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At least 19 records

Amino Acids Dominate Diffusive Nitrogen Fluxes in Acidic Tussock Tundra

Organic nitrogen (N) is abundant in soils, but early conceptual frameworks considered it nonessential for plant growth. It is now well recognised that plants have the potential to take up organic N. However, it is still unclear whether plants supplement their N requirements by taking up organic N in situ: at what rate is organic N diffusing towards roots and are plants taking it up? In this work, we combined microdialysis with live-root uptake experiments to measure amino acid speciation and diffusion rates towards roots of Eriophorum vaginatum. Amino acid diffusion rates (321 ng N cm -2 h -1 ) were c. 3× higher than those for inorganic N. Positively charged amino acids made up 68% of the N diffusing through soils compared with neutral and negatively charged amino acids. Live-root uptake experiments confirmed that amino acids are taken up by plants (up to 1 µg N g -1 min -1 potential net uptake). Amino acids must be considered when forecasting plant-available N, especially when they dominate the N supply, and when acidity favours proteolysis over net N mineralisation. Determining amino acid production pathways and supply rates will become increasingly important in projecting the extent and consequences of shrub expansion, especially considering the higher C : N ratio of plants relative to soil.

54 ENVIRONMENTAL SCIENCES↗

Collaborative Project: Building improved optimized parameter estimation algorithms to improve methane and nitrogen fluxes in a climate model

Soils in natural and managed ecosystems and wetlands are well known sources of methane, nitrous oxides, and reactive nitrogen gases, but the magnitudes of gas flux to the atmosphere are still poorly constrained. Thus, the reasons for the large increases in atmospheric concentrations of methane and nitrous oxide since the preindustrial time period are not well understood. The low atmospheric concentrations of methane and nitrous oxide, despite being more potent greenhouse gases than carbon dioxide, complicate empirical studies to provide explanations. In addition to climate concerns, the emissions of reactive nitrogen gases from soils are important to the changing nitrogen balance in the earth system, subject to human management, and may change substantially in the future. Thus improved modeling of the emission fluxes of these species from the land surface is important. Currently, there are emission modules for methane and some nitrogen species in the Community Earth System Model’s Community Land Model (CLM-ME/N); however, there are large uncertainties and problems in the simulations, resulting in coarse estimates. In this proposal, we seek to improve these emission modules by combining state-of-the-art process modules for emissions, available data, and new optimization methods. In earth science problems, we often have substantial data and knowledge of processes in disparate systems, and thus we need to combine data and a general process level understanding into a model for projections of future climate that are as accurate as possible. The best methodologies for optimization of parameters in earth system models are still being developed. In this proposal we will develop and apply surrogate algorithms that a) were especially developed for computationally expensive simulations like CLM-ME/N models; b) were (in the earlier surrogate optimization Stochastic RBF) demonstrated to perform very well on computationally expensive complex partial differential equations in earth science with limited numbers of simulations; and, c) will be (as part of the proposed research) significantly improved both by adding asynchronous parallelism, early truncation of unsuccessful simulations, and the improvement of both serial and parallel performance by the use of derivative and sensitivity information from global and local surrogate approximations S(x) . The algorithm development and testing will be focused on the CLM-ME/N model application, but the methods are general and are expected to also perform well on optimization for parameter estimation of other climate models and other classes of continuous multimodal optimization problems arising from complex simulation models. In addition, this proposal will compile available datasets of emissions of methane, nitrous oxides and reactive nitrogen species and develop protocols for site level comparisons with the CLM-ME/N. Once the model parameters are optimized against site level data, the model will be simulated at the global level and compared to atmospheric concentration measurements for the current climate, and future emissions will be estimated using climate change as simulated by the CESM. This proposal combines experts in earth system modeling, optimization, computer science, and process level understanding of soil gas emissions in an interdisciplinary team in order to improve the modeling of methane and nitrogen gas emissions. This proposal thus meets the requirements of the SciDAC RFP, by integrating state-of-the-art computer science and earth system to build an improved earth system model.

54 ENVIRONMENTAL SCIENCES↗

Evolution and regulation of nitrogen flux through compartmentalized metabolic networks in a marine diatom

Diatoms dominate phytoplankton communities by outcompeting other taxa for nitrogen, yet little is known about the molecular underpinnings of this ability. As a consequence of their evolutionary history, diatoms possess traits that are both autotroph- and heterotroph-like expanding their metabolic capabilities beyond other model organisms. Here we use the model marine diatom Phaeodactylum tricornutum to investigate the extent to which diatoms have combined elements of nitrogen metabolism from each ancestral partner. Using a range of experimental and metabolic modeling approaches, we found compartment-specific assimilation of different forms of nitrogen, requiring the operation of metabolite shuttles to synchronize organellar metabolism. Further we identified a hybrid arginine biosynthesis pathway, clarifying the role of the urea cycle in diatoms. We conclude that metabolic integration between the major energy organelles, established post-endosymbiosis in diatoms, confers an advantage for nitrogen utilization of and contributes to their ecological success in a highly dynamic marine environment.

59 BASIC BIOLOGICAL SCIENCES↗

Elevated temperature increases carbon and nitrogen fluxes between phytoplankton and heterotrophic bacteria through physical attachment

Quantifying the contribution of marine microorganisms to carbon and nitrogen cycles and their response to predicted ocean warming is one of the main challenges of microbial oceanography. Here we present a single-cell NanoSIMS isotope analysis to quantify C and N uptake by free-living and attached phytoplankton and heterotrophic bacteria, and their response to short-term experimental warming of 4 °C. Elevated temperature increased total C fixation by over 50%, a small but significant fraction of which was transferred to heterotrophs within 12 h. Cell-to-cell attachment doubled the secondary C uptake by heterotrophic bacteria and increased secondary N incorporation by autotrophs by 68%. Warming also increased the abundance of phytoplankton with attached heterotrophs by 80%, and promoted C transfer from phytoplankton to bacteria by 17% and N transfer from bacteria to phytoplankton by 50%. Lastly, our results indicate that phytoplankton-bacteria attachment provides an ecological advantage for nutrient incorporation, suggesting a mutualistic relationship that appears to be enhanced by temperature increases.

54 ENVIRONMENTAL SCIENCES↗

Constructing the Nitrogen Flux Maps (NFMs) of Plants

The main objectives of this project are to construct plant N flux maps (NFMs) from plant genomes and to determine functionality of AT enzymes and plant N metabolic network. To address this grand challenge, this project made use of rapidly growing numbers of plant genomes, high-throughput functional characterization platforms, and computational modeling to deduce both biochemical and systems level functionality of ATs and NFMs. The obtained NFMs will provide a novel framework to advance basic understanding of plant N metabolism and facilitate rational engineering of plants with high productivity even under limited N input.

59 BASIC BIOLOGICAL SCIENCES↗

Quorum Quenching of Nitrobacter winogradskyi Suggests that Quorum Sensing Regulates Fluxes of Nitrogen Oxide(s) during Nitrification

ABSTRACT Quorum sensing (QS) is a widespread process in bacteria used to coordinate gene expression with cell density, diffusion dynamics, and spatial distribution through the production of diffusible chemical signals. To date, most studies on QS have focused on model bacteria that are amenable to genetic manipulation and capable of high growth rates, but many environmentally important bacteria have been overlooked. For example, representatives of proteobacteria that participate in nitrification, the aerobic oxidation of ammonia to nitrate via nitrite, produce QS signals called acyl-homoserine lactones (AHLs). Nitrification emits nitrogen oxide gases (NO, NO 2 , and N 2 O), which are potentially hazardous compounds that contribute to global warming. Despite considerable interest in nitrification, the purpose of QS in the physiology/ecology of nitrifying bacteria is poorly understood. Through a quorum quenching approach, we investigated the role of QS in a well-studied AHL-producing nitrite oxidizer, Nitrobacter winogradskyi . We added a recombinant AiiA lactonase to N. winogradskyi cultures to degrade AHLs to prevent their accumulation and to induce a QS-negative phenotype and then used mRNA sequencing (mRNA-Seq) to identify putative QS-controlled genes. Our transcriptome analysis showed that expression of nirK and nirK cluster genes ( ncgABC ) increased up to 19.9-fold under QS-proficient conditions (minus active lactonase). These data led to us to query if QS influenced nitrogen oxide gas fluxes in N. winogradskyi . Production and consumption of NO x increased and production of N 2 O decreased under QS-proficient conditions. Quorum quenching transcriptome approaches have broad potential to identify QS-controlled genes and phenotypes in organisms that are not genetically tractable. IMPORTANCE Bacterial cell-cell signaling, or quorum sensing (QS), is a method of bacterial communication and gene regulation that is well studied in bacteria. However, little is known about the purpose of QS in many environmentally important bacteria. Here, we demonstrate quorum quenching coupled with mRNA-Seq to identify QS-controlled genes and phenotypes in Nitrobacter winogradskyi , a nitrite-oxidizing bacterium. Nitrite oxidizers play an important role in the nitrogen cycle though their participation in nitrification, the aerobic oxidation of ammonia to nitrate via nitrite. Our quorum quenching approach revealed that QS influences production and consumption of environmentally important nitrogen oxide gases (NO, NO 2 , and N 2 O) in N. winogradskyi . This study demonstrated a novel technique for studying QS in difficult-to-work-with microorganisms and showed that nitrite oxidizers might also contribute to nitrification-dependent production of nitrogen oxide gases that contribute to global warming.

Mellbye, Brett L.↗

Nitrous oxide flux and nitrogen transformations across a landscape gradient in Amazonia

Nitrous oxide flux and nitrogen turnover were measured in three types of Amazonian forest ecosystems within Reserva Florestal Ducke near Manaus, Brazil. Nitrogen mineralization and nitrate production measured during 10-day laboratory incubations were 3-4 times higher in clay soils associated with 'terra firme' forests on ridge-top and slope positions than in 'campinarana' forests on bottomland sand soils. In contrast, nitrous oxide fluxes did not differ significantly among sites, but were highly variable in space and time. The observed frequency distribution of flux was positively skewed, with a mean overall sites and all sampling times of 1.3 ng N2O-N/sq cm per hr. Overall, the flux estimates were comparable to or greater than those of temperature forests, but less than others reported for Amazoonia. Results from a field fertilization experiment suggest that most nitrous oxide flux was associated with denitrification of soil nitrate.

Livingston, Gerald P.↗

Genome-Scale, Constraint-Based Modeling of Nitrogen Oxide Fluxes during Coculture of Nitrosomonas europaea and Nitrobacter winogradskyi

Nitrification, the aerobic oxidation of ammonia to nitrate via nitrite, emits nitrogen (N) oxide gases (NO, NO 2 , and N 2 O), which are potentially hazardous compounds that contribute to global warming. To better understand the dynamics of nitrification-derived N oxide production, we conducted culturing experiments and used an integrative genome-scale, constraint-based approach to model N oxide gas sources and sinks during complete nitrification in an aerobic coculture of two model nitrifying bacteria, the ammonia-oxidizing bacterium Nitrosomonas europaea and the nitrite-oxidizing bacterium Nitrobacter winogradskyi. The model includes biotic genome-scale metabolic models (iFC578 and iFC579) for each nitrifier and abiotic N oxide reactions. Modeling suggested both biotic and abiotic reactions are important sources and sinks of N oxides, particularly under microaerobic conditions predicted to occur in coculture. In particular, integrative modeling suggested that previous models might have underestimated gross NO production during nitrification due to not taking into account its rapid oxidation in both aqueous and gas phases. The integrative model may be found at https://github.com/chaplenf/microBiome-v2.1.

59 BASIC BIOLOGICAL SCIENCES↗

Agricultural impacts on nitrogen cycling: climate and air pollution

Ratios of key soil nitrogen fluxes from two terrestrial ecosystem models (TEMs) are compared to observational values in the literature. The fluxes included nitrification, denitrification, mineralization, immobilization and plant uptake, with the latter two fluxes partitioned into NH 4 + and NO 3 -. The two TEMs, ELMv1-ECA and CLM5.0, originated from a common model but have diverged in their handling of plant/microbe competition for soil mineral N. Both models predict unrealistically low assimilation (i.e, immobilization) of NO 3 - by soil bacteria compared to literature values and unrealistic dominance of plant uptake by a single form of mineral nitrogen (NO 3 - for ELM, NH 4 + for CLM5.0). Global ratios of net mineralization (i.e., gross mineralization – gross immobilization) appear unrealistically high in both models compared to literature values while rates of nitrification/gross mineralization appear unrealistically high (ELM) and low (CLM5.0). The ratio of denitrification/nitrification is likely high in both models (3:1 ELM; 2:1 CLM5.0), but few experimental data exist to evaluate these ratios, in part because nitrification and denitrification are quantified with different techniques and generally are not measured simultaneously. The same is true of plant N uptake and microbial N assimilation. Single grid point experiments with CLM5.0 at the Hubbard Brook long-term ecological research site in New Hampshire indicate that the parameterization of nitrification is a potentially important focus area that can bring soil nitrogen flux ratios into better agreement with observations. The current flux ratios raise questions about the credibility of present and future simulations of mineral N limitation on the carbon cycle.

54 ENVIRONMENTAL SCIENCES↗

Improving the SWAT forest module for enhancing water resource projections: A case study in the St. Croix River basin

Abstract Insufficiently calibrated forest parameters of the Soil & Water Assessment Tool (SWAT) may introduce uncertainties to water resource projections in forested watersheds. In this study, we improved SWAT forest parameterization and phosphorus cycling representations to better simulate forest ecosystems in the St. Croix River basin, and we further examined how those improvements affected model projections of streamflow, sediment, and nitrogen export under future climate conditions. Simulations with improved forest parameters substantially reduced model estimates of water, sediment, and nitrogen fluxes relative to those based on default parameters. Differences between improved and default projections can be attributed to the enhanced representation of forest water consumption, nutrient uptake, and protection of soil from erosion. Better representation of forest ecosystems in SWAT contributes to constraining uncertainties in water resource projections. Results of this study highlight the importance of improving SWAT forest ecosystem representations in projecting delivery of water, sediment, and nutrients from land to rivers in response to climate change, particularly for watersheds with large areas of forests. Improved forest parameters and the phosphorus weathering algorithms developed in this study are expected to help enhance future applications of SWAT to investigate hydrological and biogeochemical consequences of climate change.

Yang, Qichun↗

Ammonia-oxidizing bacteria and archaea exhibit differential nitrogen source preferences

Ammonia-oxidizing microorganisms (AOM) contribute to one of the largest nitrogen fluxes in the global nitrogen budget. Four distinct lineages of AOM: ammonia-oxidizing archaea (AOA), beta- and gamma-proteobacterial ammonia-oxidizing bacteria (β-AOB and γ-AOB) and complete ammonia oxidizers (comammox), are thought to compete for ammonia as their primary nitrogen substrate. In addition, many AOM species can utilize urea as an alternative energy and nitrogen source through hydrolysis to ammonia. How the coordination of ammonia and urea metabolism in AOM influences their ecology remains poorly understood. Here we use stable isotope tracing, kinetics and transcriptomics experiments to show that representatives of the AOM lineages employ distinct regulatory strategies for ammonia or urea utilization, thereby minimizing direct substrate competition. The tested AOA and comammox species preferentially used ammonia over urea, while β-AOB favoured urea utilization, repressed ammonia transport in the presence of urea and showed higher affinity for urea than for ammonia. Characterized γ-AOB co-utilized both substrates. Furthermore, these results reveal contrasting niche adaptation and coexistence patterns among the major AOM lineages.

54 ENVIRONMENTAL SCIENCES↗

Metabolic Fluxes of Nitrogen and Pyrophosphate in Chemostat Cultures of Clostridium thermocellum and Thermoanaerobacterium saccharolyticum

This study discusses the fate of pyrophosphate in the metabolism of two thermophilic anaerobes that lack a soluble irreversible pyrophosphatase as present in Escherichia coli but instead use a reversible membrane-bound proton-pumping enzyme. In such organisms, the charging of tRNA with amino acids may become more reversible. This may contribute to the observed excretion of amino acids during sugar fermentation by Clostridium thermocellum and Thermoanaerobacterium saccharolyticum . Calculation of the energetic advantage of reversible pyrophosphate-dependent glycolysis, as occurs in Clostridium thermocellum , could not be properly evaluated, as currently available genome-scale models neglect the anabolic generation of pyrophosphate in, for example, polymerization of amino acids to protein. This anabolic pyrophosphate replaces ATP and thus saves energy. Its amount is, however, too small to cover the pyrophosphate requirement of sugar catabolism in glycolysis. Consequently, pyrophosphate for catabolism is generated according to ATP + P i → ADP + PP i .

59 BASIC BIOLOGICAL SCIENCES↗

Bedrock weathering rates, reactive nitrogen influxes and effluxes, and nitrous oxide emissions rates from the Pumphouse Hillslope, East River Watershed, Colorado

Atmospheric nitrous oxide contributes directly to global warming, yet models of the nitrogen cycle do not account for bedrock, the largest pool of terrestrial nitrogen, as a source of nitrous oxide. Although it is known that release rates of nitrogen from bedrock are large, there is an incomplete understanding of the connection between bedrock-hosted nitrogen and atmospheric nitrous oxide. Here, we quantify nitrogen fluxes and mass balances at a hillslope underlain by marine shale. We found that at this site bedrock weathering contributes 78% of the subsurface reactive nitrogen, while atmospheric sources (commonly regarded as the sole sources of reactive nitrogen in pristine environments) account for only the remaining 22%. About 56% of the total subsurface reactive nitrogen denitrifies, including 14% emitted as nitrous oxide. The remaining reactive nitrogen discharges in porewaters to a floodplain where additional denitrification likely occurs. We also found that the release of bedrock nitrogen occurs primarily within the zone of the seasonally fluctuating water table and suggest that the accumulation of nitrate in the vadoes zone, often attributed to fertilization and soil leaching, may also include contributions from weathered nitrogen-rich bedrock. Our hillslope study suggests that under oxygenated and moisture-rich conditions, weathering of deep, nitrogen-rich bedrock makes an important contribution to the nitrogen cycle. The data files are in Excel, which can be accessed using Microsoft Office, and consist of many data sets from the Pumphouse Hillslope PLM (Pumphouse Lower Montane) 1, 2, 3, and 4. They include soil to rock (0-10 meters) solid phase minerals and elements compositions; time- and depth-resolved pore-water chemistry and pore-gas compositions; time- and depth-resolved water table depths and water fluxes; subsurface weathering rates; nitrogen influxes and effluxes and mass balance. The attached paper is in a Word document, which can be accessed using Microsoft Office, and in pdf format.

54 ENVIRONMENTAL SCIENCES↗

Cyanobacterial Biofertilizer Production by Guanidine-Producing Enzymes

Cyanobacterial production of a biofertilizer shows promise as an environmentally benign alternative to conventional nitrogen fertilizers, reducing environmental and energy burdens through light-driven nitrogen and carbon fixation. One route to realizing the potential for a nitrogen-rich, slow-releasing biofertilizer involves the genetic engineering of cyanobacteria to produce guanidine. Recent advances have demonstrated enzymatic guanidine production in cyanobacteria, but an understanding of cyanobacterial guanidine metabolism is still limited. This Perspective highlights strategies and opportunities for cyanobacterial guanidine production in a Design−Build−Test−Learn cycle. Exploring new guanidine-producing enzymes via phylogenetics could expand candidate enzymes, while understanding the metabolism of substrates can identify constraints and opportunities in substrate utilization. Additionally, guanidine sensing and export are crucial areas of study to enable continuous fertilizer production and stable nitrogen flux. These strategies will guide the development of advanced nitrogen biofertilizer strategies for the agricultural sector.

09 BIOMASS FUELS↗

Combined buoyancy and flow direction effects on saturated boiling critical heat flux in liquid nitrogen

Buoyancy effects on the critical heat flux and general data trends for a liquid nitrogen internal flow system were determined by comparison of upflow and downflow data under identical test conditions. The test section had a 1.28 cm diameter flow passage and a 30.5 cm heated length which was subjected to uniform heat fluxes through resistance heating. Test conditions covered a range of pressures from 3.4 to 10.2 atm, inlet velocities from 0.23 to 3.51 m/sec, with the liquid nitrogen temperature at saturated inlet conditions. Data comparisons showed that the critical heat flux for downflow could be up to 36 percent lower than for upflow. A nonmonotonic relationship between the critical heat flux and velocity was determined for upflow but not for downflow. A limiting inlet velocity of 4.12 m/sec was determined to be the minimum velocity required to completely suppress the influence of buoyancy on the critical heat flux for this saturated inlet flow system. A correlation of this limiting fluid velocity is presented that was developed from previously published subcooled liquid nitrogen data and the saturated data of this investigation.

Papell, S. S.↗

Systematic comparison of local approaches for isotopically nonstationary metabolic flux analysis

Quantification of reaction fluxes of metabolic networks can help us understand how the integration of different metabolic pathways determine cellular functions. Yet, intracellular fluxes cannot be measured directly but are estimated with metabolic flux analysis (MFA) that relies on the patterns of isotope labeling of metabolites in the network. For metabolic systems, typical for plants, where all potentially labeled atoms effectively have only one source atom pool, only isotopically nonstationary MFA can provide information about intracellular fluxes. There are several global approaches that implement MFA for an entire metabolic network and estimate, at once, a steady-state flux distribution for all reactions with identifiable fluxes in the network. In contrast, local approaches deal with estimation of fluxes for a subset of reactions, with smaller data demand for flux estimation. Here we present a systematic comparative review and benchmarking of the existing local approaches for isotopically nonstationary MFA. The comparison is conducted with respect to the required data and underlying computational problems solved on a synthetic network example. Furthermore, we benchmark the performance of these approaches in estimating fluxes for a subset of reactions using data obtained from the simulation of nitrogen fluxes in the Arabidopsis thaliana core metabolism. The findings pinpoint practical aspects that need to be considered when applying local approaches for flux estimation in large-scale plant metabolic networks.

59 BASIC BIOLOGICAL SCIENCES↗