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At least 883 records · Page 49
All sourcesNASA NTRSDOE OSTI
DOE OSTI2020

Electrogenerated Chemiluminescence of Near-Infrared-Emitting CdSe@CdSe Nanorods in Aqueous Solution

Electrogenerated chemiluminescence (ECL) was observed from TOPO-capped CdSe@CdSe nanorods (NRs) depositing on a metal electrode in phosphate buffer solution (PBS). Two ECL peaks at -1.05 and -1.33 V in pH 9.2, 0.1 M PBS were found under cyclic voltammetric conditions. Cyclic voltammetry of this solution displayed no distinctive features, on the other hand, light emission was observed during cyclic potential scans. Additionally, the photoluminescence (PL) spectrum showed an emission maximum at 692 nm. The spectrum of ECL possesses one peak which coincides very well with the PL spectrum of the nanorods film. The mechanism for ECL peaks was proposed. Electron transfer reactions between charged nanorods and molecular redox-active coreactants such as dissolved oxygen and H2O2 or between positively and negatively charged nanorods occurred that led to electron and hole annihilation, producing light.

36 MATERIALS SCIENCE↗
DOE OSTI2021

Effects of Plasticizer Content and Ceramic Addition on Electrochemical Properties of Cross-Linked Polymer Electrolyte

The development of a safe electrolyte is the key to improving energy density for next generation lithium batteries. In this work, UV-crosslinked poly(ethylene oxide) (PEO) -based polymer and composite electrolytes are systematically investigated on their ionic conductivity, mechanical and electrochemical properties. The polymer electrolytes are plasticized with non-flammable linear short-chain PEO. In the composite electrolytes, a doped lithium aluminum titanium phosphate (LATP) ceramic, LICGC™, is used as the ceramic filler. It is found that the addition of the plasticizer leads to a tradeoff between ion transport and mechanical properties. In contrast, the addition of ceramic fillers improves both the ionic conductivity and mechanical properties. The sample with 20 wt% of LICGC™ shows a conductivity of ~0.6 mS cm –1 at 50 °C. This sample also demonstrates much longer cycle life than the neat polymer electrolyte in Li platting/stripping test with a capacity of 1 mAh cm –2 . Further, a full cell made with this composite electrolyte against Li metal anode and high voltage LiNi 0.6 Mn 0.2 Co 0.2 O 2 cathode shows 94% capacity retention after 30 cycles, compared to 58% capacity retention with the neat polymer electrolyte. These results demonstrate that a hybrid of polymer/ceramic/non-flammable plasticizer is a promising path to high energy density, high voltage lithium batteries.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗
DOE OSTI2021

Ion Pairing, Clustering and Transport in a LiFSI-TMP Electrolyte as Functions of Salt Concentration using Molecular Dynamics Simulations

Battery capacity is highly related to ion-pairing mechanisms in electrolytes, since a cluster formation can lead to dead Li formation, reducing the number of charge carriers and leading to capacity fading. We use molecular dynamics simulations to model an electrolyte comprising trimethyl phosphate (TMP) solvent and a lithium bis(fluorosulfonyl)imide (LiFSI) salt, exploring effects of salt concentration on solvation and ion-transport. We simulate the LiFSI-TMP electrolyte for salt concentrations of 0.7, 1.43 and 3.82 molar. A statistical analysis was performed to study ion-pairing, clustering, diffusivity, conductivity, and coordination of Li-ions, providing insights into relations between molecular structures and transport properties. Molecular structure of ionic components changes as concentration increases, from a predominant solvent separated ion pair (SSIP) and contact ion pair (CIP) to aggregate salt (AGG) and ionic cluster formation. Given the formation of the ionic cluster, the diffusion mechanism followed by Li-ions changes from a hopping/exchange to a vehicular mechanism as concentration increases; this is reflected in a decrease of ionic conductivities. Ionicity was also calculated to reveal how the ionic motion changes from an uncorrelated to a correlated one as the salt concentration increases. Furthermore, we also compared our results with experimental calculations performed for similar electrolyte systems

25 ENERGY STORAGE↗
DOE OSTI2022

Performance of a Novel In-Situ Converted Additive for High Voltage Li-ion Pouch Cells

In search for new classes of additives for high voltage NMC/graphite lithium-ion cells, the precursor additive bis(trimethylsilyl) malonate (bTMSM) is shown to be activated via a spontaneous reaction with LiPF 6 and LiBF 4 salts in carbonate-based electrolyte to form lithium tetrafluoro(malonato)phosphate (LiTFMP), and lithium difluoro(malonato)borate (LiDFMB), respectively. The reaction schemes and rates were studied via NMR spectroscopy and GCMS. The effects of LiTFMP and LiDFMB on high voltage electrochemical performance were then examined up to 4.5 V in Li[Ni 0.4 Mn 0.4 Co 0.16 ]O 2 (NMC442)/graphite and Li[Ni 0.6 Mn 0.4 Co 0.0 ]O 2 (NMC/640)/graphite pouch cells using aggressive voltage-hold cycling, long-term charge/discharge cycling, storage experiments, electrochemical impedance spectroscopy, and gas evolution measurements. While in situ converted additives suffer from gassing issues due to the presence of trimethylfluorosilane (TMSF) gas, a side product of the in situ reaction of bTMSM with LiPF 6 , the cycling and storage capability for the activated additives under study shows competitive performance and controlled impedance when compared to other well-known high voltage additives. Micro X-ray fluorescence spectroscopy (μXRF) confirmed that LiTFMP successfully minimizes the rate of transition metal deposition on the surface of graphite apparently by forming a protective agent at the cathode surface, hence allowing for improved cycling performance at high voltages.

25 ENERGY STORAGE↗
DOE OSTI2023

Metallic Cu Surface Enables Reversible Na Metal Anodes and Stabilizes Anode-Free Sodium Metal Batteries

Anode free Na metal batteries are promising for future energy storage because they not only provide the highest energy densities but also eliminate the need of handling hazardous Na metals during battery manufacturing. However, they suffer from much faster degradation due to strong sensitivities even to trace levels of side reactions. In view of the crucial roles of surface chemistry on modulating electrochemical plating, this work systematically investigated a series of Cu surfaces for Na plating and stripping in the 1.0 M NaPF 6 diglyme electrolyte. Our results suggest that Na plating and stripping on pure Cu surface without Cu oxide species exhibits much better reversibility and smaller overpotentials across a wide range of current densities, especially for the first plating/stripping cycle. The high performance includes consistently higher than 99.8% Faradaic efficiencies, much more stable interfacial resistance, and negligible formation of mossy Na after 500 cycles. This improved performance can be explained based on the stronger Na-Cu affinity compared with the Na-CuO affinity. Anode-free Na metal batteries equipped with high-capacity sodium vanadium phosphate cathodes and pure Cu current collector exhibited at least 70% capacity retention for 100 cycles.

Electrochemistry↗
DOE OSTI2024

Bipolar Membrane Capacitive Deionization for the Selective Capture of Lithium Ions from Brines and Conversion to Lithium Hydroxide

Meeting the increasing demand for lithium in vehicle electrification and renewable energy storage requires innovations in lithium-ion (Li + ) separations. Traditional solar evaporation methods for lithium recovery are slow and consume tremendous volumes of water and secondary chemicals (acids and bases). This study introduces a bipolar membrane capacitive deionization (BPM-CDI) unit for direct lithium extraction and LiOH production without the external addition of acids and bases. Utilizing de-lithiated lithium-iron-phosphate (LFP) coated carbon cloth electrodes, the BPM-CDI unit demonstrates selective Li + capture over competing ions. Molecular dynamics simulations and H-cell experiments elucidate pH inversion mechanisms during Li + release, yielding LiOH. The BPM-CDI platform efficiently removes Li + from synthetic brines featuring 8x higher Mg 2+ concentrations (200 ppm Mg 2+ ) and 26x higher Na + concentrations (682 ppm Na + ), achieving a LiOH concentration of 124 ppm (36 ppm Li + ) after 8 cycles of recirculation. Post-mortem analysis confirms electrode integrity and stability. BPM-CDI integrated with selective electrodes is a promising electrochemical separation-reactor platform for lithium recovery while producing LiOH.

Kulkarni, Tanmay↗
DOE OSTI2025

Recent Developments in Lithium-Manganese-Nickel Oxide Electrochemistry: The Alchemy of LiMn 0.5 Ni 0.5 O 2

Efforts have been underway for some time at Argonne National Laboratory to design structurally integrated and stabilized lithium-manganese-nickel-oxide cathode materials. The goal has been to find a cobalt-free, high-capacity cathode for a Li-ion battery that is competitive, both cost- and energy-wise, with lithium-nickel-manganese-cobalt-oxide (NMC) and lithium-iron-phosphate (LFP) products. This brief synopsis highlights the remarkable alchemy of LiMn 0.5 Ni 0.5 O 2 resulting from its well-known, slightly disordered layered structure and the recently discovered lithiated spinel phase and totally disordered rock-salt configurations. All three arrangements share a structurally compatible cubic-close-packed oxygen array. The lithiated spinel and layered components provide an intersecting network of two- and three-dimensional channels that facilitate Li + -ion transport, while a minor amount of an extensively disordered rock-salt component likely serves to enhance the structural and electrochemical stability of the electrode. The synopsis also encompasses the historical development of lithiated spinel electrode materials and structurally integrated systems.

Thackeray, Michael M. [Argonne National Laboratory↗
DOE OSTI2021

Structural and Kinetic Characterization of Hyperthermophilic NADH-Dependent Persulfide Reductase from Archaeoglobus fulgidus

NADH-dependent persulfide reductase (Npsr) has been proposed to facilitate dissimilatory sulfur respiration by reducing persulfide or sulfane sulfur-containing substrates to H2S. The presence of this gene in the sulfate and thiosulfate-reducing Archaeoglobus fulgidus DSM 4304 and other hyperthermophilic Archaeoglobales appears anomalous, as A. fulgidus is unable to respire S0 and grow in the presence of elemental sulfur. To assess the role of Npsr in the sulfur metabolism of A. fulgidus DSM 4304, the Npsr from A. fulgidus was characterized. AfNpsr is specific for persulfide and polysulfide as substrates in the oxidative half-reaction, exhibiting k cat / K m on the order of 104 M-1 s-1, which is similar to the kinetic parameters observed for hyperthermophilic CoA persulfide reductases. In contrast to the bacterial Npsr, AfNpsr exhibits low disulfide reductase activity with DTNB; however, similar to the bacterial enzymes, it does not show detectable activity with CoA-disulfide, oxidized glutathione, or cystine. The 3.1 Å X-ray structure of AfNpsr reveals access to the tightly bound catalytic CoA, and the active site Cys 42 is restricted by a flexible loop (residues 60-66) that is not seen in the bacterial homologs from Shewanella loihica PV-4 and Bacillus anthracis. Unlike the bacterial enzymes, AfNpsr exhibits NADH oxidase activity and also shows no detectable activity with NADPH. Models suggest steric and electrostatic repulsions of the NADPH 2 ′ -phosphate account for the strong preference for NADH. The presence of Npsr in the nonsulfur-reducing A. fulgidus suggests that the enzyme may offer some protection against S0 or serve in another metabolic role that has yet to be identified.

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

divergence in basal transcriptomes of "Wild Type" Chlamydomonas reinhardtii strains

This is a study to compare the basal transcriptomes of several widely used laboratory strains of the Chlorophyte alga, Chlamydomonas reinhardtii. Given that there is a high degree of genetic diversity among the closely-related laboratory strains, we wished to examine how much variation there is at the transcriptome level. A panel of WT strains (CC-124, CC-125, CC-1009, CC-1690, CC-1691), all believed to be descended from a single zygospore isolated in 1945, were chosen based on their representing the oldest lineages among the standard laboratory strains. Additionally, CC-4532, which was the source for the current (v6) reference assembly, and CC-4533, which is the initial parental strain of the CLiP library collection of mutant strains, were also included in this study based on their significance to the Chlamydomonas community. All strains were grown in liquid cultures under identical, mixotrophic conditions (light + acetate) to mid-log phase before collecting mRNA for RNA-Seq analysis. Overall design: Pre-cultures of each strain (CC-124, CC-125, CC-1009, CC-1690, CC-1691, CC-4532, and CC-4533) were used to innoculate flasks of Tris-Acetate-Phosphate (TAP) media supplemented with Kropat's trace metals at a concentration of 1.5 x 10^4 cells/mL. Flasks were placed on a shaking platform at 180 RPM under 50-70 µmol x m^-2 x s^-1 of light. Cultures were grown until they reached ~2-3 x 10^6 cells/mL, about three days, before total RNA was collected and subjected RNA-Seq analysis.

Source record↗
DOE OSTI2021

Altered Cardiac Energetics and Mitochondrial Dysfunction in Hypertrophic Cardiomyopathy

Background: Hypertrophic cardiomyopathy (HCM) is a complex disease partly explained by the effects of individual gene variants on sarcomeric protein biomechanics. At the cellular level, HCM mutations most commonly enhance force production, leading to higher energy demands. Despite significant advances in elucidating sarcomeric structure–function relationships, there is still much to be learned about the mechanisms that link altered cardiac energetics to HCM phenotypes. Here, we test the hypothesis that changes in cardiac energetics represent a common pathophysiologic pathway in HCM. Methods: We performed a comprehensive multiomics profile of the molecular (transcripts, metabolites, and complex lipids), ultrastructural, and functional components of HCM energetics using myocardial samples from 27 HCM patients and 13 normal controls (donor hearts). Results: Integrated omics analysis revealed alterations in a wide array of biochemical pathways with major dysregulation in fatty acid metabolism, reduction of acylcarnitines, and accumulation of free fatty acids. HCM hearts showed evidence of global energetic decompensation manifested by a decrease in high energy phosphate metabolites (ATP, ADP, and phosphocreatine) and a reduction in mitochondrial genes involved in creatine kinase and ATP synthesis. Accompanying these metabolic derangements, electron microscopy showed an increased fraction of severely damaged mitochondria with reduced cristae density, coinciding with reduced citrate synthase activity and mitochondrial oxidative respiration. These mitochondrial abnormalities were associated with elevated reactive oxygen species and reduced antioxidant defenses. However, despite significant mitochondrial injury, HCM hearts failed to upregulate mitophagic clearance. Conclusions: Overall, our findings suggest that perturbed metabolic signaling and mitochondrial dysfunction are common pathogenic mechanisms in patients with HCM. These results highlight potential new drug targets for attenuation of the clinical disease through improving metabolic function and reducing mitochondrial injury.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2020

Methanosarcina acetivorans contains a functional ISC system for iron-sulfur cluster biogenesis

Abstract Background The production of methane by methanogens is dependent on numerous iron-sulfur (Fe-S) cluster proteins; yet, the machinery involved in Fe-S cluster biogenesis in methanogens remains largely unknown. Methanogen genomes encode uncharacterized homologs of the core components of the ISC (IscS and IscU) and SUF (SufBC) Fe-S cluster biogenesis systems found in bacteria and eukaryotes. Methanosarcina acetivorans contains three iscSU and two sufCB gene clusters. Here, we report genetic and biochemical characterization of M. acetivorans iscSU2 . Results Purified IscS2 exhibited pyridoxal 5′- phosphate-dependent release of sulfur from L-cysteine. Incubation of purified IscU2 with IscS2, cysteine, and iron (Fe 2+ ) resulted in the formation of [4Fe-4S] clusters in IscU2. IscU2 transferred a [4Fe-4S] cluster to purified M. acetivorans apo-aconitase. IscU2 also restored the aconitase activity in air-exposed M. acetivorans cell lysate. These biochemical results demonstrate that IscS2 is a cysteine desulfurase and that IscU2 is a Fe-S cluster scaffold. M. acetivorans strain DJL60 deleted of iscSU2 was generated to ascertain the in vivo importance of IscSU2. Strain DJL60 had Fe-S cluster content and growth similar to the parent strain but lower cysteine desulfurase activity. Strain DJL60 also had lower intracellular persulfide content compared to the parent strain when cysteine was an exogenous sulfur source, linking IscSU2 to sulfur metabolism. Conclusions This study establishes that M. acetivorans contains functional IscS and IscU, the core components of the ISC Fe-S cluster biogenesis system and provides the first evidence that ISC operates in methanogens.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2023

Mechanisms of metabolic adaptation in the duckweed Lemna gibba: an integrated metabolic, transcriptomic and flux analysis

Abstract Background Duckweeds are small, rapidly growing aquatic flowering plants. Due to their ability for biomass production at high rates they represent promising candidates for biofuel feedstocks. Duckweeds are also excellent model organisms because they can be maintained in well-defined liquid media, usually reproduce asexually, and because genomic resources are becoming increasingly available. To demonstrate the utility of duckweed for integrated metabolic studies, we examined the metabolic adaptation of growing Lemna gibba cultures to different nutritional conditions. Results To establish a framework for quantitative metabolic research in duckweeds we derived a central carbon metabolism network model of Lemna gibba based on its draft genome. Lemna gibba fronds were grown with nitrate or glutamine as nitrogen source. The two conditions were compared by quantification of growth kinetics, metabolite levels, transcript abundance, as well as by 13 C-metabolic flux analysis. While growing with glutamine, the fronds grew 1.4 times faster and accumulated more protein and less cell wall components compared to plants grown on nitrate. Characterization of photomixotrophic growth by 13 C-metabolic flux analysis showed that, under both metabolic growth conditions, the Calvin-Benson-Bassham cycle and the oxidative pentose-phosphate pathway are highly active, creating a futile cycle with net ATP consumption. Depending on the nitrogen source, substantial reorganization of fluxes around the tricarboxylic acid cycle took place, leading to differential formation of the biosynthetic precursors of the Asp and Gln families of proteinogenic amino acids. Despite the substantial reorganization of fluxes around the tricarboxylic acid cycle, flux changes could largely not be associated with changes in transcripts. Conclusions Through integrated analysis of growth rate, biomass composition, metabolite levels, and metabolic flux, we show that Lemna gibba is an excellent system for quantitative metabolic studies in plants. Our study showed that Lemna gibba adjusts to different nitrogen sources by reorganizing central metabolism. The observed disconnect between gene expression regulation and metabolism underscores the importance of metabolic flux analysis as a tool in such studies.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2024

Nitrogen starvation causes lipid remodeling in Rhodotorula toruloides

Abstract Background The oleaginous yeast Rhodotorula toruloides is a promising chassis organism for the biomanufacturing of value-added bioproducts. It can accumulate lipids at a high fraction of biomass. However, metabolic engineering efforts in this organism have progressed at a slower pace than those in more extensively studied yeasts. Few studies have investigated the lipid accumulation phenotype exhibited by R. toruloides under nitrogen limitation conditions. Consequently, there have been only a few studies exploiting the lipid metabolism for higher product titers. Results We performed a multi-omic investigation of the lipid accumulation phenotype under nitrogen limitation. Specifically, we performed comparative transcriptomic and lipidomic analysis of the oleaginous yeast under nitrogen-sufficient and nitrogen deficient conditions. Clustering analysis of transcriptomic data was used to identify the growth phase where nitrogen-deficient cultures diverged from the baseline conditions. Independently, lipidomic data was used to identify that lipid fractions shifted from mostly phospholipids to mostly storage lipids under the nitrogen-deficient phenotype. Through an integrative lens of transcriptomic and lipidomic analysis, we discovered that R. toruloides undergoes lipid remodeling during nitrogen limitation, wherein the pool of phospholipids gets remodeled to mostly storage lipids. We identify specific mRNAs and pathways that are strongly correlated with an increase in lipid levels, thus identifying putative targets for engineering greater lipid accumulation in R. toruloides . One surprising pathway identified was related to inositol phosphate metabolism, suggesting further inquiry into its role in lipid accumulation. Conclusions Integrative analysis identified the specific biosynthetic pathways that are differentially regulated during lipid remodeling. This insight into the mechanisms of lipid accumulation can lead to the success of future metabolic engineering strategies for overproduction of oleochemicals.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2021

Metabolic engineering of the oleaginous yeast Yarrowia lipolytica PO1f for production of erythritol from glycerol

Abstract Background Sugar alcohols are widely used as low-calorie sweeteners in the food and pharmaceutical industries. They can also be transformed into platform chemicals. Yarrowia lipolytica , an oleaginous yeast, is a promising host for producing many sugar alcohols. In this work, we tested whether heterologous expression of a recently identified sugar alcohol phosphatase (PYP) from Saccharomyces cerevisiae would increase sugar alcohol production in Y. lipolytica . Results Y. lipolytica was found natively to produce erythritol, mannitol, and arabitol during growth on glucose, fructose, mannose, and glycerol. Osmotic stress is known to increase sugar alcohol production, and was found to significantly increase erythritol production during growth on glycerol. To better understand erythritol production from glycerol, since it was the most promising sugar alcohol, we measured the expression of key genes and intracellular metabolites. Osmotic stress increased the expression of several key genes in the glycerol catabolic pathway and the pentose phosphate pathway. Analysis of intracellular metabolites revealed that amino acids, sugar alcohols, and polyamines are produced at higher levels in response to osmotic stress. Heterologous overexpression of the sugar alcohol phosphatase increased erythritol production and glycerol utilization in Y. lipolytica . We further increased erythritol production by increasing the expression of native glycerol kinase (GK), and transketolase (TKL). This strain was able to produce 27.5 ± 0.7 g/L erythritol from glycerol during batch growth and 58.8 ± 1.68 g/L erythritol during fed-batch growth in shake-flasks experiments. In addition, the glycerol utilization was increased by 2.5-fold. We were also able to demonstrate that this strain efficiently produces erythritol from crude glycerol, a major byproduct of the biodiesel production. Conclusions We demonstrated the application of a promising enzyme for increasing erythritol production in Y. lipolytica . We were further able to boost production by combining the expression of this enzyme with other approaches known to increase erythritol production in Y. lipolytica . This suggest that this new enzyme provides an orthogonal route for boosting production and can be stacked with existing designs known to increase sugar alcohol production in yeast such as Y. lipolytica . Collectively, this work establishes a new route for increasing sugar alcohol production and further develops Y. lipolytica as a promising host for erythritol production from cheap substrates such as glycerol.

09 BIOMASS FUELS↗
DOE OSTI2023

Metabolic engineering to improve production of 3-hydroxypropionic acid from corn-stover hydrolysate in Aspergillus species

Fuels and chemicals derived from non-fossil sources are needed to lessen human impacts on the environment while providing a healthy and growing economy. 3-hydroxypropionic acid (3-HP) is an important chemical building block that can be used for many products. Biosynthesis of 3-HP is possible; however, low production is typically observed in those natural systems. Biosynthetic pathways have been designed to produce 3-HP from a variety of feedstocks in different microorganisms. In this study, the 3-HP β-alanine pathway consisting of aspartate decarboxylase, β-alanine-pyruvate aminotransferase, and 3-hydroxypropionate dehydrogenase from selected microorganisms were codon optimized for Aspergillus species and placed under the control of constitutive promoters. The pathway was introduced into Aspergillus pseudoterreus and subsequently into Aspergillus niger, and 3-HP production was assessed in both hosts. A. niger produced higher initial 3-HP yields and fewer co-product contaminants and was selected as a suitable host for further engineering. Proteomic and metabolomic analysis of both Aspergillus species during 3-HP production identified genetic targets for improvement of flux toward 3-HP including pyruvate carboxylase, aspartate aminotransferase, malonate semialdehyde dehydrogenase, succinate semialdehyde dehydrogenase, oxaloacetate hydrolase, and a 3-HP transporter. Overexpression of pyruvate carboxylase improved yield in shake-flasks from 0.09 to 0.12 C-mol 3-HP C-mol -1 glucose in the base strain expressing 12 copies of the β-alanine pathway. Deletion or overexpression of individual target genes in the pyruvate carboxylase overexpression strain improved yield to 0.22 C-mol 3-HP C-mol -1 glucose after deletion of the major malonate semialdehyde dehydrogenase. Further incorporation of additional β-alanine pathway genes and optimization of culture conditions (sugars, temperature, nitrogen, phosphate, trace elements) for 3-HP production from deacetylated and mechanically refined corn stover hydrolysate improved yield to 0.48 C-mol 3-HP C-mol -1 sugars and resulted in a final titer of 36.0 g/L 3-HP. The results of this study establish A. niger as a host for 3-HP production from a lignocellulosic feedstock in acidic conditions and demonstrates that 3-HP titer and yield can be improved by a broad metabolic engineering strategy involving identification and modification of genes participated in the synthesis of 3-HP and its precursors, degradation of intermediates, and transport of 3-HP across the plasma membrane.

09 BIOMASS FUELS↗
DOE OSTI2024

Pyrophosphate-free glycolysis in Clostridium thermocellum increases both thermodynamic driving force and ethanol titers

Background: Clostridium thermocellum is a promising candidate for production of cellulosic biofuels, however, its final product titer is too low for commercial application, and this may be due to thermodynamic limitations in glycolysis. Previous studies in this organism have revealed a metabolic bottleneck at the phosphofructokinase (PFK) reaction in glycolysis. In the wild-type organism, this reaction uses pyrophosphate (PP i ) as an energy cofactor, which is thermodynamically less favorable compared to reactions that use ATP as a cofactor. Previously we showed that replacing the PP i -linked PFK reaction with an ATP-linked reaction increased the thermodynamic driving force of glycolysis, but only had a local effect on intracellular metabolite concentrations, and did not affect final ethanol titer. Results: In this study, we substituted PP i -pfk with ATP-pfk, deleted the other PPi-requiring glycolytic gene pyruvate:phosphate dikinase (ppdk), and expressed a soluble pyrophosphatase (PPase) and pyruvate kinase (pyk) genes to engineer PP i -free glycolysis in C. thermocellum. We demonstrated a decrease in the reversibility of the PFK reaction, higher levels of lower glycolysis metabolites, and an increase in ethanol titer by an average of 38% (from 15.1 to 21.0 g/L) by using PP i -free glycolysis. Conclusions: By engineering PP i -free glycolysis in C. thermocellum, we achieved an increase in ethanol production. These results demonstrate that optimizing the thermodynamic landscape through metabolic engineering can enhance product titers. While further increases in ethanol titers are necessary for commercial application, this work represents a significant step toward engineering glycolysis in C. thermocellum to increase ethanol titers.

09 BIOMASS FUELS↗
DOE OSTI2022

Adapted laboratory evolution of Thermotoga sp. strain RQ7 under carbon starvation

Abstract Objective Adaptive laboratory evolution (ALE) is an effective approach to study the evolution behavior of bacterial cultures and to select for strains with desired metabolic features. In this study, we explored the possibility of evolving Thermotoga sp. strain RQ7 for cellulose-degrading abilities. Results Wild type RQ7 strain was subject to a series of transfers over six and half years with cellulose filter paper as the main and eventually the sole carbon source. Each transfer was accompanied with the addition of 50 μg of Caldicellulosiruptor saccharolyticus DSM 8903 genomic DNA. A total of 331 transfers were completed. No cellulose degradation was observed with the RQ7 cultures. Thirty three (33) isolates from six time points were sampled and sequenced. Nineteen (19) of the 33 isolates were unique, and the rest were duplicated clones. None of the isolates acquired C. saccharolyticus DNA, but all accumulated small-scale mutations throughout their genomes. Sequence analyses revealed 35 mutations that were preserved throughout the generations and another 15 mutations emerged near the end of the study. Many of the affected genes participate in phosphate metabolism, substrate transport, stress response, sensory transduction, and gene regulation.

54 ENVIRONMENTAL SCIENCES↗
DOE OSTI2022

Ozonized biochar filtrate effects on the growth of Pseudomonas putida and cyanobacteria Synechococcus elongatus PCC 7942

Background: Biochar ozonization was previously shown to dramatically increase its cation exchange capacity, thus improving its nutrient retention capacity. The potential soil application of ozonized biochar warrants the need for a toxicity study that investigates its effects on microorganisms. Results: In the study presented here, we found that the filtrates collected from ozonized pine 400 biochar and ozonized rogue biochar did not have any inhibitory effects on the soil environmental bacteria Pseudomonas putida, even at high dissolved organic carbon (DOC) concentrations of 300 ppm. However, the growth of Synechococcus elongatus PCC 7942 was inhibited by the ozonized biochar filtrates at DOC concentrations greater than 75 ppm. Further tests showed the presence of some potential inhibitory compounds (terephthalic acid and p-toluic acid) in the filtrate of non-ozonized pine 400 biochar; these compounds were greatly reduced upon wet-ozonization of the biochar material. Nutrient detection tests also showed that dry-ozonization of rogue biochar enhanced the availability of nitrate and phosphate in its filtrate, a property that may be desirable for soil application. Conclusion: Ozonized biochar substances can support soil environmental bacterium Pseudomonas putida growth, since ozonization detoxifies the potential inhibitory aromatic molecules.

59 BASIC BIOLOGICAL SCIENCES↗