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All sourcesNASA NTRSDOE OSTI
DOE OSTI2025

Biotransformation of Phenolics in Spent Liquor from Aqueous Ammonia Pretreatment

Spent liquors of biomass pretreatment provide a source for renewable chemical production. These liquors require treatment before being discharged; otherwise, they negatively impact the environment. Herein, spent liquors from aqueous ammonia pretreatment of poplar wood are characterized for phenolic content via liquid chromatography–mass spectrometry and nuclear magnetic resonance spectroscopy. The main phenolics are phenol, p-hydroxybenzamide (pHBAm), and p-hydroxybenzoic acid (pHBA), of which pHBAm and pHBA are produced from the ester-linked p-hydroxybenzoates in poplar wood. Phenol is produced from pHBA via decarboxylation. The potential biotransformation of the extracted phenolics into 2-pyrone-4,6-dicarboxylic acid (PDC) is assessed using an engineered strain of Novosphingobium aromaticivorans DSM12444 (PDC strain). Biotransformation of pHBAm to PDC is shown to be possible in the presence of pHBA, but not when pHBAm is the sole phenolic substrate, this is the first reported observation of N. aromaticivorans producing PDC from an aromatic amide. The phenol present is not transformed to PDC and does not inhibit PDC production. This study demonstrates that the phenolic amide in spent liquor from ammonia pretreatment can be valorized via biotransformation using N. aromaticivorans, which adds to the growing versatility of N. aromaticivorans as a microbial chassis for converting plant-derived compounds to useful products.

biomass↗
DOE OSTI2026

NMR of Fully and Partially 13 C-Enriched Biomass Enhances Pendent Group Structural Characterization

Traditional solution-state NMR experiments may either fail or yield unsatisfactory results when employing fully- 13 C-labeled biomass due to complications arising from 13 C– 13 C coupling. Constant-time analogs of HSQC experiments mitigate such issues and deliver enhanced sensitivity. A rarely reported CT-HSQC-TOCSY experiment allows the proton coupling network to deliver much of the same value as the parent experiment on unlabeled or 10–15%- 13 C-labeled biomass polymers but with enhanced sensitivity. In the absence of a viable HMBC analog for long-range correlations, a relayed C–C experiment, i.e., via directly bonded 13 C-labeled networks, enables the reliable assignment of coupled carbons, with the added advantage of correlating the more elusive quaternaries. A C–C-FLOPSY experiment takes advantage of fully- 13 C-labeled materials for mapping extensive carbon networks in the complex polymer mixtures inherent in biomass. Various pendent groups (tricin units, cis- and trans-p-coumarates, and p -hydroxybenzoates) that adorn lignins, and the cis- and trans-ferulates on arabinoxylan polysaccharides, are exquisitely revealed in spectra from isolated lignins or whole-cell-wall materials from maize, sorghum, and poplar.

biopolymers↗
DOE OSTI2026

Using Novosphingobium aromaticivorans for Concurrent Production of Intracellular and Extracellular Products from Aromatics Extracted from Poplar Biomass

Achieving high biochemical production in biotransformations of renewable resources requires using concentrated cultures that not only generate the product of interest but also produce abundant microbial cell waste. We explored the concept of gaining value from microbial cells by producing intracellular products in tandem with a desired extracellular product. Specifically, we engineered a strain ofNovosphingobium aromaticivorans to extracellularly produce 2-pyrone-4,6-dicarboxylic acid (PDC) from aromatic substrates and to intracellularly accumulate astaxanthin along with coenzyme Q 10 , all of which are products of industrial interest. Achieving the goal of concurrent production of intracellular and extracellular products required the creative application of bioreactor engineering principles. Although a continuously fed membrane bioreactor (MBR) maximized extracellular product biosynthesis, it had a negative effect on intracellular product accumulation. However, operating the MBR as a sequencing batch reactor (MBR-SBR) with a step-feed resulted in stable concurrent production of both extracellular and intracellular products. With aromatics extracted from poplar biomass, we achieved productivities of 1.14 g of PDC/L-h for the extracellular product and 0.04 mg of astaxanthin/L-h and 0.64 mg of CoQ 10 /L-h for intracellular products, respectively. Our findings demonstrate that the mode of operation of a bioreactor impacts the simultaneous production of intracellular and extracellular products byN. aromaticivorans.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗
DOE OSTI2025

Elephant range and population, strontium isotopes, and genetics combine to give local-scale specificity to ivory hotspot tracking

We use Sr isotopes to increase the precision of DNA-based origin estimates of wildlife products. Population information is used to develop Sr isotope Elephant Polygons that are overlaid onto the region of origin identified by DNA assignment to determine the sources of seized ivory samples. Our approach is cognizant of isotope mixing due to isotope turnover within animals and also of the large home range of elephants or other mobile species. Genetic information from 3 different law enforcement ivory seizures suggests a region of origin confined to Kenya and Tanzania in eastern Africa. We determine characteristic 87 Sr/ 86 Sr ratios for each of 25 different Elephant Polygons within this region using analyses of more the 600 known-origin reference samples. Using both the 87 Sr/ 86 Sr ratios of the seized ivory samples and elephant population estimates from individual Elephant Polygons we find that at least 75 % of the samples likely came from a single Elephant Polygon which includes the Tsavo National Parks in Kenya and the Mkomazi National Park in Tanzania. A few samples may have come from other regions, most likely from Tanzania. This study illustrates the value of combining genetics, isotope geochemistry, and population surveys in wildlife forensics studies.

Africa↗
DOE OSTI2025

Thermodynamics shapes the in vivo enzyme burden of glycolytic pathways

Thermodynamically constrained reactions and pathways are hypothesized to impose greater protein demands on cells, requiring higher enzyme amounts to sustain a given flux compared to those with stronger thermodynamics. To test this, we quantified the absolute concentrations of glycolytic enzymes in three bacterial species— Zymomonas mobilis, Escherichia coli , and Clostridium thermocellum —which employ distinct glycolytic pathways with varying thermodynamic driving forces. By integrating enzyme concentration data with corresponding in vivo metabolic fluxes and ΔG measurements, we found that the highly favorable Entner-Doudoroff pathway in Z. mobilis requires only one-fourth the amount of enzymatic protein to sustain the same flux as the thermodynamically constrained pyrophosphate-dependent glycolytic pathway in C. thermocellum , with the Embden-Meyerhof-Parnas pathway in E. coli exhibiting intermediate thermodynamic favorability and enzyme demand. Across all three pathways, early reactions with stronger thermodynamic driving forces generally required lower enzyme investment than later, less favorable steps. Additionally, reflecting differences in glycolytic strategies, the highly reversible ethanol fermentation pathway in C. thermocellum requires 10-fold more protein to maintain the same flux as the irreversible, forward-driven ethanol fermentation pathway in Z. mobilis . Thus, protein investment across glycolytic pathways reflects differences in their thermodynamic favorability.

Clostridium thermocellum↗
DOE OSTI2026

Engineering of 2‐ketoacid Decarboxylases for Production of Isobutanol and Other Fusel Alcohols in Saccharomyces cerevisiae

Isobutanol is a fusel alcohol that can be produced microbially for use as a biofuel or upgraded into sustainable aviation fuel (SAF). A key enzyme in the isobutanol biosynthetic pathway is 2-ketoacid decarboxylase (KDC), which irreversibly decarboxylates 2-ketoisovalerate (KIV) to yield isobutyraldehyde. However, many previously characterized KDC enzymes also act promiscuously on other 2-ketoacids, (e.g., pyruvate) to produce a related aldehyde (e.g., acetaldehyde). This unwanted side reaction is especially important when isobutanol is produced in Saccharomyces cerevisiae (S. cerevisiae) because it leads to pyruvate being diverted to ethanol. In order to make S. cerevisiae a strict isobutanologen, a KDC enzyme that is specific for KIV must be deployed. In this study, we used a combination of cell-based and in vitro enzyme assays to investigate KDC substrate specificity, characterizing a large set of homologs for KIV, pyruvate, and phenylpyruvate (PPV) activity. A diverse range of substrate specificities was discovered, and some previously uncharacterized KDCs were revealed to have high KIV activity and low pyruvate activity. Multi-site saturation mutagenesis (SSM) of one of these KDCs identified mutants with increased KIV activity, while maintaining low levels of pyruvate activity. In a KIV bioconversion experiment, bioprospected and engineered KDCs allowed similar KIV consumption to when using the previously characterized Lactococcus lactis KdcA, though with some ethanol also produced. The KDCs identified here show promise for production of isobutanol and other alcohols derived from 2-ketoacids, and the dataset of newly characterized KDCs can inform future efforts to understand and engineer substrate specificity in KDCs.

2-ketoacid decarboxylase↗
DOE OSTI2025

Risk of Mortality in Family Members of Men Seeking Fertility Assessment

Objective: To assess mortality in family members of men seeking fertility assessment. Subfertility serves as a biomarker for overall somatic health, and poor semen quality is associated with increased risk of hospitalization and mortality from chronic conditions. However, it is unclear if these risks extend to family members of men with low sperm count. Design: Retrospective cohort study. Subjects: Family members, up to third-degree relatives, of men in the Subfertility, Health and Assisted Reproduction and the Environment cohort who underwent a semen analysis as part of a fertility assessment 1996–2017. Relatives of men with a recorded total sperm count who lived in Utah for ≥1 year 1904–2017 were included in the analysis (N = 22,280 families). Exposure: Individuals were classified by family membership. Families were classified as relatives of azoospermic (0M), oligozoospermic (<39M), or normozoospermic (≥39M) men. The average total sperm count of the proband (male relative) with fertility assessment was also included as a continuous exposure measure. Main Outcome Measures: The main outcomes were all-cause and cause-specific mortality risk by sex, age, and degree of relation: first-, second-, and third-degree. Cox proportional hazard models were used to test the association between fertility classification and mortality, controlling for sex, race/ethnicity, and birth year. Results: A total of 666,437 relatives of men with fertility assessment (N deaths = 183,974) were included in the analysis. Relative to normozoospermia families, all-cause mortality risk increased in oligozoospermia families (hazard ratio [HR] oligozoospermia , 1.03; 95% confidence interval [CI], 1.01–1.05). Close relatives, first- (HR oligozoospermia , 1.17; 95% CI, 1.07–1.28) and second-degree relatives (HR azoospermia , 1.11; 95% CI, 1.04–1.20; HR oligozoospermia , 1.05; 95% CI,1.01–1.09), of azoospermic and oligozoospermic men had the highest all-cause and cause-specific mortality risk, including death attributed to cardiovascular disease or congenital birth conditions. Conclusion: Our results suggest that familial all-cause and cause-specific mortality risk differ by fertility phenotype. Families of azoospermic and oligozoospermic men showed significantly increased risk, particularly for close relatives. This study provides further evidence that shared genetic and/or environmental factors could influence both fertility and somatic health.

Male fertility↗
DOE OSTI2025

Complex spin density wave ordering in La4Ni3O10

The discovery of high-temperature superconductivity in layered nickelates under pressure has recently triggered enormous interest. Studies of these compounds have revealed a density-wave-like transition at ambient pressure, though its connection with superconductivity is still not well understood. Here, we report a detailed µ⁢SR study on single crystals of trilayer nickelate La4⁢Ni3⁢O10 at ambient pressure. We have identified a spin-density-wave (SDW) transition at the temperature of 𝑇N∼130K, as well as a broad crossover around 70–100 K. Based on the temperature dependence of the muon precession amplitudes and magnetic susceptibility, we attribute this additional crossover either to a spin reorientation, or to an inhomogeneous SDW ordering.

Gardner, Jason [ORNL] (ORCID:0000000278234072)↗
DOE OSTI2025

Combining Eddy Covariance Towers, Field Measurements, and the MEMS 2 Ecosystem Model Improves Confidence in the Climate Impacts of Bioenergy With Carbon Capture and Storage

ABSTRACT Carbon dioxide removal technologies such as bioenergy with carbon capture and storage (BECCS) are required if the effects of climate change are to be reversed over the next century. However, BECCS demands extensive land use change that may create positive or negative radiative forcing impacts upstream of the BECCS facility through changes to in situ greenhouse gas fluxes and land surface albedo. When quantifying these upstream climate impacts, even at a single site, different methods can give different estimates. Here we show how three common methods for estimating the net ecosystem carbon balance of bioenergy crops established on former grassland or former cropland can differ in their central estimates and uncertainty. We place these net ecosystem carbon balance forcings in the context of associated radiative forcings from changes to soil N 2 O and CH 4 fluxes, land surface albedo, embedded fossil fuel use, and geologically stored carbon. Results from long term eddy covariance measurements, a soil and plant carbon inventory, and the MEMS 2 process‐based ecosystem model all agree that establishing perennials such as switchgrass or mixed prairie on former cropland resulted in net negative radiative forcing (i.e., global cooling) of −26.5 to −39.6 fW m −2 over 100 years. Establishing these perennials on former grassland sites had similar climate mitigation impacts of −19.3 to −42.5 fW m −2 . However, the largest climate mitigation came from establishing corn for BECCS on former cropland or grassland, with radiative forcings from −38.4 to −50.5 fW m −2 , due to its higher plant productivity and therefore more geologically stored carbon. Our results highlight the strengths and limitations of each method for quantifying the field scale climate impacts of BECCS and show that utilizing multiple methods can increase confidence in the final radiative forcing estimates.

Falvo, Grant [Department of Plant, Soil and Microb↗
DOE OSTI2026

Transgenic Mixed‐Linkage‐Glucan Enhancement Affects Root Characteristics and Decomposition in Soils of Contrasting Vegetation History

ABSTRACT Development of transgenic bioenergy sorghum [ Sorghum bicolor (L.) Moench] with increased contents of mixed‐linkage (1,3;1,4)‐β‐glucan (MLG) is an important step towards enhancing quality of bioenergy feedstocks. Since MLG‐enhancement leads to greater biomass digestibility, our overarching hypothesis is that root residues of MLG‐enhanced plants may be more readily decomposed in the soil, potentially creating new opportunities for optimizing soil carbon (C) sequestration, nutrient cycling, and overall agricultural sustainability. The study examined morphological, chemical, and enzymatic characteristics of fine and coarse roots of four bioenergy sorghum genotypes. Then, we incubated the roots within soils with contrasting vegetation histories while measuring C mineralization, microbial biomass C (MBC), and activity of hydrolytic enzymes and calculating vector length and vector angle enzymatic stoichiometry parameters. The results indicated that MLG‐enhancing transformations increased root total nitrogen (N) contents, decreased C/N ratios, and were associated with higher MLG concentrations in fine than in coarse roots. Incubations with transgenic roots led to 16%–38% higher MBC and 19%–41% lower microbial metabolic quotient (qCO 2 ). While enzyme activity differed markedly among the studied genotypes, it did not directly respond to MLG levels in root tissues. The increase in MBC without concurrent increases in C mineralization or hydrolytic enzyme activities in transgenic genotypes suggests that MLG enhancement promoted microbial anabolic retention of root‐derived C rather than stimulating catabolic decomposition. Enzymatic vector results indicated that these parameters reflect a variety of drivers behind microbial enzyme production, including availability of specific substrates, such as MLG here, and/or deficiency in specific nutrients, such as phosphorus (P). The study confirms the positive impacts from the roots of engineered MLG‐enhanced bioenergy plants on soil microbial activity and highlights the interactive influences on the MLG‐enhancement effects from root size and inherent soil properties.

Mahmoodabadi, Majid [Department of Plant, Soil, an↗
DOE OSTI2025

Confocal Raman Microscopy for Measuring In Situ Temperature-Dependent Structural Changes in Poly(Ethylene Oxide) Thin Films

Crystallization from the melt is a critical process governing the properties of semi-crystalline polymeric materials. While structural analyses of melting and crystallization transitions in bulk polymers have been widely reported, in contrast, those in thin polymer films on solid supports have been underexplored. Herein, in situ Raman microscopy and self-modeling curve resolution (SMCR) analysis are applied to investigate the temperature-dependent structural changes in poly(ethylene oxide) (PEO) films during melting and crystallization phase transitions. By resolving complex overlapping sets of spectra, SMCR analysis reveals that the thermal transitions of 50 µm thick PEO films comprise two structural phases: an ordered crystalline phase and a disordered amorphous phase. The ordered structure of the crystalline PEO film entirely disappears as the polymer is heated; conversely, the disordered structure of the amorphous PEO film reverts to the ordered structure as the polymer is cooled. Broadening of the Raman bands was observed in PEO films above the melting temperature (67 °C), while sharpening of bands was observed below the crystallization temperature (45 °C). The temperatures at which these spectral changes occurred were in good agreement with differential scanning calorimetry (DSC) measurements, especially during the melting transition. The results illustrate that in situ Raman microscopy coupled with SMCR analysis is a powerful approach for unraveling complex structural changes in thin polymer films during melting and crystallization processes. Furthermore, we show that confocal Raman microscopy opens opportunities to apply the methodology to interrogate the structural features of PEO or other surface-supported polymer films as thin as 2 µm, a thickness regime beyond the reach of conventional thermal analysis techniques.

Koh, Miharu [Department of Chemistry, University o↗
DOE OSTI2024

Intrinsic and environmental drivers of pairwise cohesion in wild Canis social groups

Animals within social groups respond to costs and benefits of sociality by adjusting the proportion of time they spend in close proximity to other individuals in the group (cohesion). Variation in cohesion between individuals, in turn, shapes important group-level processes such as subgroup formation and fission–fusion dynamics. Although critical to animal sociality, a comprehensive understanding of the factors influencing cohesion remains a gap in our knowledge of cooperative behavior in animals. We tracked 574 individuals from six species within the genus Canis in 15 countries on four continents with GPS telemetry to estimate the time that pairs of individuals within social groups spent in close proximity and test hypotheses regarding drivers of cohesion. Pairs of social canids (Canis spp.) varied widely in the proportion of time they spent together (5%–100%) during seasonal monitoring periods relative to both intrinsic characteristics and environmental conditions. The majority of our data came from three species of wolves (gray wolves, eastern wolves, and red wolves) and coyotes. For these species, cohesion within social groups was greatest between breeding pairs and varied seasonally as the nature of cooperative activities changed relative to annual life history patterns. Across species, wolves were more cohesive than coyotes. For wolves, pairs were less cohesive in larger groups, and when suitable, small prey was present reflecting the constraints of food resources and intragroup competition on social associations. Pair cohesion in wolves declined with increased anthropogenic modification of the landscape and greater climatic variability, underscoring challenges for conserving social top predators in a changing world. We show that pairwise cohesion in social groups varies strongly both within and across Canis species, as individuals respond to changing ecological context defined by resources, competition, and anthropogenic disturbance. Our work highlights that cohesion is a highly plastic component of animal sociality that holds significant promise for elucidating ecological and evolutionary mechanisms underlying cooperative behavior.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2025

Higher levels of mixed-linkage (1,3;1,4)-β-glucan in transgenic grasses may impact soil C processing

Carbohydrates, including mixed-linkage glucan (MLG), in grass cell walls make them a valuable potential feedstock for biofuel production. Hence, the development of transgenic grasses with elevated levels of MLG is being actively pursued worldwide. Changes in chemical and physical root characteristics of MLG-overproducing transgenic plants can affect processing of the root-derived carbon (C) by soil microorganisms, impacting soil C cycling. Here, this study is the first attempt to elucidate the impact of MLG-related genetic modifications on root traits, root decomposition, and soil C processing. We explored four genotypes of Brachypodium ( Brachypodium distachyon ): a wildtype, a loss-of-function mutant with low MLG, an MLG overexpressing line, and a line lacking MLG hydrolase (with high MLG), incubating their roots in soils of two contrasting vegetation histories: monoculture switchgrass and polyculture restored prairie. The four genotypes exhibited contrasting root MLG and soluble sugar concentrations, and different growth phenotypes. Roots with the highest MLG content resulted in a ∼55 % increase in microbial biomass C compared to wildtype in both soils. However, the genotype effects on C mineralization rates were influenced by the vegetation history, with significant effects observed only in the soil from switchgrass but not prairie origin. While further work is required to understand the full impact of MLG-overproducing plants on soil C accrual, our findings suggest that their influence on soil C processes cannot be discounted.

Brachypodium distachyon↗
DOE OSTI2025

Ether Cleavage Decreases the Ion Exchange Capacity of Anion Exchange Membranes

Anion exchange membranes (AEMs) are integral to fuel cells and water electrolysis systems but suffer from poor durability under alkaline conditions. Ether cleavage is an important failure pathway of poly(arylene ether)-based AEMs that compromises both mechanical stability and ion transport. While this degradation pathway is often studied in terms of polymer fragmentation, the role of newly formed hydrophilic groups has been largely overlooked. Here, we show that polymer scission leads to reduced mechanical rigidity, while the introduction of hydrophilic groups partially mitigates this loss. Under alkaline conditions, phenoxide groups formed during ether cleavage neutralize the polymer cations, leading to a previously unreported loss of ion exchange capacity (IEC). This IEC loss mechanism exacerbates the reduction in ionic conductivity, emphasizing the severity of ether cleavage as a degradation pathway. Recognizing that ether cleavage introduces significant chemical changes beyond polymer fragmentation provides critical insights into its interplay with other degradation mechanisms, such as the direct reduction of cationic sites by E2 and S N 2, and provides molecular-level interpretations for the concurrent effects of polymer scission and increased hydrophilicity on membrane performance.

crystal cleavage↗
DOE OSTI2025

Highly Active Hydrogen Evolution Reaction (HER) Catalysts Formed by Energetic Pt n Cluster Deposition: Deposition Dynamics and the HER Mechanism

Mass-selected Pt n + (n ≤ 7) were deposited at variable energies on highly oriented pyrolytic graphite (HOPG), creating highly active hydrogen evolution reaction (HER) electrocatalysts. HER mass activities were ~2 to >10 times higher than those for the surface atoms in bulk Pt and for Pt n deposited on several other supports. Thus, high activity reflects the Pt-C structures formed by energetic Pt n -HOPG impacts, in addition to high Pt surface availability. The Pt n /HOPG electrodes were probed by X-ray photoelectron spectroscopy, low energy ion scattering, and electron microscopy. Born-Oppenheimer molecular dynamics (BOMD) was used to simulate Pt n - HOPG impacts, revealing the types of structures formed at different energies, then DFT was used to probe their most important HER pathways. For low deposition energies, the Pt n deposit onto the HOPG surface with sub-unit sticking probability, aggregating at defects. With increasing deposition energy, the sticking probability initially decreases, then rises to unity as subplantation and defect creation allow formation of strongly bonded platinum-carbon structures. Barriers for HER on these structures were found to be low and weakly dependent on Pt n size, consistent with experiment. The activities were highest for small covalently-bonded Pt-C structures created at high deposition energies. The larger aggregated structures formed at low energies were less active, but still substantially better than the bulk Pt surface monolayer. The catalysts were stable in repeated potential cycling at reducing potentials, but electrodes containing subplanted Pt became more active when scanned to oxidizing potentials, due to emergence of subplanted Pt onto the surface.

08 HYDROGEN↗