Pigment composition and concentrations within the plant (Ceratophyllum demersum L.) component of the STS-89 C.E.B.A.S. Mini-Module spaceflight experiment.

The Closed Equilibrated Biological Aquatic System (C.E.B.A.S.) Mini-Module, a Space Shuttle middeck locker payload which supports a variety of aquatic inhabitants (fish, snails, plants and bacteria) in an enclosed 8.6 L chamber, was tested for its biological stability in microgravity. The aquatic plant, Ceratophyllum demersum L., was critical for the vitality and functioning of this artificial mini-ecosystem. Its photosynthetic pigment concentrations were of interest due to their light harvesting and protective functions. "Post-flight" chlorophyll and carotenoid concentrations within Ceratophyllum apical segments were directly related to the quantities of light received in the experiments, with microgravity exposure (STS-89) failing to account for any significant deviation from ground control studies.     

Aquatic modules for bioregenerative life support systems: developmental aspects based on the space flight results of the C.E.B.A.S. MIN-MODULE.

The Closed Equilibrated Biological Aquatic System (C.E.B.A.S.) is an artificial aquatic ecosystem which contains teleost fishes, water snails, ammonia oxidizing bacteria and edible non-gravitropic water plants. It serves as a model for aquatic food production modules which are not seriously affected by microgravity and other space conditions. Its space flight version, the so-called C.E.B.A.S. MINI-MODULE was already successfully tested in the STS-89 and STS-90 (NEUROLAB) missions. It will be flown a third time in space with the STS-107 mission in January 2003. All results obtained so far in space indicate that the basic concept of the system is more than suitable to drive forward its development. The C.E.B.A.S. MINI-MODULE is located within a middeck locker with limited space for additional components. These technical limitations allow only some modifications which lead to a maximum experiment time span of 120 days which is not long enough for scientifically essential multi-generation-experiments. The first necessary step is the development of "harvesting devices" for the different organisms. In the limited space of the plant bioreactor a high biomass production leads to self-shadowing effects which results in an uncontrolled degradation and increased oxygen consumption by microorganisms which will endanger the fishes and snails. It was shown already that the latter reproduce excellently in space and that the reproductive functions of the fish species are not affected. Although the parent-offspring-cannibalism of the ovoviviparous fish species (Xiphophorus helleri) serves as a regulating factor in population dynamics an uncontrolled snail reproduction will also induce an increased oxygen consumption per se and a high ammonia concentration in the water. If harvesting locks can be handled by astronauts in, e. g., 4-week intervals their construction is not very difficult and basic technical solutions are already developed. The second problem is the feeding of the animals. Although C.E.B.A.S.-based aquaculture modules are designed to be closed food loop systems (edible herbivorous fish species and edible water plants) which are already verified on Earth this will not be possible in space without devices in which the animals are fed from a food storage. This has to be done at least once daily which would waste too much crew time when done by astronauts. So, the development of a reliable automated food dispenser has highest priority. Also in this case basic technical solutions are already elaborated. The paper gives a comprehensive overview of the proposed further C.E.B.A.S.-based development of longer-term duration aquatic food production modules.     

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.     

C.E.B.A.S. MINI MODULE: test results of an artificial (man-made) aquatic ecosystem.

The original Closed Equilibrated Biological Aquatic System (C.E.B.A.S.) is a long-term multi-generation research facility for experiments with aquatic animals and plants in a space station the development of which is surrounded by a large international scientific program. In addition, a miniaturized laboratory prototype, the C.E.B.A.S. MINI MODULE, with a total volume of about 10-12 liters for a Spacelab middeck locker was developed and a first version was tested successfully for two weeks with a population of fishes (Xiphophorus helleri) in the animal tank and a Ceratophyllum spec. in the illuminated higher plant growth chamber. The water recycling system consisted of a bacteria filter and a mechanical filter and the silastic tubing gas exchanger was separated by valves for the utilization in emergency cases only. Data were collected with the acquisition module of the original C.E.B.A.S. process control system. In addition, an optimized version was tested for 7 weeks with fishes and plants and thereafter with fish and with plants only for 2 and 1 weeks, resp.. The paper presents the relevant water parameters (e.g., pH, pressure, temperature, oxygen saturation, flow rate, ion concentrations) during the test period as well as morphological and physiological data of the enclosed animals and plants. On the basis of the given results the possible role of the C.E.B.A.S. system as a scientific tool in artificial ecosystem research and for the development of a combined animal-plant intensive aquaculture system and its utilization in bioregenerative life support is discussed.     

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.     

Possible applications of aquatic bioregenerative life support modules for food production in a Martian base.

Water is the essential precondition of life in general and also for the establishment of a Martian base suitable for long duration stays of humans. It is not yet proven if there is indeed a "frozen ocean" under the surface of Mars but if this could be verified it would open innovative aspects for the construction of bioregenerative life support systems (BLSS). In a general concept higher plants will play the predominant role in a Martian BLSS. It is not clear, however, how these will grow and bring seed in reduced gravity and there may be differences in the productivity in comparison to Earth conditions. Therefore, organisms which are already adapted to low gravity conditions, namely non-gravitropic aquatic plants and also aquatic animals may be used to enhance the functionality of the Martian BLSS as a whole. It has been shown already with the so-called C.E.B.A.S. MINIMODULE in the STS-89 and STS-90 spaceshuttle missions that the water plant Ceratophyllum demersum has an undisturbed and high biomass production under space conditions. Moreover, the teleost fish species Xiphophorus helleri adapted easily to the micro-g environment and maintained its normal reproductive functions. Based on this findings a possible scenario is presented in which aquatic plant production modules and combined animal-plant production systems may be used for human food production and water and air regeneration in a Martian base.     

NASDA next generation aquatic habitat for Space Shuttle and ISS.

The National Space Development Agency of Japan (NASDA) has more than 20 years of experience developing aquatic animal experiment facilities. We are now studying the next-generation aquatic animal experiment facility or the Aquatic Habitat (AQH) for both Space Shuttle and International Space Station use. A prototype breeding system was designed and tested. Medaka adult fish were able to mate and spawn in this closed circulatory breeding system, and the larvae grew to adult fish and spawned on the 45th day after hatching. The water quality-control system using nitrifying bacteria worked well throughout the medaka breeding test. For amphibians, we also conducted the African clawed toad (Xenopus laevis) breeding test with the same specimen chambers, although a part of circulation loop was opened to air. Xenopus larvae grew and completed metamorphosis successfully in the small specimen chamber. The first metamorphic climax started on the 30th day and was completed on the 38th day.     

Aquatic food production modules in bioregenerative life support systems based on higher plants.

Most bioregenerative life support systems (BLSS) are based on gravitropic higher plants which exhibit growth and seed generation disturbances in microgravity. Even when used for a lunar or martian base the reduced gravity may induce a decreased productivity in comparison to Earth. Therefore, the implementation of aquatic biomass production modules in higher plant and/or hybrid BLSS may compensate for this and offer, in addition, the possibility to produce animal protein for human nutrition. It was shown on the SLS-89 and SLS-90 space shuttle missions with the C.E.B.A.S.-MINI MODULE that the edible non gravitropic rootless higher aquatic plant Ceratophyllum demeresum exhibits an undisturbed high biomass production rate in space and that the teleost fish species, Xiphophorus helleri, adapts rapidly to space conditions without loss of its normal reproductive functions. Based on these findings a series of ground-based aquatic food production systems were developed which are disposed for utilization in space. These are plant production bioreactors for the species mentioned above and another suitable candidate, the lemnacean (duckweed) species, Wolffia arrhiza. Moreover, combined intensive aquaculture systems with a closed food loop between herbivorous fishes and aquatic and land plants are being developed which may be suitable for integration into a BLSS of higher complexity. Grant numbers: WS50WB9319-3, IVA1216-00588.     

Readaptation of fish to 1g after long-term microgravity: behavioural results from the STS 89 mission.

The swimming behaviour of adult and neonate swordtail fish Xiphophorus helleri was qualitatively analysed from video recordings taken throughout the STS 89 spaceshuttle mission from launch to landing and thereafter. After the flight, the swimming behaviour of neonate samples was quantitatively assessed in the course of the readaptation to 1g earth gravity at days 0, 1 and 4 after recovery. Regarding the swimming behaviour during the mission, the adult fish swam thigmotactically (i.e., responding to tactile stimuli) along the walls of their aquarium, but like the neonates, they did not show any aberrant behavioural patterns. This indicates that they could easily adapt themselves to microgravity. On mission day 9, however, looping responses (most probably initiated by mechanical disturbances) occurred indicating a continuously performed "C-start" escape response (the respective body bend looks like the letter "C"). Immediately after landing (observed in videos recorded onboard the space shuttle), the adults performed a head-up swimming beating heavily with the caudal and pectoral fins; this aberrant behaviour gradually decreased during the first hours after recovery.     

Effects of CO2 concentration and light intensity on photosynthesis of a rootless submerged plant, Ceratophyllum demersum L., used for aquatic food production in bioregenerative life support systems.

In addition to green microalgae, aquatic higher plants are likely to play an important role in aquatic food production modules in bioregenerative systems for producing feed for fish, converting CO2 to O2 and remedying water quality. In the present study, the effects of culture conditions on the net photosynthetic rate of a rootless submerged plant, Ceratophyllum demersum L., was investigated to determine the optimum culture conditions for maximal function of plants in food production modules including both aquatic plant culture and fish culture systems. The net photosynthetic rate in plants was determined by the increase in dissolved O2 concentrations in a closed vessel containing a plantlet and water. The water in the vessel was aerated sufficiently with a gas containing a known concentration of CO2 gas mixed with N2 gas before closing the vessel. The CO2 concentrations in the aerating gas ranged from 0.3 to 10 mmol mol-1. Photosynthetic photon flux density (PPFD) in the vessel ranged from 0 (dark) to 1.0 mmol m-2 s-1, which was controlled with a metal halide lamp. Temperature was kept at 28 degrees C. The net photosynthetic rate increased with increasing PPFD levels and was saturated at 0.2 and 0.5 mmol m-2 s-1 PPFD under CO2 levels of 1.0 and 3.0 mmol mol-1, respectively. The net photosynthetic rate increased with increasing CO2 levels from 0.3 to 3.0 mmol mol-1 showing the maximum value, 75 nmol O2 gDW-1 s-1, at 2-3 mmol mol-1 CO2 and gradually decreased with increasing CO2 levels from 3.0 to 10 mmol mol-1. The results demonstrate that C. demersum could be an efficient CO2 to O2 converter under a 2.0 mmol mol-1 CO2 level and relatively low PPFD levels in aquatic food production modules.     

A simple closed aquatic ecosystem (CAES) for space

A closed aquatic ecosystem (CAES) was developed to study the effects of microgravity on the function of closed ecosystems aboard the Chinese retrieved satellite and on the spacecraft SHENZHOU-II. These systems housed a small freshwater snail (Bulinus australianus) and an autotrophic green algae (Chlorella pyrenoidosa). The results of the test on the satellite were that the concentration of algae changed little, but that the snails died during the experiments. We then sought to optimize the function of the control system, the cultural conditions and the data acquisition system and carried out an experiment on the spacecraft SHENZHOU-II. Using various sensors to monitor the CAES, real-time data regarding the operation of the CAES in microgravity was acquired. In addition, an on-board 1g centrifuge was included to identify gravity-related factors. It was found that microgravity is the major factor affecting the operation of the CAES in space. The change in biomass of the primary producer during each day in microgravity was larger than that of the control groups. The mean biomass concentration per day in the microgravity group decreased, but that of the control groups increased for several days and then leveled off. Space effects on the biomass of a primary producer may be a result of microgravity effects leading to increasing metabolic rates of the consumer combined with decreases in photosynthesis.     

Possible nutrient limiting factor in long term operation of closed aquatic ecosystem

To investigate nutrient limitation effect on the community metabolism of closed aquatic ecosystem and possible nutrient limiting factors in the experimental food chains, depletion of inorganic chemicals including carbon, nitrogen and phosphorous was tested. A closed aquatic ecosystem lab module consisting of Chlorella pyrenoidosa and Chlamydomonas reinhardtii, Daphnia magna and associated unidentified microbes was established. Closed ecological systems receive no carbon dioxide; therefore, we presumed carbon as a first limiting factor. The results showed that the algae population in the nutrient saturated group was statistically higher than that in the nutrient limited groups, and that the chlorophyll a content of algae in the phosphorus limited group was the highest among the limited groups. However, the nitrogen limited group supported the most Daphnia, followed by the carbon limited group, the nutrient saturated group and the phosphorus limited group. Redundancy analysis showed that the total phosphorus contents were correlated significantly with the population of algae, and that the amount of soluble carbohydrate as feedback of nutrient depletion was correlated with the number of Daphnia. Thus, these findings suggest that phosphorus is the limiting factor in the operation of closed aquatic ecosystem. The results presented herein have important indications for the future construction of long term closed ecological system.     

Controlling denitrification in closed artificial ecosystems.

Denitrification, the dissimilatory reduction of NO3- to N2O and N2, is found in a wide variety of organisms. In closed artificial systems, especially closed plant growth chambers, a significant loss of fixed-N occurs through denitrification, thereby decreasing the efficiency of the system and fouling the atmosphere with N2O. Denitrification is a form of anaerobic respiration. Whenever available, however, denitrifiers preferentially use O2 as their terminal electron acceptor. As a result, rates of denitrification and growth are a function of O2. Typically, in closed systems O2 consumption is greater than the diffusion of O2 through the medium to the cell, decreasing the O2 level near the cell and denitrification occurs. Using Pseudomonas fluorescens (ATCC # 17400) as a model organism grown in a two L bioreactor under varying levels of O2 we studied its effects on population growth and its ability to mitigate denitrification in closed systems. The results indicate that denitrification occurs in a closed system even when it is considered aerobic, that is well mixed and sparged with either air, or sufficient pure O2 to cause a complete turnover in the gaseous atmosphere in the bioreactor vessel every five minutes.     

Performance of a simple Closed Aquatic Ecosystem (CAES) in space.

A simple Closed Aquatic Ecosystem (CAES) consisting of single-celled green algae (Chlorella pyrenoidosa, producer), a spiral snail (Bulinus australianus, consumer) and a data acquisition and control unit was flown on the Chinese Spacecraft SHENZHOU-II in January 2001 for 7 days. In order to study the effect of microgravity on the operation of CAES, a 1 g centrifuge reference group in space, a ground 1 g reference group and a ground 1 g centrifuge reference group (1.4 g group) were run concurrently. Real-time data about algae biomass (calculated from transmission light intensity), temperature, light and centrifugation of the CAES were logged at minute intervals. It was found that algae biomass of both the microgravity group and the ground 1 g-centrifuge reference group (1.4 g) fluctuated during the experiment, but the algae biomass of the 1 g centrifuge reference group in space and the ground 1 g reference group increased during the experiment. The results may be attributable to influences of microgravity and 1.4 g gravity on the algae and snails metabolisms. Microgravity is the main factor to affect the operation of CAES in space and the contribution of microgravity to the effect was also estimated. These data may be valuable for the establishment of a complex CELSS in the future.     

Progress in ultrasonic bioreactors for CELSS applications.

An important issue in Controlled Ecological Life Support Systems (CELSS) is the recycling of inedible crop residues to recover inorganic plant nutrients such as nitrates, phosphates, potassium and other macro- and micro-nutrients. In a closed system in space, such regeneration is vital to the long term viability of plant growth necessary for the food production and waste handling process. Chemical approaches to recycling such as incineration and wet oxidation are not compatible with low energy and environmentally friendly regeneration of such nutrients. Biological regeneration is more acceptable environmentally, but it is a very slow process and does not typically result in complete recovery of inorganic and organic nutrients. A new approach to biological regeneration is described here involving the combined use of special enzymatic catalysts and ultrasonic energy in a bioreactor system. This new system has the potential for rapid, efficient, environmentally friendly and complete conversion of crop wastes to inorganic plant nutrients and food recovery from cellulose materials. A series of experimental tests were carried out with a soybean crop residue meal substrate. Biochemical conversion rates were significantly expedited with the addition of enzymes and further enhanced through ultrasonic stimulation of these enzymes. The difference in conversion rates was particularly increased after the initial period of soluble organics conversion. The remaining cellulose substrate is much more difficult to biodegrade, and the ultrasonically-enhanced reaction was able to demonstrate a much higher rate of substrate conversion.     

C.E.B.A.S., a closed equilibrated biological aquatic system as a possible precursor for a long-term life support system?

C.E.B.A.S.-AQUARACK is a long-term multi-generation experimental device for aquatic organisms which is disposed for utilization in a space station. It results from the basic idea of a space aquarium for maintaining aquatic animals for longer periods integrated in a AQUARACK which consists of a modular animal holding tank, a semi-biological/physical water recycling system and an electronical control unit. The basic idea to replace a part of the water recycling system by a continuous culture of unicellular algae primarily leads to a second system for experiments with algae, a botanical AQUARACK consisting of an algal reactor, a water recyling and the electronical control unit. The combination of the zoological part, and the botanical part with a common control system in the AQUARACK, however, results in a "Closed Equilibrated Biological Aquatic System" (C.E.B.A.S.) representing an closed artificial ecosystem. Although this is disposed primarily as an experimental device for basic zoological, botanical and interdisciplinary research it opens the theoretical possibility to adapt it for combined production of animal and plant biomass on ground or in space. The paper explains the basic conception of the hardware construction of the zoological part of the system, the corresponding scientific frame program including the choice of the experimental animals and gives some selected examples of the hardware-related research. It further on discusses the practical and economical relevance of the system in the development of a controlled aquatical life support system in general.     

Aquatic animal research in space station and its issues--focus on support technology on nitrate toxicity.

We studied the effects of accumulated nitrate in water on the spawning, hatching and development of medaka using a simple nitrifying filter and a combined filter having both nitrifying and denitrifying capabilities. A nitrate concentration of 100 mg NO3(-)-N/L was clearly of lethal toxicity to fish when they were exposed to nitrate in both adult and the growing phases. A nitrate concentration of 75 mg NO3(-)-N/L reduced the fertilization rate, delayed the hatching time and reduced the hatching rate of the eggs laid by adults and decreased the growth rate of juveniles. In addition, nitrate accumulations as low as 50 mg NO3(-)-N/L remarkably retarded spawning and lowered the number of eggs laid by fish exposed in the juvenile phase. The effects on the reproduction system may be initiated by a low concentration, approximately 30 mg NO3(-)-N/L.     

Basic design concept of Closed Ecology Experiment Facilities.

In order to study the relationship between the physiological metabolism of living things and the environmental factors such as the atmospheric contents and so on within the closed ecosystem, Closed Ecology Experiment Facilities (CEEF) were designed and now under construction based on the following concepts: (1) Individual sealed chambers (called modules) for the plant cultivation, animal breeding, human habitation and microbial waste treatment are to be constructed independently to be able to study the metabolic effects of each living thing on the environmental factors within closed ecosystem. (2) A chamber for the microbial waste treatment are to be replaced with two systems; wet oxidation reactors and chemical nitrogen fixation reactors. (3) Atmospheric control systems are to be independently attached to each module for stabilizing atmospheric contents in each module. (4) Any construction materials having the possibility to absorb oxygen and carbon dioxide are to be prohibited to use in each module for sustaining material balance. (5) Facilities have to be developed so that the closed plant and animal experiments can be done independently, as well as integrated experiments with plants and animals through exchanging foods, carbon dioxide, oxygen, condensed water and nutrient solution.     

Design for a bioreactor with sunlight supply and operations systems for use in the space environment.

An experiment was carried out to determine the characteristics of an operations system that can support fast cultivation of algae at high densities in the weightlessness of space. The experiment was conducted in glass bioreactor tanks, in which light was supplied by radiator rods connected to optical fiber cables. The illumination areas of the tanks were 2600 cm2, 6000 cm2, and 9200 cm2 per liter of solution. The characteristics of O2-CO2 gas exchange, concentration and separation of chlorella in the growth medium, dialysis of ionic salts in the growth medium, etc. were examined. Chloralla ellipsoidea was used in the experiment, yielding the following results: (1) By increasing the ratio of illumination area to volume, growth rates of up to approximately 0.6 g/L h could be obtained in a highly concentrated solution (one that contains 20 g/L or more of algae). (2) The most suitable proportions of carbon dioxide and oxygen gases for growing algae quickly at high concentrations were found to be 10% CO2 and 10% O2 (by volume). (3) There was a high optimum concentration for fast cultivation, and the data obtained resembled the theoretical curve postulated by P. Behrens et al. (4) It was possible to exchange carbon dioxide and oxygen using gas-permeable membrane modules. (5) It was possible to separate the chlorella from the growth medium and recycle the medium.     

Understanding plant-soil relationships using controlled environment facilities.

Although soil is a component of terrestrial ecosystems, it is comprised of a complex web of interacting organisms, and therefore can be considered itself as an ecosystem. Soil microflora and fauna derive energy from plants and plant residues and serve important functions in maintaining soil physical and chemical properties, thereby affecting net primary productivity (NPP), and in the case of contained environments, the quality of the life support system. We have been using 3 controlled-environment facilities (CEF's) that incorporate different levels of soil biological complexity and environmental control, and differ in their resemblance to natural ecosystems, to study relationships among plant physiology, soil ecology, fluxes of minerals and nutrients, and overall ecosystem function. The simplest system utilizes growth chambers and specialized root chambers with organic-less media to study the physiology of plant-mycorrhizal associations. A second system incorporates natural soil in open-top chambers to study soil bacterial and fungal population response to stress. The most complex CEF incorporates reconstructed soil profiles in a "constructed" ecosystem, enabling close examination of the soil foodweb. Our results show that closed ecosystem research is important for understanding mechanisms of response to ecosystem stresses. In addition, responses observed at one level of biological complexity may not allow prediction of response at a different level of biological complexity. In closed life support systems, incorporating soil foodwebs will require less artificial manipulation to maintain system stability and sustainability.