Dinitrogen Fixation Research Articles

Getting to the Route: The Evolution of Nitrogen-Fixing Nodules.

Root nodule symbiosis allows for plant acquisition of reactive nitrogen through fixation of atmospheric molecular dinitrogen by nitrogen-fixing bacteria. Nodulation is a complex trait, with diverse modes of bacterial infection and nodule morphologies across species, reflecting evolutionary adaptation. Understanding ancient forms of this trait may carry advantages for its current utilization, since basal states likely reflect the least complexity. In this review we focus on the evolution of nodule development, particularly on events that have led to increased complexity of this symbiosis in later adaptations. We hypothesize that the ancestral form of nodulation comprises of an evolutionary coupling of nutrient-dependent lateral root development with apoplastic intercellular bacterial growth, alongside the acquisition or evolution of an ancestral chitinaceous signaling molecule by the microbial symbiont. Uncovering the evolutionary adaptations underpinning the extant diversity of this trait allows for a better understanding of the simplest ancestral state.

Dynamics of diazotroph particle colonization in the Arctic Ocean

Global warming is causing sea ice retreat and intensifying algal blooms in the Arctic Ocean, in turn increasing nitrogen limitation in surface waters. Dinitrogen fixation by diazotrophic microorganisms, usually favored in low reactive nitrogen systems, may become an increasingly important source of nitrogen in the Arctic. Previous studies have shown that non-cyanobacterial diazotrophs are dominant in the Arctic Ocean. Lacking a photosynthetic apparatus, non-cyanobacterial diazotrophs may utilize organic particles as carbon- and energy-rich niches. However, cyanobacterial diazotrophs may also form particles by aggregation. To further understand diazotroph-particle associations, here we study the chemotactic behavior and colonization dynamics of diazotrophs on model organic particles using a modified chemotaxis assay. Artificial organic particles (agarose, alginate) were incubated with surface seawater communities from four contrasted stations in the Barents Sea, and their DNA was sequenced targeting nifH and 16S rRNA genes after 2, 36, and 72 h of incubation. Our results show that diazotroph groups have selective colonization behaviors, with Gammaproteobacteria members preferentially colonizing alginate particles derived from brown algae, a form of organic matter becoming more common in the Arctic as it warms up. We also observe niche partitioning among microbial groups, with diazotrophs colonizing nitrogen-poor, carbon-rich particles earlier than non-diazotrophic prokaryotes. As Arctic warming proceeds, increased algal blooms may expand the niches for particle-associated diazotrophs, whose dinitrogen fixation supports phytoplankton growth and primary productivity.

Silicon Supply Improves the Rhizodeposition and Transfer of Nitrogen from Trifolium incarnatum L. to Brassica napus L.

The association of legumes with other non-legume plants, such as Brassica napus L., has been reported as an agro-ecological alternative for reducing the nitrogen (N) inputs required for B. napus growth, thanks in particular to the transfer of N compounds from the legume to B. napus. Moreover, recent studies have evidenced that silicon (Si) supply can improve either N uptake by B. napus or the dinitrogen fixation capacity of T. incarnatum. However, the effect of Si supply on the N nutrition of both B. napus and T. incarnatum, especially when growing in association, has not been assessed so far. The aim of this study was to assess the effect of Si supply on the growth of B. napus and T. incarnatum cultivated in association by focusing particularly on N rhizodeposition by T. incarnatum and its transfer to B. napus. The experiment was performed for 10 weeks under a split-root system combined with an 15N labeling method. The results showed that the Si supply increased the amount of rhizo-deposited N by T. incarnatum by over 40% and enhanced its transfer to B. napus. The transferred N was allocated mainly to pods (17%), as their biomass increased under Si supply. For the first time, this study demonstrates that the association with legume plants together with the Si supply could be an effective approach to improve the agro-ecological balance of B. napus.

Ambient and Selective Electrochemical Dinitrogen Fixation to NH3 Catalyzed by Microporous Organic–Inorganic Hybrid Copper Phosphonate

Ambient and Selective Electrochemical Dinitrogen Fixation to NH₃ Catalyzed by Microporous Organic–Inorganic Hybrid Copper Phosphonate

Implicating the Role of Au-H Bonds in Photochemical N2 Fixation by Ruthenium-Doped Gold Clusters.

Dinitrogen fixation through the Nitrogen Reduction Reaction (NRR) under mild conditions without the use of sacrificial agents has its share of formidable hurdles. It has been shown recently that Ru-doped Au nanoclusters can reduce N2 molecules to NH3 only in the presence of UV-Vis light in aqueous medium. Herein, using theoretical techniques (Density Functional Theory), we shed light on the mechanistic avenues traversed to achieve this prodigious chemical feat. Our findings suggest that the bimetallic Au22Ru6 cluster successfully accomplishes the NRR process under ambient pressure and temperature conditions by the virtue of its bifunctional nature. Contrary to the existing views, we find that NRR propagates through an alternative associative pathway, where the Ru dopant assists in N2 adsorption while the Au-H bonds formed from Au-assisted water splitting are implicated in facilitating NRR.

Diazotroph-derived nitrogen release and transfer under varying light intensity: insights from co-culture studies

Biological dinitrogen (N2) fixation is a major source of new N to surface seawater, sustaining ocean productivity. However, the fate of diazotroph-derived nitrogen (DDN), specifically its release and transfer, and the factors controlling these processes, remain poorly understood. Here, we established stable co-cultures of the major diazotrophs, filamentous Trichodesmium erythraeum IMS101 and unicellular Crocosphaera watsonii WH8501, with the pico-cyanobacterium Synechococcus sp. WH8102, to explore the intrinsic differences in DDN release and transfer between diazotroph strains. We found that T. erythraeum released similar amounts of DDN as C. watsonii, but had a significantly higher DDN transfer efficiency for supporting Synechococcus cell growth. These results implied a higher bioavailability of fixed N released by T. erythraeum than by C. watsonii. Additionally, we showed that elevated light levels significantly enhanced T. erythraeum DDN release and transfer. Our results provide new insights into the fate of N fixed by different diazotrophs and the environmental factors that control the process.

Nitrogen stable isotope fractionation by biological nitrogen fixation reveals cellular nitrogenase is diffusion limited

Biological fixation of dinitrogen (N2), the primary natural source of new bioavailable nitrogen (N) on Earth, is catalyzed by the enzyme nitrogenase through a complex mechanism at its active site metal cofactor. How this reaction functions in cellular environments, including its rate-limiting step, and how enzyme structure affects functioning remain unclear. Here, we investigated cellular N2 fixation through its N isotope effect (15εfix), measured as the difference between the 15N/14N ratios of diazotroph net new fixed N and N2 substrate. The value of 15εfix underpins N cycle reconstructions and differs between diazotrophs using molybdenum-containing and molybdenum-free nitrogenases. By examining 15εfix for Azotobacter vinelandii strains with natural and mutated nitrogenases, we determined if 15εfix reflects enzyme-scale isotope effects and, thus, N2 use efficiency. Distinct and relatively stable 15εfix values for wild-type molybdenum- and vanadium-nitrogenase isoforms (2.5‰ and 5.8–6.6‰, respectively), despite changing cellular growth rate and electron availability, support 15εfix as a proxy for isoform type among extant nitrogenases. Structural mutation of active site N2 access altered molybdenum-nitrogenase 15εfix (3.0–6.8‰ for α-70VI mutant). Structure-function and isotopic modeling results indicated cellular N2 reduction is rate-limited by N2 diffusion inside nitrogenase due to highly efficient catalysis by the active site cofactor, exemplifying 15εfix as a tool to probe N2 fixation mechanisms. Diffusion-constrained reactions could reflect structural tradeoffs that protect the oxygen-sensitive cofactor from oxygen inactivation. This suggests that nitrogenase function is optimized for modern oxygenated environments and that pre-Great Oxidative Event nitrogenases were less diffusion-limited and potentially exhibited larger 15εfix values.

Unveiling the contribution of particle-associated non-cyanobacterial diazotrophs to N2 fixation in the upper mesopelagic North Pacific Gyre

Dinitrogen (N2) fixation supports marine life through the supply of reactive nitrogen. Recent studies suggest that particle-associated non-cyanobacterial diazotrophs (NCDs) could contribute significantly to N2 fixation contrary to the paradigm of diazotrophy as primarily driven by cyanobacterial genera. We examine the community composition of NCDs associated with suspended, slow, and fast-sinking particles in the North Pacific Subtropical Gyre. Suspended and slow-sinking particles showed a higher abundance of cyanobacterial diazotrophs than fast-sinking particles, while fast-sinking particles showed a higher diversity of NCDs including Marinobacter, Oceanobacter and Pseudomonas. Using single-cell mass spectrometry we find that Gammaproteobacteria N2 fixation rates were higher on suspended and slow-sinking particles (up to 67 ± 48.54 fmol N cell⁻¹ d⁻¹), while putative NCDs’ rates were highest on fast-sinking particles (121 ± 22.02 fmol N cell⁻¹ d⁻¹). These rates are comparable to previous diazotrophic cyanobacteria observations, suggesting that particle-associated NCDs may be important contributors to pelagic N2 fixation.

Global biogeography of N2-fixing microbes: nifH amplicon database and analytics workflow

Abstract. Marine dinitrogen (N2) fixation is a globally significant biogeochemical process carried out by a specialized group of prokaryotes (diazotrophs), yet our understanding of their ecology is constantly evolving. Although marine N2 fixation is often ascribed to cyanobacterial diazotrophs, indirect evidence suggests that non-cyanobacterial diazotrophs (NCDs) might also be important. One widely used approach for understanding diazotroph diversity and biogeography is polymerase chain reaction (PCR) amplification of a portion of the nifH gene, which encodes a structural component of the N2-fixing enzyme complex, nitrogenase. An array of bioinformatic tools exists to process nifH amplicon data; however, the lack of standardized practices has hindered cross-study comparisons. This has led to a missed opportunity to more thoroughly assess diazotroph diversity and biogeography, as well as their potential contributions to the marine N cycle. To address these knowledge gaps, a bioinformatic workflow was designed that standardizes the processing of nifH amplicon datasets originating from high-throughput sequencing (HTS). Multiple datasets are efficiently and consistently processed with a specialized DADA2 pipeline to identify amplicon sequence variants (ASVs). A series of customizable post-pipeline stages then detect and discard spurious nifH sequences and annotate the subsequent quality-filtered nifH ASVs using multiple reference databases and classification approaches. This newly developed workflow was used to reprocess nearly all publicly available nifH amplicon HTS datasets from marine studies and to generate a comprehensive nifH ASV database containing 9383 ASVs aggregated from 21 studies that represent the diazotrophic populations in the global ocean. For each sample, the database includes physical and chemical metadata obtained from the Simons Collaborative Marine Atlas Project (CMAP). Here we demonstrate the utility of this database for revealing global biogeographical patterns of prominent diazotroph groups and highlight the influence of sea surface temperature. The workflow and nifH ASV database provide a robust framework for studying marine N2 fixation and diazotrophic diversity captured by nifH amplicon HTS. Future datasets that target understudied ocean regions can be added easily, and users can tune parameters and studies included for their specific focus. The workflow and database are available, respectively, on GitHub (https://github.com/jdmagasin/nifH-ASV-workflow, last access: 21 January 2025; Morando et al., 2024c) and Figshare (https://doi.org/10.6084/m9.figshare.23795943.v2; Morando et al., 2024b).

Organic metabolite uptake by diazotrophs in the North Pacific Ocean.

Dinitrogen (N₂) fixation by diazotrophs supports ocean productivity. Diazotrophs include photoautotrophic cyanobacteria, non-cyanobacterial diazotrophs (NCDs), and the recently discovered N2-fixing haptophyte. While NCDs are ubiquitous in the ocean, their ecology and metabolism remain largely unknown. Unlike cyanobacterial diazotrophs and the haptophyte, NCDs are primarily heterotrophic and depend on dissolved organic matter (DOM) for carbon and energy. However, conventional DOM amendment incubations do not allow discerning how different diazotrophs use DOM molecules, limiting our knowledge on DOM-diazotroph interactions. To identify diazotrophs using DOM, we amended North Pacific microbial communities with 13C-labeled DOM from phytoplankton cultures that was molecularly characterized, revealing the dominance of nitrogen-rich compounds. After DOM additions, we observed a community shift from cyanobacterial diazotrophs like Crocosphaera and Trichodesmium to NCDs at stations where the N2-fixing haptophyte abundance was relatively low. Through DNA stable isotope probing and gene sequencing, we identified diverse diazotrophs capable of taking up DOM. Our findings highlight unexpected DOM uptake by the haptophyte's nitroplast, changes in community structure, and previously unrecognized osmotrophic behavior in NCDs, shaped by local biogeochemical conditions.

Trichodesmium metaproteomes reflect the differential influence of resource availability across ocean regions

The diazotroph Trichodesmium is an important contributor to marine dinitrogen fixation, supplying nitrogen to phytoplankton in typically nitrogen-limited ocean regions. Identifying how iron and phosphorus influence Trichodesmium activity and biogeography is an ongoing area of study, where predicting patterns of resource stress is complicated by the uncertain bioavailability of organically complexed iron and phosphorus. Here, a comparison of 26 metaproteomes from picked Trichodesmium colonies identified significantly different patterns between three ocean regions: the western tropical South Pacific, the western North Atlantic, and the North Pacific Subtropical Gyre. Trichodesmium KEGG submodule signals differed significantly across regions, and vector fitting showed that dissolved iron, dissolved inorganic phosphorus, and temperature significantly correlated with regional metaproteome patterns. Patterns of iron and phosphorus stress marker proteins previously validated in culture studies showed significant enrichment of a phosphorus stress signal in the western North Atlantic and an iron stress signal in the North Pacific. Populations in the western tropical South Pacific appeared to modulate their proteomes in response to both dissolved iron and dissolved inorganic phosphorus bioavailability, with significant enrichment of iron and phosphorus stress marker proteins, concomitant proteome restructuring, and significant decreases in the relative abundance of the dinitrogen fixation protein, NifH. These signals recapitulate established regional patterns of resource stress on phytoplankton communities released from nitrogen stress. Evaluating community stress patterns may therefore predict resource controls on diazotroph biogeography. These data highlight how Trichodesmium modulates its metabolism in the field and provide an opportunity to more accurately constrain controls on Trichodesmium biogeography and dinitrogen fixation.

Effect of environmental factors on recombinant activity of root nodule bacteria

The legume-rhizobia symbiosis is a unique natural phenomenon, which supplies the plant with the necessary mineral nitrogen via fixation of atmospheric dinitrogen. This interaction involves two partners: the legume plant and root nodule bacteria (rhizobia). In the wild, members of the Fabaceae family enter into symbiosis with a polymorphic group of rhizobia specific to them; the mechanism and reasons for the formation of heterogeneity of rhizobia are currently the subject of active research. In the present work, a Rhizobium leguminosarum strain strictly specific to Phaseolus vulgaris L. was used to show that within 30 days upon its introduction into soil, genetic rearrangements occurred in the cells, as was revealed by changes in the pattern of its genetic profile. It was also found that recombination activity of thecells was also affected by the root exudates produced during seed germination, which may indicate involvement of the plant in the formation of polymorphism of its microsymbionts. These findings suggest interpretation of this process not as a spontaneous event, but rather as the event controlled by the plant.

Cross-Coupling of Mo- and V-Nitrogenases Permits Protein-Mediated Protection from Oxygen Deactivation.

Nitrogenases catalyze dinitrogen (N2) fixation to ammonia (NH3). While these enzymes are highly sensitive to deactivation by molecular oxygen (O2) they can be produced by obligate aerobes for diazotrophy, necessitating a mechanism by which nitrogenase can be protected from deactivation. In the bacterium Azotobacter vinelandii, one mode of such protection involves an O2-responsive ferredoxin-type protein ("Shethna protein II", or "FeSII") which is thought to bind with Mo-dependent nitrogenase's two component proteins (NifH and NifDK) to form a catalytically stalled yet O2-tolerant tripartite protein complex. This protection mechanism has been reported for Mo-nitrogenase, however, in vitro assays with V-nitrogenase suggest that this mechanism is not universal to the three known nitrogenase isoforms. Here we report that the reductase of the V-nitrogenase (VnfH) can engage in this FeSII-mediated protection mechanism when cross-coupled with Mo-nitrogenase NifDK. Interestingly, the cross-coupling of the Mo-nitrogenase reductase NifH with the V-nitrogenase VnfDGK protein does not yield such protection.

Heterogeneity of nitrogen fixation in the mesopelagic zone of the South China Sea

Biological dinitrogen (N2) fixation, the energetically expensive conversion of N2 gas to ammonia, plays an essential role in balancing the nitrogen budget in the ocean. Accumulating studies show detectable N2 fixation rates below the euphotic zone in various marine systems, revealing new insights of marine N2 fixation. However, the reported rates are highly variable and frequently fall close to detection limits, raising the question of the ubiquity and significance of N2 fixation in the global dark ocean. Using highly sensitive isotopic labeling incubation including a set of control incubations, we confirm the occurrence of mesopelagic N2 fixation in the South China Sea. Interestingly, we consistently observed that ca. 30% of samples show a significant elevation of 15N in the particulate nitrogen after incubation at most depths (200 - 1000 m). Although this approach does not allow accurate quantification of N2 fixation rates, our data suggest the occurrence of dark N2 fixation yet with highly heterogeneous signals in the mesopelagic zone of the South China Sea. A data compilation of reported N2 fixation in the global dark ocean further reveals that such heterogeneity has also been observed elsewhere, unveiling the ubiquitous heterogeneity in mesopelagic N2 fixation. Thus, we call for more observations to constrain mesopelagic N2 fixation budgets and to understand the underlying mechanism for such heterogeneity.

Ionomic and proteomic changes highlight the effect of silicon supply on the nodules functioning of Trifolium incarnatum L.

Numerous studies have reported the beneficial effects of silicon (Si) in alleviating biotic or abiotic stresses in many plant species. However, the role of Si in Fabaceae facing environmental stress is poorly documented. The aim of this study is to investigate the effect of Si on physiological traits and nodulation efficiency in Trifolium incarnatum L. Si was supplied (1.7 mM in the form of Na2SiO3) plants inoculated with Rhizobium leguminosarum bv trifolii and plant physiological traits and nodule ionomic and molecular traits were monitored over 25 days. Si supply promoted shoot biomass, the quantity of both Si and N in roots and shoots, and the number, biomass and density of nodules and their nitrogenase abundance which contribute to better dinitrogen (N2) fixation. Ionomic analysis of nodules revealed that Si supply increased the amount of several macroelements (potassium, phosphorus and sulfur) and microelements (copper, zinc and molybdenum) known to improve nodulation efficiency and N2 fixation. Finally, comparative proteomic analysis (+Si versus -Si) of nodules highlighted that Si modulated the proteome of both symbionts with 989 and 212 differentially accumulated proteins (DAPs) in the infected host root cells and their symbiont bacteria, respectively. Among the DAPs, the roles of those involved in nodulation and N2 fixation are discussed. For the first time, this study provides new insights into the effects of Si on both nodular partners and paves the way for a better understanding of the impact of Si on improving nodule function, and more specifically, on the nodules' N2-fixing capacity.

Identifying the major metabolic potentials of microbial-driven carbon, nitrogen and sulfur cycling on stone cultural heritage worldwide

Identifying the major metabolic potentials of microbial-driven carbon, nitrogen and sulfur cycling on stone cultural heritage worldwide

Substrate Competition of Diazotrophic Nitrous Oxide Assimilation Over Dinitrogen Fixation

Abstract Nitrous oxide (N2O) is a potent greenhouse gas and is depleting the stratospheric ozone layer. Diazotrophic N2O assimilation to biomass represents a novel biological N2O consumption pathway in addition to canonical denitrification. Thermodynamically, N2O assimilation is more favorable than dinitrogen (N2) fixation in natural environments, especially under higher N2O concentration and cooler conditions. Via isotopic tracing experiments, N2O assimilation was detected on cultured diazotrophs Crocosphaera and Trichodesmium with specific rates from 1.27 ± 0.16 × 10−4 to 2.00 ± 0.25 × 10−4 hr−1 under elevated [N2O]/[N2] conditions (0.0005–0.01) within 24‐hr incubation. The rates of N2O assimilation during the light and dark periods were statistically insignificant compared with N2 fixation activity. In a eutrophic estuary, N2O assimilation was not detected in the absence of diazotrophic activity. A competitive substrate kinetic model with experimentally calibrated parameters successfully quantified rate ratios of N2O assimilation and N2 fixation in varying substrate concentrations. The low [N2O]/[N2] ratio in natural conditions leads to N2O assimilation rate being <0.1% of N2 fixation rate, rendering negligible impact of N2O assimilation. The model was also used to predict the time required for experimental detection of N2O assimilation in isotopic tracing experiments under varying [N2O]/[N2] ratios. This study enhances the mechanistic understanding of N2O assimilation by diazotrophs, broadening the microbial nitrogen cycle by a potential N2O sink and nitrogen source for production.

Origin and Evolution of the Azolla Superorganism.

Azolla is the only plant with a co-evolving nitrogen-fixing (diazotrophic) cyanobacterial symbiont (cyanobiont), Nostoc azollae, resulting from whole-genome duplication (WGD) 80 million years ago in Azolla's ancestor. Additional genes from the WGD resulted in genetic, biochemical, and morphological changes in the plant that enabled the transmission of the cyanobiont to successive generations via its megaspores. The resulting permanent symbiosis and co-evolution led to the loss, downregulation, or conversion of non-essential genes to pseudogenes in the cyanobiont, changing it from a free-living organism to an obligate symbiont. The upregulation of other genes in the cyanobiont increased its atmospheric dinitrogen fixation and the provision of nitrogen-based products to the plant. As a result, Azolla can double its biomass in less than two days free-floating on fresh water and sequester large amounts of atmospheric CO2, giving it the potential to mitigate anthropogenic climate change through carbon capture and storage. Azolla's biomass can also provide local, low-cost food, biofertiliser, feed, and biofuel that are urgently needed as our population increases by a billion every twelve years. This paper integrates data from biology, genetics, geology, and palaeontology to identify the location, timing and mechanism for the acquisition of a co-evolving diazotrophic cyanobiont by Azolla's ancestor in the Late Cretaceous (Campanian) of North America.

Nitrogen fixation rate and phosphorus enrichment effects on diazotrophic cyanobacteria in the Gulf of Riga

In eutrophied marine systems such as the Baltic Sea, diazotrophic cyanobacteria have the potential to add additional bioavailable nitrogen (N) to the system through fixation of atmospheric dinitrogen (N2). However, their growth is regarded to be limited by phosphorus availability (P). This study investigates the response of two cyanobacteria species, Aphanizomenon flosaquae and Nodularia spumigena, collected from the Gulf of Riga under different environmental conditions to a short-period dissolved inorganic phosphorus (DIP) enrichment. The samples were collected during the summer cyanobacterial bloom of 2022 in the central region of the Gulf of Riga. Contrary to expectations, neither species demonstrated a significant increase in biomass. The study also established that N2-fixation rates did not correlate directly with the total diazotrophic cyanobacteria biomass, but showed a significant correlation with heterocyst presence in both species addressed during this study. The findings suggest the influence of additional factors beyond DIP availability on the N2-fixing cyanobacteria growth in the Gulf of Riga.

Fronts divide diazotroph communities in the Southern Indian Ocean

Dinitrogen (N2) fixation represents a key source of reactive nitrogen in marine ecosystems. While the process has been rather well-explored in low latitudes of the Atlantic and Pacific Oceans, other higher latitude regions and particularly the Indian Ocean have been chronically overlooked. Here, we characterize N2 fixation and diazotroph community composition across nutrient and trace metals gradients spanning the multifrontal system separating the oligotrophic waters of the Indian Ocean subtropical gyre from the high nutrient low chlorophyll waters of the Southern Ocean. We found a sharp contrasting distribution of diazotroph groups across the frontal system. Notably, cyanobacterial diazotrophs dominated north of fronts, driving high N2 fixation rates (up to 13.96 nmol N l−1 d−1) with notable peaks near the South African coast. South of the fronts non-cyanobacterial diazotrophs prevailed without significant N2 fixation activity being detected. Our results provide new crucial insights into high latitude diazotrophy in the Indian Ocean, which should contribute to improved climate model parameterization and enhanced constraints on global net primary productivity projections.