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Search indexed NASA NTRS and DOE OSTI research on propulsion, heat transfer, battery materials and energy systems. Follow report and document links to the original sources.

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At least 145 records · Page 8
All sourcesNASA NTRSDOE OSTI
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 OSTI2020

Beyond Ammonia: Nitrogen–Element Bond Forming Reactions with Coordinated Dinitrogen

The functionalization of coordinated dinitrogen to form nitrogen-element bonds en route to nitrogen-containing molecules is a long-standing challenge in chemical synthesis. The strong triple bond and the non-polarity of the N 2 molecule pose thermodynamic and kinetic challenges for promoting reactivity. Although heterogeneous, homogeneous and biological catalysts are all known for catalytic nitrogen fixation to ammonia, the catalytic synthesis of more complicated nitrogen-containing organic molecules has far less precedent. Examples of silyl radical additions to coordinated nitrogen to form silylamines stands as the lone example of a catalytic reaction involving N 2 to form a product other than ammonia. Our review surveys the field of molecular transition metal complexes, as well as recent boron examples for the formation of nitrogen-element bonds. Emphasis is placed on the coordination and activation modes of N 2 in the various metal compounds from across the transition series and how these structures can rationally inform reactivity studies. Over the past few decades, the field has evolved from the addition of carbon electrophiles in a manner similar to protonation reactions to more organometallic-inspired reactivity including insertions, 1,2-additions and cycloadditions. Various N–C, N–Si and N–B bond-forming reactions have been discovered highlighting that the challenge for catalytic chemistry is not in the reactivity of coordinated dinitrogen but rather removal of the functionalized ligand from the coordination sphere of the metal.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗
DOE OSTI2024

The elite haplotype OsGATA8 -H coordinates nitrogen uptake and productive tiller formation in rice

Excessive nitrogen promotes the formation of nonproductive tillers in rice, which decreases nitrogen use efficiency (NUE). Developing high-NUE rice cultivars through balancing nitrogen uptake and the formation of productive tillers remains a long-standing challenge, yet how these two processes are coordinated in rice remains elusive. Here we identify the transcription factor OsGATA8 as a key coordinator of nitrogen uptake and tiller formation in rice. OsGATA8 negatively regulates nitrogen uptake by repressing transcription of the ammonium transporter gene OsAMT3.2. Meanwhile, it promotes tiller formation by repressing the transcription of OsTCP19, a negative modulator of tillering. We identify OsGATA8-H as a high-NUE haplotype with enhanced nitrogen uptake and a higher proportion of productive tillers. The geographical distribution of OsGATA8-H and its frequency change in historical accessions suggest its adaption to the fertile soil. Overall, this study provides molecular and evolutionary insights into the regulation of NUE and facilitates the breeding of rice cultivars with higher NUE.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2020

Ammonia emission abatement does not fully control reduced forms of nitrogen deposition

Significance Control of nitrogen oxides emissions has successfully reduced the deposition of oxidized nitrogen over the United States. Recent studies have called attention to the rapid growth of deposition of reduced forms of nitrogen, which would offset the benefits of reduction in oxidized nitrogen deposition. Our analysis, based on both monitoring and modeling approaches, illustrates that implementing ammonia emission abatement may not lead to full response in nitrogen deposition over continental regions. Here we provide important information to the community and policy makers concerning nitrogen deposition, given the urgent needs for developing control strategies on ammonia emissions.

Tan, Jiani↗
DOE OSTI2021

Does resource exchange in ectomycorrhizal symbiosis vary with competitive context and nitrogen addition?

Ectomycorrhizal symbiosis is essential for the nutrition of most temperate forest trees and helps regulate the movement of carbon and nitrogen through forested ecosystems. The factors governing the exchange of plant carbon for fungal nitrogen, however, remain obscure. Because competition and soil resources may influence ectomycorrhizal resource movement, we performed a ten month split-root microcosm study using Pinus muricata seedlings with Thelephora terrestris, Suillus pungens, or no ectomycorrhizal fungus, under two nitrogen levels in artificial soil. Fungi competed directly with roots, and indirectly with each other. We used stable isotope enrichment to track plant photosynthate and fungal nitrogen. For Thelephora, plants received N commensurate with the C given to their fungal partners. Thelephora was a superior mutualist under high nitrogen. For Suillus, plant C and fungal N exchange were not coupled. However, in low nitrogen, plants preferentially allocated C to Suillus rather than Thelephora. Our results suggest that ectomycorrhizal resource transfer depends on competitive and nutritional context. Plants can exchange C for fungal N, but coupling of these resources can depend on the fungal species and soil N. Here, understanding the diversity of fungal strategies, and how they change with environmental context, reveals mechanisms driving this important symbiosis.

stable isotope enrichment↗
DOE OSTI2025

Comparing field and lab quantitative stable isotope probing for nitrogen assimilation in soil microbes

ABSTRACT Soil microbial communities play crucial roles in nutrient cycling and can help retain nitrogen in agricultural soils. Quantitative stable isotope probing (qSIP) is a useful method for investigating taxon-specific microbial growth and utilization of specific nutrients, such as nitrogen (N). Typically, qSIP is performed in a highly controlled lab setting, so the field relevance of lab qSIP studies remains unknown. We conducted and compared tandem lab and field qSIP to quantify the assimilation of 15 N by maize-associated soil prokaryotic communities at two agricultural sites. Here, we show that field qSIP with 15 N can be used to measure taxon-specific microbial N assimilation. Relative 15 N assimilation rates were generally lower in the field, and the magnitude of this difference varied by site. Rates differed by method (lab vs field) for 19% of the top N assimilating genera. The field and lab measures were more comparable when relative assimilation rates were weighted by relative abundance to estimate the proportion of N assimilated by each genus with only ~10% of taxa differing by method. Of those that differed, the taxa consistently higher in the lab were inclined to have opportunistic lifestyle strategies, whereas those higher in the field had niches reliant on plant roots or in-tact soil structure (biofilms, mycelia). This study demonstrates that 15 N-qSIP can be successfully performed using field-incubated soils to identify microbial allies in N retention and highlights the strengths and limitations of field and lab qSIP approaches. IMPORTANCE Soil microbes are responsible for critical biogeochemical processes in natural and agricultural ecosystems. Despite their importance, the functional traits of most soil organisms remain woefully under-characterized, limiting our ability to understand how microbial populations influence the transformation of elements such as nitrogen (N) in soil. Quantitative stable isotope probing (qSIP) is a powerful tool to measure the traits of individual taxa. This method has rarely been applied in the field or with 15 N to measure nitrogen assimilation. In this study, we measured genus-specific microbial nitrogen assimilation in two agricultural soils and compared field and lab 15 N qSIP methods. Our results identify taxa important for nitrogen assimilation in agricultural soils, shed light on the field relevance of lab qSIP studies, and provide guidance for the future application of qSIP to measure microbial traits in the field.

Reed, Kinsey (ORCID:0000000155178664)↗
DOE OSTI2023

Investigating the Unique Ability of Trichodesmium To Fix Carbon and Nitrogen Simultaneously Using MiMoSA

The open ocean is an extremely competitive environment, partially due to the dearth of nutrients. Trichodesmium erythraeum, a marine diazotrophic cyanobacterium, is a keystone species in the ocean due to its ability to fix nitrogen and leak 30 to 50% into the surrounding environment, providing a valuable source of a necessary macronutrient to other species. While there are other diazotrophic cyanobacteria that play an important role in the marine nitrogen cycle, Trichodesmium is unique in its ability to fix both carbon and nitrogen simultaneously during the day without the use of specialized cells called heterocysts to protect nitrogenase from oxygen. Here, we use the advanced modeling framework called multiscale multiobjective systems analysis (MiMoSA) to investigate how Trichodesmium erythraeum can reduce dimolecular nitrogen to ammonium in the presence of oxygen. Our simulations indicate that nitrogenase inhibition is best modeled as Michealis-Menten competitive inhibition and that cells along the filament maintain microaerobia using high flux through Mehler reactions in order to protect nitrogenase from oxygen. We also examined the effect of location on metabolic flux and found that cells at the end of filaments operate in distinctly different metabolic modes than internal cells despite both operating in a photoautotrophic mode. These results give us important insight into how this species is able to operate photosynthesis and nitrogen fixation simultaneously, giving it a distinct advantage over other diazotrophic cyanobacteria because they can harvest light directly to fuel the energy demand of nitrogen fixation.

09 BIOMASS FUELS↗
DOE OSTI2020

Data from Unique Contributions of Chlorophyll and Nitrogen to Predict Crop Photosynthetic Capacity from Leaf Spectroscopy

The photosynthetic capacity or the CO2-saturated photosynthetic rate (Vmax), chlorophyll, and nitrogen are closely linked leaf traits that determine C4 crop photosynthesis and yield. Accurate, timely, rapid, and non-destructive approaches to predict leaf photosynthetic traits from hyperspectral reflectance are urgently needed for high-throughput crop monitoring to ensure food and bioenergy security. Therefore, this study thoroughly evaluated the state-of-the-art physically based radiative transfer models (RTMs), data-driven partial least squares regression (PLSR), and generalized PLSR (gPLSR) models to estimate leaf traits from leaf-clip hyperspectral reflectance, which was collected from maize (Zea mays L.) bioenergy plots with diverse genotypes, growth stages, treatments with nitrogen fertilizers, and ozone stresses in three growing seasons. The results show that leaf RTMs considering bidirectional effects can give accurate estimates of chlorophyll content (Pearson correlation r=0.95), while gPLSR enabled retrieval of leaf nitrogen concentration (r=0.85). Using PLSR with field measurements for training, the cross-validation indicates that Vmax can be well predicted from spectra (r=0.81). The integration of chlorophyll content (strongly related to visible spectra) and nitrogen concentration (linked to shortwave infrared signals) can provide better predictions of Vmax (r=0.71) than only using either chlorophyll or nitrogen individually. This study highlights that leaf chlorophyll content and nitrogen concentration have key and unique contributions to Vmax prediction.

Biomass Analytics↗
DOE OSTI2022

Quantifying Nitrogen Bioextraction by Seaweed Farms – A Real-time Modeling-Monitoring Case Study in Hood Canal, WA

Anthropogenic nitrogen input is the leading cause of increasing marine eutrophication. Cultivating seaweeds at large scales may provide a viable solution for mitigating eutrophication especially when nitrogen extraction is achieved as an added environmental benefit of biofuel and food production. As the first step to promoting and implementing this solution, it is critical to quantify and assess the effectiveness of nitrogen removal by existing seaweed farms. Pacific Northwest National Laboratory and Hood Canal Mariculture Inc. conducted a case study by applying a coupled modeling-monitoring approach to quantify nitrogen bioextraction by a one-hectare commercial sugar kelp farm in Hood Canal, WA. Through this two-year effort, the project team conducted intensive field monitoring of water quality parameters at the farm site and subsequent laboratory analysis. Meanwhile, the team applied a coupled hydrodynamic-macroalgal growth model to simulate kelp growth and nitrogen bioextraction over the two kelp growing seasons. The model results showed an overall good comparison with field monitoring data and showed the effectiveness of nitrogen removal by kelp farms. The established modeling-monitoring approach can also be transferable for supporting broader research activities in macroalgal farming and eutrophication mitigation.

09 BIOMASS FUELS↗
DOE OSTI2025

Phylogenomic discovery and engineering of nitrogen fixation into the bioenergy woody crop poplar

Biological nitrogen fixation (BNF) is a key process enabling plants in specific lineages to convert atmospheric dinitrogen (N₂) into bioavailable ammonia through symbioses with diazotrophic microbes. Expanding this capability beyond native nitrogen-fixing clades into non-nodulating crops would reduce synthetic fertilizer use, lowering energy inputs and environmental impacts in agriculture. Supported by DOE Funding Award DE-SC0018247, the NitFix project advanced foundational knowledge required to engineer root-nodule symbioses in new host species. The team generated the most comprehensive phylogenomic analysis to date of all known nodulating lineages, resolving the evolutionary history of nitrogen-fixing symbiosis and identifying core gene suites retained across nodulating taxa. Through multimodal genomics, transcriptomics, and functional analyses in Medicago truncatula and related species, the project mapped regulatory networks underlying nodule organogenesis, bacterial infection, and nitrogen-fixation efficiency. Key discoveries include the identification of conserved signaling modules for rhizobial recognition, transcription factors controlling nodule differentiation, and metabolic pathways integrating fixed nitrogen into plant growth. The project also developed enabling tools—including optimized transformation pipelines, gene-editing workflows, and imaging-based phenotyping—to accelerate engineering efforts in emerging models. Together, these results refine the mechanistic framework of symbiotic nitrogen fixation and highlight transferable components essential for rewiring these traits into non-nodulating crops.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2021

Unifying themes and distinct features of carbon and nitrogen assimilation by polysaccharide-degrading bacteria: a summary of four model systems

Our current understanding of enzymatic polysaccharide degradation has come from a huge number of in vitro studies with purified enzymes. While this vast body of work has been invaluable in identifying and characterizing novel mechanisms of action and engineering desirable traits into these enzymes, a comprehensive picture of how these enzymes work as part of a native in vivo system is less clear. Recently, several model bacteria have emerged with genetic systems that allow for a more nuanced study of carbohydrate active enzymes (CAZymes) and how their activity affects bacterial carbon metabolism. With these bacterial model systems it is now possible to not only study a single nutrient system in isolation (i.e. carbohydrate degradation and carbon metabolism), but also how multiple systems are integrated. Given that most environmental polysaccharides are carbon rich but nitrogen poor (e.g. lignocellulose), the interplay between carbon and nitrogen metabolism in polysaccharide-degrading bacteria can now be studied in a physiologically relevant manner. Therefore, in this review we have summarized what has been experimentally determined for CAZyme regulation, production, and export in relation to nitrogen metabolism for two Gram-positive (Caldicellulosiruptor bescii and Clostridium thermocellum) and two Gram-negative (Bacteroides thetaiotaomicron and Cellvibrio japonicus) polysaccharide-degrading bacteria. By comparing and contrasting these four bacteria we have highlighted the shared and unique features of each, with a focus on in vivo studies, in regard to carbon and nitrogen assimilation. Finally, we conclude with what we believe are two important questions that can act as guideposts for future work to better understand the integration of carbon and nitrogen metabolism in polysaccharide-degrading bacteria.

Cellvibrio japonicus↗
DOE OSTI2025

Variation in Biomass Yield and Cell Wall Composition in Switchgrass Natural Variants Under Two Nitrogen Regimes

Switchgrass (Panicum virgatum) is a promising lignocellulosic biofuel crop for which biomass and processing quality are important. Inherent plant variability across genotypes and environments challenges uniformity and product quality. In this study, the impact of nitrogen (N) application on switchgrass yield and quality was examined under field conditions using a highly diverse switchgrass panel over a 4-year period at Knoxville, TN. Overall, biomass production was correlated between low (0 kg of added N/ha) and moderate (135 kg of added N/ha) nitrogen treatments, suggesting that the N impact is largely uniform across the genotypes. Nonetheless, high biomass genotypes were identified with high nitrogen-use efficiency ; biomass was congruent or even higher (up to 9-fold) in the low N treatment. Genotypes were also identified with up to 94% nitrogen-remobilization efficiency. Furthermore, nitrogen application appeared to impact lignin content in whole tillers but was neutral to lignin monomer syringyl-to-guaiacyl (S/G) ratio. The same panel grown under natural conditions in Watkinsville, GA, produced significantly more biomass than the Tennessee panel in the first 3 years, but biomass was similar across both sites and treatments in year 4. Top performing genotypes overlapped between sites by 20-37%. There were low correlations in lignin content in whole tillers across the two field sites, but moderate correlations were observed for S/G ratios. The high yielding genotypes from the low N plot identified in this study can be used in breeding programs and management strategies in switchgrass to evade adverse environmental and economic effects.

09 BIOMASS FUELS↗
DOE OSTI2024

Role of nitrogen in the high-temperature oxidation of titanium alloys

Here, we characterized the oxidation of pure Ti, Ti-0.8Si, and Ti-2Nb at 800 °C in N 2 -20% O 2 and in sequential isotopically labeled N 2 -20% O 2 atmospheres to clarify how nitrogen influences the high-temperature oxidation mechanisms of titanium alloys. The alloys always exhibited a nitride layer beneath a compact oxide scale with localized nitrogen concentrations in the underlying metal. Nitrogen is found to limit the oxygen concentrations in the metal. The synergy between alloying and nitrogen helps maintain compact oxide scales, preventing the formation of the lamellar scales observed during oxidation in nitrogen-free atmosphere and the oxidation of pure Ti regardless of the atmosphere.

36 MATERIALS SCIENCE↗
DOE OSTI2021

Long-term leaf C:N ratio change under elevated CO 2 and nitrogen deposition in China: Evidence from observations and process-based modeling

Climate change, elevating atmosphere CO 2 (eCO 2 ) and increased nitrogen deposition (iNDEP) are altering the biogeochemical interactions between plants, microbes and soils, which further modify plant leaf carbon-nitrogen (C:N) stoichiometry and their carbon assimilation capability. Many field experiments have observed large sensitivity of leaf C:N ratio to eCO 2 and iNDEP. However, the large-scale pattern of this sensitivity is still unclear, because eCO 2 and iNDEP drive leaf C:N ratio toward opposite directions, which are further compounded by the complex processes of nitrogen acquisition and plant-and-microbial nitrogen competition. Here, we attempt to map the leaf C:N ratio spatial variation in the past 5 decades in China with a combination of data-driven model and process-based modeling. These two approaches showed consistent results. Over different regions, we found that leaf C:N ratio had significant but uneven changes between 2 time periods (1960-1989 and 1990-2015): a 5% ± 8% increase for temperate grasslands in northern China, a 3% ± 6% increase for boreal grasslands in western China, and by contrast, a 7% ± 6% decrease for temperate forests in southern China, and a 3% ± 5% decrease for boreal forests in northeastern China. Additionally, the structural equation models indicated that the leaf C:N change was sensitive to ΔNDEP, ΔCO 2 and ΔMAT rather than ΔMAP and ecosystem types. In this work, process-based modeling suggested that iNDEP was the main source of soil mineral nitrogen change, dominating leaf C:N ratio change in most areas in China, while eCO 2 led to leaf C:N ratio increase in low iNDEP area. This study also indicates that the long-term leaf C:N ratio acclimation was dominated by climate constraint, especially temperature, but was constrained by soil N availability over decade scale.

54 ENVIRONMENTAL SCIENCES↗
DOE OSTI2021

DNA affinity purification sequencing and transcriptional profiling reveal new aspects of nitrogen regulation in a filamentous fungus

Significance Microorganisms have evolved transcriptional networks to prioritize utilization of available nutrient sources. For filamentous fungi, such as Neurospora crassa , this entails distinguishing between a variety of organic and inorganic nitrogen sources. Here, we transcriptionally profiled the response of N. crassa to a variety of nitrogen sources and used DNA affinity purification sequencing to characterize the role of regulatory genes and their direct downstream targets. We identified a transcription factor responsible for regulating genes involved in amino acid and mannose metabolism. By comparing the genes regulated by transcription factors that regulate specific nitrogen utilization pathways and transcription factors that regulate utilization of all nitrogen sources that require metabolic processing before utilization, we revealed aspects of the nitrogen regulatory network.

59 BASIC BIOLOGICAL 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 OSTI2024

Enhancing nitrogen fixation efficiency in glow-like discharge by reducing cathode-fall voltage

In plasma nitrogen fixation devices, discharge electrodes are crucial yet susceptible to oxidation and corrosion due to plasma’s high temperatures and oxygen content, which could alter discharge modes. This research evaluates the impact of different electrode materials, including iron, chromium, nickel, copper, and 304 stainless steel, on nitrogen fixation efficiency in glow-like discharges driven by high-voltage DC power. Notably, iron and 304 stainless steel cathodes undergo a mode transition at increased currents, evident from plasma color shifts and significant voltage reductions. Fourier transform infrared spectroscopy analyses reveal that such mode changes minimally affect nitrogen oxide production rates, leading to a notable decrease in energy consumption for nitrogen fixation by up to 40%. OES and SEM-EDS measurements suggest that iron oxide, with its higher secondary electron emission, replaces metal as the cathode material, facilitating mode transitions and maintaining discharge current at lower voltages. Further, this voltage change is largely attributed to the cathode voltage drop, highlighting the minimal role of the cathode fall region in NO x synthesis. These findings underscore the potential for improving plasma nitrogen fixation energy efficiency by choosing suitable cathode materials to lower the cathode-fall voltage.

70 PLASMA PHYSICS AND FUSION TECHNOLOGY↗
DOE OSTI2024

Nanoscale elemental and morphological imaging of nitrogen-fixing cyanobacteria

Nitrogen-fixing cyanobacteria bind atmospheric nitrogen and carbon dioxide using sunlight. This experimental study focused on a laboratory-based model system, Anabaena sp., in nitrogen-depleted culture. When combined nitrogen is scarce, the filamentous prokaryotes reconcile photosynthesis and nitrogen fixation by cellular differentiation into heterocysts. To better understand the influence of micronutrients on cellular function, 2D and 3D synchrotron X-ray fluorescence mappings were acquired from whole biological cells in their frozen-hydrated state at the Bionanoprobe, Advanced Photon Source. To study elemental homeostasis within these chain-like organisms, biologically relevant elements were mapped using X-ray fluorescence spectroscopy and energy-dispersive X-ray microanalysis. Higher levels of cytosolic K + , Ca 2+ , and Fe 2+ were measured in the heterocyst than in adjacent vegetative cells, supporting the notion of elevated micronutrient demand. P-rich clusters, identified as polyphosphate bodies involved in nutrient storage, metal detoxification, and osmotic regulation, were consistently co-localized with K + and occasionally sequestered Mg 2+ , Ca 2+ , Fe 2+ , and Mn 2+ ions. Machine-learning-based k-mean clustering revealed that P/K clusters were associated with either Fe or Ca, with Fe and Ca clusters also occurring individually. In accordance with XRF nanotomography, distinct P/K-containing clusters close to the cellular envelope were surrounded by larger Ca-rich clusters. The transition metal Fe, which is a part of nitrogenase enzyme, was detected as irregularly shaped clusters. The elemental composition and cellular morphology of diazotrophic Anabaena sp. was visualized by multimodal imaging using atomic force microscopy, scanning electron microscopy, and fluorescence microscopy. This paper discusses the first experimental results obtained with a combined in-line optical and X-ray fluorescence microscope at the Bionanoprobe.

Anabaena sp↗