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At least 163 records · Page 9
All sourcesNASA NTRSDOE OSTI
DOE OSTI2024

Mo than meets the eye: genomic insights into molybdoenzyme diversity of Seleniivibrio woodruffii strain S4T

Abstract Seleniivibrio woodruffii strain S4T is an obligate anaerobe belonging to the phylum Deferribacterota. It was isolated for its ability to respire selenate and was also found to respire arsenate. The high-quality draft genome of this bacterium is 2.9 Mbp, has a G+C content of 48%, 2762 predicted genes of which 2709 are protein-coding, and 53 RNA genes. An analysis of the genome focusing on the genes encoding for molybdenum-containing enzymes (molybdoenzymes) uncovered a remarkable number of genes encoding for members of the dimethylsulfoxide reductase family of proteins (DMSOR), including putative reductases for selenate and arsenate respiration, as well as genes for nitrogen fixation. Respiratory molybdoenzymes catalyze redox reactions that transfer electrons to a variety of substrates that can act as terminal electron acceptors for energy generation. Seleniivibrio woodruffii strain S4T also has essential genes for molybdate transporters and the biosynthesis of the molybdopterin guanine dinucleotide cofactors characteristic of the active centers of DMSORs. Phylogenetic analysis revealed candidate respiratory DMSORs spanning nine subfamilies encoded within the genome. Our analysis revealed the untapped potential of this interesting microorganism and expanded our knowledge of molybdoenzyme co-occurrence.

Louie, Tiffany S.↗
DOE OSTI2022

Exploring the Role of Cryptic Nitrogen Fixers in Terrestrial Ecosystems: A Frontier in Nitrogen Cycling Research

Biological nitrogen fixation represents the largest natural flux of new nitrogen (N) into terrestrial ecosystems, providing a critical N source to support net primary productivity of both natural and agricultural systems. When they are common, symbiotic associations between plants and bacteria can add more than 100 kg N ha –1 y –1 to ecosystems. Yet, these associations are uncommon in many terrestrial ecosystems. In most cases, N inputs derive from more cryptic sources, including mutualistic and/or free-living microorganisms in soil, plant litter, decomposing roots and wood, lichens, insects, and mosses, among others. Unfortunately, large gaps remain in the understanding of cryptic N fixation. Here, we conducted a literature review to explore rates, patterns, and controls of cryptic N fixation in both unmanaged and agricultural ecosystems. Our analysis indicates that, as is common with N fixation, rates are highly variable across most cryptic niches, with N inputs in any particular cryptic niche ranging from near zero to more than 20 kg ha –1 y –1 . Such large variation underscores the need for more comprehensive measurements of N fixation by organisms not in symbiotic relationships with vascular plants in terrestrial ecosystems, as well as identifying the factors that govern cryptic N fixation rates. We highlight several challenges, opportunities, and priorities in this important research area, and we propose a conceptual model that posits an interacting hierarchy of biophysical and biogeochemical controls over N fixation that should generate valuable new hypotheses and research.

54 ENVIRONMENTAL SCIENCES↗
NASA NTRS1979

Fixation of nitrogen in the prebiotic atmosphere

Reactions between nitrogen and water in the air surrounding lightning discharges can provide an important source of nitric oxide even under conditions where oxygen is a minor atmospheric constituent. Estimates are given for the associated source of soluble nitrite and nitrate. It is shown that lightning and subsequent atmospheric chemistry can provide a source of nitrate for the primitive ocean as large as one million tons of nitrogen per year, sufficient to fill the ocean to its present level of nitrate in less than one million years.

Yung, Y. L.↗
DOE OSTI2017

Diazotroph Community Characterization via a High-Throughput nifH Amplicon Sequencing and Analysis Pipeline

The dinitrogenase reductase gene ( nifH ) is the most widely established molecular marker for the study of nitrogen-fixing prokaryotes in nature. A large number of PCR primer sets have been developed for nifH amplification, and the effective deployment of these approaches should be guided by a rapid, easy-to-use analysis protocol. Bioinformatic analysis of marker gene sequences also requires considerable expertise. In this study, we advance the state of the art for nifH analysis by evaluating nifH primer set performance, developing an improved amplicon sequencing workflow, and implementing a user-friendly bioinformatics pipeline. Furthermore, the developed amplicon sequencing workflow is a three-stage PCR-based approach that uses established technologies for incorporating sample-specific barcode sequences and sequencing adapters. Based on our primer evaluation, we recommend the Ando primer set be used with a modified annealing temperature of 58°C, as this approach captured the largest diversity of nifH templates, including paralog cluster IV/V sequences. To improve nifH sequence analysis, we developed a computational pipeline which infers taxonomy and optionally filters out paralog sequences. In addition, we employed an empirical model to derive optimal operational taxonomic unit (OTU) cutoffs for the nifH gene at the species, genus, and family levels. A comprehensive workflow script named TaxADivA (TAXonomy Assignment and DIVersity Assessment) is provided to ease processing and analysis of nifH amplicons. Our approach is then validated through characterization of diazotroph communities across environmental gradients in beach sands impacted by the Deepwater Horizon oil spill in the Gulf of Mexico, in a peat moss-dominated wetland, and in various plant compartments of a sugarcane field. IMPORTANCE: Nitrogen availability often limits ecosystem productivity, and nitrogen fixation, exclusive to prokaryotes, comprises a major source of nitrogen input that sustains food webs. The nifH gene, which codes for the iron protein of the nitrogenase enzyme, is the most widely established molecular marker for the study of nitrogen-fixing microorganisms (diazotrophs) in nature. In this study, a flexible sequencing/analysis pipeline, named TaxADivA, was developed for nifH amplicons produced by Illumina paired-end sequencing, and it enables an inference of taxonomy, performs clustering, and produces output in formats that may be used by programs that facilitate data exploration and analysis. Diazotroph diversity and community composition are linked to ecosystem functioning, and our results advance the phylogenetic characterization of diazotroph communities by providing empirically derived nifH similarity cutoffs for species, genus, and family levels. Thus, the utility of our pipeline is validated for diazotroph communities in a variety of ecosystems, including contaminated beach sands, peatland ecosystems, living plant tissues, and rhizosphere soil.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2021

Metabolic Model of the Nitrogen-Fixing Obligate Aerobe Azotobacter vinelandii Predicts Its Adaptation to Oxygen Concentration and Metal Availability

There is considerable interest in promoting biological nitrogen fixation (BNF) as a mechanism to reduce the inputs of nitrogenous fertilizers in agriculture, but considerable fundamental knowledge gaps still need to be addressed. BNF is catalyzed by nitrogenase, which requires a large input of energy in the form of ATP and low potential electrons. Diazotrophs that respire aerobically have an advantage in meeting the ATP demands of BNF but face challenges in protecting nitrogenase from inactivation by oxygen. Here, we constructed a genome-scale metabolic model of the nitrogen-fixing bacterium Azotobacter vinelandii, which uses a complex respiratory protection mechanism to consume oxygen at a high rate to keep intracellular conditions microaerobic. Our model accurately predicts growth rate under high oxygen and substrate concentrations, consistent with a large electron flux directed to the respiratory protection mechanism. While a partially decoupled electron transport chain compensates for some of the energy imbalance under high-oxygen conditions, it does not account for all substrate intake, leading to increased maintenance rates. Interestingly, the respiratory protection mechanism is required for accurate predictions even when ammonia is supplemented during growth, suggesting that the respiratory protection mechanism might be a core principle of metabolism and not just used for nitrogenase protection. We have also shown that rearrangement of flux through the electron transport system allows A. vinelandii to adapt to different oxygen concentrations, metal availability, and genetic disruption, which cause an ammonia excretion phenotype. Accurately determining the energy balance in an aerobic nitrogen-fixing metabolic model is required for future engineering approaches.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2021

From inert gas to fertilizer, fuel and fine chemicals: N 2 reduction and fixation

The 100th anniversary of a leading nitrogen fixation technology developer like CASALE SA is a reason to reflect over the 20th century successful solution of the problem of world food supply, and to look out for solutions for sustainable developments with respect to ammonia production. Here, we review the role of nitrogen as essential chemical constituent in photosynthesis and biology, and component of ammonia as it is used as fertilizer for primary production by photosynthesis for farming and food supply and its future role as energy carrier. While novel synthesis methods and very advanced synchrotron based x-ray analytical techniques are being developed, we feel it is important to refer to the historical and economical context of nitrogen. The breaking of the N≡N triple bond remains a fundamental chemical and energetic problem in this context. We review the electrochemical ammonia synthesis as an energetically and environmentally benign method. Two relatively novel X-ray spectroscopy methods, which are relevant for the molecular understanding of the catalysts and biocatalysts, i.e. soft X-ray absorption spectroscopy and nuclear resonant vibration spectroscopy are presented. We illustrate the perceived reality in fertilizer usage on the field, and fertilizer production in the factory complex with photos and thus provide a contrast to the academic view of the molecular process of ammonia function and production.

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

A Surface-Strained and Geometry-Tailored Nanoreactor that Promotes Ammonia Electrosynthesis

A surface-strained and geometry-optimized TiO 2 nanoreactor enhances the performance of electrocatalytic nitrogen fixation. The nanotubular confinement allows spatial regulation of the mass transport of nitrogen during the NRR process and offers an enlarged surface area, thus boosting the ammonia production with high selectivity. Both experimental and theoretical evidence support strained Ti 3+ sites, demonstrating a more favorable pathway for the N 2 activation and selective NH 3 production with a faster kinetic rate than the pristine TiO 2 . The TiO 2 -based nanoreactor with surface and bulk structure tailoring delivered an NH 3 yield rate up to 5.50 μg h -1 cm -2 (16.67 μg h -1 mg cat -1 ) and high faradaic efficiency of 26 % under ambient aqueous conditions. Furthermore, our findings highlight the concept of lattice strain and geometry modified nanoreactors, which will have broad implications in the renewable energy catalysis and electrosynthesis of valuable products.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗
DOE OSTI2019

In-Situ Metabolomic Analysis of Setaria viridis Roots Colonized by Beneficial Endophytic Bacteria

Over the past decades, crop yields have risen in parallel with increasing use of fossil-fuel derived nitrogen (N) fertilizers, but with concomitant negative impacts on climate and water resources. There is a need for more sustainable agricultural practices, and biological nitrogen fixation (BNF) could be part of the solution. A variety of nitrogen-fixing, epiphytic and endophytic plant growth promoting bacteria (PGPB) are known to stimulate plant growth. However, compared to the rhizobium-legume symbiosis, little mechanistic information is available as to how PGPB affect plant metabolism. Therefore, we investigated the metabolic changes in roots of the model grass species Setaria viridis upon endophytic colonization by Herbaspirillum seropedicae SmR1 (fix + ) or a fix - mutant strain (SmR54), compared to uninoculated roots. Endophytic colonization of the root is highly localized and, hence, analysis of whole root segments dilutes the metabolic signature of those few cells impacted by the bacteria. Therefore, we utilized in situ laser ablation electrospray ionization mass spectrometry (LAESI-MS) to sample only those root segments at or adjacent to the sites of bacterial colonization. We report netabolites involved in purine, zeatin, and riboflavin pathways were significantly more abundant in inoculated plants while metabolites indicative of nitrogen, starch, and sucrose metabolism were reduced in roots inoculated with the fix - strain or uninoculated, presumably due to N limitation. Interestingly, compounds, involved in indole-alkaloid biosynthesis were more abundant in the roots colonized by the fix - strain, perhaps reflecting a plant defense response.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2023

Influence of sulfide on diazotrophic growth of the methanogen Methanococcus maripaludis and its implications for the origin of nitrogenase

Abstract Methanogens inhabit euxinic (sulfide-rich) or ferruginous (iron-rich) environments that promote the precipitation of transition metals as metal sulfides, such as pyrite, reducing metal or sulfur availability. Such environments have been common throughout Earth’s history raising the question as to how anaerobes obtain(ed) these elements for the synthesis of enzyme cofactors. Here, we show a methanogen can synthesize molybdenum nitrogenase metallocofactors from pyrite as the source of iron and sulfur, enabling nitrogen fixation. Pyrite-grown, nitrogen-fixing cells grow faster and require 25-fold less molybdenum than cells grown under euxinic conditions. Growth yields are 3 to 8 times higher in cultures grown under ferruginous relative to euxinic conditions. Physiological, transcriptomic, and geochemical data indicate these observations are due to sulfide-promoted metal limitation, in particular molybdenum. These findings suggest that molybdenum nitrogenase may have originated in a ferruginous environment that titrated sulfide to form pyrite, facilitating the availability of sufficient iron, sulfur, and molybdenum for cofactor biosynthesis.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2017

Divergent Responses of Forest Soil Microbial Communities under Elevated CO 2 in Different Depths of Upper Soil Layers

Numerous studies have shown that the continuous increase of atmosphere CO 2 concentrations may have profound effects on the forest ecosystem and its functions. However, little is known about the response of belowground soil microbial communities under elevated atmospheric CO 2 (eCO 2 ) at different soil depth profiles in forest ecosystems. In this paper, we examined soil microbial communities at two soil depths (0 to 5 cm and 5 to 15 cm) after a 10-year eCO 2 exposure using a high-throughput functional gene microarray (GeoChip). The results showed that eCO 2 significantly shifted the compositions, including phylogenetic and functional gene structures, of soil microbial communities at both soil depths. Key functional genes, including those involved in carbon degradation and fixation, methane metabolism, denitrification, ammonification, and nitrogen fixation, were stimulated under eCO 2 at both soil depths, although the stimulation effect of eCO 2 on these functional markers was greater at the soil depth of 0 to 5 cm than of 5 to 15 cm. Moreover, a canonical correspondence analysis suggested that NO 3 -N, total nitrogen (TN), total carbon (TC), and leaf litter were significantly correlated with the composition of the whole microbial community. This study revealed a positive feedback of eCO 2 in forest soil microbial communities, which may provide new insight for a further understanding of forest ecosystem responses to global CO 2 increases. The concentration of atmospheric carbon dioxide (CO 2 ) has continuously been increasing since the industrial revolution. Understanding the response of soil microbial communities to elevated atmospheric CO 2 (eCO 2 ) is important for predicting the contribution of the forest ecosystem to global atmospheric change. This study analyzed the effect of eCO 2 on microbial communities at two soil depths (0 to 5 cm and 5 to 15 cm) in a forest ecosystem. Our findings suggest that the compositional and functional structures of microbial communities shifted under eCO 2 at both soil depths. Finally, more functional genes involved in carbon, nitrogen, and phosphorus cycling were stimulated under eCO 2 at the soil depth of 0 to 5 cm than at the depth of 5 to 15 cm.

54 ENVIRONMENTAL SCIENCES↗
NASA NTRS1999

Mesoscale Eddies, Satellite Altimetry, and New Production in the Sargasso Sea

Satellite altimetry and hydrographic observations are used to characterize the mesoscale eddy field in the Sargasso Sea near Bermuda and to address the role of physical processes on the supply of new nutrients to the euphotic zone. The observed sea level anomaly (SLA) field is dominated by the occurrence of westward propagating features with SLA signatures as large as 25 cm, Eulerian temporal scales of roughly a month, lifetimes of several months, spatial scales of approximately 200 km, and a propagation of approximately 5 cm/s. Hydrographic estimates of dynamic height anomaly (referenced to 4000 dbar) are well correlated with satellite SLA (r(exp 2) = 0.65), and at least 85% of the observed dynamic height variability is associated with the first baroclinic mode of motion. This allows us to apply the satellite observations to remotely sensed estimate isopycnal displacements and the flux of nutrients into the euphotic zone due to eddy pumping. Eddy pumping is the process by which mesoscale eddies induce isopycnal displacements that lift nutrient-replete waters into the euphotic zone, driving new primary production. A kinematic approach to the estimation of the eddy pumping results in a flux of 0.24 +/- 0.1 mol N/sq m (including a scale estimate for the small contribution due to 18 deg water eddies). This flux is more than an order of magnitude larger than the diapycnal diffusive flux as well as scale estimates for the vertical transport due to isopycnal mixing along sloping isopycnal surfaces. Eddy pumping and wintertime convection are the two dominant mechanisms transporting new nutrients into the euphotic zone, and the sum of all physical new nutrient supply fluxes effectively balances previous geochemical estimates of annual new production for this site. However, if biological transports (e.g., nitrogen fixation, etc.) are significant, the new nitrogen supply budget will be in excess of geochemical new production estimates. This suggests that the various physical and biological transport fluxes, as well as geochemical inferences of new production, still need to be reconciled and many outstanding questions remain.

Siegel, David A.↗
NASA NTRS1999

Mesoscale Eddies, Satellite Altimetry, and New Production in the Sargasso Sea

Satellite altimetry and hydrographic observations are used to characterize the mesoscale eddy field in the Sargasso Sea near Bermuda and to address the role of physical processes on the supply of new nutrients to the euphotic zone. The observed sea level anomaly (SLA) field is dominated by the occurrence of westward propagating features with SLA signatures as large as 25 cm, Eulerian temporal scales of roughly a month, lifetimes of several months, spatial scales of approximately 200 km, and a propagation of approximately 5 cm/s . Hydrographic estimates of dynamic height anomaly (referenced to 4000 dbar) are well correlated with satellite SLA (r(sup 2) = 0.65), and at least 85% of the observed dynamic height variability is associated with the first baroclinic mode of motion. This allows us to apply the satellite observations to remotely estimate isopycnal displacements and the flux of nutrients into the euphotic zone due to eddy pumping. Eddy pumping is the process by which mesoscale eddies induce isopycnal displacements that lift nutrient- replete waters into the euphotic zone, driving new primary production. A kinematic approach to the estimation of the eddy pumping results in a flux of 0.24+/-0.1 mol N/sq m/yr (including a scale estimate for the small contribution due to 18 deg water eddies). This flux is more than an order of magnitude larger than the diapycnal diffusive flux as well as scale estimates for the vertical transport due to isopycnal mixing along sloping isopycnal surfaces. Eddy pumping and wintertime convection are the two dominant mechanisms transporting new nutrients into the euphotic zone, and the sum of all physical new nutrient supply fluxes effectively balances previous geochemical estimates of annual new production for this site. However, if biological transports (e.g., nitrogen fixation, etc.) are significant, the new nitrogen supply budget will be in excess of geochemical new production estimates. This suggests that the various physical and biological transport fluxes, as well as geochemical inferences of new production, still need to be reconciled and many outstanding questions remain.

Siegel, David A.↗
DOE OSTI2022

Defining the Sphagnum Core Microbiome across the North American Continent Reveals a Central Role for Diazotrophic Methanotrophs in the Nitrogen and Carbon Cycles of Boreal Peatland Ecosystems

Peat mosses of the genus Sphagnum are ecosystem engineers that frequently predominate over photosynthetic production in boreal peatlands. Sphagnum spp. host diverse microbial communities capable of nitrogen fixation (diazotrophy) and methane oxidation (methanotrophy), thereby potentially supporting plant growth under severely nutrient-limited conditions. Moreover, diazotrophic methanotrophs represent a possible “missing link” between the carbon and nitrogen cycles, but the functional contributions of the Sphagnum-associated microbiome remain in question. A combination of metagenomics, metatranscriptomics, and dual-isotope incorporation assays was applied to investigate Sphagnum microbiome community composition across the North American continent and provide empirical evidence for diazotrophic methanotrophy in Sphagnum-dominated ecosystems. Remarkably consistent prokaryotic communities were detected in over 250 Sphagnum SSU rRNA libraries from peatlands across the United States (5 states, 17 bog/fen sites, 18 Sphagnum species), with 12 genera of the core microbiome comprising 60% of the relative microbial abundance. Additionally, nitrogenase (nifH) and SSU rRNA gene amplicon analysis revealed that nitrogen-fixing populations made up nearly 15% of the prokaryotic communities, predominated by Nostocales cyanobacteria and Rhizobiales methanotrophs. While cyanobacteria comprised the vast majority (>95%) of diazotrophs detected in amplicon and metagenome analyses, obligate methanotrophs of the genus Methyloferula (order Rhizobiales) accounted for one-quarter of transcribed nifH genes. Furthermore, in dual-isotope tracer experiments, members of the Rhizobiales showed substantial incorporation of 13 CH 4 and 15 N 2 isotopes into their rRNA. Our study characterizes the core Sphagnum microbiome across large spatial scales and indicates that diazotrophic methanotrophs, here defined as obligate methanotrophs of the rare biosphere (Methyloferula spp. of the Rhizobiales) that also carry out diazotrophy, play a keystone role in coupling of the carbon and nitrogen cycles in nutrient-poor peatlands.

54 ENVIRONMENTAL SCIENCES↗
DOE OSTI2024

Novel candidate taxa contribute to key metabolic processes in Fennoscandian Shield deep groundwaters

The continental deep biosphere contains a vast reservoir of microorganisms, although a large proportion of its diversity remains both uncultured and undescribed. In this study, the metabolic potential (metagenomes) and activity (metatranscriptomes) of the microbial communities in Fennoscandian Shield deep subsurface groundwaters were characterized with a focus on novel taxa. DNA sequencing generated 1270 de-replicated metagenome-assembled genomes and single-amplified genomes, containing 7 novel classes, 34 orders, and 72 families. The majority of novel taxa were affiliated with Patescibacteria, whereas among novel archaea taxa, Thermoproteota and Nanoarchaeota representatives dominated. Metatranscriptomes revealed that 30 of the 112 novel taxa at the class, order, and family levels were active in at least one investigated groundwater sample, implying that novel taxa represent a partially active but hitherto uncharacterized deep biosphere component. The novel taxa genomes coded for carbon fixation predominantly via the Wood–Ljungdahl pathway, nitrogen fixation, sulfur plus hydrogen oxidation, and fermentative pathways, including acetogenesis. These metabolic processes contributed significantly to the total community’s capacity, with up to 9.9% of fermentation, 6.4% of the Wood–Ljungdahl pathway, 6.8% of sulfur plus 8.6% of hydrogen oxidation, and energy conservation via nitrate (4.4%) and sulfate (6.0%) reduction. Key novel taxa included the UBA9089 phylum, with representatives having a prominent role in carbon fixation, nitrate and sulfate reduction, and organic and inorganic electron donor oxidation. These data provided insights into deep biosphere microbial diversity and their contribution to nutrient and energy cycling in this ecosystem.

Candidatus↗
DOE OSTI2020

Diazotrophs Show Signs of Restoration in Amazon Rain Forest Soils with Ecosystem Rehabilitation

Biological nitrogen fixation can be an important source of nitrogen in tropical forests that serve as a major CO 2 sink. Extensive deforestation of the Amazon is known to influence microbial communities and the biogeochemical cycles they mediate. However, it is unknown how diazotrophs (nitrogen-fixing microorganisms) respond to deforestation and subsequent ecosystem conversion to agriculture, as well as whether they can recover in secondary forests that are established after agriculture is abandoned. To address these knowledge gaps, here we combined a spatially explicit sampling approach with high-throughput sequencing of nifH genes. The main objectives were to assess the functional distance decay relationship of the diazotrophic bacterial community in a tropical forest ecosystem and to quantify the roles of various factors that drive the observed changes in the diazotrophic community structure. We observed an increase in local diazotrophic diversity (α-diversity) with a decrease in community turnover (β-diversity), associated with a shift in diazotrophic community structure as a result of the forest-to-pasture conversion. Both diazotrophic community turnover and structure showed signs of recovery in secondary forests. Changes in the diazotrophic community were primarily driven by the change in land use rather than differences in geochemical characteristics or geographic distances. The diazotroph communities in secondary forests resembled those in primary forests, suggesting that at least partial recovery of diazotrophs is possible following agricultural abandonment. IMPORTANCE The Amazon region is a major tropical forest region that is being deforested at an alarming rate to create space for cattle ranching and agriculture. Diazotrophs (nitrogen-fixing microorganisms) play an important role in supplying soil N for plant growth in tropical forests. It is unknown how diazotrophs respond to deforestation and whether they can recover in secondary forests that establish after agriculture is abandoned. Using high-throughput sequencing of nifH genes, we characterized the response of diazotrophs’ β-diversity and identified major drivers of changes in diazotrophs from forest-to-pasture and pasture-to-secondary-forest conversions. Studying the impact of land use change on diazotrophs is important for a better understanding of the impact of deforestation on tropical forest ecosystem functioning, and our results on the potential recovery of diazotrophs in secondary forests imply the possible restoration of ecosystem functions in secondary forests.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2023

Genome-Resolved Metagenomics of Nitrogen Transformations in the Switchgrass Rhizosphere Microbiome on Marginal Lands

Switchgrass (Panicum virgatum L.) remains the preeminent American perennial (C4) bioenergy crop for cellulosic ethanol, that could help displace over a quarter of the US current petroleum consumption. Intriguingly, there is often little response to nitrogen fertilizer once stands are established. The rhizosphere microbiome plays a critical role in nitrogen cycling and overall plant nutrient uptake. We used high-throughput metagenomic sequencing to characterize the switchgrass rhizosphere microbial community before and after a nitrogen fertilization event for established stands on marginal land. We examined community structure and bulk metabolic potential, and resolved 29 individual bacteria genomes via metagenomic de novo assembly. Community structure and diversity were not significantly different before and after fertilization; however, the bulk metabolic potential of carbohydrate-active enzymes was depleted after fertilization. We resolved 29 metagenomic assembled genomes, including some from the ‘most wanted’ soil taxa such as Verrucomicrobia, Candidate phyla UBA10199, Acidobacteria (rare subgroup 23), Dormibacterota, and the very rare Candidatus Eisenbacteria. The Dormibacterota (formally candidate division AD3) we identified have the potential for autotrophic CO utilization, which may impact carbon partitioning and storage. Our study also suggests that the rhizosphere microbiome may be involved in providing associative nitrogen fixation (ANF) via the novel diazotroph Janthinobacterium to switchgrass.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2019

Development and refinement of an in situ ‘molecular microscope’ utilizing ultrahigh resolution mass spectrometry

The ability to elucidate complex metabolic behavior of living organisms in situ and with cellular resolution is a bioanalytical grand challenge that is yet to be realized. Mass spectrometry imaging (MSI) using laser ablation electrospray ionization mass spectrometry (LAESI) has recently been demonstrated, and holds promise for direct spatially resolved molecular profiling in a variety of living systems of DOE relevance (e.g., plant leaf, stem, and root tissue). The LAESI MSI approach has revealed cell-to-cell heterogeneity of metabolites within living plant tissue, in a multiplexed fashion and without the need for labeling. Cellular resolution by in situ, ambient pressure MSI methods are far from routine, however, and have inadequate spatial resolution for many biological questions of interest. We propose to develop next-generation LAESI approaches, using advanced fiber optics and transmission-based geometries, in efforts to attain dynamic bioanalysis of living systems at better than 20 µm lateral resolution. In parallel with LAESI innovations, attendant increases in sensitivity and dynamic range of the mass spectrometer system will also be necessary for detection of the limited amount of material being ionized. To respond to this need, a new, high magnetic field (21 Tesla) Fourier Transform Ion Cyclotron Resonance (FTICR) MS at the EMSL/PNNL will be coupled with the newly developed LAESI source. This mass spectrometer, currently being assembled, will be one of only two such systems in the world, and the only one in a DOE laboratory or user facility. Fundamentally, all key figures of merits of FTICR MS performance improve with increased magnetic field strength, providing the ability to achieve unparalleled molecular specificity (near unequivocal molecular constituent identification) at the highest possible mass resolving power and sensitivity. The work proposed here will greatly increase the utility of this FTICR MS instrument, providing unique capabilities to support BER-relevant science. The research plan is comprised of three tasks, two of which are directed by the academic leads (University of Missouri and George Washington University), and one of which will be performed in conjunction with the BER user facility at EMSL/PNNL. In task 1, advancements of the LAESI technology will be achieved, with oversight and leadership of Professor Akos Vertes of George Washington University. In task 2, incorporation of this ion source with EMSL’s new 21T FTICR MS system will be undertaken, supplemented by global omics measurements and calibrated against more conventional MSI platforms also available at EMSL (Drs. Pasa-Tolic, Anderton, and Koppenaal team). In the final task, the resultant innovations and advancements will be tested and validated by addressing important BER relevant science questions on model systems of interest under the direction of Professor Gary Stacey of the University of Missouri. This research follows on key recommendations of the recent BER “Research in Sustainable Bioenergy” report that, for example, stressed that “The design of sustainable biofuel systems requires knowledge about key plant-microbe-environment interactions that provide a range of ecosystem services.” This task will focus on validation of the instrumentation developed through this proposal, advertising this capability to the community through demonstration and, finally, discovering new biology relevant to the BER mission. This final objective will make use of Setaria viridis, a model C4 grass species, as well as switchgrass and sorghum, two promising biofuel crops. Specifically, we will demonstrate applicability to plant-microbe rhizosphere interactions focusing on the biological nitrogen fixation by monitoring metabolic changes in real-time with nutrient, growth condition, and other perturbations.

09 BIOMASS FUELS↗