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All sources NASA NTRS DOE OSTI

DOE OSTI2020

Pseudomonas sp. Strain 273 Degrades Fluorinated Alkanes

Fluorinated organic compounds have emerged as environmental constituents of concern. We demonstrate that the alkane degrader Pseudomonas sp. strain 273 utilizes terminally monofluorinated C 7 –C 10 alkanes and 1,10-difluorodecane (DFD) as the sole carbon and energy sources in the presence of oxygen. Strain 273 degraded 1-fluorodecane (FD) (5.97 ± 0.22 mM, nominal) and DFD (5.62 ± 0.13 mM, nominal) within 7 days of incubation, and 92.7 ± 3.8 and 90.1 ± 1.9% of the theoretical maximum amounts of fluorine were recovered as inorganic fluoride, respectively. With n-decane, strain 273 attained (3.24 ± 0.14) × 10 7 cells per μmol of carbon consumed, while lower biomass yields of (2.48 ± 0.15) × 10 7 and (1.62 ± 0.23) × 10 7 cells were measured with FD or DFD as electron donors, respectively. The organism coupled decanol and decanoate oxidation to denitrification, but the utilization of (fluoro)alkanes was strictly oxygen-dependent, presumably because the initial attack on the terminal carbon requires oxygen. Fluorohexanoate was detected as an intermediate in cultures grown with FD or DFD, suggesting that the initial attack on the fluoroalkanes can occur on the terminal methyl or fluoromethyl groups. Overall, the findings indicate that specialized bacteria such as Pseudomonas sp. strain 273 can break carbon–fluorine bonds most likely with oxygenolytic enzyme systems and that terminally monofluorinated alkanes are susceptible to microbial degradation. The findings have implications for the fate of components associated with aqueous film-forming foam (AFFF) mixtures.

54 ENVIRONMENTAL SCIENCES↗

DOE OSTI2024

Cell-Free Systems Biology: Characterizing Central Metabolism of Clostridium thermocellum with a Three-Enzyme Cascade Reaction

Genetic approaches have been traditionally used to understand microbial metabolism, but this process can be slow in nonmodel organisms due to limited genetic tools. An alternative approach is to study metabolism directly in the cell lysate. This avoids the need for genetic tools and is routinely used to study individual enzymatic reactions but is not generally used to study systems-level properties of metabolism. Here we demonstrate a new approach that we call “cell-free systems biology”, where we use well-characterized enzymes and multienzyme cascades to serve as sources or sinks of intermediate metabolites. This allows us to isolate subnetworks within metabolism and study their systems-level properties. To demonstrate this, we worked with a threeenzyme cascade reaction that converts pyruvate to 2,3-butanediol. Although it has been previously used in cell-free systems, its pH dependence was not well characterized, limiting its utility as a sink for pyruvate. We showed that improved proton accounting allowed better prediction of pH changes and that active pH control allowed 2,3-butanediol titers of up to 2.1 M (189 g/L) from acetoin and 1.6 M (144 g/L) from pyruvate. The improved proton accounting provided a crucial insight that preventing the escape of CO 2 from the system largely eliminated the need for active pH control, dramatically simplifying our experimental setup. We then used this cascade reaction to understand limits to product formation in Clostridium thermocellum, an organism with potential applications for cellulosic biofuel production. We showed that the fate of pyruvate is largely controlled by electron availability and that reactions upstream of pyruvate limit overall product formation.

09 BIOMASS FUELS↗

DOE OSTI2021

Reductive dissolution of pyrite by methanogenic archaea

Abstract The formation and fate of pyrite (FeS2) modulates global iron, sulfur, carbon, and oxygen biogeochemical cycles and has done so since early in Earth’s geological history. A longstanding paradigm is that FeS2 is stable at low temperature and is unavailable to microorganisms in the absence of oxygen and oxidative weathering. Here, we show that methanogens can catalyze the reductive dissolution of FeS2 at low temperature (≤38 °C) and utilize dissolution products to meet cellular iron and sulfur demands associated with the biosynthesis of simple and complex co-factors. Direct access to FeS2 is required to catalyze its reduction and/or to assimilate iron monosulfide that likely forms through coupled reductive dissolution and precipitation, consistent with close associations observed between cells and FeS2. These findings demonstrate that FeS2 is bioavailable to anaerobic methanogens and can be mobilized in low temperature anoxic environments. Given that methanogens evolved at least 3.46 Gya, these data indicate that the microbial contribution to the iron and sulfur cycles in ancient and contemporary anoxic environments may be more complex and robust than previously recognized, with impacts on the sources and sinks of iron and sulfur and other bio-essential and thiophilic elements such as nickel and cobalt.

59 BASIC BIOLOGICAL SCIENCES↗

NASA NTRS1996

Accumulation of Dissolved DMSP by Marine Bacteria and its Degradation Via Bacterivory

Several bacterial isolates enriched from seawater using complex media were able to accumulate dimethylsulfoniopropionate (DMSP) from media into cells over several hours without degrading it. Uptake only occurred in metabolically active cells, and was repressed in some strains by the presence of additional carbon sources. Accumulation was also more rapid in osmotically-stressed cells, suggesting DMSP is used as an osmotic solute. Uptake could be blocked by inhibitors of active transport systems (2,4-dinitrophenol, azide, arsenate) and of protein synthesis (chloramphenicol). Some structural analogs such as glycine betaine and S-methyl methionine also blocked DMSP uptake, suggesting that the availability of alternate organic osmolytes may influence DMSP uptake. Stresses such as freezing, heating, or osmotic down shock resulted in partial release of DMSP back to the medium. One strain which contained a DMSP-lyase was also able to accumulate DMSP, and DMS was only produced in the absence of alternate carbon sources. Bacteria containing DMSP were prepared as prey for bacterivorous ciliates and flagellates, to examine the fate of the DMSP during grazing. In all cases, predators metabolized the DMSP in bacteria. In some cases, DMS was produced, but it is not clear if this was due to the predators or to associated bacteria in the non-axenic grazer cultures. Bacterivores may influence DMSP cycling by either modulating populations of DMSP-metabolizing bacteria, or by metabolizing DMSP accumulated by bacterial prey.

Wolfe, Gordon V.↗

DOE OSTI2020

In-field bioreactors demonstrate dynamic shifts in microbial communities in response to geochemical perturbations

Subsurface microbial communities mediate the transformation and fate of redox sensitive materials including organic matter, metals and radionuclides. Few studies have explored how changing geochemical conditions influence the composition of groundwater microbial communities over time. We temporally monitored alterations in abiotic forces on microbial community structure using 1L in-field bioreactors receiving background and contaminated groundwater at the Oak Ridge Reservation, TN. Planktonic and biofilm microbial communities were initialized with background water for 4 days to establish communities in triplicate control reactors and triplicate test reactors and then fed filtered water for 14 days. On day 18, three reactors were switched to receive filtered groundwater from a contaminated well, enriched in total dissolved solids relative to the background site, particularly chloride, nitrate, uranium, and sulfate. Biological and geochemical data were collected throughout the experiment, including planktonic and biofilm DNA for 16S rRNA amplicon sequencing, cell counts, total protein, anions, cations, trace metals, organic acids, bicarbonate, pH, Eh, DO, and conductivity. We observed significant shifts in both planktonic and biofilm microbial communities receiving contaminated water. This included a loss of rare taxa, especially amongst members of the Bacteroidetes, Acidobacteria, Chloroflexi, and Betaproteobacteria, but enrichment in the Fe- and nitrate- reducing Ferribacterium and parasitic Bdellovibrio. These shifted communities were more similar to the contaminated well community, suggesting that geochemical forces substantially influence microbial community diversity and structure. These influences can only be captured through such comprehensive temporal studies, which also enable more robust and accurate predictive models to be developed.

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2024

Comprehensive Cradle to Grave Life Cycle Analysis of On-Road Vehicles in the United States based on GREET

To properly compare and contrast the environmental performance of one vehicle technology against another, it is necessary to consider their production, operation, and end-of-life fates. Since 1995, Argonne’s GREET® life cycle analysis model (Greenhouse gases, Regulated Emissions, and Energy use in Technologies) has been annually updated to model and refine the latest developments in fuels and materials production, as well as vehicle operational and composition characteristics. Updated cradle-to-grave life cycle analysis results from the model’s latest release are described for a wide variety of fuel and powertrain options for U.S. light-duty and medium/heavy-duty vehicles. Light-duty vehicles include a passenger car, sports utility vehicle (SUV), and pick-up truck, while medium/heavy-duty vehicles include a Class 6 pickup-and-delivery truck, Class 8 day-cab (regional) truck, and Class 8 sleeper-cab (long-haul) truck. Powertrain coverage includes internal combustion (spark ignition and compression ignition) engines, hybrid electric, plug-in hybrid, full battery electric, and fuel cell vehicles powered by conventional and low carbon energy sources. The results offer insights into the current state of these technologies, as well as a projection of the likely environmental implications of future fuel and vehicle advancements through a time-series evaluation of life cycle greenhouse gas emissions.

Kelly, Jarod C.↗

DOE OSTI2022

Effective Mechanisms for Improving Seed Oil Production in Pennycress (Thlaspi arvense L.) Highlighted by Integration of Comparative Metabolomics and Transcriptomics

Pennycress is a potentially lucrative biofuel crop due to its high content of long-chain unsaturated fatty acids, and because it uses non-conventional pathways to achieve efficient oil production. However, metabolic engineering is required to improve pennycress oilseed content and make it an economically viable source of aviation fuel. Research is warranted to determine if further upregulation of these non-conventional pathways could improve oil production within the species even more, which would indicate these processes serve as promising metabolic engineering targets and could provide the improvement necessary for economic feasibility of this crop. To test this hypothesis, we performed a comparative biomass, metabolomic, and transcriptomic analyses between a high oil accession (HO) and low oil accession (LO) of pennycress to assess potential factors required to optimize oil content. An evident reduction in glycolysis intermediates, improved oxidative pentose phosphate pathway activity, malate accumulation in the tricarboxylic acid cycle, and an anaplerotic pathway upregulation were noted in the HO genotype. Additionally, higher levels of threonine aldolase transcripts imply a pyruvate bypass mechanism for acetyl-CoA production. Nucleotide sugar and ascorbate accumulation also were evident in HO, suggesting differential fate of associated carbon between the two genotypes. An altered transcriptome related to lipid droplet (LD) biosynthesis and stability suggests a contribution to a more tightly-packed LD arrangement in HO cotyledons. In addition to the importance of central carbon metabolism augmentation, alternative routes of carbon entry into fatty acid synthesis and modification, as well as transcriptionally modified changes in LD regulation, are key aspects of metabolism and storage associated with economically favorable phenotypes of the species.

59 BASIC BIOLOGICAL SCIENCES↗

NASA NTRS2000

atonal regulates neurite arborization but does not act as a proneural gene in the Drosophila brain

Drosophila atonal (ato) is the proneural gene of the chordotonal organs (CHOs) in the peripheral nervous system (PNS) and the larval and adult photoreceptor organs. Here, we show that ato is expressed at multiple stages during the development of a lineage of central brain neurons that innervate the optic lobes and are required for eclosion. A novel fate mapping approach shows that ato is expressed in the embryonic precursors of these neurons and that its expression is reactivated in third instar larvae (L3). In contrast to its function in the PNS, ato does not act as a proneural gene in the embryonic brain. Instead, ato performs a novel function, regulating arborization during larval and pupal development by interacting with Notch.

NASA Discipline Developmental Biology↗

DOE OSTI2021

Inside out: Measuring the effect of wood anatomy on the efflux and assimilation of xylem–transported CO 2

Carbon dioxide concentrations (including aqueous CO 2 , carbonic acid, bicarbonate, and carbonate) in woody tissues can be up to 750 times higher than atmospheric CO 2 concentrations, ranging from <1% to >26% vs atmospheric ~0.04% (Fig. 1) (Teskey et al. 2008). CO 2 formed through respiration is generally assumed to diffuse to the atmosphere from tissues adjacent to where it is produced. Here, this CO 2 buildups in the stem due to the diffusional barriers in woody and bark tissues. CO 2 in the stem has three fates: 1) it can be refixed for photosynthesis, 2) it can be used for anaplerotic reactions, or 3) it can exit the plant either adjacent to where it was produced (radial diffusion) or in an area remote from its point of origin (xylem-transported CO 2 ). Not accounting for assimilation of xylem-transported CO 2 may result in underestimating total plant photosynthesis. Alternatively, overlooking the transport of CO 2 away from its point of origin complicates the estimation of respiration in stems, branches, or even leaves. For example, CO 2 efflux from the stem may not solely represent stem respiration, but it may represent CO 2 generated through respiration in areas remote from the point of efflux (Stutz et al., 2017). Additionally, wood anatomy and branching architecture likely influence how much and how far xylem-transported CO 2 travels (Fig. 2). Few studies have combined measurements of both the efflux of xylem-transported CO 2 from trees together and the assimilation of xylem-transported CO 2 . Excitingly in this issue of Plant, Cell & Environment, Salomón et al. (pp. ) demonstrate the importance of xylem-transported CO 2 in plants with different wood anatomies by comparing the amount of xylem-transported CO 2 used for photosynthesis to the amount effluxed.

54 ENVIRONMENTAL SCIENCES↗

DOE OSTI2021

Anaerobic Microbial Metabolism of Dichloroacetate

Dichloroacetate (DCA) commonly occurs in the environment due to natural production and anthropogenic releases, but its fate under anoxic conditions is uncertain. Mixed culture RM comprising “Candidatus Dichloromethanomonas elyunquensis” strain RM utilizes DCA as an energy source, and the transient formation of formate, H 2 , and carbon monoxide (CO) was observed during growth. Only about half of the DCA was recovered as acetate, suggesting a fermentative catabolic route rather than a reductive dechlorination pathway. Sequencing of 16S rRNA gene amplicons and 16S rRNA gene-targeted quantitative real-time PCR (qPCR) implicated “Candidatus Dichloromethanomonas elyunquensis” strain RM in DCA degradation. An (S)-2-haloacid dehalogenase (HAD) encoded on the genome of strain RM was heterologously expressed, and the purified HAD demonstrated the cofactor-independent stoichiometric conversion of DCA to glyoxylate at a rate of 90 ± 4.6 nkat mg -1 protein. Differential protein expression analysis identified enzymes catalyzing the conversion of DCA to acetyl coenzyme A (acetyl-CoA) via glyoxylate as well as enzymes of the Wood-Ljungdahl pathway. Glyoxylate carboligase, which catalyzes the condensation of two molecules of glyoxylate to form tartronate semialdehyde, was highly abundant in DCA-grown cells. The physiological, biochemical, and proteogenomic data demonstrate the involvement of an HAD and the Wood-Ljungdahl pathway in the anaerobic fermentation of DCA, which has implications for DCA turnover in natural and engineered environments, as well as the metabolism of the cancer drug DCA by gut microbiota.

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2025

Unraveling metabolism underpinning biomass composition shift in Scenedesmus obliquus under simulated outdoor conditions using 13 C-fluxomics

To render the resulting biomass more attractive and amenable for utilization as the basis for low-carbon intensity bioproducts, single-celled algae need to be biochemically and metabolically poised to assimilate and store the delivered carbon in the fastest and most efficient manner. Accelerating biochemical carbon storage, as primarily carbohydrates or lipids, is critical to achieve the high carbon capture potential that is assigned to algae. To guide strain optimization and engineering for maximizing carbon capture and storage, it is essential to elucidate the link between carbon metabolism and biomass composition. Most published metabolomics work in algae remains largely restricted to ideal and simplified environmental conditions in model organisms, thereby limiting their translation to outdoor implementation. In this work, we utilize 13 C isotopic labeling to characterize distinct intracellular metabolic fluxes before, during, and after nitrogen depletion-induced compositional shifts in Scenedesmus obliquus UTEX 393. The results indicate that a transition to carbohydrates is characterized by diverting flux to starch instead of replenishing the Calvin cycle for CO 2 fixation whereas the subsequent transition to lipids is fueled by NADPH produced by upregulating the phosphoenolpyruvate carboxylase (PEPC)–malic enzyme (ME) cycle flux. Our work highlights bottlenecks to carbohydrate- and lipid-rich biomass and can guide implementable strategies to control the fate of fixed carbon in S. obliquus.

09 BIOMASS FUELS↗

DOE OSTI2024

Subject-specific multi-scale modeling of the fate of inhaled aerosols

Determining the fate of inhaled aerosols in the respiratory system is essential in assessing the potential toxicity of inhaled airborne materials, responses to airborne pathogens, or in improving inhaled drug delivery. The availability of high-resolution clinical lung imaging and advances in the reconstruction of lung airways from CT images have led to the development of subject-specific in-silico 3D models of aerosol dosimetry, often referred to as computational fluid-particle-dynamics (CFPD) models. As CFPD models require extensive computing resources, they are typically confined to the upper and large airways. These models can be combined with lower-dimensional models to form multiscale models that predict the transport and deposition of inhaled aerosols in the entire respiratory tract. Understanding where aerosols deposit is only the first of potentially several key events necessary to predict an outcome, being a detrimental health effect or a therapeutic response. To that end, multiscale approaches that combine CFPD with physiologically-based pharmacokinetics (PBPK) models have been developed to evaluate the absorption, distribution, metabolism, and excretion (ADME) of toxic or medicinal chemicals in one or more compartments of the human body. CFPD models can also be combined with host cell dynamics (HCD) models to assess regional immune system responses. Here, this paper reviews the state of the art of these different multiscale approaches and discusses the potential role of personalized or subject-specific modeling in respiratory health.

60 APPLIED LIFE SCIENCES↗

NASA NTRS2019

Energetic Particles of Cosmic Accelerators II: Active Galactic Nuclei and Gamma-ray Bursts

The high-energy universe has revealed that energetic particles are ubiquitous in the cosmos and play a vital role in the cultivation of cosmic environments on all scales. Our pursuit of more than a century to uncover the origins and fate of these cosmic energetic particles has given rise to some of the most interesting and challenging questions in astrophysics. Within our own galaxy, we have seen that energetic particles engage in a complex interplay with the galactic environment and even drive many of its key characteristics (for more information, see the first white paper in this series). On cosmological scales, the energetic particles supplied by the jets of active galactic nuclei (AGN) are an important source of energy for the intracluster and intergalactic media, providing a mechanism for regulating star formation and black hole growth and cultivating galaxy evolution (AGN feedback). Gamma-ray burst (GRB) afterglows encode information about their circumburst environment, which has implications for massive stellar winds during previous epochs over the stellar lifecycle. As such, GRB afterglows provide a means for studying very high-redshift galaxies since GRBs can be detected even if their host galaxy cannot. It has even been suggest that GRB could be used to measure cosmological distance scales if they could be shown to be standard candles. Though they play a key role in cultivating the cosmological environment and/or enabling our studies of it, there is still much we do not know about AGNs and GRBs, particularly the avenue in which and through which they supply radiation and energetic particles, namely their jets. Despite the enormous progress in particle-in-cell and magnetohydrodynamic simulations, we have yet to pinpoint the processes involved in jet formation and collimation and the conditions under which they can occur. For that matter, we have yet to identify the mechanism(s) through which the jet accelerates energetic particles – is it the commonly invoked diffusive shock acceleration process or is another mechanism, such as magnetic reconnection, required? Do AGNs and GRBs accelerate hadrons, and if so, do they accelerate them to ultra-high energies and are there high-energy neutrinos associated with them? MeV gamma-ray astronomy, enabled by technological advances that will be realized in the coming decade, will provide a unique and indispensable perspective on the persistent mysteries of the energetic universe. This White Paper is the second of a two-part series highlighting the most well-known high-energy cosmic accelerators and contributions that MeV gamma-ray astronomy will bring to understanding their energetic particle phenomena. Specifically, MeV astronomy will: 1. Determine whether AGNs accelerate CRs to ultra-high energies; 2. Provide the missing pieces for the physics of the GRB prompt emission; 3. Measure magnetization in cosmic accelerators and search for acceleration via reconnection.

Venters, Tonia M.↗

DOE OSTI2023

Predicting partner fitness based on spatial structuring in a light-driven microbial community

Microbial communities have vital roles in systems essential to human health and agriculture, such as gut and soil microbiomes, and there is growing interest in engineering designer consortia for applications in biotechnology ( e . g ., personalized probiotics, bioproduction of high-value products, biosensing). The capacity to monitor and model metabolite exchange in dynamic microbial consortia can provide foundational information important to understand the community level behaviors that emerge, a requirement for building novel consortia. Where experimental approaches for monitoring metabolic exchange are technologically challenging, computational tools can enable greater access to the fate of both chemicals and microbes within a consortium. In this study, we developed an in-silico model of a synthetic microbial consortia of sucrose-secreting Synechococcus elongatus PCC 7942 and Escherichia coli W. Our model was built on the NUFEB framework for Individual-based Modeling (IbM) and optimized for biological accuracy using experimental data. We showed that the relative level of sucrose secretion regulates not only the steady-state support for heterotrophic biomass, but also the temporal dynamics of consortia growth. In order to determine the importance of spatial organization within the consortium, we fit a regression model to spatial data and used it to accurately predict colony fitness. We found that some of the critical parameters for fitness prediction were inter-colony distance, initial biomass, induction level, and distance from the center of the simulation volume. We anticipate that the synergy between experimental and computational approaches will improve our ability to design consortia with novel function.

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2020

Investigation of Mass Transfer and Sorption in CO 2 /Brine/Rock Systems via In Situ FT-IR

CO 2 geological storage in deep saline formations is considered a promising method to mitigate anthropogenic CO 2 emissions and, thereby, minimize changes to the Earth’s atmosphere. A fundamental understanding of CO 2 mass transfer and sorption phenomena in brine-saturated reservoir formations is necessary to understand the long-term fate of injected CO 2 as it is subjected to different (physical, dissolution, and mineral) trapping mechanisms. In this work, we investigate CO 2 sorption in brine-saturated and dry Mt. Simon sandstone samples via in-situ Fourier Transform infrared spectroscopy (FT-IR) at elevated pressures, ranging from 0.3 MPa to 8.3 MPa, at a temperature of 50 ⁰C. The FT-IR spectra of bulk-phase CO 2 were simultaneously recorded under the same conditions. For bulk-phase CO 2 , we observed, in agreement with past studies, a doublet peak at 2361 cm -1 and 2336 cm -1 and another peak (ν 2 bending mode) at 667 cm -1 . With increasing pressure, the position of the peak at 667 cm -1 remains invariant, however, when crossing into the supercritical region the doublet peak degenerates onto a single peak at 2336 cm -1 with a barely visible shoulder at 2361 cm -1 . The bulk CO 2 data provide a perfect fit for Beer’s law for the whole range of pressure conditions. For the dry sample, the IR spectrum is experimentally indistinguishable from the bulk CO 2 spectrum, signifying that if physical adsorption occurs to any significant extent, the adsorbed CO 2 molecules are not substantially more rotationally constrained than the dense bulk CO 2 molecules. For the brine-saturated sample, we observe a strong band centered at 2342 cm -1 and a small companion peak at 2360-2361 cm -1 that degenerates into a barely visible shoulder peak at the higher pressures. The 2342 cm -1 band has been previously observed by other investigators for CO 2 dissolved in bulk water/brine as well during its adsorption on a variety of other wet natural porous media. We observe no peaks corresponding to bicarbonate or carbonate bulk species, which correlates well with the prior literature on similar low-pH aqueous solutions. The integrated peak area for the CO 2 sorbed in the brine-saturated sample correlates linearly with its solubility in the same bulk brine, as measured separately via a PVT-cell approach. Here, this validates the accuracy of both techniques, and the potential of the FT-IR method to be used in the study of mass transfer and adsorption in such systems. To that effect, a simple mathematical model is presented to analyze the FT-IR data to determine the CO 2 effective diffusivity in the brine-saturated sandstone sample.

58 GEOSCIENCES↗