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At least 127 records · Page 7
All sourcesNASA NTRSDOE OSTI
DOE OSTI2021

Improving the Methanol Tolerance of an Escherichia coli Methylotroph via Adaptive Laboratory Evolution Enhances Synthetic Methanol Utilization

There is great interest in developing synthetic methylotrophs that harbor methane and methanol utilization pathways in heterologous hosts such as Escherichia coli for industrial bioconversion of one-carbon compounds. While there are recent reports that describe the successful engineering of synthetic methylotrophs, additional efforts are required to achieve the robust methylotrophic phenotypes required for industrial realization. Here, we address an important issue of synthetic methylotrophy in E. coli : methanol toxicity. Both methanol, and its oxidation product, formaldehyde, are cytotoxic to cells. Methanol alters the fluidity and biological properties of cellular membranes while formaldehyde reacts readily with proteins and nucleic acids. Thus, efforts to enhance the methanol tolerance of synthetic methylotrophs are important. Here, adaptive laboratory evolution was performed to improve the methanol tolerance of several E. coli strains, both methylotrophic and non-methylotrophic. Serial batch passaging in rich medium containing toxic methanol concentrations yielded clones exhibiting improved methanol tolerance. In several cases, these evolved clones exhibited a > 50% improvement in growth rate and biomass yield in the presence of high methanol concentrations compared to the respective parental strains. Importantly, one evolved clone exhibited a two to threefold improvement in the methanol utilization phenotype, as determined via 13 C-labeling, at non-toxic, industrially relevant methanol concentrations compared to the respective parental strain. Whole genome sequencing was performed to identify causative mutations contributing to methanol tolerance. Common mutations were identified in 30S ribosomal subunit proteins, which increased translational accuracy and provided insight into a novel methanol tolerance mechanism. This study addresses an important issue of synthetic methylotrophy in E. coli and provides insight as to how methanol toxicity can be alleviated via enhancing methanol tolerance. Coupled improvement of methanol tolerance and synthetic methanol utilization is an important advancement for the field of synthetic methylotrophy.

Bennett, R. Kyle↗
DOE OSTI2021

Molecular dynamics simulation of vacancy and void effects on strain-induced martensitic transformations in Fe-50 at.% Ni model concentrated solid solution alloy

Strain-induced martensitic transformations can improve the strength and ductility of some face centered cubic (FCC) metals and alloys. Irradiation-induced defects such as vacancies, dislocation loops, and voids activate martensitic transformations over a wider range of mechanical loading conditions than in pristine material. Furthermore, the mechanisms underlying irradiation-enabled martensite transformations remain unclear. In this work, we use molecular dynamics simulations to study the effect of vacancies and voids on strain-induced martensitic transformations in a model concentrated solid solution alloy Fe-50 at.% Ni. It has been found that single vacancies have no resolvable effect on the transformation because they reduce the stacking fault energy by a relatively insignificant margin and do so only if the vacancy is located on the stacking fault plane. Voids, however, activate the martensite transformation through shear strain accumulation around the void due to dislocation pileup. The larger the void, the more pronounced this effect.

36 MATERIALS SCIENCE↗
DOE OSTI2022

Optimization of sporulation and purification methods for sporicidal efficacy assessment on Bacillus spores

Abstract Validating the efficacy of sporicidal agents is a critical step in current good manufacturing practices for disinfection requirements. A limitation is that the poor quality of spores can lead to false positive sporicidal results. The aim of this study was to explore optimal sporulation and purification methods in Bacillus spores. Spores of 7 Bacillus strains were produced in 5 different sporulation media. After density centrifugation, spore yields were measured by phase-contrast microscopy and enumeration assays. Effects of purification methods including heat, sonication and lysozyme, and maturation on spore qualities were determined by sodium hypochlorite sporicidal assay. Difco sporulation media was identified as the preferred sporulation medium for 4 out of 7 tested Bacillus strains. Sporulation rates in B. cereus, B. sphaericus, and B. thuringiensis were higher at 30°C than the rates at 37°C at a difference of 5%, 65%, and 20%, respectively. Bacillus licheniformis favored Mn2+-amended 10% Columbia Broth at 37°C for sporulation with 40–72% higher sporulation rates than other media. The maximum sporulation rates of B. cereus and B. thuringiensis were observed on double-strength Schaeffer's-glucose broth. All studied purification methods improved the spore purity with strain variations. However, intense heat (80°C for 20 min) and lysozyme (100 μg/mL) treatment impaired the spore quality of specific Bacillus strains by sensitizing them against sodium hypochlorite. The length of the maturation period had an impact on the spore resistance, and the most optimal maturation periods ranged from 7 to 21 days in Bacillus strains. The results of this study will pave the way for further evaluation of the sporicidal activity of disinfectants.

Biotechnology & Applied Microbiology↗
DOE OSTI2025

Construction of MoS 2 /MoO 3 Heterostructure with Ultrafast-Charged and Superior Low-Temperature Sodium Storage Properties

The electrochemical reaction kinetics of sodium-ion batteries (SIBs) become sluggish at low temperatures, resulting in significant reductions in energy density and power density. Rational design of anode materials with excellent low-temperature performance is of great significance for promoting the application of SIBs under extreme conditions. Here, in this study, the spontaneous hydrolysis and oxidation reactions of MoS 2 in aqueous solution are used to successfully construct the MoS 2 /MoO 3 heterostructure. The formation of built-in electric fields at the MoS 2 /MoO 3 heterointerfaces improves the electrochemical reaction kinetics, thereby enhancing the rate performance. In addition, the dual-phase material can effectively buffer the volume strain during the cycle process, thereby improving the cycle stability. Thus, the MoS 2 /MoO 3 displays ultrafast charging properties at room temperature (up to 244.6 mAh g –1 at 40 A g –1 , discharge/charge in 22 s). Even at −40 °C, it also exhibits a high capacity of 303.7 mAh g –1 and superior cycling performance (capacity retention rate up to 92.9% after 900 cycles at 2 A g –1 ).

MoS2↗
DOE OSTI2026

Characteristics of Fluid‐Solid Interaction Constitutive Models Within Poroelastodynamics at Higher Strain‐Rates and Large Deformations Implemented in 1D

The large deformation, mixed formulation, finite element (FE) modeling approach presented in Irwin et al. 2024 is extended herein to include improved constitutive models for representing dynamic solid-fluid interactions at higher strain rates (𝒪⁢(1⁢0 2 −1⁢0 3 )⁢s −1 ) and larger overpressure magnitudes (𝒪⁡(1⁢0 2 )⁢kPa) within a biphasic soft porous material using Theory of Porous Media (TPM) at finite strain. Specifically, these constitutive modeling improvements are the following: (i) a more physically robust constitutive model for pore fluid seepage velocity via inclusion of pore fluid viscous stress, and (ii) a modified deformation-dependent-permeability model and updated hyperelastic constitutive model better suited for handling larger volumetric compressions and extensions. The novelty of the present work is mainly the contribution (i): inclusion of pore fluid viscous stress at higher strain-rate and large deformations, which requires 𝐶 1 continuity in the weak formulation, accomplished by employing Hermite cubic interpolation functions within a mixed nonlinear poromechanical finite element formulation. In (ii), the model is updated to weakly enforce solid phase incompressibility, such that this assumption is not violated numerically, which provides improved numerical stability for achieving larger overpressure magnitudes on 𝒪⁡(1⁢0 2 ) kPa, which were not achievable with the previous Kozeny–Carman model in Irwin et al. 2024. Also in (ii), the volumetric part of the solid skeleton free energy function is modified to ensure proper bounds on the solid skeleton Jacobian of deformation 𝐽 s related to incompressibility of the solid phase. Uniaxial strain, unidirectional flow examples at higher strain rates (𝒪⁢(1⁢0 2 −1⁢0 3 )⁢s −1 ) and larger deformations (up to 0.2 (or 20%) nominal axial strain) demonstrate the improved physical representation—and numerical stability—of these constitutive model improvements.

42 ENGINEERING↗
DOE OSTI2020

Transcriptional Regulatory Networks Involved in C3–C4 Alcohol Stress Response and Tolerance in Yeast

Alcohol toxicity significantly impacts the titer and productivity of industrially produced biofuels. To overcome this limitation, we must find and use strategies to improve stress tolerance in production strains. Previously, we developed a multiplex navigation of a global regulatory network (MINR) library that targeted 25 regulatory genes that are predicted to modify global regulation in yeast under different stress conditions. Herein, we expanded this concept to target the active sites of 47 transcriptional regulators using a saturation mutagenesis library. The 47 targeted regulators interact with more than half of all yeast genes. We then screened and selected for C3–C4 alcohol tolerance. We identified specific mutants that have resistance to isopropanol and isobutanol. Notably, the WAR1_K110N variant improved tolerance to both isopropanol and isobutanol. In addition, we investigated the mechanisms for improvement of isopropanol and isobutanol stress tolerance and found that genes related to glycolysis play a role in tolerance to isobutanol, while changes in ATP synthesis and mitochondrial respiration play a role in tolerance to both isobutanol and isopropanol. Overall, this work sheds light on basic mechanisms for isopropanol and isobutanol toxicity and demonstrates a promising strategy to improve tolerance to C3–C4 alcohols by perturbing the transcriptional regulatory network.

09 BIOMASS FUELS↗
DOE OSTI2023

High-Throughput Directed Evolution of Marine Microalgae and Phototrophic Consortia for Improved Biomass Yields (Final Report)

Primary project achievements include using selective pressures (O 2 , light, temperature) and developing culturing regimes for the diatom Nitzschia inconspicua str. hildebrandi to attain enrichments with an ~90% increase in areal biomass productivity relative to the parental strain under pond-mimicking conditions with high O 2 stress in laboratory bioreactors. The resulting strain (GAI-337) was tested further for dilution time, culture density, CO 2 supplementation, pH, temperature, and dissolved O 2 concentration under outdoor pond-mimicking conditions to improve areal productivities. These experiments yielded an optimum harvest and dilution time just after sunset, ~0.45 g AFDW L -1 initial culture density for maximal productivities, no requirement for CO 2 supplementation or pH control, maximal performance under a diel temperature curve going from 24 °C at night to 36 °C during the day, and benefits from some O 2 removal from the culture by bubbling with air. Using pond-mimicking laboratory bioreactors, N. inconspicua GAI-337 achieved ~42 g AFDW m -2 d -1 . Nutrient limitation experiments resulted in a biomass composition that equated to ~160 Gallons of Gasoline Equivalent energy per ton AFDW, highlighting the potential of GAI-337 as a promising renewable fuel feedstock strain. Genome resequencing has revealed genome alterations potentially contributing to the improved growth of GAI-337 in the laboratory. Based on the comparative analyses of the GAI-337 and GAI-229 (reference) strains, we identified 144 single nucleotide substitutions that resulted in amino acid change, 7 single nucleotide substitutions that resulted in protein truncation; 5 deletions; and 1 frameshift mutation. From the mutations that potentially affect expression of functionally annotated genes, particular interest was noted for an interferon-induced 6-16 family protein that may be involved in the host immune response against microbe invasion; the chaperone protein DnaK, which may function to protect the folding of proteins within the cell; and SPRY domain protein that is found in many eukaryotic proteins important in cell signaling pathways. Transcriptome analysis revealed over 1000 genes with increased transcript levels. Many of these and many of the genes with mutations are not yet functionally annotated and an increased bioinformatics effort is necessary to more completely analyze the Nitzschia inconspicua genome. Adaptive laboratory evolution (ALE) was performed for over 300 days using consecutive 0.5°C temperature increases in a constant temperature incubator to attain greater thermal tolerance in Nitzschia inconspicua. The adapted strain was able to grow at a constant temperature of 37.5°C; whereas this constant temperature was lethal to the parental control, which had an upper temperature boundary of 35.5°C prior to adaptive evolution. Several high-temperature clonal isolates were obtained from the evolved population following ALE, and increased temperature tolerance was observed in clonal adapted cultures. The final temperature adaptation was maintained through cryopreservation and was observed in multiple clonal isolates, including multiple clonal isolates with significantly increased cell size, indicating the potential occurrence of a sexual cycle during the clonal isolation process. A survey of Nannochloropsis strains was conducted for tolerances to high pH and high bicarbonate media. Nannochloropsis granulata showed promising growth in diel bioreactors and was successfully grown at the GAI Kauai farm site in long-term growth campaigns. Co-culturing using Nitzschia inconspicua, Nannochloropsis and a cyanobacterium were assembled in the laboratory to determine if productivity synergies could be attained. Although all strains grew well in the laboratory high-bicarbonate media individually, the cyanobacterium quickly outgrew the other strains in the laboratory consortium pushing the co-culture away from a diverse (and potentially synergistic assemblage) phototroph culture towards a monoculture dominated by the cyanobacterium. Several outdoor growth campaigns were conducted, with productivities ranging between 10-20 g/m 2 /d of biomass. The best performing strain in the laboratory (GAI-337) did not outperform reference strains at the Kauai farm under the conditions used. Addition growth campaigns are necessary under conditions that result in higher biomass (>20 g/m 2 /d) and that attain higher O 2 levels are likely necessary. Initial data indicate that the thermally adapted strain did slightly better than the control strain at higher temperatures; however, additional campaigns are necessary to establish statistical significance. In summary, Nitzschia inconspicua is able to attain exemplary biomass and lipid yields in the laboratory bioreactors. Strain evolution to both O 2 and temperature resulted in targeted strain improvements. Additional outdoor campaigns are necessary to determine if laboratory improvements translate to the field.

09 BIOMASS FUELS↗
DOE OSTI2024

Establishing Co‐Continuous Network of Conjugated Polymers and Elastomers for High‐Performance Polymer Solar Cells with Extreme Stretchability

High power conversion efficiency (PCE) and mechanical robustness are prerequisites for wearable applications of organic solar cells (OSCs). However, stretchability of present active systems (i.e., crack-onset strain (COS) < 30%) should be improved. While introducing elastomers into active systems is considered a simple method for improving stretchability, the inclusion of elastomers typically results in a decrease in PCE of the OSC with a limited enhancement in the stretchability due to lack of interconnected electrical and mechanical pathways. In this study, it is developed efficient and intrinsically stretchable (IS)-OSCs with exceptional mechanical robustness, by constructing co-continuous networks of conjugated polymers (D18) and elastomers (SEBS) within active layers. It is demonstrated that the blend film with a specific ratio (40:60 w/w) of D18:SEBS is crucial for forming co-continuous structures, establishing well-connected mechanical and electrical channels. Consequently, D18 0.4 :SEBS 0.6 /L8-BO OSCs achieve 16-times higher stretchability (COS = 126%) than the OSCs based on D18/L8-BO (COS = 8%), while achieving 4-times higher PCE (12.13%) compared to the OSCs based on SEBS-rich active layers (D18 0.2 :SEBS 0.8 /L8-BO, PCE = 3.15%). Furthermore, D18 0.4 :SEBS 0.6 -based IS-OSCs preserve 86 and 90% of original PCEs at 50% strain and after 200 stretching/releasing cycles with 15% strain, respectively, demonstrating the highest mechanical robustness among reported IS-OSCs.

14 SOLAR ENERGY↗
DOE OSTI2020

Engineering Atomically Dispersed FeN 4 Active Sites for CO 2 Electroreduction

Atomically dispersed FeN 4 active sites have exhibited exceptional catalytic activity and selectivity for the electrochemical CO 2 reduction reaction (CO2RR) to CO. However, the understanding behind the intrinsic and morphological factors contributing to the catalytic properties of FeN 4 sites is still lacking. By using a Fe-N-C model catalyst derived from the ZIF-8, we deconvoluted three key morphological and structural elements of FeN 4 sites, including particle sizes of catalysts, Fe content, and Fe-N bond structures. Furthermore, their respective impacts on the CO2RR were comprehensively elucidated. Engineering the particle size and Fe doping is critical to control extrinsic morphological factors of FeN 4 sites for optimal porosity, electrochemically active surface areas, and the graphitization of the carbon support. In contrast, the intrinsic activity of FeN 4 sites was only tunable by varying thermal activation temperatures during the formation of FeN 4 sites, which impacted the length of the Fe-N bonds and the local strains. The structural evolution of Fe-N bonds was examined at the atomic level. First-principles calculations further elucidated the origin of intrinsic activity improvement associated with the optimal local strain of the Fe-N bond.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗
DOE OSTI2022

Strain-retardant coherent perovskite phase stabilized Ni-rich cathode

The use of state-of-the-art Ni-rich layered oxides (LiNi x Co y Mn 1-x-y O 2 , x > 0.5) as the cathode material for lithium-ion batteries can push the energy and power density to a higher level than is currently available. However, volume variation associated with anisotropic lattice strain and stress that is being developed during lithium (de) intercalation induces severe structural instability and electrochemical decay of the cathode materials, which is amplified further when the battery is operating at a high voltage (above 4.5 V), which is essential for unlocking its high energy. Even after much effort by the research community, an intrinsic strain-retardant method for directly alleviating the continuous accumulation of lattice strain remains elusive. By introducing a coherent perovskite phase into the layered structure functioning as a ‘rivet’, we significantly mitigate the pernicious structural evolutions by a pinning effect. The lattice strain evolution in every single cycle is markedly reduced by nearly 70% when compared with conventional materials, which significantly enhances morphological integrity leading to a notable improvement in battery cyclability. This strain-retardant approach broadens the perspective for lattice engineering to release the strain raised from lithium (de)intercalation and paves the way for the development of high-energy-density cathodes with long durability.

25 ENERGY STORAGE↗
DOE OSTI2020

Production of Biocrude in an Advanced Photobioreactor-Based Biorefinery

Algenol Biotech, the National Renewable Energy Laboratory (NREL), Georgia Institute of Technology, Arizona State University, and Reliance Industries formed a team to advance the state of the art in algal biomass production and downstream processing technologies, with the end goal of a sustainable, economically viable biofuel intermediate (BFI, biocrude) product. The project included examination of high value co-product production as a market entry strategy and for enhancing the economics of a biorefinery for BFI production. The project targeted innovations in biology, operations, and engineering. The goals of the project were: BFI productivity greater than 4,000 gal-BFI/acre-yr on an annualized basis; energy efficient innovations in downstream operations resulting in an energy expenditure less than 10% of the BFI energy content and a carbon footprint reduction of more than 60% compared to fossil alternatives; and a comprehensive Techno-Economic Analysis (TEA) that identifies limiting factors for commercial viability of a photobioreactor (PBR)-based biofuel product. The project achieved the overall objectives. Strain development efforts led to the identification of a strain (Cyanobacterium sp. AB1166) that, relative to the previous best strain (Cyanobacterium sp. AB1), exhibited a ~10% increase in productivity under commercially-relevant cultivation conditions and also resulted in cultures with a >50% reduction in viscosity such that harvesting efficiency was improved; these results represent achievement of key project milestones. Progress was also made at NREL in altering the biochemical composition of algal biomass to improve the yield of BFI produced via HTL. These strain enhancements coupled with improved outdoor cultivation practices, including semi-continuous operation, increased areal biomass productivity by nearly 80% over the established baseline productivity. The annualized productivity achieved (26.8 g/m2-d), paired with HTL conversion yields realized at NREL and RIL (38% ± 2% BFI), translates to 4,100 gal-BFI/acre-yr, exceeding the FY20 BETO goal of 3,700 gal-BFI/acre-yr. Significant progress was also demonstrated in large scale PBR-based production system design, operability, and cost reduction. A 24,000-L production module comprised of 240 interlinked PBRs was constructed and successfully operated outdoors for over one year in Fort Myers, Florida. Aided by a state-of-the-art productivity model, the productivities achieved convincingly demonstrated scalability of laboratory results determined at the mL to L scale to large-scale outdoor operations exceeding 20,000 L. The system was used to cultivate Arthrospira platensis (Spirulina), an industrially-relevant cyanobacterium and source for phycocyanin, an approved blue food colorant that Algenol is developing as a risk reduction strategy for future biofuel projects and as a potential business opportunity. A key project milestone to develop phycocyanin extraction and purification technologies was achieved ahead of schedule, and product samples received positive feedback from potential customers. The production and downstream operations data generated in this project were used to conduct and refine Techno-Economic and Life Cycle Assessments to provide research guidance for reducing the costs and environmental footprint of algal biofuel and co-product manufacturing plants. Several CO 2 supply scenarios for an algal biorefinery were identified as being capable of providing a large (>60%) reduction in carbon footprint in comparison to gasoline. The TEA assessments incorporated detailed comparisons of PBR versus open pond production systems, yielding a 3-fold higher areal productivity for PBRs and suggesting overall production cost parity for the two systems. The progress in this ABY2 project addressed many of the the barriers identified for the Advanced Algal Systems R&D Program and are directly relevant to achieving the established BETO goals associated with large scale biofuel production and cost reduction.

09 BIOMASS FUELS↗
DOE OSTI2020

Design of a V–Ti–Ni alloy with superelastic nano-precipitates

Stress-induced martensitic transformations enable metastable alloys to exhibit enhanced strain hardening capacity, leading to improved formability and toughness. As is well-known from transformation-induced plasticity (TRIP) steels, however, the resulting martensite can limit ductility and fatigue life due to its intrinsic brittleness. In this work, we explore an alloy design strategy that utilizes stress-induced martensitic transformations but does not retain the martensite phase. This strategy is based on the introduction of superelastic nano-precipitates, which exhibit reverse transformation after initial stress-induced forward transformation. To this end, utilizing ab-initio simulations and thermodynamic calculations we designed and produced a V 45 Ti 30 Ni 25 (at%) alloy. In this alloy, TiNi is present as nano-precipitates uniformly distributed within a ductile V-rich base-centered cubic (bcc) β matrix, as well as being present as a larger matrix phase. We characterized the microstructure of the produced alloy using various scanning electron microscopy (SEM) and transmission electron microscopy (TEM) methods. Furthermore, the bulk mechanical properties of the alloy are demonstrated through tensile tests, and the reversible transformation in each of the TiNi morphologies were confirmed by in-situ TEM micro-pillar compression experiments, in-situ high-energy diffraction synchrotron cyclic tensile tests, indentation experiments, and differential scanning calorimetry experiments. The observed transformation pathways and variables impacting phase stability are critically discussed.

59 BASIC BIOLOGICAL SCIENCES↗
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 OSTI2025

Fluoropolymer Composites from Partially Perfluoroalkylated Waste Polyethylene

Chemically modified plastics have emerged as practical solutions to plastic waste increases. Here, the inherent novelty of decorating polymer chains with chemical functionality results in distinct properties that expand the available application space. Nevertheless, developing designer materials for specific applications beyond compatibilization or mild property enhancement is difficult due to the synergistic effects of both the polar functionality imparted and the parent materials' intrinsic properties. By incorporating perfluoro-alkyl side-chains onto the backbone of dehydrogenated waste HDPE, unique surface properties intermediate between polytetrafluoroethylene (PTFE, the model fluoropolymer) and HDPE become apparent, while the overall material mechanical and thermal properties result in more LLDPE-like materials. This is demonstrated through moderate decreases in the surface free energy of the perfluoroalkylated polyolefin surface (increase in H 2 O contact angle of ~ 6°) and increased ordering under shear when blended with PTFE nanoparticles where the crossover point occurred at higher strains. Critically, perfluoroalkylated HDPE possesses improved rheological modification properties at elevated temperatures with PTFE nanoparticles, resulting in more thermally robust and stable composite materials.

fluoropolymer↗
DOE OSTI2025

Laboratory evolution in Novosphingobium aromaticivorans enables rapid catabolism of a model lignin-derived aromatic dimer

Lignin contains a variety of interunit linkages, leading to a range of potential decomposition products that can be used as carbon and energy sources by microbes. β-O-4 linkages are the most common in native lignin, and associated catabolic pathways have been well characterized. However, the fate of the mono-aromatic intermediates that result from β-O-4 dimer cleavage has not been fully elucidated. Here, we used experimental evolution to identify mutant strains of Novosphingobium aromaticivorans with improved catabolism of a model aromatic dimer containing a β-O-4 linkage, guaiacylglycerol-β-guaiacyl ether (GGE). We identified several parallel causal mutations, including a single nucleotide polymorphism in the promoter of an uncharacterized gene that roughly doubled the growth yield with GGE. We characterized the associated enzyme and demonstrated that it oxidizes an intermediate in GGE catabolism, β-hydroxypropiovanillone, to vanilloyl acetaldehyde. Identification of this enzyme and its key role in GGE catabolism furthers our understanding of catabolic pathways for lignin-derived aromatic compounds.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2021

Further engineering of R. toruloides for the production of terpenes from lignocellulosic biomass

Abstract Background Mitigation of climate change requires that new routes for the production of fuels and chemicals be as oil-independent as possible. The microbial conversion of lignocellulosic feedstocks into terpene-based biofuels and bioproducts represents one such route. This work builds upon previous demonstrations that the single-celled carotenogenic basidiomycete, Rhodosporidium toruloides , is a promising host for the production of terpenes from lignocellulosic hydrolysates. Results This study focuses on the optimization of production of the monoterpene 1,8-cineole and the sesquiterpene α-bisabolene in R. toruloides . The α-bisabolene titer attained in R. toruloides was found to be proportional to the copy number of the bisabolene synthase (BIS) expression cassette, which in turn influenced the expression level of several native mevalonate pathway genes. The addition of more copies of BIS under a stronger promoter resulted in production of α-bisabolene at 2.2 g/L from lignocellulosic hydrolysate in a 2-L fermenter. Production of 1,8-cineole was found to be limited by availability of the precursor geranylgeranyl pyrophosphate (GPP) and expression of an appropriate GPP synthase increased the monoterpene titer fourfold to 143 mg/L at bench scale. Targeted mevalonate pathway metabolite analysis suggested that 3-hydroxy-3-methyl-glutaryl-coenzyme A reductase (HMGR), mevalonate kinase (MK) and phosphomevalonate kinase (PMK) may be pathway bottlenecks are were therefore selected as targets for overexpression. Expression of HMGR, MK, and PMK orthologs and growth in an optimized lignocellulosic hydrolysate medium increased the 1,8-cineole titer an additional tenfold to 1.4 g/L. Expression of the same mevalonate pathway genes did not have as large an impact on α-bisabolene production, although the final titer was higher at 2.6 g/L. Furthermore, mevalonate pathway intermediates accumulated in the mevalonate-engineered strains, suggesting room for further improvement. Conclusions This work brings R. toruloides closer to being able to make industrially relevant quantities of terpene from lignocellulosic biomass.

09 BIOMASS FUELS↗
DOE OSTI2026

Integrating CO2 Electrolysis with Gas Fermentation to Produce Valuable Fuels and Chemicals

Many industrial activities squander CO2, decreasing process yield. We envision a future where this waste carbon is instead captured, upgraded, and valorized directly at the point of emission. Within the CO2 Reduction and Upgrading Consortium (a collaboration of seven US national laboratories and industrial partners), we are pursuing this goal by developing and de-risking new technologies for low temperature CO2 electrolysis, coupled with biological upgrading of intermediates into more valuable compounds. One such process involves electrocatalytic reduction of CO2 to generate carbon monoxide (CO). As both a carbon and energy source, CO represents an attractive feedstock for microbial upgrading by certain syngas-fermenting species, such as the autotrophic bacterium Clostridium autoethanogenum. Our team has developed new genetic tools and optimized cultivation techniques to enhance C. autoethanogenum as a platform host for the biological conversion of syngas into value-added products. For example, we have created novel CRISPR-based genetic engineering techniques to build new, genome-reduced, platform strains of C. autoethanogenum with improved growth rates. Further, we ve introduced heterologous biochemical pathways into C. autoethanogenum to enable the production of high-value compounds from syngas, such as the isoprenoid precursor mevalonic acid. With the tools of electrochemistry and synthetic biology, there is virtually no limit to the spectrum of products that could be sustainably manufactured from CO2.

09 BIOMASS FUELS↗