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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
DOE OSTI2023

RNAseq analysis of Cellvibrio japonicus during starch utilization differentiates between genes encoding carbohydrate active enzymes controlled by substrate detection or growth rate

ABSTRACT Bacterial utilization of starch is increasingly of interest as the importance and contributions of animal gut microbiomes become more defined. Consequently, identifying and characterizing the bacterial enzymes responsible for the degradation, transport, and metabolism of starch will enable developments in pharmaceutical, biotechnological, and culinary industries searching for novel prebiotics, carrier molecules, and low glycemic index sweeteners. The current challenge is that bacteria proficient at starch utilization often have hundreds of carbohydrate active enzymes, and it is unclear which are essential for starch utilization using only homology-based bioinformatics or computational methods. Complementary experimental data are also needed, especially to understand the regulation of bacterial starch utilization. We have completed an RNAseq analysis of the Gram-negative bacterium Cellvibrio japonicus and found that it has sophisticated regulation that includes substrate sensing and growth rate components for genes that encode starch-degrading enzymes. Among the 22 genes predicted to encode starch-active enzymes, C. japonicus has 10 alpha-amylases, 4 alpha-glucosidases, 2 pullulnases, and 2 cyclomaltodextrin glucanotransferases, 15 of which were up-regulated during exponential growth on starch and 8 up-regulated in stationary phase. Growth analyses with an enzyme secretion deficient mutant of C. japonicus suggested that secreted amylases are essential for this bacterium to degrade starch. Our approach of coupling a physiological growth assay with transcriptomic data provides a platform to identify targets for further genetic or biochemical analysis that can be broadly applied to other starch-utilizing bacteria. IMPORTANCE Understanding the bacterial metabolism of starch is important as this polysaccharide is a ubiquitous ingredient in foods, supplements, and medicines, all of which influence gut microbiome composition and health. Our RNAseq and growth data set provides a valuable resource to those who want to better understand the regulation of starch utilization in Gram-negative bacteria. These data are also useful as they provide an example of how to approach studying a starch-utilizing bacterium that has many putative amylases by coupling transcriptomic data with growth assays to overcome the potential challenges of functional redundancy. The RNAseq data can also be used as a part of larger meta-analyses to compare how C. japonicus regulates carbohydrate active enzymes, or how this bacterium compares to gut microbiome constituents in terms of starch utilization potential.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2022

Transformation and gene editing in the bioenergy grass Miscanthus

Miscanthus, a C4 member of Poaceae, is a promising perennial crop for bioenergy, renewable bioproducts, and carbon sequestration. Species of interest include nothospecies M. x giganteus and its parental species M. sacchariflorus and M. sinensis. Use of biotechnology-based procedures to genetically improve Miscanthus, to date, have only included plant transformation procedures for introduction of exogenous genes into the host genome at random, non-targeted sites. We developed gene editing procedures for Miscanthus using CRISPR/Cas9 that enabled the mutation of a specific (targeted) endogenous gene to knock out its function. Classified as paleo-allopolyploids (duplicated ancient Sorghum-like DNA plus chromosome fusion event), design of guide RNAs (gRNAs) for Miscanthus needed to target both homeologs and their alleles to account for functional redundancy. Prior research in Zea mays demonstrated that editing the lemon white1 (lw1) gene, involved in chlorophyll and carotenoid biosynthesis, via CRISPR/Cas9 yielded pale green/yellow, striped or white leaf phenotypes making lw1 a promising target for visual confirmation of editing in other species. Using sequence information from both Miscanthus and sorghum, orthologs of maize lw1 were identified; a multi-step screening approach was used to select three gRNAs that could target homeologs of lw1. Embryogenic calli of M. sacchariflorus, M. sinensis and M. x giganteus were transformed via particle bombardment (biolistics) or Agrobacterium tumefaciens introducing the Cas9 gene and three gRNAs to edit lw1. Leaves on edited Miscanthus plants displayed the same phenotypes noted in maize. Sanger sequencing confirmed editing; deletions in lw1 ranged from 1 to 26 bp in length, and one deletion (433 bp) encompassed two target sites. Confocal microscopy verified lack of autofluorescence (chlorophyll) in edited leaves/sectors. We developed procedures for gene editing via CRISPR/Cas9 in Miscanthus and, to the best of our knowledge, are the first to do so. This included five genotypes representing three Miscanthus species. Designed gRNAs targeted all copies of lw1 (homeologous copies and their alleles); results also confirmed lw1 made a good editing target in species other than Z. mays. The ability to target specific loci to enable endogenous gene editing presents a new avenue for genetic improvement of this important biomass crop.

09 BIOMASS FUELS↗
DOE OSTI2026

Data for Comparison of Genotyping Assays for Detection of Targeted CRISPR/Cas Mutagenesis in Highly Polyploid Sugarcane

Sugarcane ( Saccharum spp.) is an important biofuel feedstock and a leading source of global table sugar. Saccharum hybrid cultivars are highly polyploid (2n = 100–130), containing large numbers of functionally redundant hom(e)ologs in their genomes. Genome editing with sequence-specific nucleases holds tremendous promise for sugarcane breeding. However, identification of plants with the desired level of co-editing within a pool of primary transformants can be difficult. While DNA sequencing provides direct evidence of targeted mutagenesis, it is cost-prohibitive as a primary screening method in sugarcane and most other methods of identifying mutant lines have not been optimized for use in highly polyploid species. In this study, non-sequencing methods of mutant screening, including capillary electrophoresis (CE), Cas9 RNP assay, and high-resolution melt analysis (HRMA), were compared to assess their potential for CRISPR/Cas9-mediated mutant screening in sugarcane. These assays were used to analyze sugarcane lines containing mutations at one or more of six sgRNA target sites. All three methods distinguished edited lines from wild type, with co-mutation frequencies ranging from 2% to 100%. Cas9 RNP assays were able to identify mutant sugarcane lines with as low as 3.2% co-mutation frequency, and samples could be scored based on undigested band intensity. CE was highlighted as the most comprehensive assay, delivering precise information on both mutagenesis frequency and indel size to a 1 bp resolution across all six targets. This represents an economical and comprehensive alternative to sequencing-based genotyping methods which could be applied in other polyploid species.

Genomics↗
DOE OSTI2024

Thiol-based pathways in the thylakoid lumen and their role in photoprotection

The long-term goal of this research is to elaborate the catalysis of thiol-disulfide transactions in the thylakoid lumen, a compartment required for transducing energy via photophosphorylation. The molecular identity of the redox players catalyzing thiol-disulfide exchanges, their relevant targets of action in vivo and how thiol-disulfide chemistry in general controls photosynthesis are the experimental questions this research aims to address. Through previous work, we documented the requirement of catalyzed disulfide formation and reduction for the biogenesis of two photosynthetic enzymes, namely Photosystem II and the cytochrome b6f complex. We established that dedicated trans-thylakoid pathways operate in these processes by delivering reducing and oxidizing power to cysteine-containing subunits of the photosynthetic complexes, which are localized in the lumen compartment. Here we focused on CCDA, CCS4 and CCS5/HCF164, the components of the disulfide-reducing pathway which maintains the heme binding cysteines of apoforms of plastid cytochrome c in the reduced form prior to covalent heme ligation. We showed that CCS4 and CCS5 operate in functionally redundant pathways for the supply of reducing power and postulate a role for CCS4 in recruiting the source of reductants to CCDA or controlling the CCDA-dependent transduction of reducing equivalents. We also demonstrate the disulfide reducing pathway operates to counter thiol oxidation of the heme-binding cysteines by disulfide forming enzyme LTO1 in the thylakoid lumen.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2024

Comparison of genotyping assays for detection of targeted CRISPR/Cas mutagenesis in highly polyploid sugarcane

Sugarcane (Saccharum spp.) is an important biofuel feedstock and a leading source of global table sugar. Saccharum hybrid cultivars are highly polyploid (2n = 100–130), containing large numbers of functionally redundant hom(e)ologs in their genomes. Genome editing with sequence-specific nucleases holds tremendous promise for sugarcane breeding. However, identification of plants with the desired level of co-editing within a pool of primary transformants can be difficult. While DNA sequencing provides direct evidence of targeted mutagenesis, it is cost-prohibitive as a primary screening method in sugarcane and most other methods of identifying mutant lines have not been optimized for use in highly polyploid species. In this study, non-sequencing methods of mutant screening, including capillary electrophoresis (CE), Cas9 RNP assay, and high-resolution melt analysis (HRMA), were compared to assess their potential for CRISPR/Cas9-mediated mutant screening in sugarcane. These assays were used to analyze sugarcane lines containing mutations at one or more of six sgRNA target sites. All three methods distinguished edited lines from wild type, with co-mutation frequencies ranging from 2% to 100%. Cas9 RNP assays were able to identify mutant sugarcane lines with as low as 3.2% co-mutation frequency, and samples could be scored based on undigested band intensity. CE was highlighted as the most comprehensive assay, delivering precise information on both mutagenesis frequency and indel size to a 1 bp resolution across all six targets. This represents an economical and comprehensive alternative to sequencing-based genotyping methods which could be applied in other polyploid species.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2022

In vivo Functional Characterization of Hydrophilic X2 Modules in the Cellulosomal Scaffolding Protein

As part of free cellulases or scaffolding proteins in cellulosomes, the hydrophilic non-catalytic X2 module is widely distributed in cellulolytic Clostridia or other Firmicutes bacteria. Previous biochemical studies suggest that X2 modules might increase the solubility and substrate binding affinity of X2-bearing proteins. However, their in vivo biological functions remain elusive. Here we employed CRISPR-Cas9 editing to genetically modify X2 modules by deleting the conserved motif (NGNT) from the CipC scaffoldin. Both single and double X2 mutants (X2-N: near the N terminus of CipC; X2-C: near the C terminus of CipC) presented similar stoichiometric compositions in isolated cellulosomes as the wildtype strain (WT). These X2 mutants had an elongated adaptation stage during growth on cellulose compared to cellobiose. Compared to WT, the double mutant ΔX2-NC reduced cellulose degradation by 15% and the amount of released soluble sugars by 63%. Since single X2 mutants did not present such obvious physiological changes as ΔX2-NC, there seems to be a functional redundancy between X2 modules in CipC. The in vivo adhesion assay revealed that ΔX2-NC decreased cell attachment to cellulose by 70% but a weaker effect was also overserved in single X2 mutants. These results highlight the in vivo biological role of X2 in increasing cellulose degradation efficiency by enhancing the binding affinity between cells and cellulose, which provides new perspectives for microbial engineering.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2022

Listeria monocytogenes requires cellular respiration for NAD+ regeneration and pathogenesis

Cellular respiration is essential for multiple bacterial pathogens and a validated antibiotic target. In addition to driving oxidative phosphorylation, bacterial respiration has a variety of ancillary functions that obscure its contribution to pathogenesis. We find here that the intracellular pathogen Listeria monocytogenes encodes two respiratory pathways which are partially functionally redundant and indispensable for pathogenesis. Loss of respiration decreased NAD + regeneration, but this could be specifically reversed by heterologous expression of a water-forming NADH oxidase (NOX). NOX expression fully rescued intracellular growth defects and increased L. monocytogenes loads >1000-fold in a mouse infection model. Consistent with NAD + regeneration maintaining L. monocytogenes viability and enabling immune evasion, a respiration-deficient strain exhibited elevated bacteriolysis within the host cytosol and NOX expression rescued this phenotype. These studies show that NAD + regeneration represents a major role of L. monocytogenes respiration and highlight the nuanced relationship between bacterial metabolism, physiology, and pathogenesis.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2023

Diazotroph diversity across a land-management gradient

Soil diazotrophs are ubiquitous bacteria and archaea responsible for converting atmospheric nitrogen into plant-available ammonium. This process, if properly harnessed, can ameliorate excessive fertilizer inputs in conventional agricultural systems. However, the effect of diazotroph biodiversity on free-living nitrogen fixation (FLNF) is poorly understood. Here, we characterized diazotroph biodiversity across a land-management intensity gradient, determining the interactive effects of land management and diazotroph biodiversity on their ecosystem functioning (FLNF). Subsequently, we evaluated the functional redundancy of diazotroph biodiversity on FLNF in-vitro by performing a biodiversity reduction assay with chloroform fumigation, incubating soils with chloroform for either 24 or 72 hours. We estimated diazotroph biodiversity for both in-situ and chloroform-fumigated samples by preparing and sequencing nifH gene libraries. The raw sequencing data of our nifH library is deposited here.

Kristy, Brandon↗
NASA NTRS1973

The OSO-7 mission

Addition of a gyro and star sensor to the wheel section of OSO-G provided nighttime pointing capability and functional redundancy in correcting an anomaly that put the combined OSO spacecraft and launch vehicle into a flat spin.

Thole, J. M.↗
NASA NTRS1973

Assuring reliability program effectiveness.

An attempt is made to provide simple identification and description of techniques that have proved to be most useful either in developing a new product or in improving reliability of an established product. The first reliability task is obtaining and organizing parts failure rate data. Other tasks are parts screening, tabulation of general failure rates, preventive maintenance, prediction of new product reliability, and statistical demonstration of achieved reliability. Five principal tasks for improving reliability involve the physics of failure research, derating of internal stresses, control of external stresses, functional redundancy, and failure effects control. A final task is the training and motivation of reliability specialist engineers.

Ball, L. W.↗
NASA NTRS1976

A dual-mode generalized likelihood ratio approach to self-reorganizing digital flight control system design

Analytic techniques have been developed for detecting and identifying abrupt changes in dynamic systems. The GLR technique monitors the output of the Kalman filter and searches for the time that the failure occured, thus allowing it to be sensitive to new data and consequently increasing the chances for fast system recovery following detection of a failure. All failure detections are based on functional redundancy. Performance tests of the F-8 aircraft flight control system and computerized modelling of the technique are presented.

Source record↗
NASA NTRS1981

Design and verification of a multiple fault tolerant control system for STS applications using computer simulation

General Dynamics/Convair is under NASA contract to integrate the Centaur upper stage into the space transportation system for future planetary missions. This requires that control of all safety critical functions be two-failure tolerant. The control system developed consists of five asynchronous computers, each contributing at their outputs to a 3-out-of-5 voting plane. Subsystem control is based on an end function redundancy management scheme. Analysis of multiple component failures and worst-case time-phase asynchrony among the computers is performed by a real-time computer simulation. The simulation emulates the hardware and subsystem interfaces, wire by wire, providing assessibility to any component for the insertion of preprogrammed failures. Observability is provided via a graphics system and diagnostic software. The simulation provides an engineering tool where the integrity of control system hardware and imbedded software can be demonstrated.

Szatkowski, G. P.↗
NASA NTRS1985

Failure analysis and performance evaluation of NASA inertial reference unit (DRIRU 2) after 50 months of orbital operation

The first production DRIRU 2 (NASA standard high performance inertial reference unit) system was launched as a subsystem of the Modular Attitude Control System for the Solar Maximum Mission (SMM) spacecraft in February 1980. This hardware was retrieved during the repair of the SMM during Shuttle Flight 41-C in April 1984 and returned to Teledyne Systems Company (manufacturer) for investigation and performance measurements as directed by Goddard Space Flight Center. A failure of one of the three gyro channels occurred approximately 6.5 months after launch. The built in redundancy functioned properly, the DRIRU 2 continued to provide the required attitude control function without performance degradation. Subsequent failure of other attitude control subsystems made the SMM a candidate for the first demonstration of the shuttle in-orbit repair capabilty. The in-orbit DRIRU 2 II failure scenario and the results of the analyses/tests conducted after retrieval are discussed. Comparison of this data with similar data prior to launch demonstates the excellent stability of performance parameters achieveable with DRIRU 2.

Green, K. N.↗
NASA NTRS1986

Environmental Control Life Support for the Space Station

The preliminary design of the nation's Space Station is presently being developed. The Environmental Control and Life Support System (ECLSS), consisting of regenerative and nonregenerative technologies, has progressed through a series of trade studies including evaluation of the closure and distribution within the evolutionary Space Station configuration. The analysis has included the identification of the time-critical functions, redundancy (backup) management, definition of common subsystem interfaces and quantification of technology options for the process equipment. Each technology has been characterized by its physical characteristics of weight, power, volume, heat rejection, etc. Summaries of the trade study findings for the overall ECLSS in terms of physical characteristics and the impact of selected technologies is presented.

Miller, Craig W.↗
NASA NTRS1989

Control of intelligent robots in space

In view of space activities like International Space Station, Man-Tended-Free-Flyer (MTFF) and free flying platforms, the development of intelligent robotic systems is gaining increasing importance. The range of applications that have to be performed by robotic systems in space includes e.g., the execution of experiments in space laboratories, the service and maintenance of satellites and flying platforms, the support of automatic production processes or the assembly of large network structures. Some of these tasks will require the development of bi-armed or of multiple robotic systems including functional redundancy. For the development of robotic systems which are able to perform this variety of tasks a hierarchically structured modular concept of automation is required. This concept is characterized by high flexibility as well as by automatic specialization to the particular sequence of tasks that have to be performed. On the other hand it has to be designed such that the human operator can influence or guide the system on different levels of control supervision, and decision. This leads to requirements for the hardware and software concept which permit a range of application of the robotic systems from telemanipulation to autonomous operation. The realization of this goal requires strong efforts in the development of new methods, software and hardware concepts, and the integration into an automation concept.

Freund, E.↗
NASA NTRS1995

Linear and nonlinear trending and prediction for AVHRR time series data

The variability of AVHRR calibration coefficient in time was analyzed using algorithms of linear and non-linear time series analysis. Specifically we have used the spline trend modeling, autoregressive process analysis, incremental neural network learning algorithm and redundancy functional testing. The analysis performed on available AVHRR data sets revealed that (1) the calibration data have nonlinear dependencies, (2) the calibration data depend strongly on the target temperature, (3) both calibration coefficients and the temperature time series can be modeled, in the first approximation, as autonomous dynamical systems, (4) the high frequency residuals of the analyzed data sets can be best modeled as an autoregressive process of the 10th degree. We have dealt with a nonlinear identification problem and the problem of noise filtering (data smoothing). The system identification and filtering are significant problems for AVHRR data sets. The algorithms outlined in this study can be used for the future EOS missions. Prediction and smoothing algorithms for time series of calibration data provide a functional characterization of the data. Those algorithms can be particularly useful when calibration data are incomplete or sparse.

Smid, J.↗
NASA NTRS1998

Human Exploration of Mars: The Reference Mission of the NASA Mars Exploration Study Team

The Reference Mission was developed over a period of several years and was published in NASA Special Publication 6107 in July 1997. The purpose of the Reference Mission was to provide a workable model for the human exploration of Mars, which is described in enough detail that alternative strategies and implementations can be compared and evaluated. NASA is continuing to develop the Reference Mission and expects to update this report in the near future. It was the purpose of the Reference Mission to develop scenarios based on the needs of scientists and explorers who want to conduct research on Mars; however, more work on the surface-mission aspects of the Reference Mission is required and is getting under way. Some aspects of the Reference Mission that are important for the consideration of the surface mission definition include: (1) a split mission strategy, which arrives at the surface two years before the arrival of the first crew; (2) three missions to the outpost site over a 6-year period; (3) a plant capable of producing rocket propellant for lifting off Mars and caches of water, O, and inert gases for the life-support system; (4) a hybrid physico-chemical/bioregenerative life-support system, which emphasizes the bioregenerative system more in later parts of the scenario; (5) a nuclear reactor power supply, which provides enough power for all operations, including the operation of a bioregenerative life-support system as well as the propellant and consumable plant; (6) capability for at least two people to be outside the habitat each day of the surface stay; (7) telerobotic and human-operated transportation vehicles, including a pressurized rover capable of supporting trips of several days' duration from the habitat; (7) crew stay times of 500 days on the surface, with six-person crews; and (8) multiple functional redundancies to reduce risks to the crews on the surface. New concepts are being sought that would reduce the overall cost for this exploration program and reducing the risks that are indigenous to Mars exploration. Among those areas being explored are alternative space propulsion approaches, solar vs. nuclear power, and reductions in the size of crews.

Connolly, John↗
NASA NTRS2000

Advances in Space Transportation Technology Toward the NASA Goals

NASA has set goals to increase safety by one hundred times while reducing cost tenfold over the next decade. This dramatic increase in safety will come by departing from a past emphasis on cost and performance to a new paradigm of safety and reliability, which, in turn, will drive down cost. To accomplish this, vehicle systems must be inherently reliable, functionally redundant wherever practical and designed to minimize or eliminate catastrophic failure modes. Over the next twenty-five years, NASA has set goals to increase safety ten thousand times while reducing cost one hundred fold. Safety will increase towards today's airline safety and the low price per flight has the potential to enable a 15-fold increase in the size of the current projected space launch market. This level of improvement is comparable to the developments in the 1970s in the computer microprocessor when microchips went from hundreds of thousands to tens of thousands of dollars - hailing the era of the personal computer. In order to achieve such ambitious cost goals, today's partial and fully expendable rockets must be replaced by fully reusable systems. A RLV can eliminate assembly and checkout of the large number of complex interfaces on today's Space Shuttle. Full reusability will eliminate the necessity to throw away expensive hardware and the need for on-going production. Systems in ten years will have to accommodate 50 to 100 missions per year and could be commercially certified for hundreds of flights. In twenty-five years, the number of flights per year could jump to over 1,000, which will require certification for thousands of flights. The large increase in flights per year will demand that current operations and maintenance procedures be revolutionized. Unlike the current shuttle, which requires several thousand personnel over five months to process, the next generation system must be turned around in one week with less than one hundred personnel. In contrast to the rigorous disassembly and inspections required for the Space Shuttle's subsystems, the next generation vehicle's on-board health monitoring systems will could tell the ground crews which systems need replacement before landing. In twenty-five years, vehicles will be re-flown within one with crews numbering less than one hundred. Fully automated ground processing systems must require only a handful of personnel to launch the vehicle. Due to the increased intelligence of on-board systems, only cursory walk-around inspections would be required between flights An assessment of the progress in breakthrough technologies toward these goals by the NASA Advanced Space Transportation Program is presented. These breakthrough technologies include combined rocket and air breathing propulsion, high strength lightweight structures, high temperature materials, vehicle health management, and flight operations.

Lyles, Garry M.↗