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DOE OSTI2025

A cobalamin-dependent pathway of choline demethylation from the human gut acetogen Eubacterium limosum

Elevated serum levels of trimethylamine N-oxide (TMAO) are reported to promote the development of atherosclerosis. TMAO is produced by hepatic oxidation of trimethylamine (TMA) produced by the gut microbiome from dietary quaternary amines such as choline. Net TMA production in the gut depends on microbial enzymes that either produce or consume TMA and its precursors. Here we report the elucidation of a novel microbial pathway consuming choline without TMA production. The human gut acetogen Eubacterium limosum grows by demethylating choline to N-N-dimethylaminoethanol. Quantitative mass spectral analysis of the proteome revealed a multi-protein choline to tetrahydrofolate (THF) methyltransferase system present only in choline-grown cells. The components are encoded in a gene cluster on the genome and include MthB, an MttB superfamily member; MthC, homologous to methylotrophic cobalamin-binding proteins; MthA, homologous to cobalamin:THF methyltransferases; and MthK, a protein related to serine kinases. Together, MthB, MthC, and MthA methylate THF with phosphocholine, but not choline or other quaternary amines. MthB specifically methylates Co(I)-MthC with phosphocholine. MthK acts as a bifunctional choline kinase which can utilize ATP or the MthB demethylation product, N,N-dimethylaminoethanol phosphate, to phosphorylate choline. Together, MthK, MthB, MthC, and MthA are proposed to carry out the methylation of THF with choline. These results outline a THF methylation pathway in which choline is first activated with ATP to phosphocholine prior to demethylation to form N,N-dimethylaminoethanol phosphate. Furthermore, the latter can be recycled by MthK to form more phosphocholine without expending additional ATP, thus minimizing energy utilization during choline-dependent acetogenesis.

acetogenesis↗

NASA NTRS2018

Steps Toward Improved Integration, Search, and Analysis of Heterogeneous Data in the Astrobiology Habitable Environments Database

The Astrobiology Habitable Environments Database (AHED) is a new data system being developed as a long-term, open-access repository for astrobiology data. AHED is intended to store user-contributed results from NASA or externally-funded research in astrobiology, and to encourage sharing and synergy within the astrobiology community. However, the interdisciplinary nature of astrobiology presents some specific challenges to data management, integration, and analysis within AHED. In some disciplines (e.g., genomics), open databases thrive because the contributed products are fairly uniform and standardized (e.g., sequence data). In astrobiology, each investigation produces a unique set of data products; this makes it difficult to search across different datasets to find similar data, or to combine results from separate investigations. With AHED, we are taking steps to ensure there is adequate metadata - both at the dataset and record levels - to facilitate search, integration, and analysis. At the dataset level, we are developing a new metadata standard for describing astrobiology datasets, with detailed information about content, funding source, and scientific relevance, along with a set of topical keywords for characterizing datasets. At the record level, we are encouraging users to provide more structured content and finer-grained metadata. In many user-contributed science data repositories, few restrictions are placed on the uploaded data format, and minimal or no record-level metadata is required; thus users are unburdened when it comes to data preparation. The tradeoff is that deep integration and search across datasets is almost impossible without standardized structures and metadata. Although AHED users are free to upload minimally-described datasets, they will be encouraged to use database authoring tools (supplied by the underlying platform - Open Data Repository's Data Publisher) plus a set of customizable astrobiology-specific templates to help structure their data and provide standardized metadata. In reward for their extra effort, AHED will be able to deliver enhanced search, discovery, and analysis capabilities.

astrobiology↗

DOE OSTI2024

Biomass yields, reproductive fertility, compositional analysis, and genetic diversity of newly developed triploid giant miscanthus hybrids

Abstract Miscanthus × giganteus (giant miscanthus), first found as a naturally occurring hybrid, has shown promise as a bioenergy/biomass crop throughout much of the temperate world. This allotriploid (2 n = 3 x = 57) hybrid resulted from a cross between tetraploid Miscanthus sacchariflorus (2 n = 4 x = 76) and diploid Miscanthus sinensis (2 n = 2 x = 38) and is particularly desirable due to its low fertility that minimizes reseeding and potential invasiveness. However, there is limited genetic diversity in commonly grown cultivars of triploid M. × giganteus and breeding and development efforts to improve and domesticate this crop have been minimal. Here, we report on newly developed M. × giganteus hybrids compared with the industry standard M. × giganteus '1993‐1780'. Dry biomass yields of new hybrids ranged from 19.5 to 32.4 Mg/ha/year for the fourth growing season, compared with 21.0 Mg/ha/year for M. × giganteus '1993‐1780'. Plant reproductive fertility remained low for all accessions with overall fertility [(seed set × seed germination)/100] ranging from 0.3% to 4.5% for new hybrids compared to 0.4% for M. × giganteus '1993‐1780'. Culm density and height varied among accessions and were positively correlated with increased biomass. Based on compositional analyses, theoretical ethanol yields ranged from 9, 740 to 16,278 L/ha/year for new hybrids compared to 10,406 L/ha/year for M. × giganteus '1993‐1780'. Relative feed value indices were low overall and ranged between 66.0 and 72.8 for new hybrids compared to M. × giganteus '1993‐1780' with 71.3. The genetic diversity of new hybrids, compared with existing cultivars, was characterized using whole genome sequences. Based on pair‐wise distances, cluster analysis clearly showed increased diversity of new hybrids compared with earlier selections. These results document new triploid hybrids of M. × giganteus with enhanced biomass and theoretical ethanol yields in combination with broader genetic diversity and lowreproductive fertility.

Touchell, Darren H.↗

NASA NTRS2016

The Biomolecule Sequencer Project: Nanopore Sequencing as a Dual-Use Tool for Crew Health and Astrobiology Investigations

Human missions to Mars will fundamentally transform how the planet is explored, enabling new scientific discoveries through more sophisticated sample acquisition and processing than can currently be implemented in robotic exploration. The presence of humans also poses new challenges, including ensuring astronaut safety and health and monitoring contamination. Because the capability to transfer materials to Earth will be extremely limited, there is a strong need for in situ diagnostic capabilities. Nucleotide sequencing is a particularly powerful tool because it can be used to: (1) mitigate microbial risks to crew by allowing identification of microbes in water, in air, and on surfaces; (2) identify optimal treatment strategies for infections that arise in crew members; and (3) track how crew members, microbes, and mission-relevant organisms (e.g., farmed plants) respond to conditions on Mars through transcriptomic and genomic changes. Sequencing would also offer benefits for science investigations occurring on the surface of Mars by permitting identification of Earth-derived contamination in samples. If Mars contains indigenous life, and that life is based on nucleic acids or other closely related molecules, sequencing would serve as a critical tool for the characterization of those molecules. Therefore, spaceflight-compatible nucleic acid sequencing would be an important capability for both crew health and astrobiology exploration. Advances in sequencing technology on Earth have been driven largely by needs for higher throughput and read accuracy. Although some reduction in size has been achieved, nearly all commercially available sequencers are not compatible with spaceflight due to size, power, and operational requirements. Exceptions are nanopore-based sequencers that measure changes in current caused by DNA passing through pores; these devices are inherently much smaller and require significantly less power than sequencers using other detection methods. Consequently, nanopore-based sequencers could be made flight-ready with only minimal modifications.

John, K. K.↗

NASA NTRS2014

Chronic Lunar Dust Exposure on Rat Cornea: Evaluation by Gene Expression Profiling

Lunar dust is capable of entering habitats and vehicle compartments by sticking to spacesuits or other objects that are transferred into the spacecraft from the lunar surface and has been reported to cause irritation upon exposure. During the Apollo missions, crewmembers reported irritation specifically to the skin and eyes after contamination of the lunar and service modules. It has since been hypothesized that ocular irritation and abrasion might occur as a result of such exposure, impairing crew vision. Recent work has shown that both ultrafine and unground lunar dust exhibited minimal irritancy of the ocular surface (i.e., cornea); however, the assessment of the severity of ocular damage resulting from contact of lunar dust particles to the cornea has focused only on macroscopic signs of mechanical irritancy and cytotoxicity. Given the chemical reactive properties of lunar dust, exposure of the cornea may contribute to detrimental effects at the molecular level including but not limited to oxidative damage. Additionally, low level chronic exposures may confound any results obtained in previous acute studies. We report here preliminary results from a tissue sharing effort using 10‐week‐old Fischer 344 male rats chronically exposed to filtered air or jet milled lunar dust collected during Apollo 14 using a Jaeger‐NYU nose‐only chamber for a total of 120 hours (6 hours daily, 5 days a week) over a 4‐week period. RNA was isolated from corneas collected from rats at 1 day and 7 days after being exposed to concentrations of 0, 20, and 60 mg/m3 of lunar dust. Microarray analysis was performed using the Affymetrix GeneChip Rat Genome 230 2.0 Array with Affymetrix Expression Console and Transcriptome Analysis Console used for normalization and secondary analysis. An Ingenuity iReport"TM" was then generated for canonical pathway identification. The number of differentially expressed genes identified increases with dose compared to controls suggesting a more severe response to the lunar dust insult at higher levels. Pathways of interests that have been identified in all exposed samples include oxidative stress response, mitochondrial dysfunction, fibrosis, epithelial healing, TGF-Beta signaling, and extracellular matrix remodeling. Several biological processes related to cell migration, cellular proliferation, and eye development were also identified to be altered by exposure to lunar dust. Our preliminary results suggest that even a chronic insult of lunar dust as low as 20 mg/m(exp 3) elicits a molecular response in cornea tissue. Lunar dust on the surface of the moon would have the added properties of ionization and activation potentially leading to further damage to the cornea and greater sensitivity to any other environmental insult such as exposure to radiation. Additional studies are required to fully assess the risk of vision impairment and the mechanistic responses initiated in cornea exposed to lunar dust as well as the potential for long‐term effects to astronaut health

Theriot, C. A.↗

NASA NTRS2023

Molecular Identification of Microbial Contaminants

Microorganisms can have significant impacts on the success of NASA’s missions, including the integrity of materials, the reliability of scientific results, and maintenance of crew health. Robust cleaning and sterilization protocols are currently in place in NASA facilities, but agency experts agree that microbial contamination is unavoidable and its impact on NASA’s missions and science must be minimized. Therefore, it is critical to understand: 1) what specific microorganisms are present, 2) how they may impact scientific objectives, and 3) how to select appropriate mitigation strategies. The Marshall Space Flight Center (MSFC) Planetary Protection (PP) microbiology lab historically relied solely upon enumeration of culturable microbial contamination associated with spacecraft materials or cleanrooms. However, this process is time consuming, many microbes cannot be cultured, and very few can be identified with any fidelity using NASA standard microbiological methods. The work described in this white paper includes the establishment of molecular identification capabilities at MSFC, including DNA isolation, amplification, purification, and Sanger sequencing. This capability will not only improve planetary protection efforts at MSFC (i.e. by identifying contaminating microorganisms in cleanrooms or on spacecraft) but also offers a service center-wide for the identification of contaminants that arise in other projects, processing locations, or during set up and roll out of spacecraft. This work also lays the foundation for higher throughput efforts to identify large populations of microbes across the lifetime of a project and serves as the starting point for future work into whole genome sequencing, non-culture based methods, or additional characterization studies. Ultimately, accurate identification informs appropriate mitigation strategies, increasing the chances of success for NASA’s missions and objectives.

C. D. Cassilly↗

NASA NTRSDate not supplied

Elevating the Quality of Space Omics Sequencing Data: Innovations and Methodologies from NASA GeneLab Sample Processing Laboratory

NASA’s GeneLab, part of the NASA Open Science Data Repository, is a space-related database that hosts a diverse range of transcriptomics, proteomics, epigenomics and genomics data. The NASA GeneLab Sample Processing Laboratory (SPL) generates omics data from biological experiments conducted aboard the International Space Station, Space Shuttle and space related ground experiments, this omics data then hosted on the GeneLab repository. Samples generated such experiments pose numerous technical challenges such as small experimental sample size, variance in dissection times, limited tissue preservation methods, prolonged storage time, and more. GeneLab SPL team had developed specialized expertise in nucleic acid extraction, library preparation and sequencing of such biological samples via extensive training and years of experience. In order to ensure data accuracy and consistency across experiments, SPL has developed standardized protocols for each species and tissue type. These protocols in conjunction with quality control metrics and data standards are crucial in generating of high-quality data. SPL protocols and standards have been developed in collaboration with the scientific community and had been made publicly available on the GeneLab portal, guaranteeing comparability of datasets across spaceflight experiments. To ensure reliability of data generation, SPL leverages cutting-edge innovations in laboratory automation for sample processing. By leveraging these state-of-the-art platforms, SPL achieves high levels of data reproducibility while significantly minimizing sources of bias and variability, especially across experiments with large numbers of samples. Over the past few years, the space biology investigator community has accessed SPL-generated data from the Open Science Data Repository for a myriad of data re-analysis and re-use studies. We observe a trend that in-house SPL-generated data consistently outperforms outsourced sequencing data in terms of technical standards, quality control metrics, timeliness of data delivery, and sequencing and reagent efficiency. Superior data generation has and will continue to enable discoveries in disease, diagnostic tools, and the biological effects of long duration spaceflight.

GeneLab↗

NASA NTRSDate not supplied

Elevating the Quality of Space Omics Sequencing Data: Innovations and Methodologies from NASA GeneLab Sample Processing Laboratory

GeneLab↗

NASA NTRS2004

Malaria Modeling using Remote Sensing and GIS Technologies

Malaria has been with the human race since the ancient time. In spite of the advances of biomedical research and the completion of genomic mapping of Plasmodium falciparum, the exact mechanisms of how the various strains of parasites evade the human immune system and how they have adapted and become resistant to multiple drugs remain elusive. Perhaps because of these reasons, effective vaccines against malaria are still not available. Worldwide, approximately one to three millions deaths are attributed to malaria annually. With the increased availability of remotely sensed data, researchers in medical entomology, epidemiology and ecology have started to associate environmental and ecological variables with malaria transmission. In several studies, it has been shown that transmission correlates well with certain environmental and ecological parameters, and that remote sensing can be used to measure these determinants. In a NASA project, we have taken a holistic approach to examine how remote sensing and GIs can contribute to vector and malaria controls. To gain a better understanding of the interactions among the possible promoting factors, we have been developing a habitat model, a transmission model, and a risk prediction model, all using remote sensing data as input. Our objectives are: 1) To identify the potential breeding sites of major vector species and the locations for larvicide and insecticide applications in order to reduce costs, lessen the chance of developing pesticide resistance, and minimize the damage to the environment; 2) To develop a malaria transmission model characterizing the interactions among hosts, vectors, parasites, landcover and environment in order to identify the key factors that sustain or intensify malaria transmission, and 3) To develop a risk model to predict the occurrence of malaria and its transmission intensity using epidemiological data and satellite-derived or ground-measured environmental and meteorological data.

Kiang, Richard↗

DOE OSTI2025

Signal sequences target enzymes and structural proteins to bacterial microcompartments and are critical for microcompartment formation

ABSTRACT Spatial organization of pathway enzymes has emerged as a promising tool to address several challenges in metabolic engineering, such as flux imbalances and off-target product formation. Bacterial microcompartments (MCPs) are a spatial organization strategy used natively by many bacteria to encapsulate metabolic pathways that produce toxic, volatile intermediates. Several recent studies have focused on engineering MCPs to encapsulate heterologous pathways of interest, but how this engineering affects MCP assembly and function is poorly understood. In this study, we investigated the role of signal sequences, short domains that target proteins to the MCP core, in the assembly of 1,2-propanediol utilization (Pdu) MCPs. We characterized two novel Pdu signal sequences on the structural proteins PduM and PduB, which constitute the first report of metabolosome signal sequences on structural proteins rather than enzymes. We then explored the role of enzymatic and structural Pdu signal sequences on MCP assembly by deleting their encoding sequences from the genome alone and in combination. Deleting enzymatic signal sequences decreased the MCP formation, but this defect could be recovered in some cases by overexpressing genes encoding the knocked-out signal sequence fused to a heterologous protein. By contrast, deleting structural signal sequences caused similar defects to knocking out the genes encoding the full-length PduM and PduB proteins. Our results contribute to a growing understanding of how MCPs form and function in bacteria and provide strategies to mitigate assembly disruption when encapsulating heterologous pathways in MCPs. IMPORTANCE Spatially organizing biosynthetic pathway enzymes is a promising strategy to increase pathway throughput and yield. Bacterial microcompartments (MCPs) are proteinaceous organelles that many bacteria natively use as a spatial organization strategy to encapsulate niche metabolic pathways, providing significant metabolic benefits. Encapsulating heterologous pathways of interest in MCPs could confer these benefits to industrially relevant pathways. Here, we investigate the role of signal sequences, short domains that target proteins for encapsulation in MCPs, in the assembly of 1,2-propanediol utilization (Pdu) MCPs. We characterize two novel signal sequences on structural proteins, constituting the first Pdu signal sequences found on structural proteins rather than enzymes, and perform knockout studies to compare the impacts of enzymatic and structural signal sequences on MCP assembly. Our results demonstrate that enzymatic and structural signal sequences play critical but distinct roles in Pdu MCP assembly and provide design rules for engineering MCPs while minimizing disruption to MCP assembly.

Johnson, Elizabeth R. (ORCID:0000000179236881)↗

DOE OSTI2026

Integrative analysis of CAM photosynthesis reveals its impact on primary metabolism in Yucca

Crassulacean Acid Metabolism (CAM) is an adaptation that temporally separates carbon uptake at night from photosynthesis during the day. CAM has evolved repeatedly across vascular plants, as its emergence may depend on simple regulatory changes to deeply conserved metabolic pathways. Modern CAM research relies heavily on interpretation of transcriptomic data, though regulation occurs at multiple levels following transcription. Additionally, while most research to date has focused on a handful of genes and metabolites in the core CAM pathway, the co-option of conserved regulatory and functional genes is bound to have wide-ranging effects on other aspects of primary metabolism. In this study, we integrate transcriptomic, proteomic, and metabolomic data to compare primary metabolism between the CAM species Yucca aloifolia and closely related C 3 species, Y. filamentosa. We observe minimal correlation between protein abundance and mRNA expression, suggesting significant post-transcriptional regulation in CAM species. We also find evidence of shifts in gene expression and metabolite accumulation outside of the central CAM pathway, suggesting that the shift to CAM has cascading effects across primary metabolism, especially nitrogen metabolism. Our findings provide insights into the metabolic shifts associated with CAM evolution, and highlight the complexity of its regulation at multiple biological levels.

59 BASIC BIOLOGICAL SCIENCES↗

NASA NTRS2026

Does the International Space Station Leak DNA? Preliminary Results from the ISS External Microorganisms Payload

Existing crewed spacecraft like the ISS (International Space Station) leak by design. The ISS routinely releases gas to maintain life support systems and when astronauts exit the station to perform space walks. The chemical component of this leakage is well characterized, but the biological components are not. The ISS is not subject to planetary protection requirements, but planned missions to Mars will use similar systems and will be subject to planetary protection requirements. If detectable microorganisms are escaping through vents and or airlocks we may need to redesign our crewed habitats to minimize this type of contamination. To test the hypothesis that microorganisms from inside ISS are detectable on exterior surfaces an astronaut used the ISS External Microorganisms sampling kit (Rucker et al. 2018) to sample exterior surfaces of the ISS during an EVA (Extra Vehicular Activity) in January of 2025. These samples were returned to Earth for DNA extraction and sequencing. We successfully, extracted and sequenced bacterial, fungal and viral DNA from these samples that was not present in the negative controls. These results should help NASA refine the planetary protection requirements for crewed missions. Methods: The samples were collected using sterile, DNA free, buccal swabs (23 mm. diameter) housed in custom canisters. Each canister uses a 0.2 μm Teflon filter to maintain sterility as the caddy, holding 8 swabs moves in and out of vacuum. The astronaut sampled the: 1) airlock vestibule, 2) airlock thermal cover, 3) a gap in the micrometeorite shielding near the airlock, 4) a handrail near the airlock, 5) the Carbon Dioxide Removal Assembly vent, and 6) the Vacuum Exhaust System vent. The seventh swab was exposed to vacuum during the EVA without touching it to a surface. The eighth swab, a negative control, was not opened until the caddy returned to Earth. DNA was extracted from the swabs using a QIamp UCP Pathogen kit and prepared for sequencing on an Aviti (Element Biosciences) sequencer (Arslan et al. 2024). The resulting sequences were analyzed using the EDGE Bioinformatics platform (Li et al. 2017). The sequences were analyzed individually using tools like BLAST, GOTTCHA2, Kraken2, and PanGIA. The data were also assembled into metagenome assembled genomes) using tools like CONCOCT, MaxBin2 and MetaBAT2. Results: We successfully extracted and sequenced bacterial, archaeal, fungal and viral DNA from all seven samples. The handrail swab had the lowest number of reads (768,651) and the airlock thermal cover had the highest number of reads (8,819,230). These samples contain DNA from human associated bacteria (e.g. Crynebacterium riegelii ), fungi (.e.g. Penicillium rubens ), and viruses (e.g Alphapapillomavirus ). Conclusion: Preliminary interpretation suggest that the airlock and the space suits themselves are the largest sources of contaminant DNA. Most if not all of the DNA is from organisms known to be present inside the ISS. Vents attached to life support systems may be a lesser source of biological contamination. Further analysis should help NASA address planetary protection knowledge gaps for crewed missions.

Aaron B Regberg↗

DOE OSTI2026

Data for An End-to-End Pipeline for Succinic Acid Production at an Industrially Relevant Scale Using Issatchenkia orientalis

Microbial production of succinic acid (SA) at an industrially relevant scale has been hindered by high downstream processing costs arising from neutral pH fermentation for over three decades. Here, we metabolically engineer the acid-tolerant yeast Issatchenkia orientalis for SA production, attaining the highest titers in sugar-based media at low pH (pH 3) in fed-batch fermentations, i.e. 109.5 g/L in minimal medium and 104.6 g/L in sugarcane juice medium. We further perform batch fermentation using sugarcane juice medium in a pilot-scale fermenter (300×) and achieve 63.1 g/L of SA, which can be directly crystallized with a yield of 64.0%. Finally, we simulate an end-to-end low-pH SA production pipeline, and techno-economic analysis and life cycle assessment indicate our process is financially viable and can reduce greenhouse gas emissions by 34–90% relative to fossil-based production processes. We expect I. orientalis can serve as a general industrial platform for production of organic acids.

Metabolomics↗

DOE OSTI2025

Alternate routes to acetate tolerance lead to varied isoprenol production from mixed carbon sources in Pseudomonas putida

ABSTRACT Lignocellulose is a renewable resource for the production of a diverse array of platform chemicals, including the biofuel isoprenol. Although this carbon stream provides a rich source of sugars, other organic compounds, such as acetate, can be used by microbial hosts. Here, we examined the growth and isoprenol production in a Pseudomonas putida strain pre-tolerized (“PT”) background where its native isoprenol catabolism pathway is deleted, using glucose and acetate as carbon sources. We found that PT displays impaired growth in minimal medium containing acetate and often fails to grow in glucose-acetate medium. Using a mutant recovery-based approach, we generated tolerized strains that overcame these limitations, achieving fast growth and isoprenol production in the mixed carbon feed. Changes in the glucose and acetate assimilation routes, including an upregulation in PP_0154 (SpcC, succinyl-CoA:acetate CoA-transferase) and differential expression of the gluconate assimilation pathways, were key for higher isoprenol titers in the tolerized strains, whereas a different set of mechanisms were likely enabling tolerance phenotypes in media containing acetate. Among these, a coproporphyrinogen-III oxidase (HemN) was upregulated across all tolerized strains and in one isolate required for acetate tolerance. Utilizing a defined glucose and acetate mixture ratio reflective of lignocellulosic feedstocks for isoprenol production in P. putida allowed us to obtain insights into the dynamics and challenges unique to dual carbon source utilization that are obscured when studied separately. Together, this enabled the development of a P. putida bioconversion chassis able to use a more complex carbon stream to produce isoprenol. IMPORTANCE Acetate is a relatively abundant component of many lignocellulosic carbon streams and has the potential to be used together with sugars, especially in microbes with versatile catabolism such as P. putida . However, the use of mixed carbon streams necessitates additional optimization. Furthermore, the use of P. putida for the production of the biofuel target, isoprenol, requires the use of engineered strains that have additional growth and production constraints when cultivated in acetate and glucose mixtures. In this study, we generate acetate-tolerant P. putida strains that overcome these challenges and examine their ability to produce isoprenol. We show that acetate tolerance and isoprenol production, although independent phenotypes, can both be optimized in a given P. putida strain. Using proteomics and whole genome sequencing, we examine the molecular basis of both phenotypes and show that tolerance to acetate can occur via alternate routes and result in different impacts on isoprenol production.

de Siqueira, Guilherme M. V. (ORCID:00000002364563↗

DOE OSTI2025

Fast event-based electron counting for small-molecule structure determination by MicroED

Electron counting helped realize the resolution revolution in single-particle cryoEM and is now accelerating the determination of MicroED structures. Its advantages are best demonstrated by new direct electron detectors capable of fast (kilohertz) event-based electron counting (EBEC). This strategy minimizes the inaccuracies introduced by coincidence loss (CL) and promises rapid determination of accurate structures. We used the Direct Electron Apollo camera to leverage EBEC technology for MicroED data collection. Given its ability to count single electrons, the Apollo collects high-quality MicroED data from organic small-molecule crystals illuminated with incident electron beam flux densities as low as 0.01–0.045 e − /Å 2 /s. Under even the lowest flux density (0.01 e − /Å 2 /s) condition, fast EBEC data produced ab initio structures of a salen ligand (268 Da) and biotin (244 Da). Each structure was determined from a 100° wedge of data collected from a single crystal in as few as 50 s, with a delivered fluence of only ∼0.5 e − /Å 2 . Fast EBEC data collected with a fluence of 2.25 or 3.33 e − /Å 2 also facilitated a 1.5 Å structure of thiostrepton (1665 Da). While refinement of these structures appeared unaffected by CL, a CL adjustment applied to EBEC data further improved the distribution of intensities measured from the salen ligand and biotin crystals. However, CL adjustment only marginally improved the refinement of their corresponding structures, signaling the already high counting accuracy of detectors with counting rates in the kilohertz range. Overall, by delivering low-dose structure-worthy data, fast EBEC collection strategies open new possibilities for high-throughput MicroED.

EBEC↗

DOE OSTI2025

Degradable Biocomposite Thermoplastic Polyurethanes

In this project, the team developed tough and degradable biocomposite thermoplastic polyurethanes (TPUs) by incorporating bacterial spores into TPUs as a biofunctional living filler. The team screened various bacteria and selected the Bacillus subtilis ATCC 6633 strain as the final candidate, primarily due to its genomic availability, sporulation ability and TPU assimilation activity. The heat-shock tolerance of ATCC 6633 spores was further improved through evolutionary engineering via Adaptive Laboratory Evolution (ALE), demonstrating a 17.7-fold enhanced germination efficiency post heat-shock treatment compared to the wild-type strain (WT). The team fabricated biocomposite TPUs by incorporating lyophilized powder of heat-shock tolerized (HST) spores during the hot melt extrusion (HME) of TPU at 135 °C. The baseline TPU used in this project is a commercially available soft-grade TPU (BCF45) manufactured by BASF. Colony forming unit (CFU) assays quantified that WT and HST spores in the TPU matrix retained approximately 20% and 100% survivability, respectively, after HME. Tensile testing demonstrated that the spores behaved as a polymer-reinforcing filler, positively affecting the overall tensile properties of the biocomposite TPU. For example, biocomposite TPU with WT and HST spores (BC TPU WT and BC TPU HST , respectively) exhibited up to 25% and 37% improved toughness, respectively, compared to TPU without spores. BC TPU HST showed remarkably improved disintegration in autoclaved compost (92% mass loss in 5 months), which simulated a microbially poor environment for TPU degradation. When compared to TPU without spores (44% mass loss in 5 months) the acceleration of degradation is marked. Respirometry confirmed that 72% of BC TPU HST was biomineralized into CO2 within 6 months, indicating that spores in the biocomposite TPU were germinated by utilizing nutrients in the autoclaved compost, facilitating TPU degradation at the end of the material's life. The team demonstrated the scale-up of biocomposite TPU fabrication using continuous extrusion and injection molding techniques. Processing conditions optimized in a lab-scale microcompounder were successfully transferred to a continuous extruder with a 30-fold increased throughput. Biocomposite TPUs prepared using these industry-relevant processes showed comparable toughness improvements to samples prepared in the lab-scale extruder. Excitingly, following compounding in the pilot-extruder the composite material could be injection molded, while retaining high spore viability and similar toughness improvements. The team also found that spores in biocomposite TPU served as antioxidants, preventing toughness decay during the recycled extrusion of BC TPU HST . Long-term storage tests over one year showed that the addition of spores had no negative effect on the longevity of the TPU. Furthermore, the team demonstrated the fabrication of spore-bearing biocomposite polymers with other polyesters such as PBAT, PLA, and PCL. We obtained promising preliminary data that showed overall toughness improvements for all polymers with spore addition. Finally, life cycle assessment (LCA) and techno-economic analysis (TEA) were carried out, which indicated minimal additional cost of fabrication. Overall, a tough and degradable biocomposite thermoplastic was successfully developed through this project, with all tasks completed successfully, achieving >100% of the objectives.

36 MATERIALS SCIENCE↗

DOE OSTI2025

Data for "Land-based Resources for Engineered Carbon Dioxide Removal in the United States Exceed the Expected Needs"

Gigatonne-scale atmospheric carbon dioxide removal (CDR), alongside deep emission cuts, is critical to stabilizing the climate. However, some of the most scalable CDR technologies are also the most land intensive. Here, we examine whether adequate land resources exist in the contiguous United States to meet CDR targets when prioritizing grid emissions reduction, food production, and the protection of sensitive ecosystems. We focus on biomass carbon removal and storage (BiCRS) and direct air capture and storage (DACS) and show that suitable lands exceed the expected needs: 37.6 million hectares of land are available for BiCRS, resulting in 0.26 GtCO2 of CDR/year, and 34 million hectares are suitable for wind- and solar-powered DACS, resulting in 4.8 GtCO2 of CDR/year if facilities are co-located with geologic CO2 storage. We identify biomass and energy supply hotspots to meet CDR targets while ensuring land protection and minimizing land competition.

carbon↗

DOE OSTI2026

Data for Multisite Field Evaluation of Oil Accumulation and Agronomic Performance in Grain and Sweet Sorghums Engineered for Lipid Hyperaccumulation

Oil sorghum (OS) has been developed by engineering grain (TX430) and sweet (Ramada) genetic backgrounds to accumulate triacylglycerols (TAG) in vegetative tissues as an energy-dense feedstock for sustainable aviation fuel (SAF) and other biofuels. This study evaluated two TX430 OS lines (TxHO-2, TxHO-3) and two Ramada OS lines (RmHO-1, RmHO-2) alongside wild-type (WT) lines in NE and IL over 2 years (2023–2024) to quantify genotype × environment effects on agronomic performance and TAG accumulation. Across four environments, TX430 OS lines showed average TAG concentrations of 15.0 g kg−1 in leaves and 12.8 g kg−1 in stems, approximately 19-fold higher than WT. Ramada OS lines accumulated 26.1 g kg−1 in leaves and 12.3 g kg−1 in stems, approximately 25-fold and 13-fold increases over WT, respectively. OS lines in TX430 exhibited an 18% reduction in biomass (8.4 vs. 9.9 Mg ha−1 for WT), while Ramada OS lines had similar WT biomass (18.3 vs. 19.9 Mg ha−1 for WT). Among TX430 OS lines, TxHO-2 achieved the highest TAG yield (190 kg ha−1), while RmHO-1 led the Ramada lines (335 kg ha−1) due to higher biomass and similar TAG concentration. Enhanced TAG accumulation increased N, P, and K removal in TX430 lines but not in Ramada lines. Structural carbohydrate and ash concentration were unaffected. Overall, results confirm vegetative lipid accumulation as a viable strategy for high-biomass sorghum, supporting its potential as a dual-purpose feedstock for SAF. Future work should focus on minimizing biomass yield penalties and improving nutrient use efficiency in oil sorghum systems.

Agronomy↗