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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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DOE OSTI2023

Enhancing isoprenol production by systematically tuning metabolic pathways using CRISPR interference in E. coli

Regulation of metabolic gene expression is crucial for maximizing bioproduction titers. Recent engineering tools including CRISPR/Cas9, CRISPR interference (CRISPRi), and CRISPR activation (CRISPRa) have enabled effective knock-out, knock-down, and overexpression of endogenous pathway genes, respectively, for advanced strain engineering. CRISPRi in particular has emerged as a powerful tool for gene repression through the use of a deactivated Cas9 (dCas9) protein and target guide RNA (gRNA). By constructing gRNA arrays, CRISPRi has the capacity for multiplexed gene downregulation across multiple orthogonal pathways for enhanced bioproduction titers. In this study, we harnessed CRISPRi to downregulate 32 essential and non-essential genes in E. coli strains heterologously expressing either the original mevalonate pathway or isopentenyl diphosphate (IPP) bypass pathway for isoprenol biosynthesis. Isoprenol remains a candidate bioproduct both as a drop-in blend additive and as a precursor for the high-performance sustainable aviation fuel, 1,4-dimethylcyclooctane (DMCO). Of the 32 gRNAs targeting genes associated with isoprenol biosynthesis, a subset was found to vastly improve product titers. Construction of a multiplexed gRNA library based on single guide RNA (sgRNA) performance enabled simultaneous gene repression, yielding a 3 to 4.5-fold increase in isoprenol titer (1.82 ± 0.19 g/L) on M9-MOPS minimal medium. We then scaled the best performing CRISPRi strain to 2-L fed-batch cultivation and demonstrated translatable titer improvements, ultimately obtaining 12.4 ± 1.3 g/L isoprenol. Our strategy further establishes CRISPRi as a powerful tool for tuning metabolic flux in production hosts and that titer improvements are readily scalable with potential for applications in industrial bioproduction.

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2025

Integration of Metabolic and Bioprocess Engineering for the Production of B-Ketoadipic Acid from Glucose and Xylose by Pseudomonas Putida

..beta..-Ketoadipic acid is a common intermediate in aerobic microbial aromatic catabolism that can be used as a monomer in performance-advantaged biopolymers. Here, we engineered Pseudomonas putida KT2440 to produce ..beta..-ketoadipate from glucose and xylose, the most prevalent carbohydrates derived from lignocellulosic polysaccharides. With the top-performing strain, P. putida GR038, we conducted bioprocess development using glucose and xylose as substrates in a 2 : 1 molar ratio to mimic lignocellulosic hydrolysate. Fed-batch cultivations achieved a titer of 65.8 g L-1 and a rate of 0.69 g L-1 h-1, with a C-mol yield of 0.52. Application of adsorptive in situ product recovery further improved the effective titer to 92.0 g L-1 and the rate to 0.83 g L-1 h-1, while also improving the downstream purity of ..beta..-ketoadipate from 88.3 wt% to 99.0 wt%. These results show promise towards industrial production of ..beta..-ketoadipate from lignocellulosic sugars.

09 BIOMASS FUELS↗

DOE OSTI2024

Genome reduction improves octanoic acid production in scale down bioreactors

Microorganisms in large-scale bioreactors are exposed to heterogeneous environmental conditions due to physical mixing constraints. Nutritional gradients can lead to transient expression of energetically wasteful stress responses and as a result, can reduce the titres, rates and yields of a bioprocess at larger scales. To what extent these process parameters are impacted is often unknown and therefore bioprocess scale-up comes with major risk. Designing platform strains to account for these intermittent stresses before introducing synthesis pathways is one strategy for de-risking bioprocess development. For example, Escherichia coli strain RM214 is a derivative of wild-type MG1655 that has had several genes and whole operons removed from its genome based on their metabolic cost. In this study, we engineered E. coli strain RM214 (referred to as WG02) to produce octanoic acid from glycerol in batch-flask and fed-batch bioreactor cultivations and compared it to an octanoic acid-producing E. coli MG1655 (WG01). In batch flask cultivations, the two strains performed similarly. However, in carbon limited fed-batch bioreactor cultivations, WG02 provided a greater than 22% boost to biomass compared to WG01 while maintaining similar titres of octanoic acid. Reducing the biomass accumulation of WG02 with nitrogen limited fed-batch cultivation resulted in a 16% improvement in octanoic acid titre over WG01. Finally, in a scale-down system consisting of a stirred tank reactor (representing a well-mixed zone) and plug flow reactor (representing an intermittent carbon starvation zone), WG02 again improved octanoic acid titre by almost 18% while maintaining similar biomass concentrations as WG01.

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2026

Upcycling plastic waste into polyhydroxyalkanoates with high carbon conversion via CO 2 plasma-enabled deconstruction

Plastic deconstruction into fermentable intermediates is a key step for microbial bio-upcycling into value-added products. In this study, CO 2 plasma deconstruction was used as an electrified route to convert polyethylene into oxygenated intermediates and liquid (OIL). The resulting OIL was rich in fatty acids, fatty alcohols, and hydrocarbons, making it a suitable feedstock for medium-chain-length polyhydroxyalkanoate (mcl-PHA) production by Pseudomonas putida NRRL B-14688 and Pseudomonas resinovorans NRRL B-2649. Compared with batch fermentation and monocultures, fed-batch co-cultivation markedly improved biomass formation and PHA accumulation, likely due to complementary substrate utilization, particularly hydrocarbon conversion by P. resinovorans. Using virgin polyethylene-derived OIL (Vir-OIL), the co-culture achieved 40.11% mcl-PHA content and about 18% PHA yield based on total OIL fed. More importantly, OIL produced from post-consumer single-use plastic films (PCR-OIL) was directly fermented and well supported the cell growth and PHA accumulation, achieving 38.11% PHA content and about 14.7% PHA yield. Based on emulsified OIL fractions, PHA yields for Vir-OIL and PCR-OIL were comparable (∼28%). PHA granules were extracted from PCR-OIL-grown cells with high recovery (88.25%) and purity (94.8%). Five monomers were identified in the polymer, including 3-hydroxyhexanoate (3HHx), 3-hydroxyoctanoate (3HO), 3-hydroxydecanoate (3HD), 3-hydroxydodecanoate (3HDD), and 3-hydroxytetradecanoate (3HTD), with 3HO (44.28%) and 3HD (40.60%) as the dominant units. The polymer exhibited moderate molecular weight and narrow dispersity (M n = 65.4 kDa, M w = 92.1 kDa, Đ = 1.40) and low crystallinity (T m ≈ 76.6 °C, X c ≈ 15.5%). These characteristics indicate elastomer-like behavior, making the material suitable for flexible applications such as films, coatings, adhesives, and blend modifiers. Overall, this study establishes a CO 2 plasma-assisted route for generating fermentable polyethylene-derived intermediates and demonstrates that fed-batch co-culture fermentation can effectively funnel plastic-derived carbon into mcl-PHA.

42 ENGINEERING↗

NASA NTRSDate not supplied

Bioreactor Development for CO2-Based In Situ Resource Utilization Manufacturing

Sustainable long-duration manned missions on both the Moon and Mars will require in situ resource utilization (ISRU). Carbon dioxide (CO2) has great potential as a harvestable resource, making up 95% of the atmosphere on Mars and being produced as respiratory waste in spacecraft and future planetary habitats. Through ISRU, biomanufacturing has the capability to produce a near limitless array of products from local space resources, which include pharmaceuticals, bioplastics, chemical feedstocks, and industrial enzymes. Here, a CO2-based ISRU recombinant protein bioreactor and associated biomanufacturing organisms were designed to produce a highly stable carbonic anhydrase (CA). Initial work characterized candidate organisms for growth on acetate and formic acid, carbon substrates that can be synthesized via electrochemical conversion of CO2. To improve growth on the CO2 producing substrate formic acid and for direct integration of ISRU CO2, a synthetic Calvin-Benson-Bassam cycle was designed for use in Cyberlindnera jadinii and Escherichia coli. Genetic modifications in E. coli will be facilitated by a tailored CRISPR/Cas9 and λ red recombineering two-vector system. For expression of CA, a blue light regulated T7 promoter was employed for dynamic and small molecule free induction. Efficient bioproduction through a fed-batch exponential feeding strategy was determined via mass balance calculations from ISRU substrates to biomass and CA yield. Flux balance analysis was used to model ISRU substrate metabolism and metabolic pathway engineering in candidate organisms under cultivation strategy conditions for both metabolism reconstruction and pathway design optimization. Finally, a small-scale, disposable bag bioreactor concept for use in the NASA Bioculture System infrastructure was designed to enable CO2-based CA production in reduced-gravity environments.

Biomanufacturing, Pathway Engineering, Flux Balanc↗

NASA NTRSDate not supplied

Bioreactor Development for CO2-Based In Situ Resource Utilization Manufacturing

Sustainable long-duration manned missions on both the Moon and Mars will require in situ resource utilization (ISRU). Carbon dioxide (CO2) has great potential as a harvestable resource, making up 95% of the atmosphere on Mars and being produced as respiratory waste in spacecraft and future planetary habitats. Through ISRU, biomanufacturing has the capability to produce a near limitless array of products from local space resources. Here, a CO2-based ISRU recombinant protein producing bioreactor and associated biomanufacturing organisms were designed to produce a highly stable carbonic anhydrase (CA). Candidate organisms were selected by growth characterization on acetate and formic acid, carbon substrates that are synthesized via electrochemical conversion of CO2. To improve growth on the CO2 producing substrate formic acid and for direct integration of ISRU CO2, a synthetic Calvin-Benson-Bassam cycle was designed for use in Escherichia coli. Multiplex genetic modification in E. coli was facilitated by a tailored CRISPR/Cas9 and λ red recombineering two-vector system. For expression of CA, a blue light regulated T7 promoter was employed for dynamic and small molecule free induction. Efficient bioproduction through a fed-batch exponential feeding strategy was determined via mass balance analysis from ISRU substrates to biomass and CA yield. Flux balance analysis was used to model ISRU substrate metabolism and metabolic pathway engineering in candidate organisms under cultivation strategy conditions for both metabolism reconstruction and pathway design optimization. Finally, a small-scale, disposable bag bioreactor for use in the NASA Bioculture System infrastructure was designed to enable CO2-based CA biomanufacturing in reduced-gravity environments.

biomanufacturing↗

DOE OSTI2023

Lipids from High-Protein Algae Biomass

Biofuels from renewable and sustainable sources will be a critical component of reducing greenhouse gasses. To this end, in achieving maximum biomass productivity in outdoor ponds at a reasonable cost, algae must be grown at a maximal rate. This current algae cultivation strategy results in high-protein biomass because there is insufficient time for deplete-hold steps that increase lipids but also cost. Thus methods to convert high-protein (>50%) need to be developed for biofuels. The Consolidated Algae Processing (CAP) strategy has previously shown utility on high-carbohydrate algae biomass and has now been expanded to include methods aimed at converting the protein fraction to lipids. After acid-pretreatment, hydrolysate liquor is oxidatively treated to deaminate and convert soluble proteins and amino acids into four main carboxylate acids; formic, acetic, succinic, and propionic. These acids are readily and completely fermented into intracellular lipids using oleaginous yeast. Using a fed-batch fermentation strategy and no added nutrients, we achieved nearly 30% intracellular lipids and 1.3 g/L lipids demonstrating the potential of our expanded CAP process to generate additional lipids for conversion to biofuels as part of an algae-focused biorefinery.

AAD↗

DOE OSTI2025

Maximizing long-term biohydrogen production with Clostridium thermocellum for high solids conversion of lignocellulosic biomass

Biological hydrogen production from lignocellulosic biomass sustainably couples organic waste reduction with renewable energy generation. Efficient conversion is challenged by the structural complexity of lignocellulose and resulting recalcitrance to enzymatic degradation. Clostridium thermocellum natively breaks down biomass with highly effective hemi-/cellulases systems (i.e., cellulosomes) and generates hydrogen in anaerobic cultivation, creating a compelling platform for lignocellulosic biohydrogen production. Achieving commercially viable production rates requires balancing high biomass loading and throughput against uniform mixing conditions required for enzyme dispersion, pH and temperature control, and efficient hydrogen and metabolite removal in continuous operation. To address these barriers to process intensification, we implemented novel reactor and process designs for high-solids lignocellulosic biomass fermentations using the C. thermocellum KJC19-9 strain, genetically engineered for co-utilization of cellulose and hemicellulose sugars (i.e., xylose). Via computational fluid dynamics (CFD) modeling and experimental validation, we achieved a >50% improvement in biohydrogen production with an improved anchor-type impeller morphology, coupled to a threefold reduction in agitation rate. To further reduce rheological constraints and accumulation of toxic metabolites, we then transitioned the process to sequencing fed-batch operation. The resulting process generated 24.87 L H 2 L −1 from 160 g L −1 of deacetylated and mechanically refined (DMR)-pretreated corn stover biomass over 16 days while solubilizing >95% of influent cellulose and hemicellulose, setting a new performance benchmark for continuous production of biohydrogen from lignocellulose.

08 HYDROGEN↗

DOE OSTI2023

Butyric Acid Production from Delignified Corn Stover Using Thermophilic Bacterial Co-Cultures

Butyric acid (BA) is a valuable platform chemical in the food and pharmaceutical industries, and it is also a potential precursor for the production of biobutanol and sustainable aviation fuels (SAFs). BA is mainly synthesized from petroleum; thus, cost-effective, and sustainable alternatives for its production are attracting the interest of several sectors. The present work proposes a solids-to-acids bioprocess to produce BA from corn stover utilizing a co-culture that consists of two thermophilic bacteria, Clostridium thermocellum, a well-known efficient degrader of insoluble and oligomeric cellulosic substrates, and Clostridium thermobutyricum, a highly efficient BA producer from monomeric sugars. After initial proof of concept experiments, a series of optimization studies were carried out to evaluate the process limits of this co-culture. First, the co-cultivation of both microorganisms at different inoculum sizes in deacetylated and mechanically refined corn stover (DMR) was evaluated. No significant differences were found on the solids deconstruction and carbohydrates utilization among all the treatments. In addition, BA production was similar under all conditions, ranging between 2.1 and 2.4 g/L. Next, the deconstruction, and BA production capabilities of the co-culture at increased DMR solids contents (3, 4.5, and 6% (w/v)) were tested. Although no difference was observed in the solids deconstruction and carbohydrates utilization among treatments, BA production increased concomitantly to solids loading; the maximum values observed were 2.8, 3.6, and 6.2 g/L in the 3, 4.5, and 6% treatments, respectively. In fact, BA production did not reach its absolute maximum in the 6% treatment after 160 h of fermentation. Lastly, a fed-batch experiment was performed to investigate the co-culture capabilities and possible system constraints to achieve higher BA titers. Data on substrate modifications and product formation as well as on the growth of each microorganism will be shown. Results from this work demonstrate a promising bioprocess approach for the production of BA from lignocellulosic biomass using a thermophilic bacterial co-culture.

bacteria↗