Transport phenomena and crystal growth of the GeSe-Xenon system for different gravitational conditions

Previous chemical vapor transport experiments of the GeSe-GeI₄ system performed under reduced gravity conditions /1/ yielded crystals of considerably improved surface and bulk morphology. In addition, the mass transport rates observed in microgravity environment were significantly greater than predicted. A quantitative thermodynamic analysis of the solid-gas phase reactions of the GeSe-GeI₄ system revealed the multi-component, multi-reaction nature of the vapor phase /2/. Continued transport studies on ground of the GeSe-GeI₄ system in the presence of inert gases provided experimental evidence for the existence of a boundary layer /2/ and its thickness dependence on GeI₄ pressure in closed tube systems. Systematic transport rate measurements for different orientations of the density gradient relative to the gravity vector demonstrated the effects of ampoule inclination on mass flux /3/. Based on a computational model for simultaneous chemical vapor transport, sublimation, and Stefan flow /3/, the excellent agreement of predicted with ground-based experimental mass transport rates over wide pressure ranges /3/ confirmed the validity of the model and the discrepancy between observed and expected transport rates of the GeSe-GeI₄ system in microgravity.     

Possible use of a 3-D clinostat to analyze plant growth processes under microgravity conditions.

A three-dimensional (3-D) clinostat equipped with two rotation axes placed at right angles was constructed, and various growth processes of higher plants grown on this clinostat were compared with ground controls, with plants grown on the conventional horizontal clinostat, and with those under real microgravity in space. On the 3-D clinostat, cress roots developed a normal root cap and the statocytes showed the typical polar organization except a random distribution of statoliths. The structural features of clinostatted statocytes were fundamentally similar to those observed under real microgravity. The graviresponse of cress roots grown on the 3-D clinostat was the same as the control roots. On the 3-D clinostat, shoots and roots exhibited a spontaneous curvature as well as an altered growth direction. Such an automorphogenesis was sometimes exaggerated when plants were subjected to the horizontal rotation, whereas the curvature was suppressed on the vertical rotation. These discrepancies in curvature between the 3-D clinostat and the conventional ones appear to be brought about by the centrifugal force produced. Thus, the 3-D clinostat was proven as a useful device to simulate microgravity.     

Automorphogenesis and gravitropism of plant seedlings grown under microgravity conditions.

Plant seedlings exhibit automorphogenesis on clinostats. The occurrence of automorphogenesis was confirmed under microgravity in Space Shuttle STS-95 flight. Rice coleoptiles showed an inclination toward the caryopsis in the basal region and a spontaneous curvature in the same adaxial direction in the elongating region both on a three-dimensional (3-D) clinostat and in space. Both rice roots and Arabidopsis hypocotyls also showed a similar morphology in space and on the 3-D clinostat. In rice coleoptiles, the mechanisms inducing such an automorphic curvature were studied. The faster-expanding convex side of rice coleoptiles showed a higher extensibility of the cell wall than the opposite side. Also, in the convex side, the cell wall thickness was smaller, the turnover of the matrix polysaccharides was more active, and the microtubules oriented more transversely than the concave side, and these differences appear to be causes of the curvature. When rice coleoptiles grown on the 3-D clinostat were placed horizontally, the gravitropic curvature was delayed as compared with control coleoptiles. In clinostatted coleoptiles, the corresponding suppression of the amyloplast development was also observed. Similar results were obtained in Arabidopsis hypocotyls. Thus, the induction of automorphogenesis and a concomitant decrease in graviresponsiveness occurred in plant shoots grown under microgravity conditions.     

Kinetics of amyloplast movement in cress root statocytes under different gravitational loads.

In order to investigate the movement of a statolith complex along the longitudinal axis of root cap statocytes under different mass accelerations, a series of experiments with Lepidium sativum L. in an automatically operating centrifuge during the Bion-11 satellite flight and on a centrifuge-clinostat have been performed. During spaceflight, roots were grown for 24 h under root-tip-directed centrifugal 1-g acceleration, then exposed to microgravity for 6, 12 and 24 min and chemically fixed. During the first 6 min of microgravity, the statoliths moved towards the cell center with a mean velocity of 0.31 +/- 0.04 micrometers/min, which decreased to 0.12 +/- 0.01 micrometers/min within subsequent 12-24 min period. The mean relative position of the statolith complex in respect to the distal cell wall (% of total cell length) increased from 24.0 +/- 0.5% in 1 g-grown roots to 38.8 +/- 0.8% in roots exposed for 24 min to microgravity, but remained smaller than in roots grown continuously in microgravity (48.0 +/- 0.7%). The properties of the statolith movement away from the distal pole of the statocyte were studied in roots grown for 24 h vertically under 1 g and then placed for 6 min on a fast rotating clinostat (50 rpm) or 180 degrees inverted. After 2 min of both treatments, the mean relative position of the statoliths increased by about 10% versus its initial position. Later on, the proximal displacement of amyloplasts slowed down under simulated weightlessness, while it proceeded at a constant velocity under 1 g inversion. In roots grown on the clinostat and then exposed to 1 g in the longitudinal direction, amyloplast sedimentation away from the central region of statocyte was similar at the beginning of distal and proximal 6-min 1-g stimulation. However, at the end of this period statolith displacement was more pronounced in proximal direction as compared to distal. It is proposed that statolith position in the statocyte of a vertical root is controlled by the force of gravity, however, the intracellular forces, first of all those generated by the network of the cytoskeleton, are manifested when an usual orientation of the organ is changed or the statocytes are exposed to microgravity and clinorotation.     

Influence of nonuniform magnetic fields on orientation of plant seedlings in microgravity conditions.

Experiments on the spatial behavior of the flax (Linum usitatissimum, L.) seedlings in a nonuniform magnetic field were conducted on the orbital space stations "Salut" and "Mir". This field can displace sensory organelles (statoliths) inside receptor cells and such displacement should cause a physiological reaction of the plant-tropistic curvature. Experiments were conducted in the custom-built "Magnetogravistat" facility, where seeds were germinated and grown for 3-4 days in a magnetic field with the dynamic factor grad (H2/2) approximately equal to 10(7) Oe2/cm, then fixed on orbit and returned to Earth for analysis. It was found, that 93% of the seedlings were oriented in the field consistently with curvature in response to displacement of statoliths along the field gradient by ponderomotive magnetic forces, while control seedlings grew in the direction of the initial orientation of the seed. This suggests, that gravity receptors of plants recognized magnetic forces on statoliths as gravity, and that gravity stimulus can be substituted for plants by a force of a different physical nature.     

Development of a plant growth unit for growing plants over a long-term life cycle under microgravity conditions.

To study the effect of the space environment on plant growth including the reproductive growth and genetic aberration for a long-term plant life cycle, we have initiated development of a new type of facility for growing plants under microgravity conditions. The facility is constructed with subsystems for controlling environmental elements. In this paper, the concept of the facility design is outlined. Subsystems controlling air temperature, humidity, CO2 concentration, light and air circulation around plants and delivering recycled water and nutrients to roots are the major concerns. Plant experiments for developing the facility and future plant experiments with the completed facility are also overviewed. We intend to install this facility in the Japan Experiment Facility (JEM) boarded on the International Space Station.     

Leaf senescence under various gravity conditions: relevance to the dynamics of plant hormones.

Effects of simulated microgravity and hypergravity on the senescence of oat leaf segments excised from the primary leaves of 8-d-old green seedlings were studied using a 3-dimensional (D) clinostat as a simulator of weightlessness and a centrifuge, respectively. During the incubation with water under 1-g conditions at 25 degrees C in the dark, the loss of chlorophyll of the segments was found dramatically immediately after leaf excision, and leaf color completely turned to yellow after 3-d to 4-d incubation. In this case kinetin (10 micromolar) was effective in retarding senescence. The application of simulated microgravity conditions on a 3-D clinostat enhanced chlorophyll loss in the presence or absence of kinetin. The loss of chlorophyll was also enhanced by hypergravity conditions (ca. 8 to 16 g), but the effect was smaller than that of simulated microgravity conditions on the clinostat. Jasmonates (JAs) and abscisic acid (ABA) promoted senescence under simulated microgravity conditions on the clinostat as well as under 1-g conditions. After 2-d incubation with water or 5-d incubation with kinetin, the endogenous levels of JAs and ABA of the segments kept under simulated microgravity conditions on the clinostat remained higher than those kept under 1-g conditions. These findings suggest that physiological processes of leaf senescence and the dynamics of endogenous plant hormone levels are substantially affected by gravity.     

Spaceflight hardware for conducting plant growth experiments in space: the early years 1960-2000.

The best strategy for supporting long-duration space missions is believed to be bioregenerative life support systems (BLSS). An integral part of a BLSS is a chamber supporting the growth of higher plants that would provide food, water, and atmosphere regeneration for the human crew. Such a chamber will have to be a complete plant growth system, capable of providing lighting, water, and nutrients to plants in microgravity. Other capabilities include temperature, humidity, and atmospheric gas composition controls. Many spaceflight experiments to date have utilized incomplete growth systems (typically having a hydration system but lacking lighting) to study tropic and metabolic changes in germinating seedlings and young plants. American, European, and Russian scientists have also developed a number of small complete plant growth systems for use in spaceflight research. Currently we are entering a new era of experimentation and hardware development as a result of long-term spaceflight opportunities available on the International Space Station. This is already impacting development of plant growth hardware. To take full advantage of these new opportunities and construct innovative systems, we must understand the results of past spaceflight experiments and the basic capabilities of the diverse plant growth systems that were used to conduct these experiments. The objective of this paper is to describe the most influential pieces of plant growth hardware that have been used for the purpose of conducting scientific experiments during the first 40 years of research.     

Instrumentation for plant health and growth.

Comprehensive spectroscopic monitoring of plant health and growth in bioregenerative life support system environments is possible using a variety of spectrometric technologies. Absorption spectrometry and atomic emission spectrometry in combination allow for direct, on-line, reagentless monitoring of plant nutrients from nitrate and potassium to micronutrients such as copper and zinc. Fluorometric spectrometry is ideal for the on-line detection, identification and quantification of bacteria and fungi. Liquid Atomic Emission Spectrometry (LAES) is a new form of spectrometry that allows for direct measurement of atomic emission spectra in liquids. An electric arc is generated by a pair of electrodes in the liquid to provide the energy necessary to break molecular bonds and reduce the substance to atomic form. With a fiber probe attached to the electrodes, spectral light can be transmitted to a photodiode array spectrometer for light dispersion and analysis. Ultraviolet (UV) absorption spectrometry is a long-established technology, but applications typically have required specific reagents to produce an analyte-specific absorption. Nitrate and iron nutrients have native UV absorption spectra that have been used to accurately determine nutrient concentrations at the +/- 5% level. Fluorescence detection and characterization of microbes is based upon the native fluorescent signatures of most microbiological species. Spectral and time-resolved fluorometers operating with remote fiber-optic probes will be used for on-line microbial monitoring in plant nutrient streams.     

Development of tissue culture techniques and hardware to study mineralization under microgravity conditions.

To study the effects of weightlessness on mouse fetal long bone rudiment growth and mineralization we have developed a tissue culture system for the Biorack facility of Spacelab. The technique uses standard liquid tissue culture medium, supplemented with NA-beta-glycerophosphate, confined in gas permeable polyethylene bags mounted inside ESA Biorack Type I experiment containers. The containers can be flushed with an air/5% CO2 gas mixture necessary for the physiological bicarbonate buffer used. Small amounts of fluid can be introduced at the beginning (e.g. radioactive labels for incorporation studies) or at the end of the experiment (fixatives). A certain form of mechanical stimulation (continuous compression) can be used to counteract the, possibly, adverse effect of microgravity. Using 16 day old metatarsals the in vitro calcification process under microgravity conditions can be studied for a 4 day period.     

Ultrastructural and growth indices of Chlorella culture in multicomponent aquatic systems under space flight conditions.

Submicroscopic organization of Chlorella cells cultivated under space flight conditions in three-component aquatic system has been studied. Comparison of the experimental cells with that of the controls revealed certain rearrangements of cell organelles particularly, a reduction in the amount of reserve polysaccharides in chloroplasts, increase of cell vacuolization and mitochondrion volume, complication configuration of plasmalemma evaginations and invaginations, and also disturbances in the process of cytokinesis. More over an increase in the number of Chlorella cells infected by bacteria was shown in the experimental variant. No considerable differences were established in the growth characteristics of the experimental and control populations. A comparative cytological analysis revealed general regularities of organelles in Chlorella cells cultivated under space flight condition in the uni- and multicomponent systems.     

Improvements in the re-flight of spaceflight experiments on plant tropisms

In order to effectively study phototropism, the directed growth in response to light, we performed a series of experiments in microgravity to better understand light response without the “complications” of a 1-g stimulus. These experiments were named TROPI (for tropisms) and were performed on the European Modular Cultivation System (EMCS), a laboratory facility on the International Space Station (ISS). TROPI-1 was performed in 2006, and while it was a successful experiment, there were a number of technical difficulties. We had the opportunity to perform TROPI-2 in 2010 and were able to optimize experimental conditions as well as to extend the studies of phototropism to fractional gravity created by the EMCS centrifuge. This paper focuses on how the technical improvements in TROPI-2 allowed for a better experiment with increased scientific return. Major modifications in TROPI-2 compared to TROPI-1 included the use of spaceflight hardware that was off-gassed for a longer period and reduced seed storage (less than 2 months) in hardware. These changes resulted in increased seed germination and more vigorous growth of seedlings. While phototropism in response to red illumination was observed in hypocotyls of seedlings grown in microgravity during TROPI-1, there was a greater magnitude of red-light-based phototropic curvature in TROPI-2. Direct downlinking of digital images from the ISS in TROPI-2, rather than the use of analog tapes in TROPI-1, resulted in better quality images and simplified data analyses. In TROPI-2, improved cryo-procedures and the use of the GLACIER freezer during transport of samples back to Earth maintained the low temperature necessary to obtain good-quality RNA required for use in gene profiling studies.     

Energetic metabolism response in algae and higher plant species from simulation experiments with the clinostat.

Adenylate state is acknowledged to be among the most convenient approaches in the study of physiological changes in plant cells under simulation of altered gravity condition with the clinostat. Adenylate levels and the ATP/ADP ratio in cytoplasmic and mitochondrial extracts of cultivated cells of Haplopappus gracilis and algae cells of Chlorella vulgaris under initial stages of the fast-rotating and slow-rotating clinorotation, as well as the long-term clinorotation, have been investigated. For analysis of ATP and ADP levels in the plant cells under the clinorotation, we applied a high-sensitive bioluminescence method using the luciferase and piruvate kinase enzyme systems. It has been shown that the adenylate ratio is already increased during at the start of clinorotation with the different speed of rotation in the biological material tested. The considerable changes in mitochondrial ultrastructure of Chlorella cells, as well as the rising ATP level and dropping of the ATP/ADP ratio appear after long-duration clinorotation if compared to control material. It is probably connected with the distinctions in ATP-synthetase functioning in mitochondria of the cells under the clinorotation conditions.     

Achieving and documenting closure in plant growth facilities.

As NASA proceeds with its effort to develop a Controlled Ecological Life Support System (CELSS) that will provide life support to crews during long duration space missions, it must address the question of facility and system closure. Here we discuss the concept of closure as it pertains to CELSS and describe engineering specifications, construction problems and monitoring procedures used in the development and operation of a closed plant growth facility for the CELSS program. A plant growth facility is one of several modules required for a CELSS. A prototype of this module at Kennedy Space Center is the large (7m tall x 3.5m diameter) Biomass Production Chamber (BPC), the central facility of the CELSS Breadboard Project. The BPC is atmospherically sealed to a leak rate of approximately 5% of its total volume per 24 hours. This paper will discuss the requirements for atmospheric closure in this facility, present CO2 and trace gas data from initial tests of the BPC with and without plants, and describe how the chamber was sealed atmospherically. Implications that research conducted in this type of facility will have for the CELSS program are discussed.     

Population dynamics of transgenic microorganisms in the different microecosystem conditions.

The role of key environmental factors in adaptation of spore-forming and non-spore-forming transgenic microorganisms (TM) have been studied in model ecosystems. Model TM Escherichia coli Z905 (bearing plasmid genes of bacterial luminescence Ap (r) Lux+) has been found to have a higher adaptation potential than TM Bacillus subtilis 2335/105 (bearing genes of human alpha 2-interferon Km (r) Inf+), planned for employment as a living vaccine under varying environmental conditions. Effects of abiotic factors on migration of natural and recombinant plasmids between microorganisms under model ecosystem conditions has been estimated. The transgenic microorganisms with low copy number survived better under introduction conditions in the microcosms studied. This trend has been shown to be independent of the microcosm type and its complexity. Grant numbers: 99-04-96017, 25, 00-07-9011.     

Biological space experiments for the simulation of Martian conditions: UV radiation and Martian soil analogues.

The survivability of resistant terrestrial microbes, bacterial spores of Bacillus subtilis, was investigated in the BIOPAN facility of the European Space Agency onboard of Russian Earth-orbiting FOTON satellites (BIOPAN I -III missions). The spores were exposed to different subsets of the extreme environmental parameters in space (vacuum, extraterrestrial solar UV, shielding by protecting materials like artificial meteorites). The results of the three space experiments confirmed the deleterious effects of extraterrestrial solar UV radiation which, in contrast to the UV radiation reaching the surface of the Earth, also contains the very energy-rich, short wavelength UVB and UVC radiation. Thin layers of clay, rock or meteorite material were shown to be only successful in UV-shielding, if they are in direct contact with the spores. On Mars the UV radiation climate is similar to that of the early Earth before the development of a protective ozone layer in the atmosphere by the appearance of the first aerobic photosynthetic bacteria. The interference of Martian soil components and the intense and nearly unfiltered Martian solar UV radiation with spores of B. subtilis will be tested with a new BIOPAN experiment, MARSTOX. Different types of Mars soil analogues will be used to determine on one hand their potential toxicity alone or in combination with solar UV (phototoxicity) and on the other hand their UV protection capability. Two sets of samples will be placed under different cut-off filters used to simulate the UV radiation climate of Mars and Earth. After exposure in space the survival of and mutation induction in the spores will be analyzed at the DLR, together with parallel samples from the corresponding ground control experiment performed in the laboratory. This experiment will provide new insights into the principal limits of life and its adaptation to environmental extremes on Earth or other planets which and will also have implications for the potential for the evolution and distribution of life.     

Approaches in the determination of plant nutrient uptake and distribution in space flight conditions.

The effective growth and development of vascular plants rely on the adequate availability of water and nutrients. Inefficiency in either the initial absorption, transportation, or distribution of these elements are factors which impinge on plant structure and metabolic integrity. The potential effect of space flight and microgravity conditions on the efficiency of these processes is unclear. Limitations in the available quantity of space-grown plant material and the sensitivity of routine analytical techniques have made an evaluation of these processes impractical. However, the recent introduction of new plant cultivating methodologies supporting the application of radionuclide elements and subsequent autoradiography techniques provides a highly sensitive investigative approach amenable to space flight studies. Experiments involving the use of gel based 'nutrient packs' and the radionuclides calcium-45 and iron-59 were conducted on the Shuttle mission STS-94. Uptake rates of the radionuclides between ground and flight plant material appeared comparable.