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.     

The "C.E.B.A.S. MINI-MODULE": a self-sustaining closed aquatic ecosystem for spaceflight experimentation.

The C.E.B.A.S. MINI-MODULE is the miniaturized space flight version of the Closed Equilibrated Biological Aquatic System (C.E.B.A.S.). It fits into a large middeck locker tray and is scheduled to be flown in the STS 85 and in the NEUROLAB missions. Its volume is about 9 liters and it consists of two animal tanks, a plant cultivator, and a bacteria filter in a monolithic design. An external sensor unit is connected to a data acquisition/control unit. The system integrates its own biological life support. The CO2 exhaled by the consumers (fishes, snails, microorganisms) is assimilated by water plants (Ceratophyllum demersum) which provide them with oxygen. The products of biomass degradation and excretion (mainly ammonia ions) are converted by bacteria into nitrite and nitrate. The latter is taken up by the plants as a nitrogen source together with other ions like phosphate. The plants convert light energy into chemical energy and their illumination is regulated via the oxygen concentration in the water by the control unit. In ground laboratory tests the system exhibited biological stability up to three month. The buffer capacity of the biological filter system is high enough to eliminate the degradation products of about one half of the dead animal biomass as shown in a "crash test". A test series using the laboratory model of the flight hardware demonstrated the biological stability and technical reliability with mission-identical loading and test duration. A comprehensive biological research program is established for the C.E.B.A.S. MINI-MODULE in which five German and three U.S.-American universities as well as the Russian Academy of Sciences are involved.     

Changes in operational procedures to improve spaceflight experiments in plant biology in the European Modular Cultivation System

The microgravity environment aboard orbiting spacecraft has provided a unique laboratory to explore topics in basic plant biology as well as applied research on the use of plants in bioregenerative life support systems. Our group has utilized the European Modular Cultivation System (EMCS) aboard the International Space Station (ISS) to study plant growth, development, tropisms, and gene expression in a series of spaceflight experiments. The most current project performed on the ISS was termed Seedling Growth-1 (SG-1) which builds on the previous TROPI (for tropisms) experiments performed in 2006 and 2010. Major technical and operational changes in SG-1 (launched in March 2013) compared to the TROPI experiments include: (1) improvements in lighting conditions within the EMCS to optimize the environment for phototropism studies, (2) the use of infrared illumination to provide high-quality images of the seedlings, (3) modifications in procedures used in flight to improve the focus and overall quality of the images, and (4) changes in the atmospheric conditions in the EMCS incubator. In SG-1, a novel red-light-based phototropism in roots and hypocotyls of seedlings that was noted in TROPI was confirmed and now can be more precisely characterized based on the improvements in procedures. The lessons learned from sequential experiments in the TROPI hardware provide insights to other researchers developing space experiments in plant biology.     

The influence of microbial associations on germination of wheat seeds and growth of seedlings under impact of zinc salts

The life support systems (LSS) for long-term missions are to use cycling-recycling systems, including biological recycling. Higher plants are the traditional regenerator of air and producer of food. They should be used in many successive generations of their reproduction in LSS.     

Changes in symbiotic and associative interrelations in a higher plant-bacterial system during space flight.

The miniature cenosis consisting of the water fern Azolla with its associated symbiotic nitrogen-fixing cyanobacterium Anabaena and the concomitant bacteria was investigated. Ecological closure was shown to produce sharp quantitative and qualitative changes in the number and type of concomitant bacteria. Changes in the distribution of bacterial types grown on beef-extract broth after space flight were recorded. Anabaena azollae underwent the most significant changes under spaceflight conditions. Its cell number per Azolla biomass unit increased substantially. Thus closure of cenosis resulted in a weakening of control over microbial development by Azolla. This tendency was augmented by spaceflight factors. Reduction in control exerted by macro-organisms over development of associated micro-organisms must be taken into account in constructing closed ecological systems in the state of weightlessness.     

Biomass recycle as a means to improve the energy efficiency of CELSS algal culture systems.

Algal cultures can be very rapid and efficient means to generate biomass and regenerate the atmosphere for closed environmental life support systems. However, as in the case of most higher plants, a significant fraction of the biomass produced by most algae cannot be directly converted to a useful food product by standard food technology procedures. This waste biomass will serve as an energy drain on the overall system unless it can be efficiently recycled without a significant loss of its energy content. We report experiments in which cultures of the algae Scenedesmus obliquus were grown in the light and at the expense of an added carbon source, which either replaced or supplemented the actinic light. As part of these experiments we tested hydrolyzed waste biomass from these same algae to determine whether the algae themselves could be made part of the biological recycling process. Results indicate that hydrolyzed algal (and plant) biomass can serve as carbon and energy sources for the growth of these algae, suggesting that the efficiency of the closed system could be significantly improved using this recycling process.     

Modification of plant growth and development by acceleration and vibration: concerns and opportunities for plant experimentation in orbiting spacecraft.

Growth, development, and orientation of higher plants is altered by physical disturbances such as shaking, touching, or vibration. Plant growth responses to thigmic (contact rubbing) forces are almost always negative, whereas growth responses to periodic seismic (shaking) or vibric (vibrational) disturbances may be positive or negative, depending on intensity and duration of force, and prevailing environmental conditions. Seedlings are most sensitive to mechanical stress when grown in darkness or under the low-light conditions typically available in plant flight hardware. Brief exposure to physical perturbation causes immediate growth inhibition of dark-grown seedlings followed by gradual recovery of growth rate beginning 10-12 minutes later. For mild vibration, growth rate may overshoot that of undisturbed control plants within an hour of a stress episode, whereas for thigmic stress recovery may remain incomplete for 24 hours or longer. Lack of physical stimulation by gravity should make plants even more responsive to random physical perturbation. Threshold growth response of seedlings to vibrational parameters needs to be determined under real spaceflight conditions.     

Biological effects of weightlessness and clinostatic conditions registered in cells of root meristem and cap of higher plants.

Research in cellular reproduction, differentiation and vital activity, i.e. processes underlying the development and functioning of organisms, plants included, is essential for solving fundamental and applied problems of space biology. Detailed anatomical analysis of roots of higher plants grown on board the Salyut 6 orbital research station show that under conditions of weightlessness for defined duration mitosis, cytokinesis and tissue differentiation in plant vegetative organs occur essentially normally. At the same time, certain rearrangements in the structural organization of cellular organelles--mainly the plastid apparatus, mitochondria, Golgi apparatus and nucleus--are established in the root meristem and cap of the experimental plants. This is evidence for considerable changes in cellular metabolism. The structural changes in the subcellular level arising under spaceflight conditions are partially absent in clinostat experiments designed to simulate weightlessness. Various clinostatic conditions have different influences on the cell structural and functional organization than does space flight. It is suggested that alterations of cellular metabolism under weightlessness and clinostatic conditions occur within existing genetic programs.     

Instrumentation for plant health and growth in space.

The present-day plant growth facilities ("greenhouses") for space should be equipped with monitors and controllers of ambient parameters within the chamber because spacecraft environmental variations can be unfavorable to plants. Moreover, little is known about the effects of spaceflight on the greenhouse and rooting media. Lack of information about spaceflight effects on plants necessitates supplying space greenhouses with automatic, non-invasive monitors of, e.g., gas exchange rate, water and nutrient ion uptake, plant mass, temperature and water content of leaves. However, introduction of an environmental or plant sensor into the monitoring system may be reasonable only if it is justified by quantitative evaluation of the influence of a measured parameter on productivity, efficacy of illumination, or some other index of greenhouse efficiency. The multivariate adaptive optimization in terrestrial phytotrons appears to be one of the best methods to assess environmental impacts on crops. Two modifications of greenhouses with the three-dimensional adaptive optimization of crop photosynthetic characteristics include: (1) irradiation, air temperature and carbon dioxide using a modified simplex algorithm; and (2) using irradiation, air temperature, and humidity with sensitivity algorithms with varying frequency of test exposures that have been experimentally developed. As a result, during some stages of plant ontogensis, the photosynthetic productivity of wheat, tomatoes, and Chinese cabbage in these systems was found to increase by a factor of 2-3.     

Biological dosimetry in Russian and Italian astronauts.

Large uncertainties are associated with estimates of equivalent dose and cancer risk for crews of long-term space missions. Biological dosimetry in astronauts is emerging as a useful technique to compare predictions based on quality factors and risk coefficients with actual measurements of biological damage in-flight. In the present study, chromosomal aberrations were analyzed in one Italian and eight Russian cosmonauts following missions of different duration on the MIR and the international space station (ISS). We used the technique of fluorescence in situ hybridization (FISH) to visualize translocations in chromosomes 1 and 2. In some cases, an increase in chromosome damage was observed after flight, but no correlation could be found between chromosome damage and flight history, in terms of number of flights at the time of sampling, duration in space and extra-vehicular activity. Blood samples from one of the cosmonauts were exposed in vitro to 6 MeV X-rays both before and after the flight. An enhancement in radiosensitivity induced by the spaceflight was observed.     

Pituitary cells in space.

Cells of the mammalian pituitary gland synthesize and secrete several protein hormones which regulate a number of organ systems throughout the body. These include the musculoskeletal, immune, vascular and endocrine systems. Since changes occur in these tissues as a result of spaceflight, and since pituitary growth hormone (GH) and prolactin (PRL) play a role in the control of these systems on earth, we have focused attention over the last 10 years on GH and PRL cell function during and after spaceflight. The cumulative results of 4 spaceflight missions and several mimicked microgravity experiments establish 1) that production and release of biologically active GH and PRL is repeatedly and significantly attenuated (usually > 50%) and 2) that changes in cell morphology also occur. In this paper we describe our results within the framework of methodologies and approaches frequently used to study pituitary cell function on earth. In so doing we hope to develop future flight experiments aimed at uncovering possible microgravity "sensing systems" within the pituitary cell.     

Light intensity and production parameters of phytocenoses cultivated on soil-like substrate under controlled [correction of controled] environment conditions.

To increase the degree of closure of biological life support systems of a new generation, we used vermicomposting to involve inedible phytomass in the intra-system mass exchange. The resulting product was a soil-like substrate, which was quite suitable for growing plants (Manukovsky et al. 1996, 1997). However, the soil like substrate can be regarded as a candidate for inclusion in a system only after a comprehensive examination of its physical, chemical, and other characteristics. An important criterion is the ability of the soil-like substrate to supply the necessary mineral elements to the photosynthesizing component under the chosen cultivation conditions. Thus, the purpose of this work was to study the feasibility of enhancing the production activity of wheat and radish crops by varying the intensity of photosynthetically active radiation, without decreasing the harvest index. The increase of light intensity from 920 to 1150 micromoles m-2 s-1 decreased the intensity of apparent photosynthesis of the wheat crops and slightly increased the apparent photosynthesis of the radish crops The maximum total and grain productivity (kg/m2) of the wheat crops was attained at the irradiance of 920 micromoles m-2 s-1. Light intensity of 1150 micromoles m-2 s-1 decreased the productivity of wheat plants and had no significant effect on the productivity of the radish crops (kg/m2) as compared to 920 micromoles m-2 s-1. The qualitative and quantitative composition of microflora of the watering solution and substrate was determined by the condition of plants, developmental phase and light intensity. By the end of wheat growth under 1150 micromoles m-2 s-1 the numbers of bacteria of the coliform family and phytopathogenic bacteria in the watering solution and substrate were an order of magnitude larger than under other illumination conditions. The obtained data suggest that the cultivation of plants in a life support system on soil-like substrate from composts has a number of advantages over the cultivation on neutral substrates, which require continual replenishment of the plant nutrient solution from the system's store to complement the macro- and micro-elements. Yet, a number of problems arise, including those related to the controlling of the production activity of the plants by the intensity of photosynthetically active radiation. It is essential to understand why the intensity of production processes is limited at higher irradiation levels and to overcome the factors responsible for this, so that the soil-like substrate could have an even better chance in the competition for the best plant cultivation technology to be used in biological life support systems.     

The closed equilibrated biological aquatic system: a 12 months test of an artificial aquatic ecosystem.

The Closed Equilibrated Biological Aquatic System" (C.E.B.A.S.) is finally disposed for long-term multi-generation experiments with aquatic organisms in a space station. Therefore a minimum operation time of three months is required. It is verified in three versions of laboratory prototypes. The third one passed successfully a 12 months mid-term test in 1995/96 thus demonstrating its high biological stability. The third version of the C.E.B.A.S. consists of a 100 l animal tank, two plant cultivators with a volume of 15 l each with independent illuminations, a 3.0 l semibiological "mechanical" filter, a 3.0 l bacteria filter, a heating/cooling device and a dummy filter unit. The live-bearing teleost Xiphophorus helleri is the vertebrate and the pulmonate water snail Biomphalana glabrata the invertebrate experimental animal in the system. The rootless higher water plant Ceratophyllum demersum is the producer organism. Ammonia oxidizing bacteria and other microorganisms settle in the filters. A sample data acquisition is combined with temperature and plant illumination control. Besides of the space aspects the C.E.B.A.S. proved to be an extremely suitable tool to investigate the organism and subcomponent interactions in a well defined terrestrial aquatic closed ecosystem by providing physical, chemical and biological data which allow an approach to a comprehensive system analysis. Moreover the C.E.B.A.S. is the base for the development of innovative combined animal-plant aquaculture systems for human nutrition on earth which could be implemented into bioregenerative life support systems with a higher degree of complexity suitable for lunar or planetary bases.     

Dynamics of microorganism populations in recirculating nutrient solutions.

This overview covers the basic microbial ecology of recirculating hydroponic solutions. Examples from NASA and Soviet CELSS tests and the commercial hydroponic industry will be used. The sources of microorganisms in nutrient solutions include air, water, seeds, plant containers and plumbing, biological vectors, and personnel. Microbial fates include growth, death, and emigration. Important microbial habitats within nutrient delivery systems are root surfaces, hardware surfaces (biofilms), and solution suspension. Numbers of bacteria on root surfaces usually exceed those from the other habitats by several orders of magnitude. Gram negative bacteria dominate the microflora with fungal counts usually much lower. Trends typically show a decrease in counts with increasing time unless stressed plants increase root exudates. Important microbial activities include carbon mineralization and nitrogen transformations. Important detrimental interactions include competition with plants, and human and plant pathogenesis.     

载人深空探测活动中的尿液处理回收技术分析

生物再生生命保障系统(Bioregenerative Life Support System,BLSS)是人类进行深空探测活动,实现长期载人空间飞行必需的关键技术,对于太空的探索开发具有重要意义。在BLSS系统内,航天员尿液废水的处理回收是非常重要的一部分。将尿液中所含有的大量的水分和丰富的营养物质回收用于系统内植物生长所需营养液的配制,既可以保证植物的正常生长,也有助于实现系统内物质的循环利用进而提高BLSS的闭合度。尿液中所含的大量盐分会威胁植物生长,所以需通过一定的技术手段处理尿液废水并回收其中的水分和营养。为了探索适用于BLSS中的尿液处理回收技术,首先分析了几种面向空间站应用的尿液处理技术,如蒸馏技术等;然后基于回收营养物质的需求,分析了面向民用的、发展较为成熟的尿液处理回收技术,如离子交换吸附技术、氨气吹脱技术和鸟粪石沉淀技术,并讨论了这些尿液处理回收技术在BLSS中的应用前景。最后基于BLSS的实际需求,提出了有望用于BLSS中的尿液处理回收技术流程。     

Animal models for the study of the effects of spaceflight on the immune system.

Animal models have been used to determine the effects of spaceflight on the immune system. Rats and rhesus monkeys have been the primary animals used for actual space flight studies, but mice have also been utilized for studies in ground-based models. The primary ground based model used has been hindlimb unloading of rodents, which is similar to the chronic bed-rest model for humans. A variety of immune responses have been shown to be modified when animals are hindlimb unloaded. These results parallel those observed when animals are flown in space. In general, immune responses are depressed in animals maintained in the hindlimb unloading model or flown in space. These results raise the possibility that spaceflight could result in decreased resistance to infection in animals.     

The C.E.B.A.S.-Minimodule: behaviour of an artificial aquatic ecological system during spaceflight.

The C.E.B.A.S.-Minimodule, a closed aquatic ecosystem integrated into a middeck locker and consisting of a Zoological (animal tanks), a Botanical (plant bioreactor), a Microbial (bacteria filter) and an Electronic Component (data acquisition/control system) was flown on the STS-89 spaceshuttle mission in January 1998 for 9 days. Preflight the plant bioreactor was loaded with 53 g of Ceratophyllum demersum (coontail) and the animal tanks with 4 adult pregnant females of the fish, Xiphophorus helleri (sword-tails), 200 juveniles of the same species less than 1 week of age, 38 large and 30 juvenile Biomphalaria glabrata water snails. The filter compartment was filled with 200 g of lava grain inoculated with laboratory strains of ammonia-oxidizing bacteria. A ground reference was undertaken with the same biological setup with a delay of 4 d. After an adaptation period of 5 d the system was closed and integrated into the spaceshuttle one day before launch. Video recordings of the animals were automatically taken for 10 minutes in 2-hour periods; the tapes were changed daily by the astronauts. The chemical and physical data for the aquatic system were within the expected range and were closely comparable in comparison to the ground reference. After 9 d under space conditions, the plant biomass increased to 117 g. The plants were all found in very good condition. All 4 adult female fish were retrieved in a good physiological condition. The juvenile fishes had a survival rate of about 33%. Almost 97% of the snails had survived and produced more than 250 neonates and 40 spawning packs. All samples were distributed according to a defined schedule and satisfied all scientific needs of the involved 12 principal investigators. This was the first successful spaceflight of an artificial aquatic ecosystem containing vertebrates, invertebrates, higher plants and microorganisms self-sustained by its inhabitants only. C.E.B.A.S. in a modified form and biological setup is a promising candidate for the early space station utilization as a first midterm experiment.     

Spaceflight experiments with Arabidopsis starch-deficient mutants support a statolith-based model for graviperception.

In order to help resolve some of the controversy associated with ground-based research that has supported the starch-statolith theory of gravity perception in plants, we performed spaceflight experiments with Arabidopsis in Biorack during the January 1997 and May 1997 missions of the Space Shuttle. Seedlings of wild-type (WT) Arabidopsis, two reduced-starch strains, and a starchless mutant were grown in microgravity and then were given either a 30, 60, or 90 minute gravity stimulus on a centrifuge. By the 90 min 1-g stimulus, the WT exhibited the greatest magnitude of curvature and the starchless mutant exhibited the smallest curvature while the two reduced starch mutants had an intermediate magnitude of curvature. In addition, space-grown plants had two structural features that distinguished them from the controls: a greater number of root hairs and an anomalous hypocotyl hook structure. However, the morphological changes observed in the flight seedlings are likely to be due to the effects of ethylene present in the spacecraft. (Additional ground-based studies demonstrated that this level of ethylene did not significantly affect gravitropism nor did it affect the relative gravitropic sensitivity among the four strains.) Nevertheless, this experiment on gravitropism was performed the "right way" in that brief gravitational stimuli were provided, and the seedlings were allowed to express the response without further gravity stimuli. Our spaceflight results support previous ground-based studies of these and other mutants since increasing amounts of starch correlated positively with increasing sensitivity to gravity.     

ASTROCULTURE (TM) root metabolism and cytochemical analysis.

Physiology of the root system is dependent upon oxygen availability and tissue respiration. During hypoxia nutrient and water acquisition may be inhibited, thus affecting the overall biochemical and physiological status of the plant. For the Astroculture (TM) plant growth hardware, the availability of oxygen in the root zone was measured by examining the changes in alcohol dehydrogenase (ADH) activity within the root tissue. ADH activity is a sensitive biochemical indicator of hypoxic conditions in plants and was measured in both spaceflight and control roots. In addition to the biochemical enzyme assays, localization of ADH in the root tissue was examined cytochemically. The results of these analyses showed that ADH activity increased significantly as a result of spaceflight exposure. Enzyme activity increased 248% to 304% in dwarf wheat when compared with the ground controls and Brassica showed increases between 334% and 579% when compared with day zero controls. Cytochemical staining revealed no differences in ADH tissue localization in any of the dwarf wheat treatments. These results show the importance of considering root system oxygenation in designing and building nutrient delivery hardware for spaceflight plant cultivation and confirm previous reports of an ADH response associated with spaceflight exposure.