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At least 379 records · Page 21
All sourcesNASA NTRSDOE OSTI
DOE OSTI2024

Species-specific ribosomal RNA-FISH identifies interspecies cellular-material exchange, active-cell population dynamics and cellular localization of translation machinery in clostridial cultures and co-cultures

ABSTRACT The development of synthetic microbial consortia in recent years has revealed that complex interspecies interactions, notably the exchange of cytoplasmic material, exist even among organisms that originate from different ecological niches. Although morphogenetic characteristics, viable RNA and protein dyes, and fluorescent reporter proteins have played an essential role in exploring such interactions, we hypothesized that ribosomal RNA-fluorescence in situ hybridization (rRNA-FISH) could be adapted and applied to further investigate interactions in synthetic or semisynthetic consortia. Despite its maturity, several challenges exist in using rRNA-FISH as a tool to quantify individual species population dynamics and interspecies interactions using high-throughput instrumentation such as flow cytometry. In this work, we resolve such challenges and apply rRNA-FISH to double and triple co-cultures of Clostridium acetobutylicum, Clostridium ljungdahlii, and Clostridium kluyveri . In pursuing our goal to capture each organism’s population dynamics, we demonstrate dynamic rRNA, and thus ribosome, exchange between the three species leading to the formation of hybrid cells. We also characterize the localization patterns of the translation machinery in the three species, identifying distinct, dynamic localization patterns among them. Our data also support the use of rRNA-FISH to assess the culture’s health and expansion potential, and, here again, our data find surprising differences among the three species examined. Taken together, our study argues for rRNA-FISH as a valuable and accessible tool for quantitative exploration of interspecies interactions, especially in organisms which cannot be genetically engineered or in consortia where selective pressures to maintain recombinant species cannot be used. IMPORTANCE Though dyes and fluorescent reporter proteins have played an essential role in identifying microbial species in co-cultures, we hypothesized that ribosomal RNA-fluorescence in situ hybridization (rRNA-FISH) could be adapted and applied to quantitatively probe complex interactions between organisms in synthetic consortia. Despite its maturity, several challenges existed before rRNA-FISH could be used to study Clostridium co-cultures of interest. First, species-specific probes for Clostridium acetobutylicum and Clostridium ljungdahlii had not been developed. Second, “state-of-the-art” labeling protocols were tedious and often resulted in sample loss. Third, it was unclear if FISH was compatible with existing fluorescent reporter proteins. We resolved these key challenges and applied the technique to co-cultures of C. acetobutylicum , C. ljungdahlii , and Clostridium kluyveri . We demonstrate that rRNA-FISH is capable of identifying rRNA/ribosome exchange between the three organisms and characterized rRNA localization patterns in each. In combination with flow cytometry, rRNA-FISH can capture sub-population dynamics in co-cultures.

Hill, John D.↗
DOE OSTI2026

The anaerobic fungus Caecomyces churrovis produces H 2 via a non-bifurcating NADH-dependent enzyme complex

ABSTRACT Hydrogenosomes are mitochondria-derived organelles that produce ATP and H 2 to support energy metabolism in anaerobic eukaryotes. H 2 production allows reoxidation of reduced cofactors generated during fermentative metabolism; however, the metabolic mechanisms for H 2 production in anaerobic eukaryotes remains incompletely understood. In particular, it remains unclear whether anaerobic fungi (AF) hydrogenosomes use a ferredoxin-dependent pathway or a distinct mechanism to regenerate NAD(P) + and link electron transfer to H 2 formation. Here, by combining genomic search, proteomic analysis, and enzymology, we reveal the molecular mechanism for H 2 production in the AF strain Caecomyces churrovis . Our enzyme assays on the organelle fraction of C. churrovis revealed the activity of H 2 :NAD + oxidoreductase but not pyruvate:ferredoxin oxidoreductase, which is usually linked to H 2 formation. We identified genes encoding [FeFe] hydrogenase (Hyd) and NADH dehydrogenase subunits E and F (NuoE, NuoF) in C. churrovis , and confirmed their expression in the isolated hydrogenosomal fractions by proteomic analysis. Combining the individually purified enzymes, we found Hyd and NuoEF proteins formed H 2 directly from NADH independently of ferredoxin, functioning as a non-bifurcating NADH-dependent enzyme rather than an electron-bifurcating enzyme. We identified homologs of hydrogenosomal NuoE, NuoF, and Hyd in many other AF, indicating this pathway is commonly shared among the AF. This work demonstrates the existence of a non-bifurcating NADH-dependent enzyme complex in eukaryotes. Moreover, this complex could potentially be exploited as a target for controlling AF H 2 production and altering fungal metabolism. IMPORTANCE H 2 production is a prominent feature of anaerobic energy metabolism, yet our understanding of eukaryotic mechanisms remains limited. Anaerobic fungi (AF) are key decomposers of lignocellulose and contribute to hydrogen flux in anaerobic environments. Although it has been more than 40 years since the H 2 production from AF was first reported, the molecular mechanism for hydrogenosomal H 2 production and redox balance remains unclear. We demonstrate that AF produce H 2 from NADH utilizing a non-bifurcating NADH-dependent enzyme complex rather than an electron-bifurcating, ferredoxin-dependent variant. We show that this enzyme complex is conserved across multiple AF lineages and thus demonstrate the occurrence of a non-bifurcating NADH-dependent enzyme in eukaryotes. This discovery expands our understanding of eukaryotic hydrogenosomal metabolism, reveals a previously unknown strategy for redox balancing, and highlights potential targets for manipulating H 2 production. These insights have broad implications for microbial energy metabolism, anaerobic ecosystems, and bioengineering of H 2 -producing systems.

Zhang, Bo [Department of Chemical Engineering, Uni↗
DOE OSTI2025

De Novo Design of High‐Affinity Miniprotein Binders Targeting Francisella Tularensis Virulence Factor

Abstract Francisella tularensis poses considerable public health risk due to its high infectivity and potential for bioterrorism. Francisella‐like lipoprotein (Flpp3), a key virulence factor unique to Francisella, plays critical roles in infection and immune evasion, making it a promising target for therapeutic development. However, the lack of well‐defined binding pockets and structural information on native interactions has hindered structure‐guided ligand discovery against Flpp3. Here, we used a combination of physics‐based and deep‐learning methods to design high‐affinity miniprotein binders targeting two distinct sites on Flpp3. We identified four binders for site I with binding affinities ranging between 24–110 nM. For the second site, an initial binder showed a dissociation constant ( K D ) of 81 nM, and subsequent site saturation mutagenesis yielded variants with sub‐nanomolar affinities. Circular dichroism confirmed the topology of designed miniproteins. The X‐ray crystal structure of Flpp3 in complex with a site I binder is nearly identical to the design model (Cα root‐mean‐square deviation (RMSD): 0.9 Å). These designed miniproteins provide research tools to explore the roles of Flpp3 in tularemia and should enable the development of new therapeutic candidates.

Gokce‐Alpkilic, Gizem [Molecular Engineering and S↗
DOE OSTI2021

The Anaerobic Fungi: Challenges and Opportunities for Industrial Lignocellulosic Biofuel Production

Lignocellulose is a promising feedstock for biofuel production as a renewable, carbohydrate-rich and globally abundant source of biomass. However, challenges faced include environmental and/or financial costs associated with typical lignocellulose pretreatments needed to overcome the natural recalcitrance of the material before conversion to biofuel. Anaerobic fungi are a group of underexplored microorganisms belonging to the early diverging phylum Neocallimastigomycota and are native to the intricately evolved digestive system of mammalian herbivores. Anaerobic fungi have promising potential for application in biofuel production processes due to the combination of their highly effective ability to hydrolyse lignocellulose and capability to convert this substrate to H2 and ethanol. Furthermore, they can produce volatile fatty acid precursors for subsequent biological conversion to H2 or CH4 by other microorganisms. The complex biological characteristics of their natural habitat are described, and these features are contextualised towards the development of suitable industrial systems for in vitro growth. Moreover, progress towards achieving that goal is reviewed in terms of process and genetic engineering. In addition, emerging opportunities are presented for the use of anaerobic fungi for lignocellulose pretreatment; dark fermentation; bioethanol production; and the potential for integration with methanogenesis, microbial electrolysis cells and photofermentation.

pretreatment↗
DOE OSTI2023

Electrons to Molecules by Engineering and Evolution: Biological Upgrading of Formate by Cupriavidus necator

Waste carbon from industrial point sources can be captured, stored, and/or transformed using electrochemical conversion or "electrons to molecules" technologies using low-cost renewable electricity. One such process involves electrocatalytic reduction of CO2 to generate formate/formic acid, a C1 carboxylic acid. Formate is a promising potential feedstock for microbial upgrading, as it is water soluble and can be consumed as the sole source of carbon and energy by some microbial species, such as the soil bacterium Cupriavidus necator. Here we will present progress toward improving C. necator as a host for biological conversion of formate to value-added products. Using the power of adaptive laboratory evolution, we were able to isolate mutants of C. necator with significantly faster growth rates on formate. We then sequenced the genomes of these strains, elucidated the metabolic role of the mutations we found, and then used these insights to build rationally engineered strains that outperform even the best evolved isolates. These results highlight the utility of "genome streamlining" as a route for generating platform strains with potential industrial applications.

adaptive laboratory evolution↗
DOE OSTI2026

Microbial valorization of lignin to malic acid by Aspergillus niger

Lignin is the largest renewable source of aromatic carbon, yet its heterogeneity and recalcitrance limit its use in higher-value bioconversion processes. In this study, Aspergillus niger was engineered to enable the bioconversion of lignin-derived aromatics and base-catalyzed depolymerized (BCD) lignin streams into malic acid, a value-added C4 dicarboxylic acid with broad industrial relevance. Overexpression of the C4 dicarboxylate transporter C4T318 from Aspergillus oryzae enhanced malic acid secretion, while medium optimization under buffered conditions further improved the production. The engineered strain efficiently assimilated representative lignin-derived aromatics, including 4-hydroxybenzoic acid and p-coumaric acid, producing up to 3.9 g/L malic acid. Conversion of BCD lignin liquors from poplar and sorghum demonstrated effective utilization of heterogeneous aromatic mixtures, generating up to 0.82 g/L malic acid. This work demonstrates direct fungal conversion of real lignin streams into malic acid and establishes A. niger as a promising platform for sustainable lignin valorization.

Aromatic bioconversion↗
DOE OSTI2026

Bioadaptive Ni single atoms unlock high rate microbial electrosynthesis of isopropanol from CO 2

Hybrid systems that integrate electrochemical CO 2 reduction with microbial upgrading offer a viable route to high value organic compounds from CO 2 at ambient conditions. However, electrocatalyst deactivation in microbial growth media remains a key barrier, limiting efficiency and increasing cost. Here we show that a bioadaptive single-atom nickel catalyst (Ni SAC), coupled with genetically engineered Clostridium ljungdahlii, enables robust electrosynthesis of isopropanol (IPA) from CO 2 via a CO-mediated pathway. Instead of relying on H 2 as an electron carrier, the system applies high-rate CO formation in complex growth media, maintaining a tunable CO Faradaic efficiency up to 92%, which is 9.4 to 52.7 times greater than conventional Ag catalysts. This performance supports stable IPA production at current density of 10.8 A/m 2 and production rate of 161.3 mg/L/day. In situ Raman and X-ray absorption spectroscopy, together with theoretical calculations, indicate that the Ni SAC can resist competing organic adsorption and retain its coordination structure during CO 2 reduction in bioelectrolytes, providing a mechanistic basis for the catalyst stability and integrated process performance.

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

Williston Basin Resource Study for Commercial-Scale Subsurface Hydrogen Storage

This closeout presentation summarizes a Department of Energy–funded study that evaluated whether large amounts of hydrogen can be safely stored underground in the North Dakota portion of the Williston Basin. The project combined lab testing, computer simulations, and basin‑wide analysis to assess saline formations, depleted oil and gas reservoirs, and salt formations for hydrogen storage capacity, recovery, and risks. Results show that underground hydrogen storage is technically feasible across multiple formation types, with depleted oil and gas reservoirs offering higher recovery and saline formations providing large long‑term storage potential. The study also identifies key challenges—such as wellbore material durability and gas purity management—and recommends pilot projects and further site‑specific studies to support future commercialization.

08 HYDROGEN↗
DOE OSTI2020

Recent Advances in Domesticating Non-model Microorganisms

Non-model microorganisms have been increasingly explored as microbial cell factories for production of chemicals, fuels, and materials owing to their unique physiology and metabolic capabilities. Yet, these microorganisms often lack facile genetic tools for strain development, which hinders their adoption as production hosts. In this review, we describe recent advances in domestication of non-model microorganisms, including bacteria, actinobacteria, cyanobacteria, yeast, and fungi, with a focus on the development of genetic tools. Furthermore, we highlight some successful applications of non-model microorganisms as microbial cell factories.

synthetic biology↗
DOE OSTI2022

Engineering Citrobacter freundii using CRISPR/Cas9 system

The CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats/CRISPR associated proteins) system is a useful tool to edit genomes quickly and efficiently. However, the use of CRISPR/Cas9 to edit bacterial genomes has been limited to select microbial chassis primarily used for bioproduction of high value products. Thus, expansion of CRISPR/Cas9 tools to other microbial organisms is needed. Here, our aim was to assess the suitability of CRISPR/Cas9 for genome editing of the Citrobacter freundii type strain ATCC 8090. We evaluated the commonly used two plasmid pCas/pTargetF system to enable gene deletions and insertions in C. freundii and determined editing efficiency. The CRISPR/Cas9 based method enabled high editing efficiency (~91%) for deletion of galactokinase (galk) and enabled deletion with various single guide RNA (sgRNA) sequences. To assess the ability of CRISPR/Cas9 tools to insert genes, we used the fluorescent reporter mNeonGreen, an endopeptidase (yebA), and a transcriptional regulator (xylS) and found successful insertion with high efficiency (81-100%) of each gene individually. These results strengthen and expand the use of CRISPR/Cas9 genome editing to C. freundii as an additional microbial chassis.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2021

Efficient production of oxidized terpenoids via engineering fusion proteins of terpene synthase and cytochrome P450

The functionalization of terpenes using cytochrome P450 enzymes is a versatile route to the production of useful derivatives that can be further converted to value-added products. Many terpenes are hydrophobic and volatile making their availability as a substrate for P450 enzymes significantly limited during microbial production. In this study, we developed a strategy to improve the accessibility of terpene molecules for the P450 reaction by linking terpene synthase and P450 together. As a model system, fusion proteins of 1,8-cineole synthase (CS) and P450 cin were investigated and it showed an improved hydroxylation of the monoterpenoid 1,8-cineole up to 5.4-fold. Structural analysis of the CS-P450 cin fusion proteins by SEC-SAXS indicated a dimer formation with preferred orientations of the active sites of the two domains. We also applied the enzyme fusion strategy to the oxidation of a sesquiterpene epi-isozizaene and the fusion enzymes significantly improved albaflavenol production in engineered E. coli. From the analysis of positive and negative examples of the fusion strategy, we proposed key factors in structure-based prediction and evaluation of fusion enzymes. Developing fusion enzymes for terpene synthase and P450 presents an efficient strategy toward oxidation of hydrophobic terpene compounds. This strategy could be widely applicable to improve the biosynthetic titer of the functionalized products from hydrophobic terpene intermediates.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2026

Bio-based oxalic acid production in Issatchenkia orientalis enables sustainable rare earth recovery

The growing demand for rare earth elements (REEs) in clean energy and high-tech industries underscores the need for sustainable recovery methods and a reliable supply of processing chemicals. Here, we establish a microbial platform using the acid-tolerant yeast Issatchenkia orientalis SD108 to produce bio-oxalic acid for REE recovery. By introducing an oxaloacetate cleavage pathway and applying metabolic engineering, the engineered strain produces 39.53 g·L -1 oxalic acid at pH 4.0 in fed-batch fermentation. The crude fermentation broth, used without purification, efficiently precipitates over 99% neodymium (Nd), 99% dysprosium (Dy), and 98% lanthanum (La) from individual REE chloride solutions. Recovery from a low-grade ore leachate achieves over 99% total recovery. X-ray diffraction (XRD) and Fourier transform infrared spectroscopy (FTIR) confirm that REE oxalates precipitated with bio-oxalic acid closely resemble those obtained using commercial oxalic acid. Techno-economic analysis (TEA) and life cycle assessment (LCA) further demonstrate that bio-oxalic acid can be produced at a competitive price of $1.79·kg -1 while reducing carbon intensity (CI) by 112% to 63.5% with and without electricity displacement, respectively, relative to the fossil-based benchmark. These results highlight bio-oxalic acid as a green, economically viable alternative to synthetic oxalate for sustainable REE recovery.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2021

Vapor-Fed Cathode Microbial Electrolysis Cells with Closely Spaced Electrodes Enables Greatly Improved Performance

Hydrogen can be electrochemically produced in microbial electrolysis cells (MECs) by current generated from bacterial anodes with a small added voltage. MECs typically use a liquid catholyte containing a buffer or salts. However, anions in these catholytes result in charge being balanced predominantly by ions other than hydroxide or protons, leading to anode acidification. To enhance only hydroxide ion transport to the anode, we developed a novel vapor-fed MEC configuration lacking a catholyte with closely spaced electrodes and an anion exchange membrane to limit the acidification. This MEC design produced a record-high sustained current density of 43.1 ± 0.6 A/m 2 and a H 2 production rate of 72 ± 2 LH 2 /L-d (cell voltage of 0.79 ± 0.00 V). There was minimal impact on MEC performance of increased acetate concentrations, solution conductivity, or anolyte buffer capacity at applied voltages up to 1.1 V, as shown by a nearly constant internal resistance of only 6.8 ± 0.3 mΩ m 2 . At applied external voltages >1.1 V, the buffer capacity impacted performance, with current densities increasing from 28.5 ± 0.6 A/m 2 (20 mM phosphate buffer solution (PBS)) to 51 ± 1 A/m 2 (100 mM PBS). Furthermore, these results show that a vapor-fed MEC can produce higher and more stable performance than liquid-fed cathodes by enhancing transport of hydroxide ions to the anode.

anion exchange membrane↗
DOE OSTI2022

Advances in systems metabolic engineering of autotrophic carbon oxide-fixing biocatalysts towards a circular economy

High levels of anthropogenic CO 2 emissions are driving the warming of global climate. If this pattern of increasing emissions does not change, it will cause further climate change with severe consequences for the human population. On top of this, the increasing accumulation of solid waste within the linear economy model is threatening global biosustainability. The magnitude of these challenges requires several approaches to capture and utilize waste carbon and establish a circular economy. Microbial gas fermentation presents an exciting opportunity to capture carbon oxides from gaseous and solid waste streams with high feedstock flexibility and selectivity. Here we discuss available microbial systems and review in detail the metabolism of both anaerobic acetogens and aerobic hydrogenotrophs and their ability to utilize C1 waste feedstocks. More specifically, we provide an overview of the systems-level understanding of metabolism, key metabolic pathways, scale-up opportunities and commercial successes, and the most recent technological advances in strain and process engineering. Finally, we also discuss in detail the gaps and opportunities to advance the understanding of these autotrophic biocatalysts for the efficient and economically viable production of bioproducts from recycled carbon.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2021

Designing a Zn–Ag Catalyst Matrix and Electrolyzer System for CO 2 Conversion to CO and Beyond

CO 2 emissions can be transformed into high-added-value commodities through CO 2 electrocatalysis; however, efficient low-cost electrocatalysts are needed for global scale-up. Inspired by other emerging technologies, the authors report the development of a gas diffusion electrode containing highly dispersed Ag sites in a low-cost Zn matrix. Here, this catalyst shows unprecedented Ag mass activity for CO production: –614 mA cm –2 at 0.17 mg of Ag. Subsequent electrolyte engineering demonstrates that halide anions can further improve stability and activity of the Zn–Ag catalyst, outperforming pure Ag and Au. Membrane electrode assemblies are constructed and coupled to a microbial process that converts the CO to acetate and ethanol. Combined, these concepts present pathways to design catalysts and systems for CO 2 conversion toward sought-after products.

36 MATERIALS SCIENCE↗