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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 19 records
All sourcesNASA NTRSDOE OSTI
DOE OSTI2021

Non-destructive quantification of anaerobic gut fungi and methanogens in co-culture reveals increased fungal growth rate and changes in metabolic flux relative to mono-culture

Abstract Background Quantification of individual species in microbial co-cultures and consortia is critical to understanding and designing communities with prescribed functions. However, it is difficult to physically separate species or measure species-specific attributes in most multi-species systems. Anaerobic gut fungi (AGF) (Neocallimastigomycetes) are native to the rumen of large herbivores, where they exist as minority members among a wealth of prokaryotes. AGF have significant biotechnological potential owing to their diverse repertoire of potent lignocellulose-degrading carbohydrate-active enzymes (CAZymes), which indirectly bolsters activity of other rumen microbes through metabolic exchange. While decades of literature suggest that polysaccharide degradation and AGF growth are accelerated in co-culture with prokaryotes, particularly methanogens, methods have not been available to measure concentrations of individual species in co-culture. New methods to disentangle the contributions of AGF and rumen prokaryotes are sorely needed to calculate AGF growth rates and metabolic fluxes to prove this hypothesis and understand its causality for predictable co-culture design. Results We present a simple, microplate-based method to measure AGF and methanogen concentrations in co-culture based on fluorescence and absorbance spectroscopies. Using samples of < 2% of the co-culture volume, we demonstrate significant increases in AGF growth rate and xylan and glucose degradation rates in co-culture with methanogens relative to mono-culture. Further, we calculate significant differences in AGF metabolic fluxes in co-culture relative to mono-culture, namely increased flux through the energy-generating hydrogenosome organelle. While calculated fluxes highlight uncertainties in AGF primary metabolism that preclude definitive explanations for this shift, our method will enable steady-state fluxomic experiments to probe AGF metabolism in greater detail. Conclusions The method we present to measure AGF and methanogen concentrations enables direct growth measurements and calculation of metabolic fluxes in co-culture. These metrics are critical to develop a quantitative understanding of interwoven rumen metabolism, as well as the impact of co-culture on polysaccharide degradation and metabolite production. The framework presented here can inspire new methods to probe systems beyond AGF and methanogens. Simple modifications to the method will likely extend its utility to co-cultures with more than two organisms or those grown on solid substrates to facilitate the design and deployment of microbial communities for bioproduction and beyond.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2022

DISCOVR strain pipeline screening – Part II: Winter and summer season areal productivities and biomass compositional shifts in climate-simulation photobioreactor cultures

Assessing the seasonal biomass productivity and compositional shift dynamics under simulated outdoor culture conditions of the top 22 algae strains selected during Tier I flask screening is an important step in the further prioritization of strains with regard to outdoor pond cultivation. These top 22 strains were subjected to Tier II testing in the PNNL Laboratory Environmental Algae Pond Simulator (LEAPS) photobioreactors, simulating light and temperature conditions of 20 cm deep outdoor ponds during the Arizona winter and summer season. All strains were grown in two consecutive nutrient-replete batch culture experiments at their particular optimal medium salinity to quantify their respective seasonal linear-phase areal biomass productivities. To determine biomass compositional shifts in response to nutrient-depletion, the LEAPS cultures were allowed to enter a 9-day nutrient depletion phase at the end of the second batch run. The following strains were evaluated in winter-season climate-simulated cultures and are listed in the order from highest (7.9 g m -2 day -1 ) to lowest (2.3 g m -2 day -1 ) areal N-replete biomass productivity: Monoraphidium minutum 26B-AM, Tetraselmis striata LANL1001, Chlorella vulgaris LRB AZ-1201, Micractinium reisseri NREL14-F2, Monoraphidium sp. MONOR1, Chlorella vulgaris NREL4-C12, Scenedesmus obliquus UTEX393, Scenedesmus acutus LRB-AP-0401, Nannochloropsis oceanica CCAP849/10, and Stichococcus minutus CCALA727. The following strains were evaluated in summer-season climate-simulated cultures and are listed in the order from highest (31.8 g m -2 day -1 ) to lowest (14.2 g m -2 day -1 ) areal N-replete biomass productivity: Picochlorum renovo NREL39-A8, Scenedesmus obliquus UTEX393, Porphyridium cruentum CCMP675, Picochlorum celeri TG2-WT-CSM/EMRE, Chlorella sorokiniana DOE1116, Stichococcus minor CCMP819, Picochlorum oklahomensis CCMP2329, Chlorella sorokiniana DOE1412 (UTEXB3016), Scenedesmus rubescens NREL46B-D3, Picochlorum soloecismus DOE101, Tetraselmis striata LANL1001, Scenedesmus obliquus DOE 0152.z, and Agmenellum quadruplicatum UTEX2268. All LEAPS cultures experienced a significant reduction in areal biomass productivity in response to nutrient-depletion, from 7-16% in the winter season simulation and up to 1-60% in the summer season simulation. Finally, for 10 of the strains tested, the carbohydrate content more than doubled upon nutrient depletion, and for 9 strains, the lipid content increased by over 50% of the initial content.

09 BIOMASS FUELS↗
NASA NTRS2000

Differentiation of mammalian skeletal muscle cells cultured on microcarrier beads in a rotating cell culture system

The growth and repair of adult skeletal muscle are due in part to activation of muscle precursor cells, commonly known as satellite cells or myoblasts. These cells are responsive to a variety of environmental cues, including mechanical stimuli. The overall goal of the research is to examine the role of mechanical signalling mechanisms in muscle growth and plasticity through utilisation of cell culture systems where other potential signalling pathways (i.e. chemical and electrical stimuli) are controlled. To explore the effects of decreased mechanical loading on muscle differentiation, mammalian myoblasts are cultured in a bioreactor (rotating cell culture system), a model that has been utilised to simulate microgravity. C2C12 murine myoblasts are cultured on microcarrier beads in a bioreactor and followed throughout differentiation as they form a network of multinucleated myotubes. In comparison with three-dimensional control cultures that consist of myoblasts cultured on microcarrier beads in teflon bags, myoblasts cultured in the bioreactor exhibit an attenuation in differentiation. This is demonstrated by reduced immunohistochemical staining for myogenin and alpha-actinin. Western analysis shows a decrease, in bioreactor cultures compared with control cultures, in levels of the contractile proteins myosin (47% decrease, p < 0.01) and tropomyosin (63% decrease, p < 0.01). Hydrodynamic measurements indicate that the decrease in differentiation may be due, at least in part, to fluid stresses acting on the myotubes. In addition, constraints on aggregate size imposed by the action of fluid forces in the bioreactor affect differentiation. These results may have implications for muscle growth and repair during spaceflight.

Non-NASA Center↗
NASA NTRS1998

Use of an adaptable cell culture kit for performing lymphocyte and monocyte cell cultures in microgravity

The results of experiments performed in recent years on board facilities such as the Space Shuttle/Spacelab have demonstrated that many cell systems, ranging from simple bacteria to mammalian cells, are sensitive to the microgravity environment, suggesting gravity affects fundamental cellular processes. However, performing well-controlled experiments aboard spacecraft offers unique challenges to the cell biologist. Although systems such as the European 'Biorack' provide generic experiment facilities including an incubator, on-board 1-g reference centrifuge, and contained area for manipulations, the experimenter must still establish a system for performing cell culture experiments that is compatible with the constraints of spaceflight. Two different cell culture kits developed by the French Space Agency, CNES, were recently used to perform a series of experiments during four flights of the 'Biorack' facility aboard the Space Shuttle. The first unit, Generic Cell Activation Kit 1 (GCAK-1), contains six separate culture units per cassette, each consisting of a culture chamber, activator chamber, filtration system (permitting separation of cells from supernatant in-flight), injection port, and supernatant collection chamber. The second unit (GCAK-2) also contains six separate culture units, including a culture, activator, and fixation chambers. Both hardware units permit relatively complex cell culture manipulations without extensive use of spacecraft resources (crew time, volume, mass, power), or the need for excessive safety measures. Possible operations include stimulation of cultures with activators, separation of cells from supernatant, fixation/lysis, manipulation of radiolabelled reagents, and medium exchange. Investigations performed aboard the Space Shuttle in six different experiments used Jurkat, purified T-cells or U937 cells, the results of which are reported separately. We report here the behaviour of Jurkat and U937 cells in the GCAK hardware in ground-based investigations simulating the conditions expected in the flight experiment. Several parameters including cell concentration, time between cell loading and activation, and storage temperature on cell survival were examined to characterise cell response and optimise the experiments to be flown aboard the Space Shuttle. Results indicate that the objectives of the experiments could be met with delays up to 5 days between cell loading into the hardware and initial in flight experiment activation, without the need for medium exchange. Experiment hardware of this kind, which is adaptable to a wide range of cell types and can be easily interfaced to different spacecraft facilities, offers the possibility for a wide range of experimenters successfully and easily to utilise future flight opportunities.

short duration↗
DOE OSTI2024

Species-specific ribosomal RNA-FISH identifies interspecies cellular-material exchange, active-cell population dynamics and cellular localization of translation machinery in clostridial cultures and co-cultures

ABSTRACT The development of synthetic microbial consortia in recent years has revealed that complex interspecies interactions, notably the exchange of cytoplasmic material, exist even among organisms that originate from different ecological niches. Although morphogenetic characteristics, viable RNA and protein dyes, and fluorescent reporter proteins have played an essential role in exploring such interactions, we hypothesized that ribosomal RNA-fluorescence in situ hybridization (rRNA-FISH) could be adapted and applied to further investigate interactions in synthetic or semisynthetic consortia. Despite its maturity, several challenges exist in using rRNA-FISH as a tool to quantify individual species population dynamics and interspecies interactions using high-throughput instrumentation such as flow cytometry. In this work, we resolve such challenges and apply rRNA-FISH to double and triple co-cultures of Clostridium acetobutylicum, Clostridium ljungdahlii, and Clostridium kluyveri . In pursuing our goal to capture each organism’s population dynamics, we demonstrate dynamic rRNA, and thus ribosome, exchange between the three species leading to the formation of hybrid cells. We also characterize the localization patterns of the translation machinery in the three species, identifying distinct, dynamic localization patterns among them. Our data also support the use of rRNA-FISH to assess the culture’s health and expansion potential, and, here again, our data find surprising differences among the three species examined. Taken together, our study argues for rRNA-FISH as a valuable and accessible tool for quantitative exploration of interspecies interactions, especially in organisms which cannot be genetically engineered or in consortia where selective pressures to maintain recombinant species cannot be used. IMPORTANCE Though dyes and fluorescent reporter proteins have played an essential role in identifying microbial species in co-cultures, we hypothesized that ribosomal RNA-fluorescence in situ hybridization (rRNA-FISH) could be adapted and applied to quantitatively probe complex interactions between organisms in synthetic consortia. Despite its maturity, several challenges existed before rRNA-FISH could be used to study Clostridium co-cultures of interest. First, species-specific probes for Clostridium acetobutylicum and Clostridium ljungdahlii had not been developed. Second, “state-of-the-art” labeling protocols were tedious and often resulted in sample loss. Third, it was unclear if FISH was compatible with existing fluorescent reporter proteins. We resolved these key challenges and applied the technique to co-cultures of C. acetobutylicum , C. ljungdahlii , and Clostridium kluyveri . We demonstrate that rRNA-FISH is capable of identifying rRNA/ribosome exchange between the three organisms and characterized rRNA localization patterns in each. In combination with flow cytometry, rRNA-FISH can capture sub-population dynamics in co-cultures.

Hill, John D.↗
DOE OSTI2022

13 C-Isotope-Assisted Assessment of Metabolic Quenching During Sample Collection from Suspension Cell Cultures

Metabolomics and fluxomics are core approaches to directly profile and interrogate cellular metabolism in response to various genetic or environmental perturbations. In order to accurately measure the abundance and isotope enrichment of intracellular metabolites, cell culture samples must be rapidly harvested and cold-quenched to preserve the in vivo metabolic state of the cells at the time of sample collection. When dealing with suspension cultures, this process is complicated by the need to separate the liquid culture media from cellular biomass prior to metabolite extraction. Here, we examine the efficacy of several commonly used metabolic quenching methods, using the model cyanobacterium Synechocystis sp. PCC 6803 as an example. Multiple 13 C-labeled compounds, including 13 C-bicarbonate, 13 C-glucose and 13 C-glutamine, were used as tracers during the sample collection and cold-quenching process to assess the extent of metabolic turnover after cells were harvested from culture flasks. We show that the combination of rapid filtration followed by 100% cold (–80°C) methanol quenching exhibits the highest quenching efficiency, while mixing cell samples with a partially frozen 30% methanol slurry (–24°C) followed by centrifugation is slightly less effective at quenching metabolism but enables less laborious sample processing. By contrast, rapidly mixing the cells with a saline ice slurry (~0°C) is less effective as indicated by high isotope-labeling rates after sample harvest, while mixing the cells with 60% cold methanol (–65°C) prior to centrifugation causes significant metabolite loss. Furthermore, this study demonstrates a rigorous, quantitative, and broadly applicable method for assessing the metabolic quenching efficacy of protocols used for sample collection in metabolomics and fluxomics studies.

59 BASIC BIOLOGICAL SCIENCES↗
NASA NTRS2004

Space Station Biological Research Project (SSBRP) Cell Culture Unit (CCU) and incubator for International Space Station (ISS) cell culture experiments

The CCU and Incubator are habitats under development by SSBRP for gravitational biology research on ISS. They will accommodate multiple specimen types and reside in either Habitat Holding Racks, or the Centrifuge Rotor, which provides selectable gravity levels of up to 2 g. The CCU can support multiple Cell Specimen Chambers, CSCs (18, 9 or 6 CSCs; 3, 10 or 30 mL in volume, respectively). CSCs are temperature controlled from 4-39 degrees C, with heat shock to 45 degrees C. CCU provides automated nutrient supply, magnetic stirring, pH/O2 monitoring, gas supply, specimen lighting, and video microscopy. Sixty sample containers holding up to 2 mL each, stored at 4-39 degrees C, are available for automated cell sampling, subculture, and injection of additives and fixatives. CSCs, sample containers, and fresh/spent media bags are crew-replaceable for long-term experiments. The Incubator provides a 4-45 degrees C controlled environment for life science experiments or storage of experimental reagents. Specimen containers and experiment unique equipment are experimenter-provided. The Specimen Chamber exchanges air with ISS cabin and has 18.8 liters of usable volume that can accommodate six trays and the following instrumentation: five relocatable thermometers, two 60 W power outlets, four analog ports, and one each relative humidity sensor, video port, ethernet port and digital input/output port.

NASA Center ARC↗
NASA NTRS1973

Man made culture interpretation and culture revision of small scale maps

The author has identified the following significant results. A major interstate highway added to the Wilmington, Del., quadrangle by another revision method was determined by the ERTS-1 data to have been misplotted from its true position. In addition to the experiment, where ERTS-1 data is available and small-scale revision is in progress, the ERTS-1 data is routinely being used to make additions and deletions of hydrographic features.

Kosco, W. J.↗
NASA NTRSDate not supplied

Safety Culture at the World’s Premier Multi-User Spaceport

NASA’s Agency-wide Safety Culture is implemented at the Kennedy Space Center (KSC) using a unique strategy due an unparalleled approach in making human spaceflight history. Kennedy Space Center, the world’s premier multi-user spaceport, enables U.S. government and commercial space access, while providing the world a resource to allow the exploration of and the ability to work in space. A consistently healthy safety culture at KSC is imperative for mission success: desired achievements, protection of space flight hardware, and ultimately, the preservation of human life requires the support of a healthy safety culture. Emphasis on the NASA Agency-wide development of Safety Culture began with the conception of the NASA Agency Safety Culture Working Group. After the devastating loss of life and mission of the Space Shuttle Colombia, a broken safety culture was identified as an organizational cause by the Columbia Accident Investigation Board Report. Thus, the Agency Safety Culture Working Group was developed in 2009 to assess the status of the Agency’s Safety Culture, while addressing safety culture concerns at the NASA Center-level. A Five-factor model was developed to serve as the guiding principles for Safety Culture: 1) Reporting Culture, 2) Just Culture, 3) Flexible Culture, 4) Learning Culture, and 5) Engaged Culture. These five factors are included in the NASA Safety Culture logo, which was intentionally designed as a DNA double helix to prompt the permeation of safety into day-to-day work. KSC specifically implements the NASA Agency-wide Safety Culture principles in a tailored approach that is relevant to the diversity of work being performed. An emphasis is placed on the implementation to include safety at home, not exclusively at work. This emphasis is a KSC-specific element that has been intentionally added to promote a closed loop Safety Culture. To advertise the safety culture, various safety and health events are held throughout the calendar year, providing innovative speakers and engaging activities, while also promoting a wide range of curated safety initiatives. Development and continuous improvement of the KSC safety tracking database, allows for advanced tracking-to-closure, along with providing data sets used to identify areas of emphasis. Other safety initiatives rely solely on employee participation, such as the photo challenges; participants are encouraged to identify and capture themselves, coworkers, or family members participating in safe or healthy activities to share with others within the Center and at Agency levels. Fabrication of exclusive videos and graphics are utilized to advertise and inform employee of safety initiatives, upcoming safety events, and general dispersion of safety information. In addition, an anonymous Agency-wide Safety Culture Survey is advertised, administered, and analyzed at Kennedy Space Center, with the purpose of receiving basic feedback on Safety Culture perceptions to help prevent future incidents from occurring. Through these briefly identified means, and many other forms of employee engagement, Kennedy Space Center aims to maintain safety in the forefront, while creating an environment where everyone trusts that safety is a priority.

Larrin E. Moody↗
NASA NTRS2001

Optimized suspension culture: the rotating-wall vessel

Suspension culture remains a popular modality, which manipulates mechanical culture conditions to maintain the specialized features of cultured cells. The rotating-wall vessel is a suspension culture vessel optimized to produce laminar flow and minimize the mechanical stresses on cell aggregates in culture. This review summarizes the engineering principles, which allow optimal suspension culture conditions to be established, and the boundary conditions, which limit this process. We suggest that to minimize mechanical damage and optimize differentiation of cultured cells, suspension culture should be performed in a solid-body rotation Couette-flow, zero-headspace culture vessel such as the rotating-wall vessel. This provides fluid dynamic operating principles characterized by 1) solid body rotation about a horizontal axis, characterized by colocalization of cells and aggregates of different sedimentation rates, optimally reduced fluid shear and turbulence, and three-dimensional spatial freedom; and 2) oxygenation by diffusion. Optimization of suspension culture is achieved by applying three tradeoffs. First, terminal velocity should be minimized by choosing microcarrier beads and culture media as close in density as possible. Next, rotation in the rotating-wall vessel induces both Coriolis and centrifugal forces, directly dependent on terminal velocity and minimized as terminal velocity is minimized. Last, mass transport of nutrients to a cell in suspension culture depends on both terminal velocity and diffusion of nutrients. In the transduction of mechanical culture conditions into cellular effects, several lines of evidence support a role for multiple molecular mechanisms. These include effects of shear stress, changes in cell cycle and cell death pathways, and upstream regulation of secondary messengers such as protein kinase C. The discipline of suspension culture needs a systematic analysis of the relationship between mechanical culture conditions and biological effects, emphasizing cellular processes important for the industrial production of biological pharmaceuticals and devices.

Non-NASA Center↗
NASA NTRS1997

Neonatal rat heart cells cultured in simulated microgravity

In vitro characteristics of cardiac cells cultured in simulated microgravity are reported. Tissue culture methods performed at unit gravity constrain cells to propagate, differentiate, and interact in a two-dimensional (2D) plane. Neonatal rat cardiac cells in 2D culture organize predominantly as bundles of cardiomyocytes with the intervening areas filled by nonmyocyte cell types. Such cardiac cell cultures respond predictably to the addition of exogenous compounds, and in many ways they represent an excellent in vitro model system. The gravity-induced 2D organization of the cells, however, does not accurately reflect the distribution of cells in the intact tissue. We have begun characterizations of a three-dimensional (3D) culturing system designed to mimic microgravity. The NASA-designed High-Aspect Ratio Vessel (HARV) bioreactors provide a low shear environment that allows cells to be cultured in static suspension. HARV-3D cultures were prepared on microcarrier beads and compared to control-2D cultures using a combination of microscopic and biochemical techniques. Both systems were uniformly inoculated and medium exchanged at standard intervals. Cells in control cultures adhered to the polystyrene surface of the tissue culture dishes and exhibited typical 2D organization. Cells cultured in HARVs adhered to microcarrier beads, the beads aggregated into defined clusters containing 8 to 15 beads per cluster, and the clusters exhibited distinct 3D layers: myocytes and fibroblasts appeared attached to the surfaces of beads and were overlaid by an outer cell type. In addition, cultures prepared in HARVs using alternative support matrices also displayed morphological formations not seen in control cultures. Generally, the cells prepared in HARV and control cultures were similar; however, the dramatic alterations in 3D organization recommend the HARV as an ideal vessel for the generation of tissuelike organization of cardiac cells in vitro.

Non-NASA Center↗
NASA NTRS1994

Neonatal rat heart cells cultured in simulated microgravity

In vitro characteristics of cardiac cells cultured in simulated microgravity are reported. Tissue culture methods performed at unit gravity constrain cells to propagate, differentiate, and interact in a two dimensional (2D) plane. Neonatal rat cardiac cells in 2D culture organize predominantly as bundles of cardiomyocytes with the intervening areas filled by non-myocyte cell types. Such cardiac cell cultures respond predictably to the addition of exogenous compounds, and in many ways they represent an excellent in vitro model system. The gravity-induced 2D organization of the cells, however, does not accurately reflect the distribution of cells in the intact tissue. We have begun characterizations of a three-dimensional (3D) culturing system designed to mimic microgravity. The NASA designed High-Aspect-Ratio-Vessel (HARV) bioreactors provide a low shear environment which allows cells to be cultured in static suspension. HARV-3D cultures were prepared on microcarrier beads and compared to control-2D cultures using a combination of microscopic and biochemical techniques. Both systems were uniformly inoculated and medium exchanged at standard intervals. Cells in control cultures adhered to the polystyrene surface of the tissue culture dishes and exhibited typical 2D organization. Cells in cultured in HARV's adhered to microcarrier beads, the beads aggregated into defined clusters containing 8 to 15 beads per cluster, and the clusters exhibited distinct 3D layers: myocytes and fibroblasts appeared attached to the surfaces of beads and were overlaid by an outer cell type. In addition, cultures prepared in HARV's using alternative support matrices also displayed morphological formations not seen in control cultures. Generally, the cells prepared in HARV and control cultures were similar, however, the dramatic alterations in 3D organization recommend the HARV as an ideal vessel for the generation of tissue-like organizations of cardiac cells in simulated microgravity.

Akins, Robert E.↗
DOE OSTI2020

Functional and transcriptional characterization of complex neuronal co-cultures

Brain-on-a-chip systems are designed to simulate brain activity using traditional in vitro cell culture on an engineered platform. It is a noninvasive tool to screen new drugs, evaluate toxicants, and elucidate disease mechanisms. However, successful recapitulation of brain function on these systems is dependent on the complexity of the cell culture. In this study, we increased cellular complexity of traditional (simple) neuronal cultures by co-culturing with astrocytes and oligodendrocyte precursor cells (complex culture). We evaluated and compared neuronal activity (e.g., network formation and maturation), cellular composition in long-term culture, and the transcriptome of the two cultures. Compared to simple cultures, neurons from complex co-cultures exhibited earlier synapse and network development and maturation, which was supported by localized synaptophysin expression, up-regulation of genes involved in mature neuronal processes, and synchronized neural network activity. Also, mature oligodendrocytes and reactive astrocytes were only detected in complex cultures upon transcriptomic analysis of age-matched cultures. Functionally, the GABA antagonist bicuculline had a greater influence on bursting activity in complex versus simple cultures. Collectively, the cellular complexity of brain-on-a-chip systems intrinsically develops cell type-specific phenotypes relevant to the brain while accelerating the maturation of neuronal networks, important features underdeveloped in traditional cultures.

59 BASIC BIOLOGICAL SCIENCES↗