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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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At least 73 records · Page 4
All sourcesNASA NTRSDOE OSTI
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 NTRS1997

Non-standard amino acid recognition by Escherichia coli leucyl-tRNA synthetase

Recombinant E. coli leucyl-tRNA synthetase was screened for amino acid-dependent pyrophosphate exchange activity using noncognate aliphatic amino acids including norvaline, homocysteine, norleucine, methionine, and homoserine. [32P]-labeled reaction products were separated by thin layer chromatography using a novel solvent system and then quantified by phosphorimaging. Norvaline which differs from leucine by only one methyl group stimulated pyrophosphate exchange activity as did both homocysteine and norleucine to a lesser extent. The KM parameters for leucine and norvaline were measured to be 10 micromoles and 1.5 mM, respectively. Experiments are in progress to determine if norvaline is transferred to tRNA(Leu) and/or edited by a pre- or post-transfer mechanism.

Non-NASA Center↗
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↗
NASA NTRS2001

Microbial detection with low molecular weight RNA

The need to monitor microorganisms in the environment has increased interest in assays based on hybridization probes that target nucleic acids (e.g., rRNA). We report the development of liquid-phase assays for specific bacterial 5S rRNA sequences or similarly sized artificial RNAs (aRNAs) using molecular beacon technology. These beacons fluoresce only in the presence of specific target sequences, rendering as much as a 27-fold fluorescence enhancement. The assays can be used with both crude cell lysates and purified total RNA preparations. Minimal sample preparation (e.g., heating to promote leakage from cells) is sufficient to detect many Gram-negative bacteria. Using this approach it was possible to detect an aRNA-labeled Escherichia coli strain in the presence of a large background of an otherwise identical E. coli strain. Finally, by using a longer wavelength carboxytetramethylrhodamine beacon it was possible to reduce the fraction of the signal due to cellular autofluorescence to below 0.5%.

NASA Discipline Life Sciences Technologies↗
NASA NTRS1995

Plasmid acquisition in microgravity

In microgravity, bacteria often show an increased resistance to antibiotics. Bacteria can develop resistance to an antibiotic after transformation, the acquisition of DNA, usually in the form of a plasmid containing a gene for resistance to one or more antibiotics. In order to study the capacity of bacteria to become resistant to antibiotics in microgravity, we have modified the standard protocol for transformation of Escherichia coli for use in the NASA-flight-certified hardware package, The Fluid Processing Apparatus (FPA). Here we report on the ability of E. coli to remain competent for long periods of time at temperatures that are readily available on the Space Shuttle, and present some preliminary flight results.

Juergensmeyer, Margaret A.↗
NASA NTRS1995

A bacteria antibiotic system in space (23-F ANTIBIO)

In order to evaluate the effects of weightlessness and cosmic radiations on the bacteria resistance to antibiotics, the Antibio 23F experiment was undertaken onboard Discovery during the 1st International Microgravity Laboratory (IML-1) mission. The effects of various antibiotic concentrations (dihydrostreptomycin) on Escherichia coli growth and cell division behavior were studied. The antibiotic binding was investigated using a radioactive tracer (tritium). The results showed that microgravity did not affect E. coli cells in regards the growth and the cell division. The antibiotic added to the culture medium induced an inhibition of the cultures both in the flight and ground controls. However, the antibiotic was less efficient in flight. The behavior of bacteria was modified, and the exponential growth rate was increased in flight. The incorporation of radioactive antibiotics in flight was comparatively different to ground incorporation, which indicated some perturbations in antibiotic binding. The experiments performed in the 1 g centrifuge did not show any difference in the cultures developed on the static rack, and could support a radiative effect of cosmic radiation to explain the results.

Tixador, Rene↗
NASA NTRS2003

Compaction agent clarification of microbial lysates

Recombinant proteins are often purified from microbial lysates containing high concentrations of nucleic acids. Pre-purification steps such as nuclease addition or precipitation with polyethyleneimine or ammonium sulfate are normally required to reduce viscosity and to eliminate competing polyanions before anion exchange chromatography. We report that small polycationic compaction agents such as spermine selectively precipitate nucleic acids during or after Escherichia coli lysis, allowing DNA and RNA to be pelleted with the insoluble cell debris. Analysis by spectrophotometry and protein assay confirmed a significant reduction in the concentration of nucleic acids present, with preservation of protein. Lysate viscosity is greatly reduced, facilitating subsequent processing. We have used 5mM spermine to remove nucleic acids from E. coli lysate in the purification of a hexahistidine-tagged HIV reverse transcriptase.

NASA Discipline Environmental Health↗
NASA NTRS2015

Effects of Polyhydroxybutyrate Production on Cell Division

Synthetic biological engineering can be utilized to aide the advancement of improved long-term space flight. The potential to use synthetic biology as a platform to biomanufacture desired equipment on demand using the three dimensional (3D) printer on the International Space Station (ISS) gives long-term NASA missions the flexibility to produce materials as needed on site. Polyhydroxybutyrates (PHBs) are biodegradable, have properties similar to plastics, and can be produced in Escherichia coli using genetic engineering. Using PHBs during space flight could assist mission success by providing a valuable source of biomaterials that can have many potential applications, particularly through 3D printing. It is well documented that during PHB production E. coli cells can become significantly elongated. The elongation of cells reduces the ability of the cells to divide and thus to produce PHB. I aim to better understand cell division during PHB production, through the design, building, and testing of synthetic biological circuits, and identify how to potentially increase yields of PHB with FtsZ overexpression, the gene responsible for cell division. Ultimately, an increase in the yield will allow more products to be created using the 3D printer on the ISS and beyond, thus aiding astronauts in their missions.

Synthetic Biology↗
NASA NTRS1975

A comparison of certain extracting agents for extraction of adenosine triphosphate (ATP) from microorganisms for use in the firefly luciferase ATP assay

Firefly luciferase ATP assay is used in clinical and industrial applications, such as determination of urinary infection levels, microbial susceptibility testing, and monitoring of yeast levels in beverages. Three categories of extractants were investigated for their extracting efficiency. They were ionizing organic solvents, nonionizing organic solvents, and inorganic acids. Dimethylsulfoxide and formamide represented the ionizing organic solvents, while n-butanol, chloroform, ethanol, acetone, and methylene chloride were used for the nonionizing organic solvents. Nitric acid and perchloric acid were chosen for the inorganic acids category. Pathogens were tested with each solvent. They included: Saccharomyces carlsbergensis, E. coli, Staphylococcus aureus, Klebsiella pneumoniae, Enterobacter species, Proteus mirabilis, Proteus vulgaris, Staphylococcus epidermidis, Streptococcus faecalis, Pseudomonas aeruginosa, and Candida albicans. These results are shown in graphic representations.

Knust, E. A.↗
NASA NTRS2004

Escherichia coli growth under modeled reduced gravity

Bacteria exhibit varying responses to modeled reduced gravity that can be simulated by clino-rotation. When Escherichia coli was subjected to different rotation speeds during clino-rotation, significant differences between modeled reduced gravity and normal gravity controls were observed only at higher speeds (30-50 rpm). There was no apparent affect of removing samples on the results obtained. When E. coli was grown in minimal medium (at 40 rpm), cell size was not affected by modeled reduced gravity and there were few differences in cell numbers. However, in higher nutrient conditions (i.e., dilute nutrient broth), total cell numbers were higher and cells were smaller under reduced gravity compared to normal gravity controls. Overall, the responses to modeled reduced gravity varied with nutrient conditions; larger surface to volume ratios may help compensate for the zone of nutrient depletion around the cells under modeled reduced gravity.

Non-NASA Center↗
NASA NTRS1999

Autolysis of Escherichia coli and Bacillus subtilis cells in low gravity

The role of gravity in the autolysis of Bacillus subtilis and Escherichia coli was studied by growing cells on Earth and in microgravity on Space Station Mir. Autolysis analysis was completed by examining the death phase or exponential decay of cells for approximately 4 months following the stationary phase. Consistent with published findings, the stationary-phase cell population was 170% and 90% higher in flight B. subtilis and E. coli cultures, respectively, than in ground cultures. Although both flight autolysis curves began at higher cell densities than control curves, the rate of autolysis in flight cultures was identical to that of their respective ground control rates.

Flight Experiment↗
NASA NTRS1993

The evolution of energy-transducing systems: Studies with archaebacteria

N-ethylmaleimide (NEM) inhibits the ATPase of H. saccharovorum in a nucleotide protectable manner. The bulk of 14C-NEM is incorporated into subunit 1. Inhibition kinetics indicated a single binding site. To determine the sequence around this site, cyanogen bromide peptides of NEM-labeled ATPase enzyme were prepared and separated on Tris-Tricine gels. Autoradiography indicated that the NEM binding site is probably located in a fragment of Mr 10-12 K. This result will be confirmed by N-terminal sequencing of the peptide. Since the cysteinyl residue, to which NEM is bound, may be located at the C-terminal end, purification and proteolytic treatment of the 10 K peptide will be required. One inhibitor of V-type ATPases, fluoresceinisothiocyanate (FITC) inhibited also the ATPase of H. saccharovorum. Preliminary results indicated protection against inhibition by nucleotides. Localization of the binding sited to the major subunits is in progress. An extraction procedure for the membrane sector of the ATPase complex of H. saccharovorum yielded a preparation which was enriched in a peptide of Mr 5 500. Experiments to test the immunological crossreaction with subunit c from the Escherichia coli F-type ATPase and the labeling with 14C-DCCD are currently carried out. Polyclonal antiserum to the smaller of the major subunits of the ATPase from H. saccharovorum (subunit ll) reacts in Western blots strongly with the alpha and beta subunits of the F1 ATPase of E. coli, suggesting highly conserved regions on both types of ATPases. To elucidate further the regions of homology, cyanogen bromide peptides of the beta subunits were prepared for sequence analysis.

Stan-Lotter, Helga↗
NASA NTRS2005

Population dynamics of transgenic strain Escherichia coli Z905/pPHL7 in freshwater and saline lake water microcosms with differing microbial community structures

Populations of Escherichia coli Z905/pPHL7, a transgenic microorganism, were heterogenic in the expression of plasmid genes when adapting to the conditions of water microcosms of various mineralization levels and structure of microbial community. This TM has formed two subpopulations (ampicillin-resistant and ampicillin-sensitive) in every microcosm. Irrespective of mineralization level of a microcosm, when E. coli Z905/pPHL7 alone was introduced, the ampicillin-resistant subpopulation prevailed, while introduction of the TM together with indigenous bacteria led to the dominance of the ampicillin-sensitive subpopulation. A high level of lux gene expression maintained longer in the freshwater microcosms than in sterile saline lake water microcosms. A horizontal gene transfer has been revealed between the jointly introduced TM and Micrococcus sp. 9/pSH1 in microcosms with the Lake Shira sterile water. c2005 COSPAR. Published by Elsevier Ltd. All rights reserved.

Ecosystem↗
NASA NTRSDate not supplied

Harnessing Synthetic Biology for Biomanufacturing in Space: ​from Fermented Foods to Industrial Enzymes

Biomanufacturing can provide on-demand production of mission-critical compounds and materials to support long-duration space exploration while circumventing the challenges of transporting materials from Earth. Synthetic Biology Project is developing two biomanufacturing capabilities: BioNutrients and CO2-Based Manufacturing. BioNutrients is an ongoing mission aboard the International Space Station focused on the production of perishable nutrients in an on demand for direct for consumption. The first flight experiment of this project targeted the production of carotenoids: β-carotene and zeaxanthin, in recombinant yeast strains. Since then, the project has expanded to encompass the production of the fermented consumables like yogurt and kefir for use as a nutrient delivery mechanism. The CO2-Based Manufacturing system aims to use in situ resources to allow for biomanufacturing with minimal re-supply required. The manufacturing platform is combined with an electrochemical CO2 conversion system which can produce simple carbon substrates to support microbial based biomanufacturing. A comprehensive ground-based platform for recombinant protein purification is in development with the goal of producing a thermostable carbonic anhydrase enzyme from E. coli utilizing CO2-derived acetate. Our group hopes to propel advancements in space biomanufacturing for long duration space flight by harnessing the tools of synthetic biology.

synthetic biology↗