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.     

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.     

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.     

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.     

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.     

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 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.     

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.     

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.     

AToxMss--from a space proven payload to a validated testsystem in ecotoxicology.

The C.E.B.A.S.-Minimodule (Closed Equilibrated Biological Aquatic System) is a space qualified aquatic microcosm of 8.6 liters volume of water. Several aquatic species can be reared in parallel. Based on its characteristics (closed system, highly standardized, testruns longer than 4 weeks are possible, organisms of different trophic levels can be investigated) an improved module (C.B.R.U.=Closed Biological Research Unit) is under development for scientific and commercial use in ecotoxicology. In a two year project named AToxMss (Aquatic EcoToxicology in a Multispecies System) this goal can be reached. AToxMss is a R&D project of an industry team and two teams of the University of Bremen, funded by industry (OHB-System AG) and the state of Bremen, Germany. Three project phases are already completed: The parameter determination to indicate potential impact of chemicals on biological systems, the selection of test substances, as well as the manufacturing of 3 functional modules, each verified for use in ecotoxicological research. The next phase starts with a series of test runs calibrating the system by using well known toxic substances and chemicals.     

Stratospheric ozone loss, ulraviolet effects and action spectroscopy

The major effect of stratospheric ozone loss will be an increase in the amount of ultraviolet radiation reaching the ground. This increase will be entirely contained within the UV-B (290–320nm). How this will impact life on Earth will be determined by the UV-B photobiology of exposed organisms, including humans. One of the analytical methods useful in estimating these effects is Action Spectroscopy (biological effect as a function of wavelength). Carefully constructed action spectra will allow us to partially predict the increase in bio-effect due to additional UV exposure. What effect this has on the organism and the system in which the organism resides is of paramount importance. Suitable action spectra already exist for human skin cancer, human cell mutation and killing, and for one immune response. Comprehensive and widely applicable action spectra for terrestrial and aquatic plant responses are being generated but are not yet suitable for extensive analysis. There is little data available for animals, other than those experiments completed in the laboratory as model systems for human studies. Some polychromatic action spectra have proven useful in determining the possible impact of ozone loss on biological systems. The pitfalls and limits of this approach will be addressed.     

Experimental and mathematical modeling of the consumer's influence on productivity of algae in a model aquatic ecosystem.

A "producer-consumer" (Chlorella vulgaris-Paramecium caudatum) closed aquatic system has been investigated experimentally and theoretically. It has been found that there is a direct relationship between the growth of the paramecia population and their release of ammonia nitrogen, which is the best form of nitrogen for Chlorella growth. The theoretical study of a model of a "producer-consumer" aquatic biotic cycle with spatially separated compartments has confirmed the contribution of paramecia to nitrogen cycling. It has been shown that an increase in the concentration of nitrogen released as metabolites of paramecia is accompanied by an increase in the productivity of microalgae.     

NASDA aquatic animal experiment facilities for Space Shuttle and ISS.

National Space Development Agency of Japan (NASDA) has developed aquatic animal experiment facilities for NASA Space Shuttle use. Vestibular Function Experiment Unit (VFEU) was firstly designed and developed for physiological research using carp in Spacelab-J (SL-J, STS-47) mission. It was modified as Aquatic Animal Experiment Unit (AAEU) to accommodate small aquatic animals, such as medaka and newt, for second International Microgravity Laboratory (IML-2, STS-65) mission. Then, VFEU was improved to accommodate marine fish and to perform neurobiological experiment for Neurolab (STS-90) and STS-95 missions. We have also developed and used water purification system which was adapted to each facility. Based on these experiences of Space Shuttle missions, we are studying to develop advanced aquatic animal experiment facility for both Space Shuttle and International Space Station (ISS).     

Functional, regulatory and indicator features of microorganisms in man-made ecosystems.

Functional, regulatory and indicator features of microorganisms in development and functioning of the systems and sustaining stability of three man-made ecosystem types has been studied. 1) The functional (metabolic) feature was studied in aquatic ecosystems of biological treatment of sewage waters for the reducer component. 2) The regulatory feature of bacteria for plants (producer component) was studied in simple terrestrial systems "wheat plants-rhizospheric microorganisms-artificial soil" where the behavior of the system varied with activity of the microbial component. For example with atmospheric carbon dioxide content elevated microbes promote intensification of photosynthesis processes, without binding the carbon in the plant biomass. 3) The indicator feature for the humans (consumer component) was studied in Life Support Systems (LSS). High sensitivity of human microflora to system conditions allowed its use as an indicator of the state of both system components and the entire ecosystem. Grant numbers: N99-04-96017, N15.     

Mutagenic effects of heavy ions in bacteria.

Various mutagenic effects by heavy ions were studied in bacteria, irradiated at accelerators in Dubna, Prague, Berkeley or Darmstadt. Endpoints investigated are histidine reversion (B. subtilis, S. typhimurium), azide resistance (B. subtilis), mutation in the lactose operon (E. coli), SOS chromotest (E. coli) and lambda-prophage induction (E. coli). It was found that the cross sections of the different endpoints show a similar dependence on energy. For light ions (Z < or = 4) the cross section decreases with increasing energy. For ions of Z = 10, it is nearly independent of energy. For heavier ions (Z > or = 26) it increases with energy up to a maximum or saturation. The increment becomes steeper with increasing Z. This dependence on energy suggests a "mutagenic belt" inside the track that is restricted to an area where the density of departed energy is low enough not to kill the cell, but high enough to induce mutations.     

Life support for aquatic species--past; present; future.

Life Support is a basic issue since manned space flight began. Not only to support astronauts and cosmonauts with the essential things to live, however, also animals which were carried for research to space etc. together with men need support systems to survive under space conditions. Most of the animals transported to space participate at the life support system of the spacecraft. However, aquatic species live in water as environment and thus need special developments. Research with aquatic animals has a long tradition in manned space flight resulting in numerous life support systems for them starting with simple plastic bags up to complex support hardware. Most of the recent developments have to be identified as part of a technological oriented system and can be described as small technospheres. As the importance arose to study our Earth as the extraordinary Biosphere we live in, the modeling of small ecosystems began as part of ecophysiological research. In parallel the investigations of Bioregenerative Life Support Systems were launched and identified as necessity for long-term space missions or traveling to Moon and Mars and beyond. This paper focus on previous developments of Life Support Systems for aquatic animals and will show future potential developments towards Bioregenerative Life Support which additionally strongly benefits to our Earth's basic understanding.     

Population dynamics of an algal-bacterial cenosis in closed ecological system.

The paper deals with microalgae-bacteria interrelationships in the "autotroph-heterotroph" aquatic biotic cycle. Explanations of why and how algal-bacterial ecosystems are formed still remain controversial. The paper presents results of experimental and theoretical investigations of the functioning of the algal-bacterial cenosis (the microalga Chlorella vulgaris and concomitant microflora). The Chlorella microbial community is dominated by representatives of the genus Pseudomonas. Experiments with non-sterile batch cultures of Chlorella on Tamiya medium showed that the biomass of microorganisms increases simultaneously with the increase in microalgal biomass. The microflora of Chlorella can grow on organic substances released by photosynthesizing Chlorella. Microorganisms can also use dying Chlorella cells, i.e. form a "producer-reducer" biocycle. To get a better insight into the cenosis-forming role of microalgae, a mathematical model of the "autotroph-heterotroph" aquatic biotic cycle has been constructed, taking into account the utilization of Chlorella photosynthates and dead cells by microorganisms and the contribution of the components to the nitrogen cycle. A theoretical study showed that the biomass of concomitant bacteria grown on glucose and detritus is larger than the biomass of bacteria utilizing only microalgal photosynthates, which agrees well with the experimental data.     

An integrated Engineered Closed/Controlled EcoSystem for a lunar base.

Long-term human missions in space, such as the establishment of a human-tended lunar base, require autonomous life support systems. A Lunar Engineered Closed/Controlled EcoSystem (LECCES) can provide autonomy by integrating a human module with support plant and animal modules, and waste treatment subsystems. Integration of physical/chemical (P/C) and biological waste treatment subsystems can lead to viable and operational bioregenerative systems that minimize resupply requirements from Earth. A top-level diagram for LECCES is developed based on the human module requirements. The proposed diagram is presented and its components are discussed.     

Plant-module for autonomous space support (P-MASS).

A wide variety of technical and science questions arise when attempting to envision the long-term support of plants, algae and bacteria in space. Currently, spaceflight data remain elusive since there are no U.S. carriers for investigating either the germane technical or scientific issues. The first flight of the Commercial Experiment Transporter (COMET) will provide a nominal 30 day orbital opportunity to evaluate such issues. The P-MASS is a small payload that is designed to meet the mass (40 lbs.), volume (1.5 cu.ft.), and power (120 W) constraints of one of several COMET payloads while enabling flight evaluations of plants, algae and bacteria. Various P-MASS subsystems have been subjected to extensive ground tests as well as KCl35 tests. Various biological sub-systems have been similarly evaluated. Through a variety of sensors coupled with color video, the P-MASS performance and the supported biological systems will be compared for terrestrial controls versus spaceflight materials. This small, low cost payload should return valuable information regarding the requirements for hardware and biological systems needed to move toward bioregenerative life support systems in space. In addition, it should be possible to accurately identify major unresolved difficulties that may arise in the long-term, spaceflight support of various biological systems. Finally, this generic spaceflight capability should enable a variety of plant research programs focused on the use of microgravity to modulate and exploit plant products for commercial applications ranging from new agricultural products to pharmacological feedstocks and new controlled agricultural strategies.