Metabolic engineering of a diazotrophic bacterium improves ammonium release and biofertilization of plants and microalgae

The biological nitrogen fixation carried out by some Bacteria and Archaea is one of the most attractive alternatives to synthetic nitrogen fertilizers. However, with the exception of the symbiotic rhizobia-legumes system, progress towards a more extensive realization of this goal has been slow. In this study we manipulated the endogenous regulation of both nitrogen fixation and assimilation in the aerobic bacterium Azotobacter vinelandii. Substituting an exogenously inducible promoter for the native promoter of glutamine synthetase produced conditional lethal mutant strains unable to grow diazotrophically in the absence of the inducer. This mutant phenotype could be reverted in a double mutant strain bearing a deletion in the nifL gene that resulted in constitutive expression of nif genes and increased production of ammonium. Under GS non-inducing conditions both the single and the double mutant strains consistently released very high levels of ammonium (>20 mM) into the growth medium. The double mutant strain grew and excreted high levels of ammonium under a wider range of concentrations of the inducer than the single mutant strain. Induced mutant cells could be loaded with glutamine synthetase at different levels, which resulted in different patterns of extracellular ammonium accumulation afterwards. Inoculation of the engineered bacteria into a microalgal culture in the absence of sources of C and N other than N₂ and CO₂ from the air, resulted in a strong proliferation of microalgae that was suppressed upon addition of the inducer. Both single and double mutant strains also promoted growth of cucumber plants in the absence of added N-fertilizer, while this property was only marginal in the parental strain. This study provides a simple synthetic genetic circuit that might inspire engineering of optimized inoculants that efficiently channel N₂ from the air into crops.     

Delivery of N2 to bacteroids in simulated soybean nodule cells: consideration of gradients of concentration of dissolved N2 in cell walls,cytoplasm, and symbiosomes

Summary The previously published simulation of physiological functions occurring in infected cells of soybean nodules has been extended to include consideration of the diffusion of N₂ from the outside of a nodule to the nitrogen-fixing bacteroids, in relation to published values for the apparentK _m(N₂) for nitrogen fixation in the soybean nodule system. Nitrogen fixation is driven by bacteroid respiration, so increases in the average relative oxygenation (Y) of cytoplasmic leghaemoglobin lead to increased bacteroid respiration, increased nitrogen fixation, and greater differences in concentration of dissolved N₂ between the cell surface and the innermost bacteroids (d[N₂]). Over the range ofY considered, values for d[N₂] were from 5.2- to 6.2-fold greater than the corresponding values for d[O₂], because of facilitation of O₂ flux by cytoplasmic leghaemoglobin. Gradients of [N₂] within symbiosomes are small relative to cytoplasmic values and at the symbiosome surface [N₂] was greater than 0.4 mol/m³ at the greatest rates of nitrogen fixation calculated. Therefore, it is unlikely that values for [N₂] anywhere in the infected cell are low enough to affect rates of nitrogen fixation significantly, unless low external atmospheric N₂ pressures are used experimentally.Abbreviations Lb leghaemoglobin - LbO₂ oxyleghaemoglobin - [O₂], [N₂ concentrations of free, dissolved oxygen and nitrogen - Y fractional oxygenation of leghaemoglobin     

Nitrogen fixation varies spatially and seasonally in linked stream-lake ecosystems

We performed surveys of nitrogen (N₂)-fixation in three oligotrophic lake-stream systems in the Sawtooth Mountains of central Idaho to address two questions: (1) Which habitat types within linked lake-stream systems (lake pelagic, lake benthic, and stream) exhibit the highest rates of N₂ fixation?, and (2) How does N₂ fixation compare to the hydrologic flux of nitrogen? A seasonal survey showed that N₂ fixation in a single lake and its outlet stream peaked in late summer, when hydrologic N fluxes were lowest. Benthic lake N₂-fixation rates by epiphytes were highest at mid-lake depths, where their percent cover was highest, while rates by epipelon were greatest at shallow lake depths. Pelagic N₂ fixation was below detection. Stream N₂-fixation rates were greatest on rock substrates and in the lake outlet stream. These patterns were supported by a baseflow survey (late July) in three lake-stream ecosystems which confirmed that N₂-fixation rates peaked in the lake benthos at shallow depths and on rock substrates in outlet streams. Scaling N₂-fixation rates to whole lake and stream areas revealed that N₂ fixation could exceed the nitrate, and sometimes the total dissolved nitrogen flux during baseflow in lakes and outlet streams. Despite low rates, total N₂-fixation contributions (kg/day) from lakes were greater because they had far larger surface areas than the stream environments. Fixed nitrogen contributions from stream outlets were also relatively high because of high N₂-fixation rates and despite low surface areas. This study suggests that N₂ fixation could be a seasonally important nitrogen source to nutrient deficient subalpine lake-stream ecosystems. In addition, the frequency and location of lakes could control N₂-fixation contributions to watersheds by providing a large area for within-lake N₂ fixation, and creating conditions favorable for N₂ fixation in outlet streams.     

Isolation,improvement and characterization of an ammonium excreting mutant strain of the heterocytous cyanobacterium,Anabaena variabilis PCC 7937

Besides potential applications in the agriculture field as natural nitrogen fertilizer, N₂-fixing cyanobacteria have recently gained some attentions for new applications linked to the potential production of biologically active molecules or biohydrogen. Ammonium bioproduction is also gaining attention with the potential use of microalgae in biofuels production and the concerns about the increasing needs for nitrogen substrates. This study has investigated some phenotypic traits linked to biomass production and ammonium release in multicellular cyanobacteria, Anabaena variabilis PCC 7937. It confirms that this wild-type strain has no natural ability for ammonium excretion under diazotrophic conditions. A mutant strain, A. variabilis PCC 7937-C9, was obtained after double random mutagenesis treatments with ethyl methane–sulfonate and screening in batch cultures for resistance to the effect of a glutamine synthetase inhibitor, l-methionine-d,l-sulfoximine (MSX). Although significantly characterized by shorter cell filaments, the growth parameters in photobioreactors of the mutant strain cultures were in the same range of values than those of the wild type. In the presence of MSX this strain was shown to produce extracellular ammonium, with specific rates up to 4.9 μmol NH₄⁺ mg Chl a⁻¹ h⁻¹. The efficiency of this strain, estimated by its specific rate of ammonium excretion, was shown to be improved after consecutive batch cultures with increasing concentrations of MSX. Such mutant strains are of potential use for investigating ways to improve extracellular ammonium bioproduction.     

Photosynthetic Assimilation of NO(3) by Intact Cells of the Cyanobacterium Anacystis nidulans: Influence of NO(3) and NH(4) Assimilation on CO(2) Fixation

Illuminated suspensions of Anacystis nidulans, supplied with saturating concentrations of CO₂ evolved O₂ at a greater rate when nitrate was simultaneously present. The extent of the stimulation of noncyclic electron flow induced by nitrate was dependent on light intensity, being maximal under light saturating conditions. Accordingly, nitrate depressed the rate of CO₂ fixation at limiting but not at saturating light, this depression reflecting the competition between both processes for assimilatory power. In contrast, ammonium stimulated CO₂ fixation at any light intensity assayed, the stimulation being dependent on the incorporation of ammonium to carbon skeletons. The positive effect of ammonium on CO₂ fixation also appeared to occur when nitrate was the nitrogen source, since with either nitrogen source an increase in the incorporation of newly fixed carbon into acid-soluble metabolites took place. From these results, the in vivo partitioning of assimilatory power between photosynthetic nitrogen and carbon assimilation and the quantitative and qualitative effects of inorganic nitrogen assimilation on CO₂ fixation are discussed.     

The effect of nutrients on carbon and nitrogen fixation by the UCYN-A–haptophyte symbiosis

Symbiotic relationships between phytoplankton and N₂-fixing microorganisms play a crucial role in marine ecosystems. The abundant and widespread unicellular cyanobacteria group A (UCYN-A) has recently been found to live symbiotically with a haptophyte. Here, we investigated the effect of nitrogen (N), phosphorus (P), iron (Fe) and Saharan dust additions on nitrogen (N₂) fixation and primary production by the UCYN-A–haptophyte association in the subtropical eastern North Atlantic Ocean using nifH expression analysis and stable isotope incubations combined with single-cell measurements. N₂ fixation by UCYN-A was stimulated by the addition of Fe and Saharan dust, although this was not reflected in the nifH expression. CO₂ fixation by the haptophyte was stimulated by the addition of ammonium nitrate as well as Fe and Saharan dust. Intriguingly, the single-cell analysis using nanometer scale secondary ion mass spectrometry indicates that the increased CO₂ fixation by the haptophyte in treatments without added fixed N is likely an indirect result of the positive effect of Fe and/or P on UCYN-A N₂ fixation and the transfer of N₂-derived N to the haptophyte. Our results reveal a direct linkage between the marine carbon and nitrogen cycles that is fuelled by the atmospheric deposition of dust. The comparison of single-cell rates suggests a tight coupling of nitrogen and carbon transfer that stays balanced even under changing nutrient regimes. However, it appears that the transfer of carbon from the haptophyte to UCYN-A requires a transfer of nitrogen from UCYN-A. This tight coupling indicates an obligate symbiosis of this globally important diazotrophic association.     

Estuarine intertidal sandflat benthic microalgal responses to in situ and mesocosm nitrogen additions

Benthic microalgal communities are important components of estuarine food webs and make substantial contributions to coastal materials cycling. Nitrogen is generally the limiting factor for marine primary production; however other factors can limit benthic primary producers because of their access to the additional nutrients found in sediment porewater. Field and laboratory experiments were conducted to test the hypothesis that water column nitrogen supply affects estuarine sandflat benthic microalgal community structure and function. Our field and mesocosm experiments assessed changes at both the population and functional group levels. Simulated water column nitrogen additions increased maximum community photosynthesis in most cases (Pb_max from photosynthesis vs. irradiance curves). Additional changes that resulted from nitrogen additions were decreases in porewater phosphate, increases in porewater ammonium, shifts in community composition from N₂ fixing cyanobacteria toward diatoms, and detectable, though not statistically significant increases in biomass (as chlorophyll a). Results from field and laboratory experiments were quite similar, suggesting that laboratory experiments support accurate predictions of the response of intertidal benthic microalgae to changes in water column nutrient conditions.     

Comparison of Nitrogen Fixation Activity in Tall and Short Spartina alterniflora Salt Marsh Soils

A comparison of the N₂ fixers in the tall Spartina alterniflora and short S. alterniflora marsh soils was investigated. Zero-order kinetics and first-order kinetics of acetylene reduction were used to describe the activity of the N₂ fixers in marsh soil slurries. It was found that the V_max values were approximately 10 times greater for the N₂ fixers in the tall Spartina than in the short Spartina marsh when raffinose was used as the energy source. In addition, the (K_s + S_n) values were approximately 4 to 15 times lower for the N₂ fixers in the tall Spartina than in short Spartina marsh. First-order kinetics of nitrogen fixation for several substrates indicate that the N₂ fixers in the tall Spartina marsh were two to seven times more active than those in the short Spartina marsh. Ammonium chloride (25 μg/ml) did not inhibit nitrogen fixation in the tall Spartina marsh, but there was a 50% inhibition in nitrogen fixation in the short Spartina marsh. On the other hand, sodium nitrate inhibited nitrogen fixation almost 100% at 25 μg/ml in both soil environments. Amino nitrogen (25 to 100 μg/ml) had little or no effect on nitrogen fixation. The results indicate that the N₂ fixers in the tall Spartina marsh were physiologically more responsive to nutrient addition than those in the short Spartina marsh. This difference in the two populations may be related to the difference in daily tidal influence in the respective areas and thus provide another explanation for the enhanced S. alterniflora production in the creek bank soil system.     

Relationship between growth,nitrogen fixation and assimilation in a legume (Medicago sativa L.)

Summary Symbiotic N₂ fixation, NO ₃ ⁻ assimilation and protein accumulation in the shoots were measured simultaneously in alfalfa (Medicago sativa L.) grown in the field or in pots, in order to study how the balance between the two modes of nitrogen nutrition could be influenced by agronomic factors, such as harvest, mineral nitrogen supply and drought stress. During periods of rapid growth, fixation and assimilation may function simultaneously; they are antagonistic at the beginning and at the end of the growth cycle, when the nitrogen requirement of the plant is lower. When nitrogen nutrition does not limit growth, mineral nitrogen supply favours assimilation at the expense of fixation, but does not modify the amount of nitrogen accumulated, which is adjusted to the growth capacity of the plant. After cutting, nitrate assimilation compensated for the decrease in fixation and supplied the plant with the nitrogen required by the regrowth, the proliferation of which determined the fixation recovery. Drought stress decreased N₂ fixation much more than NO ₃ ⁻ assimilation. The latter made growth recovery possible when water supply conditions became normal again. These results suggested the existence of an optimum level of nitrate assimilation, which differed depending on the age of the plants and allowed both maximum growth and fixing activity.     

Functional plasticity of Trifolium repens L. in response to sulphur and nitrogen availability

Recent control of atmospheric SO₂ pollution is leading to important soil sulphur impoverishment. Plasticity could be a mechanism allowing species to adapt to this rapid global change. Trifolium repens L. is a key grassland species whose performances in community are strongly linked to nitrogen availability. Plasticity of three white clover lines contrasting in their ability to use atmospheric N₂ or soil N was assessed by analysing a set of functional traits along a gradient of nitrogen and sulphur fertilisation applied on a poor soil. White clover traits showed high morphological and physiological plasticity. Nitrogen appeared to be the most limiting factor for the VLF (Very Low Fixation) line. S was the element that modulated the most traits for the nitrogen fixing lines NNU (Normal Nitrate Uptake) and LNU (Low Nitrate Uptake). As expected, N fertilisation inhibited white clover fixation, but we also observed that N₂ fixation was enhanced when S was added. S fertilisation increased nodule length as well as the proportion of nodules containing leghaemoglobin. S fertilisation, with a direct effect and an indirect effect through N₂ fixation, increases white clover performances particularly with regards to photosynthesis and potential vegetative reproduction. The important plasticity in response to S availability should allow it to adapt to a large range of abiotic conditions, but its sensitivity to S nutrition would be a disadvantage for competition in a situation of soil sulphur impoverishment. In contrast, S fertilisation could help maintain this species when nitrogen status is against it.     

Nitrogen fixaton in Klebsiella pneumoniae during osmotic stress effect of exogenous proline or a proline overproducing plasmid

Osmotic stress, imposed by 0.5 M NaCl or other electrolytes and non-electrolytes, caused over a 100-fold reduction in the whole-cell nitrogen fixation activity in Klebsiella pneumoniae, wild-type strain M₅A₁. This reduction of nitrogen fixation activity could be reversed by the addition of proline to the culture medium at 0.5 mM concentration. With 0.5 M NaCl, in the presence of proline, nitrogenase activity was 47-fold greater than in the absence of proline. A mutation, originally isolated in Salmonella typhimurium, which resulted in proline over-production and enhanced osmotolerance, was transferred into K. pneumoniae by F′ conjugation. Intracellular proline, synthesized at high levels because of the mutation, had similar stimulatory effects on nitrogen fixation under osmotic stress as proline provided exogenously. In the overproducing strain, the cellular level of proline is elevated as much as 125-fold during stress over that seen in the control strain. To determine the mechanism of stimulation of nitrogen fixaton by proline during stress, the biosynthesis of nitrogenase polypeptides was studied. Net nitrogenase biosynthesis and the biosynthesis of other unidentified peptides, is strongly inhibited during osmotic stress; proline reverses the inhibition. The role of proline in enhancing nitrogen fixation during osmotic stress is discussed.     

Delayed inoculation and starter nitrogen for enhancing early growth and nitrogen status ofLespedeza cuneata

Summary Two growth chamber experiments were conducted to determine the response ofLespedeza cuneata (Dumont) G. Don. (sericea lespedeza) to delayed inoculation and low levels of nitrogen fertilization. Nitrogen was supplied either as NH ₄ ⁺ or as NO ₃ ^– in solution. At 0.5 and 5.0 ppm nitrogen early growth and N₂(C₂H₂) fixation was inhibited by NH ₄ ⁺ and promoted by NO ₃ ^– . Inoculation at seeding did not negatively affect growth prior to the onset of N₂(C₂H₂) fixation. Delayed inoculation until the trifoliate stage thus did not increase growth or N₂ fixation during the first 40 days of growth. After 40 days, specific nitrogenase activity was highest for plants inoculated at the first trifoliate stage of growth. In contrast, growth and total shoot nitrogen accumulation were higher in plants inoculated at planting. The experimental results suggest that delaying inoculation is not a useful technique for improving early growth ofL. cuneata for surface mine reclamation.     

Methods for Growing Spirillum lipoferum and for Counting It in Pure Culture and in Association with Plants

Methods are described for growing Spirillum lipoferum in quantities sufficient to serve as inoculant in field trials of its associative N₂-fixing ability with higher plants and as a source of cells for the preparation of nitrogenase, cytochromes, respiratory enzymes, etc. A heavy inoculum of S. lipoferum grown on NH₄⁺ was transferred to a medium of minimal nitrogen content, and initial rapid growth at the expense of residual combined nitrogen was replaced later by slower growth on N₂. Conversion to N₂ fixation was prompt upon exhaustion of fixed nitrogen; growth on N₂ was most rapid at a pO₂ of 0.005 to 0.007 atm. Numbers of S. lipoferum can be estimated by diluting soil, crushed roots, or other material, and inoculating diluted samples into a stagnant semisolid medium. Development of a characteristic subsurface layer of organisms and demonstration the these organisms can reduce C₂H₂ are presumptive evidence that they are S. lipoferum. With most-probable-number tables the observations can be converted to numbers of S. lipoferum in the samples. The most-probable-number method indicated that numbers of S. lipoferum may increase 100-fold or more in roots of maize removed from the plant and incubated for 24 h at 30°C at a pO₂ initially adjusted to 0.01 atm.     

In silico structural homology modeling of nif A protein of rhizobial strains in selective legume plants

Symbiosis is a complex genetic regulatory biological evolution which is highly specific pertaining to plant species and microbial strains. Biological nitrogen fixation in legumes is a functional combination of nodulation by nod genes and regulation by nif, fix genes. Three rhizobial strains (Rhizobium leguminosarum, Bradyrhizobium japonicum, and Mesorhizobium ciceri) that we considered for in silico analysis of nif A are proved to be the best isolates with respect to N₂ fixing for ground nut, chick pea and soya bean (in vitro) out of 47 forest soil samples. An attempt has been made to understand the structural characteristics and variations of nif genes that may reveal the factors influencing the nitrogen fixation. The primary, secondary and tertiary structure of nif A protein was analyzed by using multiple bioinformatics tools such as chou-Fasman, GOR, ExPasy ProtParam tools, Prosa -web. Literature shows that the homology modeling of nif A protein have not been explored yet which insisted the immediate development for better understanding of nif A structure and its influence on biological nitrogen fixation. In the present predicted 3D structure, the nif A protein was analyzed by three different software tools (Phyre2, Swiss model, Modeller) and validated accordingly which can be considered as an acceptable model. However further in silico studies are suggested to determine the specific factors responsible for nitrogen fixing in the present three rhizobial strains.     

Stable Isotope Probing with 15N2 Reveals Novel Noncultivated Diazotrophs in Soil

Biological nitrogen fixation is a fundamental component of the nitrogen cycle and is the dominant natural process through which fixed nitrogen is made available to the biosphere. While the process of nitrogen fixation has been studied extensively with a limited set of cultivated isolates, examinations of nifH gene diversity in natural systems reveal the existence of a wide range of noncultivated diazotrophs. These noncultivated diazotrophs remain uncharacterized, as do their contributions to nitrogen fixation in natural systems. We have employed a novel ¹⁵N₂-DNA stable isotope probing (⁵N₂-DNA-SIP) method to identify free-living diazotrophs in soil that are responsible for nitrogen fixation in situ. Analyses of 16S rRNA genes from ¹⁵N-labeled DNA provide evidence for nitrogen fixation by three microbial groups, one of which belongs to the Rhizobiales while the other two represent deeply divergent lineages of noncultivated bacteria within the Betaproteobacteria and Actinobacteria, respectively. Analysis of nifH genes from ¹⁵N-labeled DNA also revealed three microbial groups, one of which was associated with Alphaproteobacteria while the others were associated with two noncultivated groups that are deeply divergent within nifH cluster I. These results reveal that noncultivated free-living diazotrophs can mediate nitrogen fixation in soils and that ¹⁵N₂-DNA-SIP can be used to gain access to DNA from these organisms. In addition, this research provides the first evidence for nitrogen fixation by Actinobacteria outside of the order Actinomycetales.     

Control of nitrogen fixation and ammonia excretion in Azorhizobium caulinodans

Due to the costly energy demands of nitrogen (N) fixation, diazotrophic bacteria have evolved complex regulatory networks that permit expression of the catalyst nitrogenase only under conditions of N starvation, whereas the same condition stimulates upregulation of high-affinity ammonia (NH₃) assimilation by glutamine synthetase (GS), preventing excess release of excess NH₃ for plants. Diazotrophic bacteria can be engineered to excrete NH₃ by interference with GS, however control is required to minimise growth penalties and prevent unintended provision of NH₃ to non-target plants. Here, we tested two strategies to control GS regulation and NH₃ excretion in our model cereal symbiont Azorhizobium caulinodans AcLP, a derivative of ORS571. We first attempted to recapitulate previous work where mutation of both P_II homologues glnB and glnK stimulated GS shutdown but found that one of these genes was essential for growth. Secondly, we expressed unidirectional adenylyl transferases (uATs) in a ΔglnE mutant of AcLP which permitted strong GS shutdown and excretion of NH₃ derived from N₂ fixation and completely alleviated negative feedback regulation on nitrogenase expression. We placed a uAT allele under control of the NifA-dependent promoter PnifH, permitting GS shutdown and NH₃ excretion specifically under microaerobic conditions, the same cue that initiates N₂ fixation, then deleted nifA and transferred a rhizopine nifA_L94Q/D95Q-rpoN controller plasmid into this strain, permitting coupled rhizopine-dependent activation of N₂ fixation and NH₃ excretion. This highly sophisticated and multi-layered control circuitry brings us a step closer to the development of a "synthetic symbioses” where N₂ fixation and NH₃ excretion could be specifically activated in diazotrophic bacteria colonising transgenic rhizopine producing cereals, targeting delivery of fixed N to the crop while preventing interaction with non-target plants.     

Genetic specificity of nitrogen nutrition in leguminous plants

Summary Mineral nitrogen did not increase grain yield and seed protein levels ofVicia faba L. andLupinus luteus L. in field trials and pot experiments. Fixed N₂ was substituted by mineral nitrogen in these cases because of inhibition of N₂ fixation by mineral nitrogen. Contrary to these results mineral nitrogen increased grain yields and seed protein amounts ofLupinus albus L.,Pisum sativum L., andGlycine max. (L.) Merr. The nitrogen effect was caused at an early stage by saving energy due to inhibition of N₂ fixation (measurement of gas exchange by means of IRGA). In case of the N application after flowering grain, yields and seed protein levels increased because the mineral N was an additional nitrogen source for plants. At this stage the plants had ceased fixing atmospheric nitrogen. The high sink activity of growing fruits induced a lack of assimilates in nodules (determined by means of¹⁴CO₂ application). The N effect was therefore the consequence of the lower assimilate pool for supplying root nodules in these plants in comparison withVicia faba L. andLupinus luteus L. Hence it follows that response to mineral nitrogen can be a criterion for discovering more effective Rhizobium-host combinations.     

The relationship between nitrogen fixation and the production of HD from D2 by cell-free extracts of soya-bean nodule bacteroids

1. Cell-free extracts prepared from soya-bean nodule bacteroids produced HD from D₂ in the presence of dithionite, an ATP-generating system and nitrogen. 2. Crude extracts of bacteroids or of Azotobacter vinelandii showed some background D₂ exchange when any one of these was omitted. 3. Partial purification of bacteroid extracts diminished this background activity and gave increased D₂ exchange and nitrogen fixation. 4. Although increasing pN₂ stimulated both reactions, the apparent K_m (N₂) for nitrogen fixation was much higher than the apparent K_m (N₂) for D₂ exchange when partially purified bacteroid extracts were used. 5. Carbon monoxide was a competitive inhibitor of nitrogen fixation by partially purified bacteroid extracts, but D₂ exchange was inhibited in a non-competitive fashion. 6. These results are discussed in relation to the possible existence of enzyme-bound intermediates of nitrogen fixation.     

Net sediment N2 fluxes in a southern New England estuary: variations in space and time

Over the past three decades, Narragansett Bay has undergone various ecological changes, including significant decreases in water column chlorophyll a concentrations, benthic oxygen uptake, and benthic nutrient regeneration rates. To add to this portrait of change, we measured the net flux of N₂ across the sediment–water interface over an annual cycle using the N₂/Ar technique at seven sites in the bay for comparison with measurements made decades ago. Net denitrification rates ranged from about 10–90 μmol N₂–N m^?2 h^?1 over the year. Denitrification rates were not significantly different among sites and had no clear correlation with temperature. Net nitrogen fixation (?5 to ?650 μmol N₂–N m^?2 h^?1) was measured at three sites and only observed in summer (June–August). Neither denitrification nor nitrogen fixation exhibited a consistent relationship with sediment oxygen demand or with fluxes of nitrite, nitrate, ammonium, total dissolved inorganic nitrogen, or dissolved inorganic phosphate across all stations. In contrast to the mid-bay historical site where denitrification rates have declined, denitrification rates in the Providence River Estuary have not changed significantly over the past 30 years.