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DOE OSTI2021

Developments in algal processing

Macroalgae and microalgae, including cyanobacteria, are primarily photosynthetic organisms grown for the production of renewable biomass for foods, feedstocks, and high-value specialty chemicals. While there are differences in cultivating and harvesting macro- and microalgae due to scale, many of the same downstream techniques can be used for processing algal biomass. This chapter will outline various developments in processing technologies used for the treatment of algal biomass with discussion of scalability, cost, time, and efficacy. Topics will cover methods for harvesting and dewatering algae, drying algal biomass, biomass disruption, and nutrient recovery, including case studies with lessons learned. Here, the final section presents biomass applications and product considerations. While there is no universally-adopted approach for processing algal biomass, these studies provide the foundation for making informed decisions, considering the unique properties of the algae and the integrity of the desired end products.

09 BIOMASS FUELS↗

DOE OSTI2023

Superior Capacitive Energy-Storage Performance in Pb-Free Relaxors with a Simple Chemical Composition

Chemical design of lead-free relaxors with simultaneously high energy density (W rec ) and high efficiency (η) for capacitive energy-storage has been a big challenge for advanced electronic systems. The current situation indicates that realizing such superior energy-storage properties requires highly complex chemical components. Herein, we demonstrate that, via local structure design, an ultrahigh W rec of 10.1 J/cm 3 , concurrent with a high η of 90%, as well as excellent thermal and frequency stabilities can be achieved in a relaxor with a very simple chemical composition. By introducing 6s 2 lone pair stereochemical active Bi into the classical BaTiO 3 ferroelectric to generate a mismatch between A- and B-site polar displacements, a relaxor state with strong local polar fluctuations can be formed. Through advanced atomic-resolution displacement mapping and 3D reconstructing the nanoscale structure from neutron/X-ray total scattering, it is revealed that the localized Bi enhances the polar length largely at several perovskite unit cells and disrupts the long-range coherent Ti polar displacements, resulting in a slush-like structure with extremely small size polar clusters and strong local polar fluctuations. This favorable relaxor state exhibits substantially enhanced polarization, and minimized hysteresis at a high breakdown strength. This work offers a feasible avenue to chemically design new relaxors with a simple composition for high-performance capacitive energy-storage.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗

NASA NTRS2017

Development of Storage Methods for Saccharomyces Strains to be Utilized for In situ Nutrient Production in Long-Duration Space Missions

Long-duration space missions will benefit from closed-loop life support technologies that minimize mass, volume, and power as well as decrease reliance on Earth-based resupply. A system for In situ production of essential vitamins and nutrients can address the documented problem of degradation of stored food and supplements. Research has shown that the edible yeast Saccharomyces cerevisiae can be used as an on-demand system for the production of various compounds that are beneficial to human health. A critical objective in the development of this approach for long-duration space missions is the effective storage of the selected microorganisms. This research investigates the effects of different storage methods on survival rates of the non-sporulating probiotic S. boulardii, and S. cerevisiae spores and vegetative cells. Dehydration has been shown to increase long-term yeast viability, which also allows increased shelf-life and reduction in mass and volume. The process of dehydration causes detrimental effects on vegetative cells, including oxidative damage and membrane disruption. To maximize cell viability, various dehydration methods are tested here, including lyophilization (freeze-drying), air drying, and dehydration by vacuum. As a potential solution to damage caused by lyophilization, the efficacy of various cryoprotectants was tested. Furthermore, in an attempt to maintain higher survival rates, the effect of temperature during long-term storage was investigated. Data show spores of the wild-type strain to be more resilient to dehydration-related stressors than vegetative cells of either strain, and maintain high viability rates even after one year at room temperature. In the event that engineering the organism to produce targeted nutrient compounds interferes with effective sporulation of S. cerevisiae, a more robust method for improving vegetative cell storage is being sought. Therefore, anhydrobiotic engineering of S. cerevisiae and S. boulardii is being conducted

nutrient↗

NASA NTRS2017

Development of Storage Methods for Saccharomyces Strains to be Utilized for In Situ Nutrient Production in Long-Duration Space Missions

life support↗

NASA NTRSDate not supplied

Ground-based Characterization of Plant Water Management (PWM) Hydroponic Root Modules for Spaceflight

Hydroponic crop production in space is crucial for long-term space travel but faces numerous challenges – one of which is providing sufficient dissolved oxygen (DO) in nutrient solution. In microgravity environments, surface tension is the primary force acting on liquids, causing water to form into suspended spherical droplets. This can suffocate plants as the liquid clings onto plant roots and the lack of aeration deprives the plant of oxygen needed for growth. To overcome these challenges, plant water management (PWM) systems explore options of growing plants in space autonomously and passively through capillary forces. From 2018-2023, there have been 6 PWM experiments conducted on the ISS. In past experiments conducted in microgravity, bubbles formed in test cells have disrupted fluid dynamics and may adversely affect plant growth. The accumulation of bubbles may lead to inconsistent nutrient delivery, break prime in tubing, and suspend plant roots in air, leading to plant stress and eventually death. Current research efforts focus on the oxygenation capabilities of the PWM system along with a comprehensive sensor array that will improve nutrient and DO monitoring capabilities.

L Wang↗

NASA NTRS2003

Plasma membrane disruption: repair, prevention, adaptation

Many metazoan cells inhabit mechanically stressful environments and, consequently, their plasma membranes are frequently disrupted. Survival requires that the cell rapidly repair or reseal the disruption. Rapid resealing is an active and complex structural modification that employs endomembrane as its primary building block, and cytoskeletal and membrane fusion proteins as its catalysts. Endomembrane is delivered to the damaged plasma membrane through exocytosis, a ubiquitous Ca2+-triggered response to disruption. Tissue and cell level architecture prevent disruptions from occurring, either by shielding cells from damaging levels of force, or, when this is not possible, by promoting safe force transmission through the plasma membrane via protein-based cables and linkages. Prevention of disruption also can be a dynamic cell or tissue level adaptation triggered when a damaging level of mechanical stress is imposed. Disease results from failure of either the preventive or resealing mechanisms.

NASA Discipline Cell Biology↗

DOE OSTI2022

Disruption of the tagF Orthologue in the epa Locus Variable Region of Enterococcus faecalis Causes Cell Surface Changes and Suppresses an eep -Dependent Lysozyme Resistance Phenotype

The disease-producing capacity of the opportunistic pathogen Enterococcus faecalis is enhanced by the ability of the bacterium to evade killing by antimicrobial agents. Survival of E. faecalis in the presence of the human antimicrobial enzyme lysozyme is mediated in part by the site 2 metalloprotease Eep; however, a complete model of enterococcal lysozyme resistance has not been elucidated. To better understand the molecular basis for lysozyme resistance in E. faecalis, we analyzed Δeep suppressor mutants that acquire resistance to lysozyme through mutation of the gene OG1RF_11713, a predicted teichoic acid biosynthesis-encoding gene located within the variable region of the enterococcal polysaccharide antigen (epa) locus. Sequence comparisons revealed that OG1RF_11713 is most similar to the cytidine-5'-diphosphate (CDP)-glycerol:poly-(glycerolphosphate)glycerophosphotransferase TagF from Staphylococcus epidermidis. Inactivation of OG1RF_11713 in both the wild-type and Δeep genetic backgrounds was sufficient to increase the resistance of E. faecalis OG1RF to lysozyme. Minimal amounts of N-acetylgalactosamine were detectable in cell wall carbohydrate extracts of OG1RF_11713 deletion mutants, and this was associated with a reduction in negative cell surface charge. Targeted disruption of OG1RF_11713 was also associated with increased susceptibility to the antibiotic polymyxin B and membrane-targeting detergents and decreased susceptibility to the lantibiotic nisin. Furthermore, this work implicates OG1RF_11713 as a major determinant of cell envelope integrity and provides further validation that lysozyme resistance is intrinsically linked to the modification of enterococcal cell wall polysaccharides.

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2022

Ablating Lgr5-expressing prostatic stromal cells activates the ERK-mediated mechanosensory signaling and disrupts prostate tissue homeostasis

The heterogeneity of the prostate stromal cells is widely appreciated but the functional implication remains incompletely understood. Using genetic lineage tracing and light-sheet imaging, we show that some fibroblast cells near the junction of the mouse proximal prostatic ducts and prostatic urethra highly express Lgr5. Genetic ablation of these anatomically restricted stromal cells, but not nonselective ablation of prostatic stromal cells, rapidly induces prostate epithelial turnover and dedifferentiation that are reversed following spontaneous restoration of the Lgr5 + stromal cells. RNA-Seq analysis implies that ablating the Lgr5 + stromal cells activates a mechanosensory response. We show that ablating the Lgr5 + stromal cells impairs the control of prostatic ductal outlet, increases prostate tissue stiffness, and activates the mitogen-activated protein kinase (MAPK). Suppressing MAPK overrides the increased epithelial proliferation. This study demonstrates that the Lgr5 + stromal cells regulate tissue homeostasis in a long-distance manner by maintaining anatomic integrity and implies that the cells near the transitional regions between organs likely control organ homeostasis by sustaining a balanced mechanoforce.

59 BASIC BIOLOGICAL SCIENCES↗

NASA NTRS2003

Elevated levels of N-lauroylethanolamine, an endogenous constituent of desiccated seeds, disrupt normal root development in Arabidopsis thaliana seedlings

N-Acylethanolamines (NAEs) are prevalent in desiccated seeds of various plant species, and their levels decline substantially during seed imbibition and germination. Here, seeds of Arabidopsis thaliana (L.) Heynh. were germinated in, and seedlings maintained on, micromolar concentrations of N-lauroylethanolamine (NAE 12:0). NAE 12:0 inhibited root elongation, increased radial swelling of root tips, and reduced root hair numbers in a highly selective and concentration-dependent manner. These effects were reversible when seedlings were transferred to NAE-free medium. Older seedlings (14 days old) acclimated to exogenous NAE by increased formation of lateral roots, and generally, these lateral roots did not exhibit the severe symptoms observed in primary roots. Cells of NAE-treated primary roots were swollen and irregular in shape, and in many cases showed evidence, at the light- and electron-microscope levels, of improper cell wall formation. Microtubule arrangement was disrupted in severely distorted cells close to the root tip, and endoplasmic reticulum (ER)-localized green fluorescent protein (mGFP5-ER) was more abundant, aggregated and distributed differently in NAE-treated root cells, suggesting disruption of proper cell division, endomembrane organization and vesicle trafficking. These results suggest that NAE 12:0 likely influences normal cell expansion in roots by interfering with intracellular membrane trafficking to and/or from the cell surface. The rapid metabolism of NAEs during seed imbibition/germination may be a mechanism to remove this endogenous class of lipid mediators to allow for synchronized membrane reorganization associated with cell expansion.

Non-NASA Center↗

DOE OSTI2022

BefA, a microbiota-secreted membrane disrupter, disseminates to the pancreas and increases β cell mass

Microbiome dysbiosis is a feature of diabetes, but how microbial products influence insulin production is poorly understood. Here, we report the mechanism of BefA, a microbiome-derived protein that increases proliferation of insulin-producing β cells during development in gnotobiotic zebrafish and mice. BefA disseminates systemically by multiple anatomic routes to act directly on pancreatic islets. We detail BefA’s atomic structure, containing a lipid-binding SYLF domain, and demonstrate that it permeabilizes synthetic liposomes and bacterial membranes. A BefA mutant impaired in membrane disruption fails to expand β cells, whereas the pore-forming host defense protein, Reg3, stimulates β cell proliferation. Our work demonstrates that membrane permeabilization by microbiome-derived and host defense proteins is necessary and sufficient for β cell expansion during pancreas development, potentially connecting microbiome composition with diabetes risk.

59 BASIC BIOLOGICAL SCIENCES↗

NASA NTRS2003

The endomembrane requirement for cell surface repair

The capacity to reseal a plasma membrane disruption rapidly is required for cell survival in many physiological environments. Intracellular membrane (endomembrane) is thought to play a central role in the rapid resealing response. We here directly compare the resealing response of a cell that lacks endomembrane, the red blood cell, with that of several nucleated cells possessing an abundant endomembrane compartment. RBC membrane disruptions inflicted by a mode-locked Ti:sapphire laser, even those initially smaller than hemoglobin, failed to reseal rapidly. By contrast, much larger laser-induced disruptions made in sea urchin eggs, fibroblasts, and neurons exhibited rapid, Ca(2+)-dependent resealing. We conclude that rapid resealing is not mediated by simple physiochemical mechanisms; endomembrane is required.

NASA Discipline Cell Biology↗

DOE OSTI2023

Methods of transfection using sonoporation

The present invention relates to methods of transforming a cell by use of an acoustic transducer. In particular embodiments, the radiofrequency signal to the transducer can be tuned to provide optimal membrane disruption of the cell; and an agent, such as a nucleic acid, can then be delivered to the cell.

Ruffing, Anne↗

NASA NTRS2008

Computational Analyses in Support of Sub-scale Diffuser Testing for the A-3 Facility: Unsteady Analyses and Risk Assessment - Part 2

Simulation technology can play an important role in rocket engine test facility design and development by assessing risks, providing analysis of dynamic pressure and thermal loads, identifying failure modes and predicting anomalous behavior of critical systems. This is especially true for facilities such as the proposed A-3 facility at NASA SSC because of a challenging operating envelope linked to variable throttle conditions at relatively low chamber pressures. Design Support of the feasibility of operating conditions and procedures is critical in such cases due to the possibility of startup/shutdown transients, moving shock structures, unsteady shock-boundary layer interactions and engine and diffuser unstart modes that can result in catastrophic failure. Analyses of such systems is difficult due to resolution requirements needed to accurately capture moving shock structures, shock-boundary layer interactions, two-phase flow regimes and engine unstart modes. In a companion paper, we will demonstrate with the use of CFD, steady analyses advanced capability to evaluate supersonic diffuser and steam ejector performance in the sub-scale A-3 facility. In this paper we will address transient issues with the operation of the facility especially at startup and shutdown, and assess risks related to afterburning due to the interaction of a fuel rich plume with oxygen that is a by-product of the steam ejectors. The primary areas that will be addressed in this paper are: (1) analyses of unstart modes due to flow transients especially during startup/ignition, (2) engine safety during the shutdown process (3) interaction of steam ejectors with the primary plume i.e. flow transients as well as probability of afterburning. In this abstract we discuss unsteady analyses of the engine shutdown process. However, the final paper will include analyses of a staged startup, drawdown of the engine test cell pressure, and risk assessment of potential afterburning in the facility. Unsteady simulations have been carried out to study the engine shutdown process in the facility and understand the physics behind the interactions between the steam ejectors, the test cell and the supersonic diffuser. As a first approximation, to understand the dominant unsteady mechanisms in the engine test cell and the supersonic diffuser, the turning duct in the facility was removed. As the engine loses power a rarefaction wave travels downstream that disrupts the shock cell structure in the supersonic diffuser. Flow from the test cell is seen to expand into the supersonic diffuser section and re-pressurizes the area around the nozzle along with a upstream traveling compression wave that emanates from near the first stage ejectors. Flow from the first stage ejector expands to the center of the duct and a new shock train is formed between the first and second stage ejectors. Both stage ejectors keep the facility pressurized and prevent any large amplitude pressure fluctuations from affecting the engine nozzle. The resultant pressure loads the nozzle experiences in the shutdown process are small.

Ahuja, Vineet↗

DOE OSTI2021

SIRT3 mediates mitofusin 2 ubiquitination and degradation to suppress ischemia reperfusion-induced acute kidney injury

Ischemia reperfusion-induced acute kidney injury (IR-induced AKI) is a life-threatening disease with many complications. Mitofusin 2 (Mfn2) ubiquitination is related to AKI. But the underlying molecular mechanisms remain unknown. This study aimed to probe the mechanism of Mfn2 ubiquitination in IR-induced AKI development. In IR-induced AKI mouse models, orbital blood and urine were collected for assessing kidney function. The kidney injury, ultrastructure of mitochondria, and histopathology in mice were evaluated after injection of G5, an ubiquitination inhibitor. Oxygen glucose deprivation/reoxygenation (OGD/R) models were established in HK-2 cells, and the mitochondria were extracted. Cell viability, apoptosis, oxidative stress, inflammatory reaction, mitochondrial membrane potential, and ATP production were measured. Mfn2 ubiquitination in mouse and cell models was evaluated. si-SIRT3 and pcDNA3.1-SIRT3 were transfected into cell models. Consequently, kidney function in mice was impaired by IR-induced AKI. Mfn2 ubiquitination and degradation promoted IR-induced AKI. OGD/R induced renal tubular epithelial cell injury and disrupted mitochondrial dynamics and functions through promoting Mfn2 ubiquitination. SIRT3 knockdown led to Mfn2 ubiquitination by binding to UBC; while its overexpression alleviated tubular epithelial cell injury. Briefly, SIRT3 mediates Mfn2 ubiquitination to relieve IR-induced AKI. This investigation may offer new insights for the treatment of IR-induced AKI injury.

60 APPLIED LIFE SCIENCES↗

NASA NTRS2022

2019 ARIA Proposal Final Report Public Abstract: What Happens to Life in an Ocean World Plume?

The NASA Cassini mission to Saturn discovered persistent jets of water being ejected into space from a subsurface ocean on the small moon Enceladus and evidence that this ocean is habitable for life. The ice resulting from these plumes forms the E Ring of Saturn. Missions are under study to fly through these plumes and collect and analyze samples to look for evidence of life. To support design and instrument selection for these missions, there is a critical need to understand what happens to microbial life adapted to a liquid environment when suddenly ejected into the vacuum of space. We experimentally determined the structural changes in microbial cells in liquid medium when ejected into vacuum. We first developed a vacuum chamber fitted with a feedthrough and nozzle allowing fluid to be injected into vacuum. The nozzle dispersed the fluid into micron-sized droplets. Fluid containing a population of E. Coli microbes was injected into the chamber and droplets were collected on microscope slides and small cups for imaging with a Scanning Electron Microscope (SEM). Epifluorescence microscopy, using a lipid stain, and SEM imaging were used to evaluate the cellular integrity of E. coli after injection through the pressurized nozzle into vacuum. The experimental samples showed a 94% decrease in visible intact E. coli cells as compared with control samples but showed a fluorescent residue in the shape of the sublimated droplets that indicated the presence of lipids. E. coli was selected for testing although other cell types could vary physiologically which could affect their response to a vacuum environment. More testing is needed to determine whether other types of microbial cells can survive the plume environment. However, these results suggest that lipids may be directly detectable evidence of life in icy world plumes, but organisms are unlikely to survive due to disruption of their cell walls.

C. R. Stoker↗

DOE OSTI2025

Toward Quality Control in Perovskite Solar Cell Fabrication: Spot-Like Processing Defects Disrupt Charge Transport Layers and Promote Ag Metal Electrode Intrusion

Metal halide perovskite (MHP) photovoltaics provide high efficiencies with less stringent processing requirements than traditional photovoltaic materials. However, processing related defects must be suppressed as they can lead to decreases in initial device efficiency and potentially compromise long-term device operation. In this work we investigate morphological defects in MHP devices using luminescence imaging followed by in-depth structural and composition analysis using electron microscopy-based methods. We identify several different classes of spot-like processing-related defects and observe that a single device structure may contain multiple types of these defects. The presence of these defects in devices with different layer structures and absorber chemistries makes them relevant to the perovskite photovoltaic community as a whole. The defects are associated with voids in the perovskite layer, inclusions (glass, migrated Ag, dust), thickness variations, hole transport layer disruption with anomalous crystal growth, and electron transport layer disruptions that could allow Ag intrusion and lead to local shunts. As perovskite photovoltaic technology matures, mitigation of such defects is critical to improving not only initial performance but also the long-term stability required for industrial applications.

14 SOLAR ENERGY↗

NASA NTRS1996

Laser microsurgery of higher plant cell walls permits patch-clamp access

Plasma membranes of guard cells in epidermal peels of Vicia faba and Commelina communis can be made accessible to a patch-clamp pipet by removing a small portion (1-3 micrometers in diameter) of the guard cell wall using a microbeam of ultraviolet light generated by a nitrogen laser. Using this laser microsurgical technique, we have measured channel activity across plasma membranes of V. faba guard cells in both cell-attached and isolated patch configurations. Measurements made in the inside-out patch configuration revealed two distinct K(+)-selective channels. Major advantages of the laser microsurgical technique include the avoidance of enzymatic protoplast isolation, the ability to study cell types that have been difficult to isolate as protoplasts or for which enzymatic isolation protocols result in protoplasts not amenable to patch-clamp studies, the maintenance of positional information in single-channel measurements, reduced disruption of cell-wall-mediated signaling pathways, and the ability to investigate intercellular signaling through studies of cells remaining situated within tissue.

Non-NASA Center↗

DOE OSTI2024

Reactive oxygen species control protein degradation at the mitochondrial import gate

While reactive oxygen species (ROS) have long been known to drive aging and neurodegeneration, their persistent depletion below basal levels also disrupts organismal function. Cells counteract loss of basal ROS via the reductive stress response, but the identity and biochemical activity of ROS sensed by this pathway remain unknown. Here, we show that the central enzyme of the reductive stress response, the E3 ligase Cullin 2-FEM1 homolog B (CUL2 FEM1B ), speciﬁcally acts at mitochondrial TOM complexes, where it senses ROS produced by complex III of the electron transport chain (ETC). ROS depletion during times of low ETC activity triggers the localized degradation of CUL2 FEM1B substrates, which sustains mitochondrial import and ensures the biogenesis of the rate-limiting ETC complex IV. As complex III yields most ROS when the ETC outpaces metabolic demands or oxygen availability, basal ROS are sentinels of mitochondrial activity that help cells adjust their ETC to changing environments, as required for cell differentiation and survival.

FEM1B↗