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DOE OSTI2026

Metabolic rewiring and biomass redistribution enable optimized mixotrophic growth in Chlamydomonas

Aquatic photosynthetic systems account for approximately one-half of all global carbon assimilation and could be a significant source of renewable fuels and feedstocks. However, rapid growth and biomass production in algae have not always translated into high product yields, partly because central metabolism is context specific, with metabolic fluxes being influenced by nutrient conditions and other environmental factors. In the green microalga Chlamydomonas reinhardtii (Chlamydomonas), mixotrophic cultures (acetate + light) grow far faster than phototrophic (light only) or heterotrophic (acetate + dark) cultures, even though acetate partially suppresses photosynthesis. Here, an isotopic dilution strategy with unlabeled acetate was combined with 13 CO 2 transient labeling to perform isotopically nonstationary metabolic flux analysis (INST-MFA) and to directly compare autotrophic and mixotrophic metabolism in Chlamydomonas supported by data from transcriptomics, proteomics, and metabolomics. INST-MFA indicated that acetate induces a synergistic rewiring of metabolism, conserving carbon by using the glyoxylate cycle and suppressing gluconeogenesis, the latter of which was discordant with omics results and prior models. Additionally, our data provide a plausible rationale for the well-known suppression of photosynthesis by acetate. We propose that reduced total protein content in mixotrophic versus phototrophic cells, much of which is attributed to reduced levels of photosynthetic proteins, decreases the costly metabolic burden of protein synthesis and represents a growth rate optimization strategy.

59 BASIC BIOLOGICAL SCIENCES↗

NASA NTRS2000

Growth, pod, and seed yield, and gas exchange of hydroponically grown peanut in response to CO2 enrichment

The effects of elevated CO2 on growth, pod, and seed yield, and gas exchange of 'Georgia Red' peanut (Arachis hypogaea L.) were evaluated under controlled environmental conditions. Plants were exposed to concentrations of 400 (ambient), 800, and 1200 micromoles mol-1 CO2 in reach-in growth chambers. Foliage fresh and dry weights increased with increased CO2 up to 800 micromoles mol-1, but declined at 1200 micromoles mol-1. The number and the fresh and dry weights of pods also increased with increasing CO2 concentration. However, the yield of immature pods was not significantly influenced by increased CO2. Total seed yield increased 33% from ambient to 800 micromoles mol-1 CO2, and 4% from 800 to 1200 micromoles mol-1 CO2. Harvest index increased with increasing CO2. Branch length increased while specific leaf area decreased linearly as CO2 increased from ambient to 1200 micromoles mol-1. Net photosynthetic rate was highest among plants grown at 800 micromoles mol-1. Stomatal conductance decreased with increased CO2. Carboxylation efficiency was similar among plants grown at 400 and 800 micromoles mol-1 and decreased at 1200 micromoles mol-1 CO2. These results suggest that CO2 enrichment from 400 to 800 micromoles mol-1 had positive effects on peanut growth and yield, but above 800 micromoles mol-1 enrichment seed yield increased only marginally.

NASA Discipline Life Support Systems↗

NASA NTRS2005

Carbon Dynamics in Vegetation and Soils

The overall goals of CD-08 team in Phase I were to quantify the contributions of different components of the carbon cycle to overall ecosystem carbon balance in Amazonian tropical forests and to undertake process studies at a number of sites along the eastern LBA transect to understand how and why these fluxes vary with site, season, and year. We divided this work into a number of specific tasks: (1) determining the average rate (and variability) of tree growth over the past 3 decades; (2) determining age demographics of tree populations, using radiocarbon to determine tree age; (3) assessing the rate of production and decomposition of dead wood debris; (4) determining turnover rates for organic matter in soils and the mean age of C respired from soil using radiocarbon measurements; and (5) comparing our results with models and constructing models to predict the potential of tropical forests to function as sources or sinks of C. This report summarizes the considerable progress made towards our original goals, which have led to increased understanding of the potential for central Amazon forests to act as sources or sinks of carbon with altered productivity. The overall picture of tropical forest C dynamics emerging from our Phase I studies suggests that the fraction of gross primary production allocated to growth in these forests is only 25-30%, as opposed to the 50% assumed by many ecosystem models. Consequent slow tree growth rates mean greater mean tree age for a given diameter, as reflected in our measurements and models of tree age. Radiocarbon measurements in leaf and root litter suggest that carbon stays in living tree biomass for several years up to a decade before being added to soils, where decomposition is rapid. The time lags predicted from 14C, when coupled with climate variation on similar time scales, can lead to significant interannual variation in net ecosystem C exchange.

Trumbore, Susan↗

DOE OSTI2024

Phenotypic and genomic characterization of Methanothermobacter wolfeii strain BSEL, a CO 2 -capturing archaeon with minimal nutrient requirements

A new variant of Methanothermobacter wolfeii was isolated from an anaerobic digester using enrichment cultivation in anaerobic conditions. Here, the new isolate was taxonomically identified via 16S rRNA gene sequencing and tagged as M. wolfeii BSEL. The whole genome of the new variant was sequenced and de novo assembled. Genomic variations between the BSEL strain and the type strain were discovered, suggesting evolutionary adaptations of the BSEL strain that conferred advantages while growing under a low concentration of nutrients. M. wolfeii BSEL displayed the highest specific growth rate ever reported for the wolfeii species (0.27 ± 0.03 h –1 ) using carbon dioxide (CO 2 ) as unique carbon source and hydrogen (H 2 ) as electron donor. M. wolfeii BSEL grew at this rate in an environment with ammonium (NH 4 + ) as sole nitrogen source. The minerals content required to cultivate the BSEL strain was relatively low and resembled the ionic background of tap water without mineral supplements. Optimum growth rate for the new isolate was observed at 64°C and pH 8.3. In this work, it was shown that wastewater from a wastewater treatment facility can be used as a low-cost alternative medium to cultivate M. wolfeii BSEL. Continuous gas fermentation fed with a synthetic biogas mimic along with H 2 in a bubble column bioreactor using M. wolfeii BSEL as biocatalyst resulted in a CO 2 conversion efficiency of 97% and a final methane (CH 4 ) titer of 98.5%v, demonstrating the ability of the new strain for upgrading biogas to renewable natural gas.

09 BIOMASS FUELS↗

NASA NTRS2017

Where Is the Upper Altitude Boundary of Earth's Biosphere? An Upcoming Stratosphere Exploration Mission

Earths atmosphere provides a relatively thin barrier to the severe conditions of space. Terrestrial microorganisms from our planets surface gets swept up into the atmosphere at rates which are influenced by meteorology, season, and variety of other regionally-dependent conditions. Residence time of microbes in the atmosphere, dispersal patterns, and metabolic activity in clouds are topics of special interest in tropospheric aerobiology. But what about even higher altitudes? A century of ocean exploration has allowed the scientific community to characterize marine life at every depth. Such knowledge was enabled by routine access, specifically, submersible vehicles with sample return systems. The outer reaches of Earth's atmosphere probably contain trace signatures of microbial life swept up from the surface, but high-altitude exploration (from 18-50 km above sea level) has been severely constrained by a shortage of reliable affordable sample acquisition systems. Thus, attempts to establish the upper altitude limits of Earths biosphere boundary remain unresolved. Previous conclusions based on observations of microbial growth and DNA signatures from samples collected in the upper atmosphere have resulted in extraordinary claims for the tenacity of life in the stratosphere. This altitude range can be regarded as a natural laboratory for examining the edge of habitability, a topic of central important to the field of astrobiology; in fact, stratosphere conditions (high UV light and low extremes of temperature, pressure, and relative humidity) closely resemble stressors associated with the surface of Mars. Moreover, low biomass detection instruments developed exploring Earths upper atmosphere can contribute to future strategies for life detection with spacecraft exploring other worlds. While pioneering, many past stratospheric microbiology studies have been qualitative in nature and lacking rigorous contamination control measurements to verify system sterility. We have designed, constructed, and recently flown a novel system for collecting upper atmosphere bioaerosols using the NASA C20A aircraft. We will report preliminary mission results from the lower stratosphere (characterizing abundance, diversity and survivability or airborne microorganisms), describe our methods for reducing false positives, and outline the open scientific engineering challenges in an ongoing effort to better establish Earths high-altitude biosphere boundary.

Smith, David J.↗

DOE OSTI2020

Sooting tendencies of 20 bio-derived fuels for advanced spark-ignition engines

The sooting tendencies of 20 bio-derived fuels indicated as potential blendstocks for spark-ignition engines by the Co-Optimization of Fuels & Engines (Co-Optima) Initiative are studied. The Yield Sooting Index (YSI) is used to quantify the sooting tendencies. The Co-Optima Chemical Model is used to predict the numerical YSIs for all test fuels, and these results are compared with measurements. Experimental YSIs are newly measured for 2 furans and are taken from our previous work for the other compounds. Overall, the predicted YSIs agree well with measurements, within the experimental uncertainties, except for 2,5-dimethylfuran (2,5-DMF). It is found that Polycyclic Aromatic Hydrocarbons (PAH) growth reactions have little influence on the relative soot production from each fuel and therefore on YSI predictions. A modified sensitivity coefficient formulation is proposed to evaluate reaction sensitivities specifically for YSI. This formulation is applied to the 2,5-DMF-doped flame, and six 2,5-DMF initial decomposition reactions, which lead to different major soot precursors, are identified as the most sensitive reactions. The impact of the chemical kinetic uncertainties embedded in these reactions is quantified by randomly perturbing their reaction rates within a factor of two. The resulting prediction uncertainty in the 2,5-DMF-doped flame is found to be ± 8 YSI units (±11%), which demonstrates that the test-fuel-specific decomposition reactions indeed have considerable influence on its YSI prediction. Our research suggests that more accurate kinetic parameters for fuel initial decomposition reactions can potentially improve YSI predictions effectively, without altering the YSI predictions of other fuels with significantly different molecular structures.

09 BIOMASS FUELS↗

DOE OSTI2021

Lead tolerance and bioremoval by four strains of green algae from Nigerian fish ponds

Heavy metals from industry pose threats to human and environmental health. Microalgae are prime candidates to remove heavy metals from water and soil due to their metal-binding affinities, high growth rates, and contamination tolerance. Strain selection and composition of resulting contaminated biomass must be considered for downstream processing. In this study, we assessed the ability of four microalgae strains (Chlorophyceae) that were isolated from contaminated Nigerian fish ponds to take up dissolved lead at environmentally relevant concentrations (<0.05 mg L -1 – WHO standard). In a microplate assay, all strains grew in the presence of lead, tolerating concentrations up to 60 ppm, though the addition of dissolved lead affected the growth of the four strains. In a photobioreactor assay, all strains demonstrated the ability to take up dissolved lead, with removal efficiencies ranging from 86 to 93%. The concentrations of lead in contaminated biomass, removal efficiencies, and bioconcentration factors were greatest in one of the isolates Chlorella sp. MOW 12. Our results reinforce the fact that live microalgae have a greater biosorption capacity. Lead recovery from freeze-dried biomass using five different reagents showed that a 0.1 M hydrochloric acid solution was the most effective, with 83.4% recovery. We also showed that presence of lead in the culture medium resulted in an increase in the quantity of protein in the biomass. Elemental analysis of lead-bound biomass showed an increase in elements including boron and copper and a decrease in cobalt and sodium. Synergystic and antagonystic effect in uptake of other elements was strain specific, suggesting the possibility of tailored wastewater treatments depending on specific requirements.

59 BASIC BIOLOGICAL SCIENCES↗

NASA NTRS1984

Tomato growth as affected by root-zone temperature and the addition of gibberellic acid and kinetin to nutrient solutions

The effect of root-zone temperature on young tomato plants (Lycopersicon esculentum Mill. cv. Heinz 1350) was evaluated in controlled environments using a recirculating solution culture system. Growth rates were measured at root-zone temperatures of 15 degrees, 20 degrees, 25 degrees, and 30 degrees C in a near optimum foliar environment. Optimum growth occurred at 25 degrees to 30 degrees during the first 4 weeks of growth and 20 degrees to 25 degrees during the 5th and 6th weeks. Growth was severely restricted at 15 degrees. Four concentrations of gibberellic acid (GA3) and kinetin were added to the nutrient solution in a separate trial; root-zone temperature was maintained at 15 degrees and 25 degrees. Addition of 15 micromoles GA3 to solutions increased specific leaf area, total leaf area, and dry weight production of plants in both temperature treatments. GA3-induced growth stimulation was greater at 15 degrees than at 25 degrees. GA3 may promote growth by increasing leaf area, enhancing photosynthesis per unit leaf area, or both. Kinetic was not useful in promoting growth at either temperature.

NASA Program CELSS↗

NASA NTRSDate not supplied

Comparing the Regional Variability of Emission Factors of Greenhouse Gases Over Different Landscape During FIREX-AQ Campaign

Biomass burning (wildfires, prescribed and agricultural burning) is one of the major sources of trace gases and particulate emissions and annual variability in growth rates. Biomass burning can impact local, regional, and global air quality, as well as climate. Measurements of emissions from biomass burning are crucial to a better understanding of how it influences and interacts with biogeochemical cycles. High resolution in-situ measurements were recorded onboard the NASA DC-8 aircraft during the FIREX-AQ (Fire Influence on Regional to Global Environments and Air Quality) airborne field campaign July-September, 2019, which was conducted over the continental U.S. Fire emission factors (EF) are essential input for emissions models used to develop biomass burning emission inventories. Here we present the Emission Ratio (ER), MCE (Modified Combustion Efficiency), and EF (Emission Factor) of CO2, CO, and CH4, which constitute the majority of carbon emitted from the wildland, prescribed, and agricultural fires. EFCO2, EFCO, and EFCH4 from the Wildland fires at Williams Flats, WA (primarily Douglas Fir, Ponderosa pine, wheatgrass: 50-75%), ranged from 1527 – 1820 g/kg (Avg. 1641±42), 6.5 – 174.1 g/kg (110.5±24.1), and 0.7 – 11.3 g/kg (6.2±1.9), respectively. EFs from the Arizona CASTLE fire, with somewhat different fuel sources (primarily Ponderosa pine, Douglas fir: 40-70%) ranged from 1266 – 1667 g/kg (1596±59), 99.5 – 344.5 g/kg (136.7±36.8), and 0.4 – 9.2 g/kg (7.2±1.7), respectively. Another primary driver of EFs is likely fire weather. Detailed variability of greenhouse gas EFs will be examined and presented in accordance with different fuels and fire conditions at burned areas, specifically within unique wildland and croplands, using the FCCS (Fuel Characteristic Classification System) 30m land cover identification and the Cropland Data Layer (CDL).

Biomass burning↗

NASA NTRSDate not supplied

Comparing the regional variability of emission factors of greenhouse gases over different landscape during FIREX-AQ campaign

Biomass burning↗

DOE OSTI2022

Ecosystems and Networks Integrated with Genes and Molecular Assemblies (ENIGMA). Final report

The goal of the Stahl lab is to use a combination of isotopic, genomic, and activity-based analyses in the field, of selected field isolates, and defined synthetic communities to identify sources and sinks of different nitrogen species in the field, focusing on the FRC at the ORNL. The most general research objective is to characterize variants in the pathway for denitrification among field isolates and synthetic microbial assemblies as a foundation for predicting the biotic and abiotic controls of nitrogen and carbon transformations in the field. Specifically, we have focused on characterizing environmental controls of nitrous oxide emissions, an important greenhouse gas and ozone depleting molecule. Studies have mapped the flux of nitrous oxide and controlling genetic elements within the FRC subsurface, indicating the importance of different enzyme variants in nitrous oxide production and consumption in controlling surface emissions. We have developed methods to quantify and catalog the isotopic signatures imparted on nitrous oxide by different nitrous oxide reductase variants in isolated organisms and model synthetic communities. We have developed methods to quantify and catalog the nitrous oxide affinity of field relevant isolates and their biomass yield when respiring nitrous oxide on different carbon substrates. We continue to expand the collection of field-relevant microbial isolates within ENIGMA by focused enrichment and isolation from field material. These objectives have been facilitated by the development of new technologies, including: 1) the development of a multiplexed automated optical density measurement system for quantifying growth kinetics of microorganisms dependent on gaseous substrates in monoculture or gas exchange in synthetic communities and 2) the development of a microcalorimetric analysis format to quantify the impact of environmental stress on microbial maintenance energy. Since field relevant processes are mediated by assemblies of organisms interacting with each other and their local environment, we have also collaborated with other ENIGMA investigators on the assembly and characterization of synthetic communities. These studies have evaluated the role of organisms lacking a complete pathway for denitrification in the control of field processes, measuring process rates of assemblies of organisms that individually lack the full denitrification pathway but mediate the complete denitrification when growing together. These efforts have so far resulted in the publication of 18 peer reviewed papers ranging from ecological and physiological drivers of pathway optimization to field-scale process characterization.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗

DOE OSTI2021

Isotope ratio-based quantification of carbon assimilation highlights the role of plastidial isoprenoid precursor availability in photosynthesis

Abstract Background We report a method to estimate carbon assimilation based on isotope ratio-mass spectrometry (IRMS) of 13 CO 2 labeled plant tissue. Photosynthetic carbon assimilation is the principal experimental observable which integrates important aspects of primary plant metabolism. It is traditionally measured through gas exchange. Despite its centrality in plant research, gas exchange performs poorly with rosette growth habits typical of Arabidopsis thaliana , mutant lines with limited biomass, and accounts poorly for leaf shading. Results IRMS-based carbon assimilation values from plants labeled at different light intensities were compared to those obtained by gas exchange, and the two methods yielded similar values. Using this method, we observed a strong correlation between 13 C content and labeling time (R 2 = 0.999) for 158 wild-type plants labeled for 6 to 42 min. Plants cultivated under different light regimes showed a linear response with respect to carbon assimilation, varying from 7.38 nmol 13 C mg −1 leaf tissue min −1 at 80 PAR to 19.27 nmol 13 C mg −1 leaf tissue min −1 at 500 PAR. We applied this method to examine the link between inhibition of the 2 C -methyl- d -erythritol-4-phosphate (MEP) pathway and suppression of photosynthesis. A significant decrease in carbon assimilation was observed when metabolic activity in the MEP pathway was compromised by mutation or herbicides targeting the MEP pathway. Mutants affected in MEP pathway genes 1-DEOXY- d -XYLULOSE 5-PHOSPHATE SYNTHASE (DXS) or 1-HYDROXY-2-METHYL-2-(E)-BUTENYL 4-DIPHOSPHATE SYNTHASE (HDS) showed assimilation rates 36% and 61% lower than wild type. Similarly, wild type plants treated with the MEP pathway inhibitors clomazone or fosmidomycin showed reductions of 52% and 43%, respectively, while inhibition of the analogous mevalonic acid pathway, which supplies the same isoprenoid intermediates in the cytosol, did not, suggesting inhibition of photosynthesis was specific to disruption of the MEP pathway. Conclusions This method provides an alternative to gas exchange that offers several advantages: resilience to differences in leaf overlap, measurements based on tissue mass rather than leaf surface area, and compatibility with mutant Arabidopsis lines which are not amenable to gas exchange measurements due to low biomass and limited leaf surface area. It is suitable for screening large numbers of replicates simultaneously as well as post-hoc analysis of previously labeled plant tissue and is complementary to downstream detection of isotopic label in targeted metabolite pools.

2C‑methyl‑d‑erythritol 4‑phosphate pathway↗

DOE OSTI2022

Dry Season Transpiration and Soil Water Dynamics in the Central Amazon

With current observations and future projections of more intense and frequent droughts in the tropics, understanding the impact that extensive dry periods may have on tree and ecosystem-level transpiration and concurrent carbon uptake has become increasingly important. Here, we investigate paired soil and tree water extraction dynamics in an old-growth upland forest in central Amazonia during the 2018 dry season. Tree water use was assessed via radial patterns of sap flow in eight dominant canopy trees, each a different species with a range in diameter, height, and wood density. Paired multi-sensor soil moisture probes used to quantify volumetric water content dynamics and soil water extraction within the upper 100 cm were installed adjacent to six of those trees. To link depth-specific water extraction patterns to root distribution, fine root biomass was assessed through the soil profile to 235 cm. To scale tree water use to the plot level (stand transpiration), basal area was measured for all trees within a 5 m radius around each soil moisture probe. The sensitivity of tree transpiration to reduced precipitation varied by tree, with some increasing and some decreasing in water use during the dry period. Tree-level water use scaled with sapwood area, from 11 to 190 L per day. Stand level water use, based on multiple plots encompassing sap flow and adjacent trees, varied from ∼1.7 to 3.3 mm per day, increasing linearly with plot basal area. Soil water extraction was dependent on root biomass, which was dense at the surface (i.e., 45% in the upper 5 cm) and declined dramatically with depth. As the dry season progressed and the upper soil dried, soil water extraction shifted to deeper levels and model projections suggest that much of the water used during the month-long dry-down could be extracted from the upper 2–3 m. Results indicate variation in rates of soil water extraction across the research area and, temporally, through the soil profile. These results provide key information on whole-tree contributions to transpiration by canopy trees as water availability changes. In addition, information on simultaneous stand level dynamics of soil water extraction that can inform mechanistic models that project tropical forest response to drought.

54 ENVIRONMENTAL SCIENCES↗

NASA NTRS1997

Photomorphogenesis, photosynthesis, and seed yield of wheat plants grown under red light-emitting diodes (LEDs) with and without supplemental blue lighting

Red light-emitting diodes (LEDs) are a potential light source for growing plants in spaceflight systems because of their safety, small mass and volume, wavelength specificity, and longevity. Despite these attractive features, red LEDs must satisfy requirements for plant photosynthesis and photomorphogenesis for successful growth and seed yield. To determine the influence of gallium aluminium arsenide (GaAlAs) red LEDs on wheat photomorphogenesis, photosynthesis, and seed yield, wheat (Triticum aestivum L., cv. 'USU-Super Dwarf') plants were grown under red LEDs and compared to plants grown under daylight fluorescent (white) lamps and red LEDs supplemented with either 1% or 10% blue light from blue fluorescent (BF) lamps. Compared to white light-grown plants, wheat grown under red LEDs alone demonstrated less main culm development during vegetative growth through preanthesis, while showing a longer flag leaf at 40 DAP and greater main culm length at final harvest (70 DAP). As supplemental BF light was increased with red LEDs, shoot dry matter and net leaf photosynthesis rate increased. At final harvest, wheat grown under red LEDs alone displayed fewer subtillers and a lower seed yield compared to plants grown under white light. Wheat grown under red LEDs+10% BF light had comparable shoot dry matter accumulation and seed yield relative to wheat grown under white light. These results indicate that wheat can complete its life cycle under red LEDs alone, but larger plants and greater amounts of seed are produced in the presence of red LEDs supplemented with a quantity of blue light.

NASA Discipline Life Support Systems↗

NASA NTRS2016

The Effect of Future Ambient Air Pollution on Human Premature Mortality to 2100 Using Output from the ACCMIP Model Ensemble

Ambient air pollution from ground-level ozone and fine particulate matter (PM(sub 2.5)) is associated with premature mortality. Future concentrations of these air pollutants will be driven by natural and anthropogenic emissions and by climate change. Using anthropogenic and biomass burning emissions projected in the four Representative Concentration Pathway scenarios (RCPs), the ACCMIP ensemble of chemistry climate models simulated future concentrations of ozone and PM(sub 2.5) at selected decades between 2000 and 2100. We use output from the ACCMIP ensemble, together with projections of future population and baseline mortality rates, to quantify the human premature mortality impacts of future ambient air pollution. Future air-pollution-related premature mortality in 2030, 2050 and 2100 is estimated for each scenario and for each model using a health impact function based on changes in concentrations of ozone and PM(sub 2.5) relative to 2000 and projected future population and baseline mortality rates. Additionally, the global mortality burden of ozone and PM(sub 2.5) in 2000 and each future period is estimated relative to 1850 concentrations, using present-day and future population and baseline mortality rates. The change in future ozone concentrations relative to 2000 is associated with excess global premature mortality in some scenarios/periods, particularly in RCP8.5 in 2100 (316 thousand deaths per year), likely driven by the large increase in methane emissions and by the net effect of climate change projected in this scenario, but it leads to considerable avoided premature mortality for the three other RCPs. However, the global mortality burden of ozone markedly increases from 382000 (121000 to 728000) deaths per year in 2000 to between 1.09 and 2.36 million deaths per year in 2100, across RCPs, mostly due to the effect of increases in population and baseline mortality rates. PM(sub 2.5) concentrations decrease relative to 2000 in all scenarios, due to projected reductions in emissions, and are associated with avoided premature mortality, particularly in 2100: between 2.39 and 1.31 million deaths per year for the four RCPs. The global mortality burden of PM(sub 2.5) is estimated to decrease from 1.70 (1.30 to 2.10) million deaths per year in 2000 to between 0.95 and 1.55 million deaths per year in 2100 for the four RCPs due to the combined effect of decreases in PM(sub 2.5) concentrations and changes in population and baseline mortality rates. Trends in future air-pollution-related mortality vary regionally across scenarios, reflecting assumptions for economic growth and air pollution control specific to each RCP and region. Mortality estimates differ among chemistry climate models due to differences in simulated pollutant concentrations, which is the greatest contributor to overall mortality uncertainty for most cases assessed here, supporting the use of model ensembles to characterize uncertainty. Increases in exposed population and baseline mortality rates of respiratory diseases magnify the impact on premature mortality of changes in future air pollutant concentrations and explain why the future global mortality burden of air pollution can exceed the current burden, even where air pollutant concentrations decrease.

Silva, Raquel A.↗

NASA NTRSDate not supplied

Effect of salinity on the preservation of biomarkers in hypersaline microbial mat kerogens

Kerogen is the residue that remains after the minerals and organic compounds have been extracted from a rock using acid and organic solvents, respectively [1]. It is not only the largest organic carbon sink in the geological record but the best-preserved fraction due to its recalcitrance and ability to retain syngenetic source information during deposition [2]. Due to the complex, macromolecular, cross-linked polymeric structure of kerogen, it is difficult to analyse intact, often requiring breakdown into smaller subunits through thermal (pyrolysis) or chemical treatment (chemolysis) [3]. Saline and hypersaline environments host diverse microbial communities, which can trap and bind organic matrices such as cellular components (e.g., extracellular polymeric substances, cell wall, peptidoglycan, sheath material, etc.) with sediments, forming layered, laminated structures called microbial mats. Here we present depth profiles of two hypersaline cores through a microbial mat with differing total salinities. We looked at the composition of organic compounds released from kerogen during high temperature, hydrogen-assisted, pyrolysis to understand the effect of salinity on the composition and timing of their incorporation [4]. Field Site: The Exportadora del Sal S. A. (ESSA) saltern of Guerrero Negro (Baja California Sur, Mexico) lies within a typical sabhka environment adjacent to Laguna Ojo de Liebre. Note the spatial extent of the 13 major ponds of the ESSA system. The GN ponds thrive under well-monitored and stable conditions with steady state accretion and degradation of mat layers (around 0.5–1.4 cm per year). Organic mats typically attain up to 10 cm in thickness, depending on the location, which represents about 60 years of growth since these ponds were established. Sedimentation rate is rapid in these non-lithifying and non-mineralising mats, 1–1.5 cm per year. This environment is ideal for investigating in situ lipid preservation and diagenesis without the influence of constantly changing physical parameters. Methods: P4n5 and P5AB cores were collected as ~9 cm and 6.5 cm fresh cores, respectively, in June 2001 and September 2010, respectively from the ESSA saltern. The P4n5 core (9-9.1% salinity) was taken from Pond 4 near 5 and sub-divided into 10 layers. The P5AB core (11.2% salinity) was taken from Pond 5A near 5B and sub-divided into 8 layers. Lyophilised microbial mat powders were solvent-extracted using a modified Bligh-Dyer method and analysed by gas chromatography-mass spectrometry (GC-MS). Additionally, a subset of layers—layer 4 and layer 7—which represented active biomass and sediment-processed, respectively, were subjected to mild-acid methanolysis. The respective residues from conventional solvent extraction and acid methanolysis were loaded with a molybdenum sulfide catalyst and placed into a stainless-steel reactor. Samples were run on the hydropyrolysis set up (heated to 500 °C with 13-15 MPa of H2 pressure), extracted, and analysed by GC-MS. Results: Molecular profiles from the two ponds were significantly different and reflect the contributions of different photosynthetic and respiratory microbial communities to preserved organic matter. For example, the higher salinity P5AB core showed evidence of greater archaeal inputs (similarly observed in lab culture experiments [5]) as well as potential kerogen-bound carotenoids/carotenoid rearranged products or fragments. A hydrophobic emulsion formed during the acid methanolysis processing of layer 4 from both P4n5 and P5AB and layer 7 of P5AB. Hydropyrolysis of this hydrophobic residues released a greater abundance of polycyclic lipids relative to the pre-extracted control. Implications and Future Work: Catalytic hydropyrolysis of kerogen can rapidly generate abundant saturated pyrolysate products from the bound biomarker pool without altering the structures or stereochemistries of the products [6]. Chemical processing of solvent-extractable residues indicated that cellular matrices such as extracellular polymeric substances (EPS) may play a role in the sequestration of polycyclic lipid biomarkers such as steranes and hopanes. Their higher relative abundance compared to the pre-extracted control in the higher salinity layers and cores preliminarily aligns with this hypothesis, although further experiments are required to confirm this. It has been well-documented that EPS plays an important role in mineralisation, specifically carbonate formation, in microbial ecosystems. EPS can enhance calcium carbonate precipitation by providing diffusion-limited sites that create alkalinity gradients in response to microbial processes [7]. It has been experimentally demonstrated that salinity influences the total amount of EPS (both loosely- and tightly-bound) which increases with increasing salinity [8]. The higher salinity at Guerrero Negro switches the microbial population from filamentous Microcoleus to being dominated by Phormidium, Oscillatoria, and unicellular cyanobacteria. We demonstrated that lipid binding into kerogen via strong covalent linkages occurs at the very earliest stages of sedimentary diagenesis. We are currently investigating the influence of salinity on organic preservation through experimental and modelling approaches. Understanding how key biomarkers transform into preserved organic matter (i.e., from precursor biolipids to bound geolipids) in brine ecosystems will aid the search for organic biosignatures on other planetary bodies, especially Icy Moons and modern Mars.

hypersaline↗

DOE OSTI2021

Unbalancing Symbiotic Nitrogen Fixation: Can We Make Effectiveness More Effective?

One of the most critical uses of carbon fuels is in generating nitrogen fertilizer. Atmospheric dinitrogen is too stable to be used directly by plants, so plants need other chemical forms of nitrogen. Nitrogen fertilizer production uses ~4% of the world’s natural gas. Making N fertilizers leverages methane’s energy content by enabling additional energy to be captured into food and fiber through increased photosynthesis. Manufacturing fertilizer is essential—without it there would only be enough combined nitrogen to feed ~3 billion of the world’s ~7 billion people. How to alter this addiction is not obvious, but the best chance to secure substantial and sustainable amounts of future N might be through symbiotic nitrogen fixation (SNF). SNF describes mutualistic interactions where nitrogen-fixing bacteria provide fixed nitrogen to their plant hosts, freeing them from the need for nitrogen fertilizers. SNF already occurs on a large scale; "biological nitrogen” contributes about 5 Tg N/yr to American agriculture, a quarter of the nitrogen needed. Improving SNF and replacing inorganic fertilizers are both important goals in establishing sustainable and more energy-efficient farming. SNF has been used in agriculture for millennia, largely by using legumes like beans or alfalfa in mixed cropping systems. Legume SNF occurs in root nodules, organs that develop after a growing root is infected by bacteria called rhizobia. In the plant cells of a legume nodule, specialized forms of rhizobia fix nitrogen and can produce enough ammonia to supply the growing plant. The exchange of photosynthetically derived plant carbon compounds for nitrogen reduced by the bacteria is the engine that drives the symbiosis. Important details about the organization of development and metabolism in SNF remain to be determined, such as how plant and bacterial metabolism are coordinated as nitrogen is being fixed and what governs the overall level of nitrogen fixation. Most bacterial mutations that affect SNF completely block fixation (e.g. are Fix – ), providing a limited window into the feedback interactions that must be part of the process. We have investigated an unusual mutant of Sinorhizobium meliloti, a symbiotic partner of alfalfa, that fixes dinitrogen at a normal rate (i.e., it is Fix + ) but is not effective in supporting plant growth (i.e., it is ineffective or Eff – ). We have shown that the mutant has a specific mutation in the glnD gene, which codes for a protein that regulates the bacterial response to nitrogen stress, among other key pathways. A Fix + Eff – phenotype contains an unavoidable puzzle—how is it possible for the bacteria to fix nitrogen at a normal rate and without benefiting the nitrogen starved plant host? After eliminating other possibilities, we proposed that the bacteria synthesize a nitrogen-containing compound that the plant can’t use so that acquiring this compound does not relieve plant nitrogen stress. Extracts of nodules made by glnD mutant strains have high concentrations of pyruvate canaline oxime (PCO), a derivative of the plant defensive compound canaline, a toxic analog of ornithine, an amino acid. Many legumes produce canaline and the related arginine mimic canavanine. For reasons we do not yet understand, the mutation in glnD appears to trigger significant overproduction of canaline in a defense response that leads to substantial synthesis of PCO. We expected the bacteria to be able to use PCO, but we have not been able to grow S. meliloti on PCO under several conditions. PCO thus appears to be a metabolic dead end for both the plant and bacteria. We also examined bacterial catabolic pathways that degrade arginine and canavanine and showed that mutation of these did not alter the symbiosis in an obvious way. In related experiments to examine the metabolic development of root nodules, we carried out an ambitious experiment to simultaneously measure metabolites and protein changes during nodule maturation. For these experiments we used a related symbiotic interaction between S. medicae and Medicago truncatula. M. truncatula has a simpler genome than alfalfa so we could identify plant proteins based on predictions from the DNA sequence. Nodules formed on Medicago grow linearly, with the least mature tissues at the tip. Because development of nitrogen fixation is accompanied by the production of the pigmented plant oxygen carrier protein leghemoglobin, we were able to locate and manually separate the tip, transitional, and mature nitrogen-fixing regions from each other. Advances in proteomic and metabolomic techniques allowed us to analyze these tissues from a pool of as few as 10 nodules. We observed correlated transitions of various enzymes in the plant and bacterial proteomes from those characteristic of free-living metabolism to the microaerobic metabolism of the nitrogen-fixing tissues. The metabolome could not be separated into plant and bacterial domains but was consistent with this overall transition. A subsequent paper examined proteolysis in nodule bacteria. Protein turnover in mature regions of the nodule is significant. We were able to show complexes between various proteins and important proteases, using precipitation capture techniques to isolate the proteases and sensitive proteomic analysis to reveal the associated proteins.

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