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Search indexed NASA NTRS and DOE OSTI research on propulsion, heat transfer, battery materials and energy systems. Follow report and document links to the original sources.

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203 records · Page 12
All sourcesNASA NTRSDOE OSTI
DOE OSTI2024

Enhanced transport of bacteria along root systems by protists can impact plant health

ABSTRACT Soil protists have been shown to contribute to the structure and function of the rhizosphere in a variety of ways. Protists are key contributors to nutrient cycling through the microbial loop, where biomass is digested by protists and otherwise stored nutrients are returned to the environment. Protists have also been shown to feed on plant pathogenic bacteria and alter root microbiomes in ways that may benefit plants. Recently, a mechanism involving bacterial transport, facilitated by protists, has been hypothesized to contribute to the spatial distribution of bacteria in the rhizosphere. Here, we observe the differential abilities of three soil protists: a ciliate (Colpodasp.), a flagellate (Cercomonassp.), and a naked amoeba (Acanthamoeba castellanii) to transport nitrogen-fixingSinorhizobium melilotito infectible root tips. Co-inoculation of protists plusS. melilotiresulted in the movement of bacteria, as measured by the presence of nitrogen-fixing nodules, up to 15 cm farther down the root systems when compared to plants inoculated withS. melilotialone. Co-inoculation of the ciliate, Colpodasp., withS. meliloti, resulted in shoot weights that were similar to plants that grew in nitrogen-replete potting mix.Colpodasp.-feeding style and motility likely contributed to their success at transporting bacteria through the rhizosphere. We observed that the addition of protists alone without the co-inoculum ofS. melilotiresulted in plants with larger shoot weights than control plants. Follow-up experiments showed that protists plus their associated microbiomes were aiding in plant health, likely through means of nutrient cycling. IMPORTANCE Protists represent a significant portion of the rhizosphere microbiome and have been shown to contribute to plant health, yet they are understudied compared to their bacterial and fungal counterparts. This study elucidates their role in the rhizosphere community and suggests a mechanism by which protists can be used to move bacteria along plant roots. We found that the co-inoculation of protists with nitrogen-fixing beneficial bacteria,Sinorhizobium meliloti, resulted in nodules farther down the roots when compared to plants inoculated with S.melilotialone, and shoot weights similar to plants that received nitrogen fertilizer. These data illustrate the ability of protists to transport viable bacteria to uninhabited regions of the root system.

Biotechnology & Applied Microbiology↗
DOE OSTI2026

High yield production of 3-hydroxypropionic acid using Issatchenkia orientalis

Biomanufacturing provides a more sustainable alternative to fossil-based chemical manufacturing. 3-Hydroxypropionic acid (3HP) is a top Department of Energy value-added chemical and precursor to bioplastics, yet cost-effective microbial production remains elusive. Here, we establish the acid-tolerant yeast Issatchenkia orientalis as a robust host for low-pH 3HP biosynthesis. Genome-scale modeling identifies the β-alanine pathway as optimal, offering the highest theoretical yield and lowest oxygen requirement. Thermodynamic analysis confirms its favorability under acidic conditions. Using sequence similarity network analysis, we discover highly active aspartate 1-decarboxylase (PAND), β-alanine-pyruvate aminotransferase (BAPAT), and 3HP dehydrogenase (YDFG), which significantly improve the pathway efficiency. Next, to further elevate the production, pathway optimization through multi-copy PAND integration, byproduct elimination (knockouts of pyruvate decarboxylase and glycerol-3-phosphate dehydrogenase), and reinforcement of aspartate flux by overexpression of pyruvate carboxylase and aspartate amino transferase improves the titer to 29 g/L in shake flasks. Fed-batch fermentation at pH 4 with low-cost corn steep liquor medium further increases the production to 92 g/L with 0.7 g/g yield and 0.55 g/L/h productivity. Techno-economic analysis indicates that such performance could potentially enable a financially viable process for sustainable acrylic acid production. This work establishes I. orientalis as a next-generation platform for cost-effective 3HP production and paves the way toward industrial commercialization.

Biotechnology↗
DOE OSTI2025

Secure biosystems design in Saccharomyces cerevisiae establishes effective biocontainment strategies and mechanisms of escape

The widespread application of recombinant DNA and synthetic biology approaches for microbial metabolic engineering pursuits has motivated the development of biocontainment strategies, targeting safe and secure deployment of genetically modified microorganisms (GMMs). However, the design rules and mechanistic drivers governing biocontainment efficacy, as well as impacts of biocontainment upon microbial fitness, remain to be comprehensively evaluated, hindering predictive design and application of these strategies. We have developed a platform for high-resolution analysis of a transactivated kill switch in laboratory and industrial strains of Saccharomyces cerevisiae to assess modes of biocontainment escape and establish design rules for development of kill switch systems in diverse microbes. A camphor-regulated, RelE toxin system was systematically deployed to assess the impacts of differential kill switch copy number and ploidy in laboratory vs industrial strains. CRISPR-mediated integration of the biocontainment system at various loci revealed rapid escape events driven, in part, by mutations to both the Cam-transactivator (cam-TA) and RelE toxin. Genetic engineering enabled recapitulation of escape phenotypes, confirming mechanisms of escape and establishing structure-function relationships in the cam-TA system. Interestingly, genomic resequencing of escape mutants also revealed a series of off-target mutations, implicating additional modes of kill switch escape. Multi-copy integration of the kill switch system mitigated these effects by orders of magnitude, without compromising the biosynthetic capacity of the microbes, but proved insufficient to establish sustained biocontainment. The resultant data define a series of key design rules for next-generation biocontainment strategies and add to a growing foundational knowledge base targeting establishment of secure biosystems designs.

59 BASIC BIOLOGICAL SCIENCES↗
NASA NTRSDate not supplied

Cleanroom Microbes Survive Drying, Vacuum, and Proton Irradiation

Introduction : The goal of planetary protection at NASA is to mitigate the risk of contaminating sensitive target bodies with biological life. While many cleaning procedures have been put in place to reduce bioburden on spacecraft, microbes are experts at evolving to survive harsh conditions. Specifically, the dry, low-nutrient environment of a cleanroom (commonly used for assembly of spacecraft) can represent an environment where extremophiles can survive. Methods : Scientists at NASA MSFC wished to gather a snapshot of the microbial population within a variety of cleanrooms on site. A study was undertaken to collect air, surface, and floor samples from clean-rooms and isolate unique morphologies. From this study, 95 isolates were collected and saved in a microbial library. About 86% of these were identified at least to a genus level. Following identification, 24 microbes were selected, based on a literature review, as potential extremophiles. These were grown in liquid cultures, diluted to a set optical density, washed with water, and then applied to a sterilized Kapton coupon. Droplets were allowed to dry overnight in a biosafety cabinet. Coupons were then installed in a pelletron and pumped down to high vacuum (~1E-6 Torr). Samples were then subjected 100 keV protons at a fluence of 2x10 15 p+/cm 2 up to 4x10 15 p+/cm 2 . Following exposure, samples were returned to the microbiology lab where they were pro-cessed by submerging in water, vortexing, and then plating either droplets or spread plates. Recovery data collected was qualitative with a ranking or +, minor, or – for growth. Some selected radiotolerant strains were sequenced using the Illumina sequencing platform. The resulting genomes were annotated with the Rapid Annotations using Subsystems Technology (RAST) server and analyzed for conserved and unique stress response relevant genomic signatures to identify clues related to specific tolerances. Results and Discussion : After five rounds of proton radiation, we narrowed our isolates to five, non-spore forming bacteria that demonstrated survival: Arthrobacter koreensis, Paenarthrobacter nitroguajacolicus, Mycetocola manganoxydans , and an Erwinia sp. Furthermore, we exposed these four microbes to 254 nm wavelength light at an intensity of 80 W/m 2 at a distance of ~18 cm for 10 minutes. Only A. koreensis demonstrated survival following UV exposure. Finally, we performed whole genome sequencing on the four strains to look for genetic markers of stress resistance. When we compared the genomes of the four strains, we found that genes coding for GGDEF and EAL domains with PAS/PAC sensors were only found in A. koreensis . These domains, modulated by PAS/PAC sensors, are hypothesized to facilitate survival under drying, desiccation, and proton irradiation. Drying and Desiccation : PAS domains sense hydration changes and modulate GGDEF and EAL domain activity to adjust c-di-GMP levels, enhancing resistance to desiccation. For instance, in Pseudomonas aeruginosa , the PAS domain of RbdA modulates activity under varying hydration conditions, affecting stress responses [1]. Proton Irradiation : Proton irradiation causes oxidative stress, leading to ROS generation. PAS domains detect this stress and modulate GGDEF and EAL domains to manage oxidative stress responses. In Shewanella , EAL domain proteins modulated by PAS sensors help bacteria adapt to extreme conditions [2]. These genes upregulate other stress response genes, protecting membrane function, protein stability, DNA repair, and antioxidant defenses. The modulation of c-di-GMP by PAS domains is crucial for bacterial adaptation to stress conditions, enabling dynamic physio-logical adjustments [3]. Understanding these mechanisms provides insights into bacterial stress responses and strategies for controlling bacterial growth [4]. Conclusions : These findings indicate that clean-rooms harbor extremophile microbes that may be able to survive conditions in deep space. Furthermore, while we identified certain stress-response genes that may be at least partly responsible for the phenotypes observed in this study, there are likely unidentified genes or characteristics about A. koreensis , and other bacteria, that may allow them to survive in harsh environments. Future studies will focus on identifying these unknown genes and characteristics, further elucidating the mechanisms of extremophile survival and potentially informing the development of new biotechnologies for space exploration and other extreme environments.

Chelsi Cassilly↗
DOE OSTI2024

Catabolism of β-5 linked aromatics by Novosphingobium aromaticivorans

ABSTRACT Aromatic compounds are an important source of commodity chemicals traditionally produced from fossil fuels. Aromatics derived from plant lignin can potentially be converted into commodity chemicals through depolymerization followed by microbial funneling of monomers and low molecular weight oligomers. This study investigates the catabolism of the β-5 linked aromatic dimer dehydrodiconiferyl alcohol (DC-A) by the bacterium Novosphingobium aromaticivorans . We used genome-wide screens to identify candidate genes involved in DC-A catabolism. Subsequent in vivo and in vitro analyses of these candidate genes elucidated a catabolic pathway composed of four required gene products and several partially redundant dehydrogenases that convert DC-A to aromatic monomers that can be funneled into the central aromatic metabolic pathway of N. aromaticivorans . Specifically, a newly identified γ-formaldehyde lyase, PcfL, opens the phenylcoumaran ring to form a stilbene and formaldehyde. A lignostilbene dioxygenase, LsdD, then cleaves the stilbene to generate the aromatic monomers vanillin and 5-formylferulate (5-FF). We also showed that the aldehyde dehydrogenase FerD oxidizes 5-FF before it is decarboxylated by LigW, yielding ferulic acid. We found that some enzymes involved in the β-5 catabolism pathway can act on multiple substrates and that some steps in the pathway can be mediated by multiple enzymes, providing new insights into the robust flexibility of aromatic catabolism in N. aromaticivorans . A comparative genomic analysis predicted that the newly discovered β-5 aromatic catabolic pathway is common within the order Sphingomonadales. IMPORTANCE In the transition to a circular bioeconomy, the plant polymer lignin holds promise as a renewable source of industrially important aromatic chemicals. However, since lignin contains aromatic subunits joined by various chemical linkages, producing single chemical products from this polymer can be challenging. One strategy to overcome this challenge is using microbes to funnel a mixture of lignin-derived aromatics into target chemical products. This approach requires strategies to cleave the major inter-unit linkages of lignin to release monomers for funneling into valuable products. In this study, we report newly discovered aspects of a pathway by which the Novosphingobium aromaticivorans DSM12444 catabolizes aromatics joined by the second most common inter-unit linkage in lignin, the β-5 linkage. This work advances our knowledge of aromatic catabolic pathways, laying the groundwork for future metabolic engineering of this and other microbes for optimized conversion of lignin into products.

59 BASIC BIOLOGICAL SCIENCES↗