a glasshouse with N fertilizer (150 kg N ha-1) and without N fertilizer for 75 days, and the rhizosphere and bulk soils were collected separately for laboratory incubation and quantitative PCR analysis. Nitrogen application reduced DNRA rates by >16% in all the soils regardless of the rhizospheric zone, but it did not affect the nrfA gene abundance. Without N, the amount and proportion of NO3- reduced by DNRA (0.42 to 0.52 μg g-1 soil day-1 and 45 to 55%, respectively) were similar to or higher than the amount and proportion reduced by denitrification. However, with N the amount of NO3- reduced by DNRA (0.32 to 0.40 μg g-1 soil day-1) was 40 to 50% lower than the amount of NO3- reduced by denitrification. Denitrification loss increased by >20% with N addition and was affected by the rhizospheric zones. Nitrogen loss was minimal through anammox, while BNF added 0.02 to 0.25 μg N g-1 soil day-1 We found that DNRA plays a significant positive role in paddy soil N retention, as it accounts for up to 55% of the total NO3- reduction, but this is reduced by N application.IMPORTANCE This study provides evidence that nitrogen addition reduces nitrogen retention through DNRA and increases nitrogen loss via denitrification in a paddy soil ecosystem. DNRA is one of the major NO3- reduction processes, and it can outcompete denitrification in NO3- consumption when rice paddies are low in nitrogen. A significant level of DNRA activity in paddy soils indicates that DNRA plays an important role in retaining nitrogen by reducing NO3- availability for denitrification and leaching. Our study shows that by reducing N addition to rice paddies, there is a positive effect from reduced nitrogen loss but, more importantly, from the conversion of NO3- to NH4+, which is the favored form of mineral nitrogen for plant uptake.

Distinct Microbial Community Performing Dissimilatory Nitrate Reduction to Ammonium (DNRA) in a High C/NO3- Reactor.

A dissimilatory nitrate reduction to ammonium (DNRA) microbial community was developed under a high organic carbon to nitrate (C/NO3−) ratio in an anoxic semi-continuous sequencing batch reactor (SBR) fed with glucose as the source of carbon and NO3− as the electron acceptor. Activated sludge collected from a municipal wastewater treatment plant with good denitrification efficiency was used as the inoculum to start the system. The aim of this study was to examine the microbial populations in a high C/NO3− ecosystem for potential DNRA microorganisms, which are the microbial group with the ability to reduce NO3− to ammonium (NH4+). A low C/NO3− reactor was operated in parallel for direct comparisons of the microbial communities that developed under different C/NO3− values. The occurrence of DNRA in the high C/NO3− SBR was evidenced by stable isotope-labeled nitrate and nitrite (15NO3− and 15NO2−), which proved the formation of NH4+ from dissimilatory NO3−/NO2− reduction, in which both nitrogen oxides induced DNRA activity in a similar manner. An analysis of sludge samples with Illumina MiSeq 16S rRNA sequencing showed that the predominant microorganisms in the high C/NO3− SBR were related to Sulfurospirillum and the family Lachnospiraceae, which were barely present in the low C/NO3− system. A comparison of the populations and activities of the two reactors indicated that these major taxa play important roles as DNRA microorganisms under the high C/NO3− condition. Additionally, a beta-diversity analysis revealed distinct microbial compositions between the low and high C/NO3− SBRs, which reflected the activities observed in the two systems.

Nitrate reduction mechanisms and rates in an unconfined eogenetic karst aquifer in two sites with different redox potential

AbstractThis study integrates push‐pull tracer tests (PPTT) with microbial characterization of extracted water via quantitative polymerase chain reaction (qPCR) and reverse transcriptase qPCR (RT‐qPCR) of selected functional N transformation genes to quantify nitrate reduction mechanisms and rates in sites with different redox potential in a karst aquifer. PPTT treatments with nitrate (AN) and nitrate‐fumarate (ANC) were executed in two wells representing anoxic and oxic geochemical end‐members. Oxic aquifer zero‐order nitrate loss rates (mmol L−1 h−1) were similar for AN and ANC treatment, ranging from 0.03 ± 0.01 to 0.05 ± 0.01. Anoxic aquifer zero‐order nitrate loss rates ranged from 0.03 ± 0.02 (AN) to 0.13 ± 0.02 (ANC). Microbial characterization indicates mechanisms influencing these rates were dissimilatory nitrate reduction to ammonium (DNRA) at the anoxic site with AN treatment, assimilatory reduction of nitrate to ammonium (ANRA) with ANC treatment in the water column at both sites, and additional documented nitrate reduction that occurred in unsampled biofilms. With carbon treatment, total numbers of microbes (16S rRNA genes) significantly increased (fourteenfold to thirtyfold), supporting stimulated growth with resulting ANRA. Decreased DNRA gene concentrations (nrfA DNA) and increased DNRA activity ratio (nrfA‐cDNA/DNA) supported the assertion that DNRA occurred in the anoxic zone with AN and ANC treatment. Furthermore, decreased DNRA gene copy numbers at the anoxic site with ANC treatment suggests that DNRA microbes in the anoxic site are chemolithoautotrophic. Increased RT‐qPCR denitrification gene expression (nirK and nirS) was not observed in water samples, supporting that any observed NO3‐N loss due to denitrification may be occurring in unsampled microbial biofilms.

DNRA and Denitrification Coexist over a Broad Range of Acetate/N-NO3- Ratios, in a Chemostat Enrichment Culture.

Denitrification and dissimilatory nitrate reduction to ammonium (DNRA) compete for nitrate in natural and engineered environments. A known important factor in this microbial competition is the ratio of available electron donor and elector acceptor, here expressed as Ac/N ratio (acetate/nitrate-nitrogen). We studied the impact of the Ac/N ratio on the nitrate reduction pathways in chemostat enrichment cultures, grown on acetate mineral medium. Stepwise, conditions were changed from nitrate limitation to nitrate excess in the system by applying a variable Ac/N ratio in the feed. We observed a clear correlation between Ac/N ratio and DNRA activity and the DNRA population in our reactor. The DNRA bacteria dominated under nitrate limiting conditions in the reactor and were outcompeted by denitrifiers under limitation of acetate. Interestingly, in a broad range of Ac/N ratios a dual limitation of acetate and nitrate occurred with co-occurrence of DNRA bacteria and denitrifiers. To explain these observations, the system was described using a kinetic model. The model illustrates that the Ac/N effect and concomitant broad dual limitation range related to the difference in stoichiometry between both processes, as well as the differences in electron donor and acceptor affinities. Population analysis showed that the presumed DRNA-performing bacteria were the same under nitrate limitation and under dual limiting conditions, whereas the presumed denitrifying population changed under single and dual limitation conditions.

Influence of organic carbon and nitrate loading on partitioning between dissimilatory nitrate reduction to ammonium (DNRA) and N2 production

Biologically available nitrogen is removed from ecosystems through the microbial processes of anaerobic ammonium oxidation (anammox) or denitrification, while dissimilatory nitrate reduction to ammonium (DNRA) retains it. A mechanistic understanding of controls on partitioning among these pathways is currently lacking. The objective of this study was to conduct a manipulative experiment to determine the influence of organic C and NO3− loading on partitioning. Sediment was collected from a location on the southern New England shelf (78m water depth) and sieved. Half of the sediment was mixed with freeze-dried phytoplankton and the other half was not. Sediment was then spread into 1.5mm, “thin discs” closed at the bottom and placed in large aquarium tanks with filtered, N2/CO2 sparged seawater to maintain O2 limited conditions. Half of the discs received high NO3− loading, while the other half received low NO3− loading, resulting in a multifactorial design with four treatments: no C addition, low NO3− (−C−N); C addition, low NO3− (+C−N); no C addition, high NO3− (−C+N); and C addition, high NO3− (+C+N). Sediment discs were incubated in the tanks for 7weeks, during which time inorganic N (NH4+, NO3−, and NO2−) was monitored, and sediment discs were periodically removed from the tanks to conduct 15N isotope labeling experiments in vials to measure potential rates of anammox, denitrification, and DNRA. Temporal dynamics of inorganic N concentrations in the tanks were indicative of anoxic N metabolism, with strong response of the build up or consumption of the intermediate NO2−, depending on treatments. Vial incubation experiments with added 15NO2−+14NH4+ indicated significant denitrification and DNRA activity in sediment thin discs, but incubations with added 15NH4++14NO2− indicated anammox was not at all significant. Inorganic N concentrations in the tanks were fit to a reactive transport model assuming different N transformations. Organic C decomposition rates were inferred based on modeled rates as well as stoichiometric conversions of NH4+ production in pre-incubated vials. Based on model results, partitioning between DNRA and N2 production was positively linearly related to the ratio of C decomposition to NO3− reduction rates (C/NO3−) but not C decomposition alone. Based on vial results, partitioning was significantly related to C decomposition. Overall, this study supports the hypothesis that high organic C loading is a prerequisite for DNRA to be favored over denitrification but that N2 production may still be significant when organic C is high depending on NO3− availability.

Linking DNRA community structure and activity in a shallow lagoonal estuarine system.

Dissimilatory nitrate reduction to ammonium (DNRA) and denitrification are two nitrate respiration pathways in the microbial nitrogen cycle. Diversity and abundance of denitrifying bacteria have been extensively examined in various ecosystems. However, studies on DNRA bacterial diversity are limited, and the linkage between the structure and activity of DNRA communities has yet to be discovered. We examined the composition, diversity, abundance, and activities of DNRA communities at five sites along a salinity gradient in the New River Estuary, North Carolina, USA, a shallow temporal/lagoonal estuarine system. Sediment slurry incubation experiments with 15N-nitrate were conducted to measure potential DNRA rates, while the abundance of DNRA communities was calculated using quantitative PCR of nrfA genes encoding cytochrome C nitrite reductase, commonly found in DNRA bacteria. A pyrosequencing method targeting nrfA genes was developed using an Ion Torrent sequencer to examine the diversity and composition of DNRA communities within the estuarine sediment community. We found higher levels of nrfA gene abundance and DNRA activities in sediments with higher percent organic content. Pyrosequencing analysis of nrfA genes revealed spatial variation of DNRA communities along the salinity gradient of the New River Estuary. Percent abundance of dominant populations was found to have significant influence on overall activities of DNRA communities. Abundance of dominant DNRA bacteria and organic carbon availability are important regulators of DNRA activities in the eutrophic New River Estuary.

Response of the ubiquitous pelagic diatom Thalassiosira weissflogii to darkness and anoxia.

Thalassiosira weissflogii, an abundant, nitrate-storing, bloom-forming diatom in the world’s oceans, can use its intracellular nitrate pool for dissimilatory nitrate reduction to ammonium (DNRA) after sudden shifts to darkness and anoxia, most likely as a survival mechanism. T. weissflogii cells that stored 4 mM 15N-nitrate consumed 1.15 (±0.25) fmol NO3 - cell-1 h-1 and simultaneously produced 1.57 (±0.21) fmol 15NH4 + cell-1 h-1 during the first 2 hours of dark/anoxic conditions. Ammonium produced from intracellular nitrate was excreted by the cells, indicating a dissimilatory rather than assimilatory pathway. Nitrite and the greenhouse gas nitrous oxide were produced at rates 2-3 orders of magnitude lower than the ammonium production rate. While DNRA activity was restricted to the first few hours of darkness and anoxia, the subsequent degradation of photopigments took weeks to months, supporting the earlier finding that diatoms resume photosynthesis even after extended exposure to darkness and anoxia. Considering the high global abundance of T. weissflogii, its production of ammonium and nitrous oxide might be of ecological importance for oceanic oxygen minimum zones and the atmosphere, respectively.

Vertical activity distribution of dissimilatory nitrate reduction in coastal marine sediments

Abstract. The relative importance of two dissimilatory nitrate reduction pathways, denitrification (DEN) and dissimilatory nitrate reduction to ammonium (DNRA), was investigated in intact sediment cores from five different coastal marine field sites (Dorum, Aarhus Bight, Mississippi Delta, Limfjord and Janssand). The vertical distribution of DEN activity was examined using the acetylene inhibition technique combined with N2O microsensor measurements, whereas NH4+ production via DNRA was measured with a recently developed gel probe-stable isotope technique. At all field sites, dissimilatory nitrate reduction was clearly dominated by DEN (59–131% of the total NO3− reduced) rather than by DNRA, irrespective of the sedimentary inventories of electron donors such as organic carbon, sulfide, and iron. Highest ammonium production via DNRA, accounting for up to 8.9% of the total NO3− reduced, was found at a site with very high concentrations of total sulfide and NH4+ within and below the layer in which NO3− reduction occurred. Sediment from two field sites, one with low and one with high DNRA activity in the core incubations, was also used for slurry incubations. Now, in both sediments high DNRA activity was detected accounting for 37–77% of the total NO3− reduced. These contradictory results might be explained by enhanced NO3− availability for DNRA bacteria in the sediment slurries compared to the core-incubated sediments in which diffusion of NO3− from the water column may only reach DEN bacteria, but not DNRA bacteria. The true partitioning of dissimilatory nitrate reduction between DNRA and DEN may thus lie in between the values found in whole core (underestimation of DNRA) and slurry incubations (overestimation of DNRA).

Combined Gel Probe and Isotope Labeling Technique for Measuring Dissimilatory Nitrate Reduction to Ammonium in Sediments at Millimeter-Level Resolution

Dissimilatory NO(3)(-) reduction in sediments is often measured in bulk incubations that destroy in situ gradients of controlling factors such as sulfide and oxygen. Additionally, the use of unnaturally high NO(3)(-) concentrations yields potential rather than actual activities of dissimilatory NO(3)(-) reduction. We developed a technique to determine the vertical distribution of the net rates of dissimilatory nitrate reduction to ammonium (DNRA) with minimal physical disturbance in intact sediment cores at millimeter-level resolution. This allows DNRA activity to be directly linked to the microenvironmental conditions in the layer of NO(3)(-) consumption. The water column of the sediment core is amended with (15)NO(3)(-) at the in situ (14)NO(3)(-) concentration. A gel probe is deployed in the sediment and is retrieved after complete diffusive equilibration between the gel and the sediment pore water. The gel is then sliced and the NH(4)(+) dissolved in the gel slices is chemically converted by hypobromite to N(2) in reaction vials. The isotopic composition of N(2) is determined by mass spectrometry. We used the combined gel probe and isotopic labeling technique with freshwater and marine sediment cores and with sterile quartz sand with artificial gradients of (15)NH(4)(+). The results were compared to the NH(4)(+) microsensor profiles measured in freshwater sediment and quartz sand and to the N(2)O microsensor profiles measured in acetylene-amended sediments to trace denitrification.