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All sourcesNASA NTRSDOE OSTI
DOE OSTI2023

Structural insights into redox signal transduction mechanisms in the control of nitrogen fixation by the NifLA system

NifL is a conformationally dynamic flavoprotein responsible for regulating the activity of the σ 54 -dependent activator NifA to control the transcription of nitrogen fixation (nif) genes in response to intracellular oxygen, cellular energy, or nitrogen availability. The NifL-NifA two-component system is the master regulatory system for nitrogen fixation. NifL serves as a sensory protein, undergoing signal-dependent conformational changes that modulate its interaction with NifA, forming the NifL–NifA complex, which inhibits NifA activity in conditions unsuitable for nitrogen fixation. While NifL-NifA regulation is well understood, these conformationally flexible proteins have eluded previous attempts at structure determination. In work described here, we advance a structural model of the NifL dimer supported by a combination of scattering techniques and mass spectrometry (MS)-coupled structural analyses that report on the average structure in solution. Using a combination of small angle X-ray scattering-derived electron density maps and MS-coupled surface labeling, we investigate the conformational dynamics responsible for NifL oxygen and energy responses. Our results reveal conformational differences in the structure of NifL under reduced and oxidized conditions that provide the basis for a model for modulating NifLA complex formation in the regulation of nitrogen fixation in response to oxygen in the model diazotroph,Azotobacter vinelandii.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2021

Temporal dynamics of free‐living nitrogen fixation in the switchgrass rhizosphere

Abstract Free‐living nitrogen fixation (FLNF) represents an important terrestrial N source and is gaining interest for its potential to contribute plant available N to bioenergy cropping systems. Switchgrass, a cellulosic bioenergy crop, may be particularly reliant on FLNF when grown on low N systems, like marginal lands. However, the potential contributions of FLNF to switchgrass as well as the controls on this process are not well understood. In this study, we evaluated drivers of FLNF rates and N‐fixing microbial (diazotrophic) community composition in field‐grown switchgrass systems over two growing seasons with high temporal sampling. We found that climate variables are strong drivers of FLNF rates in switchgrass systems, compared to other environmental and biological factors including soil nutrients and diazotrophic community composition. Increased soil moisture availability generally promoted FLNF rates, but extreme rainfall events were detrimental. These climate‐related responses suggest a potential for loss of FLNF‐derived N contributions under projected climate shifts. We found a significant, but weak correlation between diazotrophic community composition and FLNF rates. We also observed a significant shift in the diazotrophic community composition between 2017 and 2018 and similarly measured a significant difference in FLNF rates between growing seasons. Lastly, we found that seasonal FLNF N contributions, based on measurement with high temporal resolution, has the potential to meet up to 80% of switchgrass N demands suggesting that FLNF measurements extrapolated from fewer time points or locations may underestimate these potential N contributions.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2024

Identification of Yarrowia lipolytica as a platform for designed consortia that incorporate in situ nitrogen fixation to enable ammonia-free bioconversion

Bioconversion processes require nitrogen for growth and production of intracellular enzymes to produce biofuels and bioproducts. Typically, this is supplied as reduced nitrogen in the form of ammonia, which is produced offsite from N 2 and H 2 via the Haber-Bosch process. While this has revolutionized industries dependent on fixed nitrogen (e.g., modern agriculture), it is highly energy-intensive and its reliance on natural gas combustion results in substantial global CO 2 emissions. Here we investigated the feasibility of in situ biological nitrogen fixation from N 2 gas as a strategy to reduce greenhouse gas impacts of aerobic bioconversion processes. We developed an efficient and cost-effective method to screen fungal bioconversion hosts for compatibility with the free-living diazotrophic bacterium Azotobacter vinelandii under nitrogen fixing conditions. Our screening revealed that the genus Yarrowia is particularly enriched during co-culture experiments. Follow-up experiments identified four Y. lipolytica strains (NRRL Y-11853, NRRL Y-7208, NRRL Y-7317, and NRRL YB-618) capable of growth in co-culture with A. vinelandii. These strains utilize ammonium secreted during diazotrophic fixation of N 2 , which is provided as a component of the air input stream during aerobic fermentation. This demonstrates the feasibly of in situ biological nitrogen fixation to support heterotrophic fermentation processes for production of fuels and chemicals.

09 BIOMASS FUELS↗
DOE OSTI2025

Global terrestrial nitrogen fixation and its modification by agriculture

Biological nitrogen fixation (BNF) is the largest natural source of new nitrogen (N) that supports terrestrial productivity, yet estimates of global terrestrial BNF remain highly uncertain. Here, in this study, we show that this uncertainty is partly because of sampling bias, as field BNF measurements in natural terrestrial ecosystems occur where N fixers are 17 times more prevalent than their mean abundances worldwide. To correct this bias, we develop new estimates of global terrestrial BNF by upscaling field BNF measurements using spatially explicit abundances of all major biogeochemical N-fixing niches. We find that natural biomes sustain lower BNF, 65 (52–77) Tg N yr −1 , than previous empirical bottom-up estimates, with most BNF occurring in tropical forests and drylands. We also find high agricultural BNF in croplands and cultivated pastures, 56 (54–58) Tg N yr −1 . Agricultural BNF has increased terrestrial BNF by 64% and total terrestrial N inputs from all sources by 60% over pre-industrial levels. Our results indicate that BNF may impose stronger constraints on the carbon sink in natural terrestrial biomes and represent a larger source of agricultural N than is generally considered in analyses of the global N cycle, with implications for proposed safe operating limits for N use.

Reis Ely, Carla R. [Oregon State Univ., Corvallis,↗
DOE OSTI2025

Structural analysis of the NifL-NifA complex reveals the molecular basis of anti-activation of nitrogen fixation gene expression in Azotobacter vinelandii

Understanding the molecular basis of regulated nitrogen (N 2 ) fixation is essential for engineering N 2 -fixing bacteria that fulfill the demand of crop plants for fixed nitrogen, reducing our reliance on synthetic nitrogen fertilizers. In Azotobacter vinelandii and many other members of Proteobacteria, the two-component system comprising the anti-activator protein (NifL) and the Nif-specific transcriptional activator (NifA)controls the expression of nif genes, encoding the nitrogen fixation machinery. The NifL-NifA system evolved the ability to integrate several environmental cues, such as oxygen, nitrogen, and carbon availability. The nitrogen fixation machinery is thereby only activated under strictly favorable conditions, enabling diazotrophs to thrive in competitive environments. While genetic and biochemical studies have enlightened our understanding of how NifL represses NifA, the molecular basis of NifA sequestration by NifL depends on structural information on their interaction. Here, we present mechanistic insights into how nitrogen fixation is regulated by combining biochemical and genetic approaches with a low-resolution cryo-electron microscopy (cryo-EM) map of the oxidized NifL-NifA complex. Our findings define the interaction surface between NifL and NifA and reveal how this interaction can be manipulated to generate bacterial strains with increased nitrogen fixation rates able to secrete surplus nitrogen outside the cell, a crucial step in engineering improved nitrogen delivery to crop plants.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2021

Data from: Temporal dynamics of free‐living nitrogen fixation in the switchgrass rhizosphere

Here we present data associated with the manuscript, Temporal dyanmics of free-living nitrogen fixation in the switchgrass rhizosphere. Free-living nitrogen fixation (FLNF) represents an important terrestrial N source and is gaining interest for its potential to contribute plant available N to bioenergy cropping systems. Switchgrass, a cellulosic bioenergy crop, may be reliant on FLNF when particularly when grown on low N marginal lands. These potential contributions of FLNF to switchgrass and the controls on this process are not well understood. We evaluated drivers of FLNF rates and N-fixing microbial community composition in field-grown switchgrass systems over two years with high temporal sampling. We found climate variables to be strong drivers of FLNF in switchgrass systems, compared to other environmental and biological factors including soil nutrients and N-fixing microbial community composition. Increased soil moisture availability tended to promote FLNF, but extreme rainfall events appeared to be detrimental. These climate-related responses suggest FLNF-derived N contributions may be reduced under projected climate shifts. We also found a significant, but weak correlation between N-fixing microbial community composition and FLNF rates including an observed shift in community composition between 2017 and 2018 and a similarly significant difference in FLNF rates between years. Lastly, we found that seasonal FLNF N contributions, based on measurement with high temporal resolution, may meet up to 80% of switchgrass N demands.

09 BIOMASS FUELS↗
DOE OSTI2022

Nitrogen fixation and mucilage production on maize aerial roots is controlled by aerial root development and border cell functions

Exploring natural diversity for biological nitrogen fixation in maize and its progenitors is a promising approach to reducing our dependence on synthetic fertilizer and enhancing the sustainability of our cropping systems. We have shown previously that maize accessions from the Sierra Mixe can support a nitrogen-fixing community in the mucilage produced by their abundant aerial roots and obtain a significant fraction of their nitrogen from the air through these associations. In this study, we demonstrate that mucilage production depends on root cap and border cells sensing water, as observed in underground roots. The diameter of aerial roots correlates with the volume of mucilage produced and the nitrogenase activity supported by each root. Young aerial roots produce more mucilage than older ones, probably due to their root cap’s integrity and their ability to produce border cells. Transcriptome analysis on aerial roots at two different growth stages before and after mucilage production confirmed the expression of genes involved in polysaccharide synthesis and degradation. Genes related to nitrogen uptake and assimilation were up-regulated upon water exposure. Altogether, our findings suggest that in addition to the number of nodes with aerial roots reported previously, the diameter of aerial roots and abundance of border cells, polysaccharide synthesis and degradation, and nitrogen uptake are critical factors to ensure efficient nitrogen fixation in maize aerial roots.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2024

Investigating biological nitrogen fixation via single-cell transcriptomics

The extensive use of nitrogen fertilizers has detrimental environmental consequences, and it is essential for society to explore sustainable alternatives. One promising avenue is engineering root nodule symbiosis, a naturally occurring process in certain plant species within the nitrogen-fixing clade, into non-leguminous crops. Advancements in single-cell transcriptomics provide unprecedented opportunities to dissect the molecular mechanisms underlying root nodule symbiosis at the cellular level. This review summarizes key findings from single-cell studies in Medicago truncatula, Lotus japonicus, and Glycine max. We highlight how these studies address fundamental questions about the development of root nodule symbiosis, including the following findings: (i) single-cell transcriptomics has revealed a conserved transcriptional program in root hair and cortical cells during rhizobial infection, suggesting a common infection pathway across legume species; (ii) characterization of determinate and indeterminate nodules using single-cell technologies supports the compartmentalization of nitrogen fixation, assimilation, and transport into distinct cell populations; (iii) single-cell transcriptomics data have enabled the identification of novel root nodule symbiosis genes and provided new approaches for prioritizing candidate genes for functional characterization; and (iv) trajectory inference and RNA velocity analyses of single-cell transcriptomics data have allowed the reconstruction of cellular lineages and dynamic transcriptional states during root nodule symbiosis.

Lotus japonicus↗
DOE OSTI2025

Reduced soil diazotroph diversity decreases nitrogen fixation rates, but depends on land management

Soil diazotrophs convert atmospheric nitrogen into plant-available ammonium through free-living nitrogen fixation (FLNF). This sustainable nitrogen source can reduce our dependence on synthetic fertilizer inputs in conventional agricultural systems. However, we know little about the effect of diazotroph diversity on FLNF, especially given that FLNF is intermediate within the broad-narrow functional spectrum. Here, we determined how management-mediated shifts in diazotroph diversity would impact their ecosystem function (FLNF) by quantifying diazotroph diversity across a long-term management gradient during and after the growing season. In addition to field observations, we leveraged the same management gradient to manipulate diversity in soil microcosms via chloroform fumigation exposure. In the field, diazotroph diversity was significantly higher after the growing season, and the biologically-based annual cropping system harbored the highest diazotroph diversity. However, perennial cropping systems maintained the highest FLNF despite lower diazotroph diversity, and both soil moisture and temperature were stronger predictors of FLNF. Based on these results, integrating diverse perennial crops into agricultural landscape could result in greater N from FLNF, particularly at the end of the growing season. When we reduced biodiversity in a manipulation experiment, the diversity-FLNF association was stronger than in the field experiment, suggesting that FLNF communities are not as functionally redundant as taxonomically ‘broad’ ecosystem functions. Here, the strength of diversity-FLNF correlation varied by previous land management. Diazotroph diversity better predicted FLNF in annual and forest soil microcosms, and microbial biomass carbon better predicted FLNF in perennial soil microcosms. Taken together, our results show that while diazotroph diversity influences FLNF, especially under extreme environmental disturbances, abiotic factors like soil moisture and temperature are stronger constraints on FLNF in the field.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2025

Overestimated natural biological nitrogen fixation translates to an exaggerated CO 2 fertilization effect in Earth system models

CO 2 fertilization of the terrestrial biosphere is limited by nitrogen. Biological nitrogen fixation (BNF) is the dominant natural nitrogen source to the terrestrial biosphere and can alleviate nitrogen limitation but is poorly constrained in Earth system models (ESMs). Here, in this study, we compare terrestrial BNF from an ensemble of ESMs of the 6th Coupled Model Intercomparison Project to a new global synthesis of observations across natural and agricultural biomes. We find that compared to observations, ESMs underestimate agricultural BNF but overestimate natural BNF in the present day by over 50%. Natural BNF is overestimated in the most productive ecosystems that contribute most to the terrestrial carbon sink (forests and grasslands). ESMs with different BNF representations yield a range of BNF responses to CO 2 enrichment. Some ESMs with phenomenological representations of BNF predict a natural BNF increase in response to a doubling of CO 2 that aligns with a meta-analysis of CO 2 enrichment experiments (31% increase) but fail to account for the substantial carbon cost of BNF. In contrast, ESMs with mechanistic representations of BNF account for its carbon cost as well as its regulation by nitrogen limitation but overestimate the BNF response to a doubling of CO 2 (135% increase). Overall, all current BNF representations in ESMs fall short of fully capturing its response to rising atmospheric CO 2 . Finally, we find a positive correlation between modeled present-day natural BNF and the CO 2 fertilization effect across ESMs, suggesting that overestimated natural BNF translates to an exaggerated CO 2 fertilization effect of approximately 11% in ESMs.

Biological nitrogen fixation↗
DOE OSTI2023

Identification of genetic and environmental factors influencing aerial root traits that support biological nitrogen fixation in sorghum

Abstract Plant breeding and genetics play a major role in the adaptation of plants to meet human needs. The current requirement to make agriculture more sustainable can be partly met by a greater reliance on biological nitrogen fixation by symbiotic diazotrophic microorganisms that provide crop plants with ammonium. Select accessions of the cereal crop sorghum (Sorghum bicolor (L.) Moench) form mucilage-producing aerial roots that harbor nitrogen-fixing bacteria. Breeding programs aimed at developing sorghum varieties that support diazotrophs will benefit from a detailed understanding of the genetic and environmental factors contributing to aerial root formation. A genome-wide association study of the sorghum minicore, a collection of 242 landraces, and 30 accessions from the sorghum association panel was conducted in Florida and Wisconsin and under 2 fertilizer treatments to identify loci associated with the number of nodes with aerial roots and aerial root diameter. Sequence variation in genes encoding transcription factors that control phytohormone signaling and root system architecture showed significant associations with these traits. In addition, the location had a significant effect on the phenotypes. Concurrently, we developed F2 populations from crosses between bioenergy sorghums and a landrace that produced extensive aerial roots to evaluate the mode of inheritance of the loci identified by the genome-wide association study. Furthermore, the mucilage collected from aerial roots contained polysaccharides rich in galactose, arabinose, and fucose, whose composition displayed minimal variation among 10 genotypes and 2 fertilizer treatments. These combined results support the development of sorghums with the ability to acquire nitrogen via biological nitrogen fixation.

59 BASIC BIOLOGICAL SCIENCES↗
NASA NTRS2011

Effect of Microgravity on Early Events of Biological Nitrogen Fixation in Medicago Truncatula: Initial Results from the SyNRGE Experiment

SyNRGE (Symbiotic Nodulation in a Reduced Gravity Environment) was a sortie mission on STS-135 in the Biological Research in Canisters (BRIC) hardware to study the effect of microgravity on a plant-microbe symbiosis resulting in biological nitrogen fixation. Medicago truncatula, a model species of the legume family, was inoculated with its bacterial symbiont, Sinorhizobium meliloti, to observe early events associated with infection and nodulation in Petri Dish Fixation Units (PDFUs). Two sets of experiments were conducted in orbit and in 24-hour delayed ground controls. Experiment one was designed to determine if S. meliloti infect M. truncatula and initiate physiological changes associated with nodule formation. Roots of five-day-old M. truncatula cultivar Jemalong A17 (Enodll::gus) were inoculated 24 hr before launch with either S. meliloti strain 1021 or strain ABS7 and integrated into BRIC-PDFU hardware placed in a 4 C Cold Bag for launch on Atlantis. Inoculated plants and uninoculated controls were maintained in the dark at ambient temperature in the middeck of STS-135 for 11 days before fixation in RNAlater(tM) by crew activation of the PDFU. Experiment two was designed to determine if microgravity altered the process of bacterial infection and host plant nodule formation. Seeds of two M. truncatula cultivar Jemalong A17 lines, the Enodll::gus used in experiment 1, and SUNN, a super-nodulating mutant of A17, were germinated on orbit for 11 days in the middeck cabin and returned to Earth alive inside of BRIC-PDFU's at 4 C. S. meliloti strains 1021 and ABS7 were cultivated separately in broth culture on orbit and also returned to Earth alive. After landing, flight- and groundgrown plants and bacteria were transferred from BRIC-PDFU's into Nunc(tm) 4-well plates for reciprocity crosses. Rates of plant growth and nodule development on Buffered Nodulation Medium (lacking nitrogen) were measured for 14 days. Preliminary analysis' of Experiment 1 confirms that legumes and bacteria cultivated in space 'initiate the symbiotic interaction leading to nitrogen fixation and that bacteria retain the ability to form nodules on M. truncatula roots. Initial assessment of experiment 2 shows 100% seed germination and excellent bacterial growth in microgravity.

Stutte, Gary W.↗
DOE OSTI2025

Nitrogen Fixation Symposium at ICCC 2024 (Final Report)

The symposium "Advances in Nitrogen Fixation" was part of the 45th International Conference on Coordinate Chemistry (ICCC45) in Fort Collins, Colorado. It was organized by Patrick Holland (US) and Sven Schneider (Germany). The symposium was one of four parallel sessions on the first full day of the conference (Monday July 29, 2024). There were also three posters associated with the symposium.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗
DOE OSTI2020

Biological nitrogen fixation across major biomes in Latin America: Patterns and global change effects

We report biological nitrogen fixation (BNF) supports terrestrial primary productivity and plays key roles in mediating human-induced changes in global nitrogen (N) and carbon cycling. However, there are still critical uncertainties in our understanding of the amount of BNF occurring across terrestrial ecosystems, and of how terrestrial BNF will respond to global change. We synthesized BNF data from Latin America, a region reported to sustain some of the highest BNF rates on Earth, but that is underrepresented in previous data syntheses. We used meta-analysis and modeling approaches to estimate BNF rates across Latin America's major biomes and to evaluate the potential effects of increased N deposition and land-use change on these rates. Unmanaged tropical and subtropical moist forests sustained observed and predicted total BNF rates of 10 ± 1 and 14 ± 1 kg N ha -1 y -1 , respectively, supporting the hypothesis that these forests sustain lower BNF rates than previously thought. Free-living BNF accounted for two-thirds of the total BNF in these forests. Despite an average 30% reduction of free-living BNF in response to experimental N-addition, our results suggest free-living BNF rate responses to current and projected N deposition across tropical and subtropical moist forests are small. In contrast, the conversion of unmanaged ecosystems to crop and pasture lands increased BNF rates across all terrestrial biomes, mostly in savannas, grasslands, and dry forests, increasing BNF rates 2-fold. The information obtained here provides a more comprehensive understanding of BNF patterns for Latin America.

54 ENVIRONMENTAL SCIENCES↗
NASA NTRS1978

Assessment of free-living nitrogen fixing microorganisms for commercial nitrogen fixation

Ammonia production by Klebsiella pneumoniae is not economical with present strains and improving nitrogen fixation to its theoretical limits in this organism is not sufficient to achieve economic viability. Because the value of both the hydrogen produced by this organism and the methane value of the carbon source required greatly exceed the value of the ammonia formed, ammonia (fixed nitrogen) should be considered the by-product. The production of hydrogen by KLEBSIELLA or other anaerobic nitrogen fixers should receive additional study, because the activity of nitrogenase offers a significant improvement in hydrogen production. The production of fixed nitrogen in the form of cell mass by Azotobacter is also uneconomical and the methane value of the carbon substrate exceeds the value of the nitrogen fixed. Parametric studies indicate that as efficiencies approach the theoretical limits the economics may become competitive. The use of nif-derepressed microorganisms, particularly blue-green algae, may have significant potential for in situ fertilization in the environment.

Stokes, B. O.↗
NASA NTRS1989

Biological nitrogen fixation under primordial Martian partial pressures of dinitrogen

One of the most striking differences between the conditions on early Mars and earth was a low (18 mb) partial pressure of N2 (pN2) on early Mars, as opposed to 780 mb N2 on earth. To investigate the possibility of biological nitrogen fixation under conditions of early Mars, experiments were carried out on the growth of Azotobacter vinelandii and Azomonas agilis in nitrogen-free synthetic medium under various partial pressures of N2 (ranging from 780 to 0 mb). It was found that, although the biomass, cell number, and growth rate of these bacteria decreased with decreasing pN2 values below pN2 of 400 mb, both microorganisms were capable of growing at pN2 as low as 5 mb (but not at of below 1 mb), indicating that biological fixation of nitrogen could have occurred on primordial Mars.

Klingler, J. M.↗