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At least 199 records · Page 11

NASA Tech Briefs, April 2006

The topics covered include: 1) Replaceable Sensor System for Bioreactor Monitoring; 2) Unitary Shaft-Angle and Shaft-Speed Sensor Assemblies; 3) Arrays of Nano Tunnel Junctions as Infrared Image Sensors; 4) Catalytic-Metal/PdO(sub x)/SiC Schottky-Diode Gas Sensors; 5) Compact, Precise Inertial Rotation Sensors for Spacecraft; 6) Universal Controller for Spacecraft Mechanisms; 7) The Flostation - an Immersive Cyberspace System; 8) Algorithm for Aligning an Array of Receiving Radio Antennas; 9) Single-Chip T/R Module for 1.2 GHz; 10) Quantum Entanglement Molecular Absorption Spectrum Simulator; 11) FuzzObserver; 12) Internet Distribution of Spacecraft Telemetry Data; 13) Semi-Automated Identification of Rocks in Images; 14) Pattern-Recognition Algorithm for Locking Laser Frequency; 15) Designing Cure Cycles for Matrix/Fiber Composite Parts; 16) Controlling Herds of Cooperative Robots; 17) Modification of a Limbed Robot to Favor Climbing; 18) Vacuum-Assisted, Constant-Force Exercise Device; 19) Production of Tuber-Inducing Factor; 20) Quantum-Dot Laser for Wavelengths of 1.8 to 2.3 micron; 21) Tunable Filter Made From Three Coupled WGM Resonators; and 22) Dynamic Pupil Masking for Phasing Telescope Mirror Segments.

Source record↗

Coupled Microbial-Conversion and Computational-Fluid-Dynamics (CFD) Models for Butanediol Production in Micro-Aerated Reactors

Microbial conversion of substrates to macromolecules has been widely used in the synthesis of value-added products in pharmaceutical and biotechnology industries. These bioreactions are also being investigated in the production of low-value commodities such as biofuels [1]. Gas-liquid mass-transfer and transport-reaction coupling are important challenges when designing and scaling up these reactor systems. Experiments in wellmixed small-scale reactors have enabled characterization of microbial reactivity, while their coupling with macroscale transport remains relatively unexplored. In this work, we use a coupled metabolic-CFD model to study the action of a genetically engineered microbe Zymomonas mobilis [2] on sugars to produce 2,3-Butanediol (BDO). BDO is an important hydrocarbon intermediate that can be catalytically upgraded to several fuels and chemicals [3]. An important aspect to this particular microbial conversion is the need for micro-aerated environments as opposed to traditional aerobic fermentation. Slight variations in oxygen concentration can result in competing reaction pathways that disable BDO production. Hence, gas-liquid mass transfer and transport need to be optimized in large-scale reactors to maximize BDO production, for which CFD is a valuable tool. The aerobic-fermentation CFD model previously developed by the authors [5] for simulating bubble-column and airlift reactors at scale was used in this study. The Reynolds-averaged mass, momentum and energy transport equations for interpenetrating gas and liquid phase are solved in this model along with the transport and interphase mass transfer of oxygen. Our previous work used a phenomenological model for microbial oxygen uptake that neglected microbial growth and other reaction pathways. In this work, a detailed metabolic model enabled prediction of product formation and inhibition pathways. In order to manage computational cost, we used a subcycling technique [6] that takes advantage of the clear separation in transport (~ 200 sec) and reaction (~ 2-3 hours) timescales. The CFD model is first solved to steady state, after which the metabolic model is advanced at every cell in the computational domain using the local oxygen concentration. The CFD model is then run to achieve a new steady state that provides a new oxygen distribution for the metabolic model. This process, where reaction and fluid updates are interleaved together, is iterated until reactants are completely exhausted. This work will examine the performance of different reactor designs such as bubble column and airlift reactors at scale (250-500 m3). Oxygen mass-transfer coefficient and distribution are critically analyzed among reactors, and optimization studies pertaining to aeration is presented. Furthermore, it has been observed in experiments that high BDO production may be achieved by manipulating the aerobic environment over the course of reaction, such that oxygen concentration is high during the growth phase, and very low as sugar is depleted. This characteristic will be addressed by our simulations for which a timedependent scheduling strategy for aeration is presented that maximizes BDO production. [1] Humbird, D., Davis, R., and McMillan, J., Aeration costs in stirred-tank and bubble column bioreactors, Biochemical Engineering Journal, 127, 161—166, 2017 [2] Yang, S., Mohagheghi, A., Franden, M. A., Chou, Y.-C., Chen, X., Dowe, N., Himmel, M. E., and Zhang, M., Metabolic engineering of zymomonas mobilis for 2, 3-butanediol production from lignocellulosic biomass sugars. Biotechnology for biofuels, 9(1):189, 2016 [3] Kim, S. J., Sim, H. J., Kim, J. W., Lee, Y. G., Park, Y. C., and Seo, J. H., Enhanced production of 2,3-butanediol from xylose by combinatorial engineering of xylose metabolic pathway and cofactor regeneration in pyruvate decarboxylase-deficient Saccharomyces cerevisiae. Bioresource Technology, 245:1551–1557, 2017 [4] Weller, H., Tabor, G., Jasak, H. and Fureby, C., A tensorial approach to computational continuum mechanics using object-oriented techniques, Computers in physics, 12, 6, 620--631, 1998

29 ENERGY PLANNING, POLICY, AND ECONOMY↗

Growing B Lymphocytes in a Three-Dimensional Culture System

A three-dimensional (3D) culture system for growing long-lived B lymphocytes has been invented. The capabilities afforded by the system can be expected to expand the range of options for immunological research and related activities, including testing of immunogenicity of vaccine candidates in vitro, generation of human monoclonal antibodies, and immunotherapy. Mature lymphocytes, which are the effectors of adaptive immune responses in vertebrates, are extremely susceptible to apoptotic death, and depend on continuous reception of survival-inducing stimulation (in the forms of cytokines, cell-to-cell contacts, and antigen receptor signaling) from the microenvironment. For this reason, efforts to develop systems for long-term culture of functional, non-transformed and non-activated mature lymphocytes have been unsuccessful until now. The bone-marrow microenvironment supports the growth and differentiation of many hematopoietic lineages, in addition to B-lymphocytes. Primary bone-marrow cell cultures designed to promote the development of specific cell types in vitro are highly desirable experimental systems, amenable to manipulation under controlled conditions. However, the dynamic and complex network of stromal cells and insoluble matrix proteins is disrupted in prior plate- and flask-based culture systems, wherein the microenvironments have a predominantly two-dimensional (2D) character. In 2D bone-marrow cultures, normal B-lymphoid cells become progressively skewed toward precursor B-cell populations that do not retain a normal immunophenotype, and such mature B-lymphocytes as those harvested from the spleen or lymph nodes do not survive beyond several days ex vivo in the absence of mitogenic stimulation. The present 3D culture system is a bioreactor that contains highly porous artificial scaffolding that supports the long-term culture of bone marrow, spleen, and lymph-node samples. In this system, unlike in 2D culture systems, B-cell subpopulations developing within 3D cultures that have been modified to foster lymphopoiesis retain an immunophenotype that closely recapitulates cells in fresh bone marrow harvests. The 3D culture system has been found to be capable of supporting long-lived (8 weeks) populations of B and T lymphocytes from peripheral lymphoid organs, in the absence of activation signals, to an extent not achievable by conventional culture techniques. Interestingly, it has been found that 3D-culture B cells display a phenotype that has characteristics of both B1a and B2 cells. These promising preliminary observations suggest that the 3D culture system could be used with success in the study of peripheral-B-lymphocyte biology and in the development of biotechnological techniques and processes.

Wu, J. H. David↗

Production of ent-kaurene from lignocellulosic hydrolysate in Rhodosporidium toruloides

Abstract Background Rhodosporidium toruloides has emerged as a promising host for the production of bioproducts from lignocellulose, in part due to its ability to grow on lignocellulosic feedstocks, tolerate growth inhibitors, and co-utilize sugars and lignin-derived monomers. Ent -kaurene derivatives have a diverse range of potential applications from therapeutics to novel resin-based materials. Results The Design, Build, Test, and Learn (DBTL) approach was employed to engineer production of the non-native diterpene ent -kaurene in R. toruloides . Following expression of kaurene synthase (KS) in R. toruloides in the first DBTL cycle, a key limitation appeared to be the availability of the diterpene precursor, geranylgeranyl diphosphate (GGPP). Further DBTL cycles were carried out to select an optimal GGPP synthase and to balance its expression with KS, requiring two of the strongest promoters in R. toruloides , ANT (adenine nucleotide translocase) and TEF1 (translational elongation factor 1) to drive expression of the KS from Gibberella fujikuroi and a mutant version of an FPP synthase from Gallus gallus that produces GGPP. Scale-up of cultivation in a 2 L bioreactor using a corn stover hydrolysate resulted in an ent -kaurene titer of 1.4 g/L. Conclusion This study builds upon previous work demonstrating the potential of R. toruloides as a robust and versatile host for the production of both mono- and sesquiterpenes, and is the first demonstration of the production of a non-native diterpene in this organism.

59 BASIC BIOLOGICAL SCIENCES↗

Results of the Alternative Water Processor Test, A Novel Technology for Exploration Wastewater Remediation

Biologically‐based water recovery systems are a regenerative, low energy alternative to physiochemical processes to reclaim water from wastewater. This paper summarizes the results of the Alternative Water Processor (AWP) test conducted over one year. The AWP recovered 90% of water from four crewmembers using (4) membrane aerated bioreactors (MABRs) to remove carbon and nitrogen from an exploration mission wastewater, including urine, hygiene, laundry and humidity condensate. Downstream, a coupled forward and reverse osmosis system removed large organics and inorganic salts from the biological system effluent. The system exceeded the overall objectives of the test by recovering 90% of the influent wastewater processed and a 29% reduction of consumables from the current state of the art water recovery system on the International Space Station (ISS). However the biological system fell short of its test goals, failing to remove 75% and 90% of the influent ammonium and organic carbon, respectively. Despite not meeting its test goals, the BWP demonstrated the feasibility of an attachedgrowth biological system for simultaneous nitrification and denitrification, an innovative, volume and consumable‐saving design that doesn't require toxic pretreatment. This paper will explain the reasons for this and will discuss steps to optimize each subsystem to increase effluent quality from the MABRs and the FOST to advance the system.

Leticia Vega↗

Results of the Alternative Water Processor Test, A Novel Technology for Exploration Wastewater Remediation

Biologically-based water recovery systems are a regenerative, low energy alternative to physiochemical processes to reclaim water from wastewater. This report summarizes the results of the Alternative Water Processor (AWP) Integrated Test, conducted from June 2013 until April 2014. The system was comprised of four (4) membrane aerated bioreactors (MABRs) to remove carbon and nitrogen from an exploration mission wastewater and a coupled forward and reverse osmosis system to remove large organic and inorganic salts from the biological system effluent. The system exceeded the overall objectives of the test by recovering 90% of the influent wastewater processed into a near potable state and a 64% reduction of consumables from the current state of the art water recovery system on the International Space Station (ISS). However, the biological system fell short of its test goals, failing to remove 75% and 90% of the influent ammonium and organic carbon, respectively. Despite not meeting its test goals, the BWP demonstrated the feasibility of an attached-growth biological system for simultaneous nitrification and denitrification, an innovative, volume- and consumable-saving design that does not require toxic pretreatment.

Meyer, Caitlin E.↗

Results of the Alternative Water Processor Test, A Novel Technology for Exploration Wastewater Remediation

Biologically-based water recovery systems are a regenerative, low energy alternative to physiochemical processes to reclaim water from wastewater. This paper summarizes the results of the Alternative Water Processor (AWP) test conducted over one year. The AWP recovered 90% of water from four crewmembers using (4) membrane aerated bioreactors (MABRs) to remove carbon and nitrogen from an exploration mission wastewater, including urine, hygiene, laundry and humidity condensate. Downstream, a coupled forward and reverse osmosis system removed large organics and inorganic salts from the biological system effluent. The system exceeded the overall objectives of the test by recovering 90% of the influent wastewater processed and a 29% reduction of consumables from the current state of the art water recovery system on the International Space Station (ISS). However the biological system fell short of its test goals, failing to remove 75% and 90% of the influent ammonium and organic carbon, respectively. Despite not meeting its test goals, the BWP demonstrated the feasibility of an attached-growth biological system for simultaneous nitrification and denitrification, an innovative, volume and consumable-saving design that doesn't require toxic pretreatment. This paper will explain the reasons for this and will discuss steps to optimize each subsystem to increase effluent quality from the MABRs and the FOST to advance the system.

Leticia Vega↗

A bioreactor system for the nitrogen loop in a Controlled Ecological Life Support System

As space missions become longer in duration, the need to recycle waste into useful compounds rises dramatically. This problem can be addressed by the development of Controlled Ecological Life Support Systems (CELSS) (i.e., Engineered Closed/Controlled Eco-Systems (ECCES)), consisting of human and plant modules. One of the waste streams leaving the human module is urine. In addition to the reclamation of water from urine, recovery of the nitrogen is important because it is an essential nutrient for the plant module. A 3-step biological process for the recycling of nitrogenous waste (urea) is proposed. A packed-bed bioreactor system for this purpose was modeled, and the issues of reaction step segregation, reactor type and volume, support particle size, and pressure drop were addressed. Based on minimization of volume, a bioreactor system consisting of a plug flow immobilized urease reactor, a completely mixed flow immobilized cell reactor to convert ammonia to nitrite, and a plug flow immobilized cell reactor to produce nitrate from nitrite is recommended. It is apparent that this 3-step bioprocess meets the requirements for space applications.

NASA Discipline Number 61-10↗

STS-62 Space Shuttle mission report

The STS-62 Space Shuttle Program Mission Report summarizes the Payload activities as well as the Orbiter, External Tank (ET), Solid Rocket Booster (SRB), Redesigned Solid Rocket Motor (RSRM), and the Space Shuttle main engine (SSHE) systems performance during the sixty-first flight of the Space Shuttle Program and sixteenth flight of the Orbiter vehicle Columbia (OV-102). In addition to the Orbiter, the flight vehicle consisted of an ET designated as ET-62; three SSME's which were designated as serial numbers 2031, 2109, and 2029 in positions 1, 2, and 3, respectively; and two SRB's which were designated BI-064. The RSRM's that were installed in each SRB were designated as 360L036A (lightweight) for the left SRB, and 36OWO36B (welterweight) for the right SRB. This STS-62 Space Shuttle Program Mission Report fulfills the Space Shuttle Program requirement as documented in NSTS 07700, Volume 8, Appendix E. That document requires that each major organizational element supporting the Program report the results of its hardware evaluation and mission performance plus identify all related in-flight anomalies. The primary objectives of the STS-62 mission were to perform the operations of the United States Microgravity Payload-2 (USMP-2) and the Office of Aeronautics and Space Technology-2 (OAST-2) payload. The secondary objectives of this flight were to perform the operations of the Dexterous End Effector (DEE), the Shuttle Solar Backscatter Ultraviolet/A (SSBUV/A), the Limited Duration Space Environment Candidate Material Exposure (LDCE), the Advanced Protein Crystal Growth (APCG), the Physiological Systems Experiments (PSE), the Commercial Protein Crystal Growth (CPCG), the Commercial Generic Bioprocessing Apparatus (CGBA), the Middeck Zero-Gravity Dynamics Experiment (MODE), the Bioreactor Demonstration System (BDS), the Air Force Maui Optical Site Calibration Test (AMOS), and the Auroral Photography Experiment (APE-B).

Fricke, Robert W., Jr.↗

Effect of hydraulic retention time on inorganic nutrient recovery and biodegradable organics removal in a biofilm reactor treating plant biomass leachate

A fixed-film (biofilm) reactor was designed and its performance was determined at various retention times. The goal was to find the optimal retention time for recycling plant nutrients in an advanced life support system, to minimize the size, mass, and volume (hold-up) of a production model. The prototype reactor was tested with aqueous leachate from wheat crop residue at 24, 12, 6, and 3 h hydraulic retention times (HRTs). Biochemical oxygen demand (BOD), nitrates and other plant nutrients, carbohydrates, total phenolics, and microbial counts were monitored to characterize reactor performance. BOD removal decreased significantly from 92% at the 24 h HRT to 73% at 3 h. Removal of phenolics was 62% at the 24 h retention time, but 37% at 3 h. Dissolved oxygen concentrations, nitric acid consumption, and calcium and magnesium removals were also affected by HRT. Carbohydrate removals, carbon dioxide (CO2) productions, denitrification, potassium concentrations, and microbial counts were not affected by different retention times. A 6 h HRT will be used in future studies to determine the suitability of the bioreactor effluent for hydroponic plant production.

NASA Center KSC↗

Scale-dependent enhancement of productivity and stability in xenic Nannochloropsis cultures

Although bacterial amendments can enhance algal productivity and stability, the development of algal-bacteria consortia for commercial-scale utilization is limited. Here, for this work, we used an established high-throughput approach to generate algal-bacteria consortia, and tested consortia performance at spatial scales from microplates to 320 L raceway ponds. We used both lab and field-reared strains of Nannochloropsis oceanica to build consortia. In some experiments, we imposed environmental perturbations to test the ability of bacteria to enhance algal culture stability. In repeated assays at the scale of well plates, flasks, and bioreactors, strong effects of bacterial amendments on N. oceanica were observed. These effects were most dramatic when cultures experienced stressors such as temperature perturbations or removal of CO 2 augmentation. Isolates that were advanced for field testing included species in the genera Algoriphagus, Oceanicaulis, and Marinobacter. When consortia were generated in the field, positive effects of bacterial amendments were not observed. The amended bacteria were outcompeted, and bacterial community composition across treatments converged after the first grow out. These results highlight the complexity of using consortia in open systems, where interactions between the existing bacterial community, inoculated bacteria, and changing environmental conditions are layered upon other differences in scale and cultivation regimes. Moreover, functionally redundant bacteria are likely present in the field. Following this work, we hypothesize that tight interactions (e.g., obligate relationships between partners) will scale more predictably to outdoor systems. We suggest relying on true synthetic ecological approaches in which the relationships between bacteria and algae partners are well understood, or synthetic ecological approaches coupled with high throughput approaches to design and test consortia. We also recommend future work to examine the effect of algae-bacteria inoculation ratios on productivity and stability, track dynamics of partners through time, and manage ponds to retain beneficial symbioses.

59 BASIC BIOLOGICAL SCIENCES↗

Data for Metabolic Engineering of β-Oxidation to Leverage Thioesterases for Production of 2-Heptanone, 2-Nonanone, and 2-Undecanone

Medium-chain length methyl ketones are potential blending fuels due to their cetane numbers and low melting temperatures. Biomanufacturing offers the potential to produce these molecules from renewable resources such as lignocellulosic biomass. In this work, we designed and tested metabolic pathways in Escherichia coli to specifically produce 2-heptanone, 2-nonanone and 2-undecanone. We achieved substantial production of each ketone by introducing chain-length specific acyl-ACP thioesterases, blocking the β-oxidation cycle at an advantageous reaction, and introducing active β-ketoacyl-CoA thioesterases. Using a bioprospecting approach, we identified 15 homologs of E. coli β-ketoacyl-CoA thioesterase (FadM) and evaluated the in vivo activity of each against various chain length substrates. The FadM variant from Providencia sneebia produced the most 2-heptanone, 2-nonanone, and 2-undecanone, suggesting it has the highest activity on the corresponding β-ketoacyl-CoA substrates. We tested enzyme variants, including acyl-CoA oxidases, thiolases, and bi-functional 3-hydroxyacyl-CoA dehydratases to maximize conversion of fatty acids to β-keto acyl-CoAs for 2-heptanone, 2-nonanone, and 2-undecanone production. In order to address the issue of product loss during fermentation, we applied a 20% (v/v) dodecane layer in the bioreactor and built an external water cooling condenser connecting to the bioreactor heat-transferring condenser coupling to the condenser. Using these modifications, we were able to generate up to 4.4 g/L total medium-chain length methyl ketones.

Metabolic Engineering↗

Metabolic engineering of β-oxidation to leverage thioesterases for production of 2-heptanone, 2-nonanone and 2-undecanone

Medium-chain length methyl ketones are potential blending fuels due to their cetane numbers and low melting temperatures. Biomanufacturing offers the potential to produce these molecules from renewable resources such as lignocellulosic biomass. Here, we designed and tested metabolic pathways in Escherichia coli to specifically produce 2-heptanone, 2-nonanone and 2-undecanone. We achieved substantial production of each ketone by introducing chain-length specific acyl-ACP thioesterases, blocking the β-oxidation cycle at an advantageous reaction, and introducing active β-ketoacyl-CoA thioesterases. Using a bioprospecting approach, we identified fifteen homologs of E. coli β-ketoacyl-CoA thioesterase (FadM) and evaluated the in vivo activity of each against various chain length substrates. The FadM variant from Providencia sneebia produced the most 2-heptanone, 2-nonanone, and 2-undecanone, suggesting it has the highest activity on the corresponding β-ketoacyl-CoA substrates. We further tested enzyme variants, including acyl-CoA oxidases, thiolases, and bi-functional 3-hydroxyacyl-CoA dehydratases to maximize conversion of fatty acids to β-keto acyl-CoAs for 2-heptanone, 2-nonanone, and 2-undecanone production. In order to address the issue of product loss during fermentation, we applied a 20% (v/v) dodecane layer in the bioreactor and built an external water cooling condenser connecting to the bioreactor heat-transferring condenser coupling to the condenser. Finally, using these modifications, we were able to generate up to 4.4 g/L total medium-chain length methyl ketones.

2-Heptanone↗

Shear and mixing effects on cells in agitated microcarrier tissue culture reactors

Tissue cells are known to be sensitive to mechanical stresses imposed on them by agitation in bioreactors. The amount of agitation provided in a microcarrier or suspension bioreactor should be only enough to provide effective homogeneity. Three distinct flow regions can be identified in the reactor: bulk turbulent flow, bulk laminar flow and boundary-layer flows. Possible mechanisms of cell damage are examined by analyzing the motion of microcarriers or free cells relative to the surrounding fluid, to each other and to moving or stationary solid surfaces. The primary mechanisms of cell damage appear to result from: (1) direct interaction between microcarriers and turbulent eddies; (2) collisions between microcarriers in turbulent flow; and (3) collisions against the impeller or other stationary surfaces. If the smallest eddies of turbulent flow are of the same size as the microcarrier beads, they may cause high shear stresses on the cells. Eddies the size of the average interbead spacing may cause bead-bead collisions which damage cells. The severity of the collisions increases when the eddies are also of the same size as the beads. Impeller collisions occur when beads cannot avoid the impeller leading edge as it advances through the liquid. The implications of the results of this analysis on the design and operation of tissue culture reactors are discussed.

Cherry, Robert S.↗

Novel Concept for LSS Based on Advanced Microalgal Biotechnologies

One of the key issues for successful human space exploration is biomedical life support in hostile space and planetary environments that otherwise cannot sustain life. Bioregenerative life support systems (LSS) are one of the options for atmospheric regeneration. To date, no bioregenerative LSS has shown capability for 100% air regeneration. Nor have these LSS been robust enough to simultaneously provide a regenerable complete food source. In contrast to microalgae, traditional plant approaches, e.g. wheat and lettuce, are lacking essential amino acids, vitamins, and micronutrients. Moreover, the rate of photosynthesis by microalgae significantly exceeds that of high plants. Nevertheless, the employment of microalgae in LSS technology was restricted, until recently, due to high water demands. Also the per person requirement of a 40L volume of microalgae in a photobioreactor, to provide daily O2 production, made an algae-based approach less attractive. By employing a vertically stacked membrane bioreactor, coupled with a solar tracker and photon-delivery system, a lightweight air revitalization system for space based applications, with minimal water requirements, can be developed. Our preliminary estimations suggest that a membrane bioreactor, 8m3 in volume, comprised of 80m2 (twenty 2m x 2m membranes, each spaced 10 cm apart), and a total of 70L of water could produce 2.7 kg of dried microalgal biomass that would supply the energy and essential amino acid requirements, as well as producing sufficient O2 for the daily needs of a 15 member crew. Research on the biochemical content of edible blue-green alga Spirulina (Arthrospira) platensis shows a wide spectrum of stable Spirulina mutants with an enhanced content of amino acids, -carotene, and phycobilliprotein c-phycocyanin. Feeding animals suffering from radiation-induced lesions, c-phycocyanin, extracted from strain 27G, led to a correction in the decrement of dehydrogenase activity and energy-rich phosphate levels, as well as improved antioxidant defense and pyruvate levels, compared to untreated animals. Experimental anemia in rats was corrected by feeding Spirulina platensis strains 198B and 27G, (with an enhanced content of methionine, phycobiliproteins and carotenoids). Spirulina was recently shown by Ananyev et al, 2005, to be an oxygenic organism with the highest level of photosystem II activity (O2 production). We propose therefore to develop a design for membrane-based photoreactors for Lunar and Mars exploration habitat LSS, for the cultivation of genetically modified strains of Spirulina to scrub CO2 and supply astronauts with O2, protein, vitamins, and immunostimulators.

Brown, I.↗

Skeletal muscle satellite cells cultured in simulated microgravity

Satellite cells are postnatal myoblasts responsible for providing additional nuclei to growing or regenerating muscle cells. Satellite cells retain the capacity to proliferate and differentiate in vitro and therefore provide a useful model to study postnatal muscle development. Most culture systems used to study postnatal muscle development are limited by the two-dimensional (2-D) confines of the culture dish. Limiting proliferation and differentiation of satellite cells in 2-D could potentially limit cell-cell contacts important for developing the level of organization in skeletal muscle obtained in vivo. Culturing satellite cells on microcarrier beads suspended in the High-Aspect-Ratio-Vessel (HARV) designed by NASA provides a low shear, three-dimensional (3-D) environment to study muscle development. Primary cultures established from anterior tibialis muscles of growing rats (approximately 200 gm) were used for all studies and were composed of greater than 75 % satellite cells. Different inoculation densities did not affect the proliferative potential of satellite cells in the HARV. Plating efficiency, proliferation, and glucose utilization were compared between 2-D flat culture and 3-D HARV culture. Plating efficiency (cells attached - cells plated x 100) was similar between the two culture systems. Proliferation was reduced in HARV cultures and this reduction was apparent for both satellite cells and non-satellite cells. Furthermore, reduction in proliferation within the HARV could not be attributed to reduced substrate availability since glucose levels in media from HARV and 2-D cell culture were similar. Morphologically, microcarrier beads within the HARVS were joined together by cells into three-dimensional aggregates composed of greater than 10 beads/aggregate. Aggregation of beads did not occur in the absence of cells. Myotubes were often seen on individual beads or spanning the surface of two beads. In summary, proliferation and differentiation of satellite cells on microcarrier beads within the HARV bioreactor results in a three dimensional level of organization that could provide a more suitable model to study postnatal muscle development.

Molnar, Greg↗

Closure of Regenerative Life Support Systems: Results of the Lunar-Mars Life Support Test Project

Future long duration human exploration missions away from Earth will require closed-loop regenerative life support systems to reduce launch mass, reduce dependency on resupply and increase the level of mission self sufficiency. Such systems may be based on the integration of biological and physiocochemical processes to produce potable water, breathable atmosphere and nutritious food from metabolic and other mission wastes. Over the period 1995 to 1998 a series of ground-based tests were conducted at the National Aeronautics and Space Administration, Johnson Space Center, to evaluate the performance of advanced closed-loop life support technologies with real human metabolic and hygiene loads. Named the Lunar-Mars Life Support Test Project (LMLSTP), four integrated human tests were conducted with increasing duration, complexity and closure. The first test, LMLSTP Phase I, was designed to demonstrate the ability of higher plants to revitalize cabin atmosphere. A single crew member spent 15 days within an atmospherically closed chamber containing 11.2 square meters of actively growing wheat. Atmospheric carbon dioxide and oxygen levels were maintained by control of the rate of photosynthesis through manipulation of light intensity or the availability of carbon dioxide and included integrated physicochemical systems. During the second and third tests, LMLSTP Phases II & IIa, four crew members spent 30 days and 60 days, respectively, in a larger sealed chamber. Advanced physicochemical life support hardware was used to regenerate the atmosphere and produce potable water from wastewater. Air revitalization was accomplished by using a molecular sieve and a Sabatier processor for carbon dioxide absorption and reduction, respectively, with oxygen generation performed by water hydrolysis. Production of potable water from wastewater included urine treatment (vapor compression distillation), primary treatment (ultrafiltration/reverse osmosis and multi-filtration) and post processing. For the Phase II test the water recovery rate ranged from 95 to 98%, depending on the processor. LMLSTP Phase III, the fourth test of the series, had a duration of 91 days and included four crew members. The test demonstrated an integration of physicochemical and biological technologies for air revitalization, water recovery and waste processing. Wheat supplemented the physicochemical air revitalization systems by providing approximately 25% of the oxygen required for the 4-person crew. The water recovery system included immobilized cell and trickling filter bioreactors for primary water treatment, reverse osmosis and air evaporation systems for secondary water treatment, followed by post processing. The 8 day initial supply of water was recycled through the chamber and crew 10 times over the course of the test. Grain from the wheat together with fresh lettuce from a small growth chamber within the crew chamber provided supplementation to the stored food system, but at a level less than 5% of the crew s caloric requirement. An incinerator was used to demonstrate mineralization of the crew s solid waste, with the combustion products (mainly carbon dioxide) returned to the wheat for conversion to oxygen.

Barta, Daniel↗

Flow field measurements in the cell culture unit

The cell culture unit (CCU) is being designed to support cell growth for long-duration life science experiments on the International Space Station (ISS). The CCU is a perfused loop system that provides a fluid environment for controlled cell growth experiments within cell specimen chambers (CSCs), and is intended to accommodate diverse cell specimen types. Many of the functional requirements depend on the fluid flow field within the CSC (e.g., feeding and gas management). A design goal of the CCU is to match, within experimental limits, all environmental conditions, other than the effects of gravity on the cells, whether the hardware is in microgravity ( micro g), normal Earth gravity, or up to 2g on the ISS centrifuge. In order to achieve this goal, two steps are being taken. The first step is to characterize the environmental conditions of current 1g cell biology experiments being performed in laboratories using ground-based hardware. The second step is to ensure that the design of the CCU allows the fluid flow conditions found in 1g to be replicated from microgravity up to 2g. The techniques that are being used to take these steps include flow visualization, particle image velocimetry (PIV), and computational fluid dynamics (CFD). Flow visualization using the injection of dye has been used to gain a global perspective of the characteristics of the CSC flow field. To characterize laboratory cell culture conditions, PIV is being used to determine the flow field parameters of cell suspension cultures grown in Erlenmeyer flasks on orbital shakers. These measured parameters will be compared to PIV measurements in the CSCs to ensure that the flow field that cells encounter in CSCs is within the bounds determined for typical laboratory experiments. Using CFD, a detailed simulation is being developed to predict the flow field within the CSC for a wide variety of flow conditions, including microgravity environments. Results from all these measurements and analyses of the CSC flow environment are presented and discussed. The final configuration of the CSC employs magnetic stir bars with angled paddles to achieve the necessary flow requirements within the CSC.

Cell Culture↗