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.     

Quantitative criteria for estimation of natural and artificial ecosystems functioning.

Using biotic turnover of substances in trophic chains, natural and artificial ecosystems are similar in functioning, but different in structure. It is necessary to have quantitative criteria to evaluate the efficiency of artificial ecosystems (AES). These criteria are dependent on the specific objectives for which the AES are designed. For example, if AES is considered for use in space, important criteria are efficiency in use of mass, power, volume (size) and human labor and reliability. Another task involves the determination of quantitative criteria for the functioning of natural ecosystems. To solve the problem, it is fruitful to use a hierarchical approach suitable for both individual links and the ecosystem as a whole. Energy flux criteria (principles) were developed to estimate the functional activities of biosystems at the population, community and ecosystem levels. A major feature of ecosystems as a whole is their biotic turnover of matter the rate of which is restricted by the lack of limiting substances. Obviously, the most generalized criterion is to take into account the energy flux used by the biosystem and the quantity of limiting substance included in its turnover. The use of energy flux by ecosystem, E(USED)--is determined from the photoassimilation of CO2 by plants (per time unit). It can be approximately estimated as the net primary production of photosynthesis (NPP). So, the ratio of CO2 photoassimilation rate (sometimes, measured as NPP) to the total mass of limiting substrate can serve as a main universal criterion (MUC). This MUC characterizes the specific cycling rate of limiting chemical elements in the system and effectiveness of every ecosystem including the global Biosphere. Comparative analysis and elaboration of quantitative criteria for estimation of natural and artificial ecosystems activities is of high importance both for theoretical considerations and for real applications.     

"SYMBIOSE" SYstem for Microgravity BIOregenerative Support of Experiments.

SYMBIOSE is an ESA supported research and development program which aims at establishing a first pilot model of a closed ecological system, compatible with operation in weightlessness conditions, and dedicated to scientific investigations in the microgravity environment. It integrates microalgal photosynthesis within an artificial ecosystem featuring a symbiotic strain of Chlorella (241 .80, Gottingen), which synthesizes and excretes substantial amounts of maltose, and is further looped on a consumer compartment. A technological concept has been developed. It is presently being integrated in order to gain knowledge on the system dynamics, and ultimately demonstrate the feasibility of such a biotechnology. Preliminary work on the photosynthetic metabolism of this microalga is being undertaken in order 1) to support later a mathematical formalisation of the dynamics of this artificial ecosystem, and, on this basis, 2) to compensate for its lack of stability with model-based external control. The most recent results are presented, along with a new design of the photobioreactor which integrates efficient light energy capture, microgravity compatible gas transfer and reduced shear stress.     

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.     

Key factors in development of man-made and natural ecosystems.

Key factors of ecosystem functioning are of the same nature for artificial and natural types. An hierarchical approach gives the opportunity for estimation of the quantitative behavior of both individual links and the system as a whole. At the organismic level we can use interactions of studied macroorganisms (man, animal, higher plant) with selected microorganisms as key indicating factors of the organisms immune status. The most informative factor for the population/community level is an age structure of populations and relationships of domination/elimination. The integrated key factors of the ecosystems level are productivity and rates of cycling of the limiting substances. The key factors approach is of great value for growth regulations and monitoring the state of any ecosystem, including the life support system (LSS)-type.     

ECOSIMP2 model: prediction of CO2 concentration changes and carbon status in closed ecosystems.

The ECOSIMP2 model, simulating the Plant-Soil-Atmosphere interactions, was developed as a tool for the management of an experimental artificial ecosystem. It consists in three main carbon compartments for production, consumption and decomposition of the biomass. The main biological parameters concern photosynthesis (apparent Km, CO2 compensation point), the harvest index, the rate of consumption, and the kinetics of litter decomposition. From realistic assumptions of kinetics of soil compartments, a steady-state case was obtained, simulating a terrestrial ecosystem. The stability of the atmospheric CO2 concentration was studied after a virtual enclosure of the system in a 20-m high greenhouse. In natural lighting the conditions of stability are severe because of the small size of the atmospheric compartment which amplifies any imbalance between carbon fluxes. The positive consequence of that amplification for research on artificial ecosystems was emphasized.     

Biological life-support systems for Mars mission.

Mars mission like the Lunar base is the first venture to maintain human life beyond earth biosphere. So far, all manned space missions including the longest ones used stocked reserves and can not be considered egress from biosphere. Conventional path proposed by technology for Martian mission LSS is to use physical-chemical approaches proved by the experience of astronautics. But the problem of man living beyond the limits of the earth biosphere can be fundamentally solved by making a closed ecosystem for him. The choice optimum for a Mars mission LSS can be substantiated by comparing the merits and demerits of physical-chemical and biological principles without ruling out possible compromise between them. The work gives comparative analysis of ecological and physical-chemical principles for LSS. Taking into consideration universal significance of ecological problems with artificial LSS as a particular case of their solution, complexity and high cost of large-scale experiments with manned LSS, it would be expedient for these works to have the status of an International Program open to be joined. A program of making artificial biospheres based on preceding experience and analysis of current situation is proposed.     

Activity of the sympathetic-adrenomedullary system in rats after space flight on the COSMOS biosatellites

The indicators of adrenomedullary activity (catecholamine content (CA) and the activity of the catecholamine-synthesizing enzymes tyrosine hydroxylase (TH) and dopamine-β-hydroxylase (DBH)) were measured in the adrenal glands of rats living in a state of weightlessness for 18.5–19.5 days on board the biosatellites COSMOS 936 and COSMOS 1129. None of these indicators was significantly changed by space flight, neither in the group living in a state of weightlessness nor in the group living in a centrifuge on board the spacecraft and exposed to artificial gravity of 1 g (COSMOS 936). Animals exposed after space flight to repeated immobilization stress on Earth showed a significant decrease of adrenal adrenaline and an appreciable increase in adrenal TH activity compared to stressed animals which were not in space. These results suggest that a prolonged state of weightlessness during space flight does not by itself represent an intensive stressful stimulus for the adrenomedullary system but potentiates the response of cosmonauts to stress after return to Earth.     

An overview of Japanese CELSS research activities.

Many research activities regarding Controlled Ecological Life Support System (CELSS) have been conducted and continued all over the world since the 1960's and the concept of CELSS is now changing from Science Fiction to Scientific Reality. Development of CELSS technology is inevitable for future long duration stays of human beings in space, for lunar base construction and for manned mars flight programs. CELSS functions can be divided into two categories, Environment Control and Material Recycling. Temperature, humidity, total atmospheric pressure and partial pressure of oxygen and carbon dioxide, necessary for all living things, are to be controlled by the environment control function. This function can be performed by technologies already developed and used as the Environment Control Life Support System (ECLSS) of Space Shuttle and Space Station. As for material recycling, matured technologies have not yet been established for fully satisfying the specific metabolic requirements of each living thing including human beings. Therefore, research activities for establishing CELSS technology should be focused on material recycling technologies using biological systems such as plants and animals and physico-chemical systems, for example, a gas recycling system, a water purifying and recycling system and a waste management system. Based on these considerations, Japanese research activities have been conducted and will be continued under the tentative guideline of CELSS research activities as shown in documents /1/, /2/. The status of the over all activities are discussed in this paper.     

Pilot CELSS based on a maltose-excreting Chlorella: concept and overview on the technological developments.

A typical ecosystem is composed of three compartments: photosynthetic producer (anabolizing processes), consumer and decomposer (catabolizing processes). It is still far too much complex, however, to form the basis on which establishing an engineered artificial ecosystem, dedicated to support life (of the consumer) in space. A simpler, two compartments, pilot model to start with has been selected. It is based on a symbiotic Chlorella (strain 241.80), which can be tuned, at low pH, to produce maltose. This feature prevents the accumulation of useless biomass, not readily edible by the consumer. Being excreted, maltose is easily recoverable, and constitutes a direct source of carbon suitable for many consumers. Since they will totally catabolize it back to CO2, the necessity for a decomposer compartment is avoided. The present status of the technological concept designed to support life of small consumers (animals, microorganisms) will be presented, taking into account the space compatibility of the technologies developed.     

MELISSA: a potential experiment for a precursor mission to the Moon.

MELISSA (Micro-Ecological Life Support System Alternative) has been conceived as a micro-organism based ecosystem intended as a tool for developing the technology for a future artificial ecosystem for long term space missions, as for example a lunar base. The driving element of MELISSA is the recovering of edible biomass from waste, CO2, and minerals with the use of sun light as energy source. In this publication, we focus our attention on the potential applications of MELISSA for a precursor mission to the Moon. We begin by a short review of the requirements for bioregenerative Life Support. We recall the concept of MELISSA and the theoretical and technical approaches of the study. We present the main results obtained since the beginning of this activity and taking into account the requirements of a mission to the Moon we propose a preliminary experiment based on the C cycle of the MELISSA loop.     

“Biospherics” approach for studies of natural and artificial ecosystems

The main unifying feature of natural and artificial ecosystems is their biotic turnover (cycling) of substances which is induced with energy fluxes. A new integrating scientific discipline – Biospherics – studies biotic cycles (both in experiments and in mathematical models) of different degree of closure and complexity. By its origin, Biospherics is to be connected with extensive studies of Biosphere by Russian academician Vladimir Vernadsky. He developed and used “empirical generalizations” based on innumerous observations, comparisons and reflections. His “bio-geo-chemical principles” of Biosphere and ecosystems development have more qualitative than quantitative nature. Quantitative criteria to evaluate the efficiency of natural and artificial ecosystems are to take into account energy fluxes and their use in ecosystems of different types. At least, three of them are of value for estimation of natural and artificial ecosystems’ functional activities. Energy principle of extensive development (EPED), energy principle of intensive development (EPID) and main universal (generalized) criterion (MUC). The last criterion (Principle) characterizes the specific cycling rate of limiting chemical elements in multi-organism systems, developing under external energy fluxes. Its value can be a quantitative measure of effectiveness for every ecosystem functioning, including our global Biosphere. Different examples of these (above-mentioned) integrated criteria actions are presented and analyzed in the paper.     

The influence of gravity on structure and function of animals

Gravity is the only environmental parameter that has remained constant during the period of evolution of living matter on Earth. Thus, it must have been a major force in shaping livimg things. The influence of gravitational loading on evolution of the vertebrate skeleton is well recognized, and scale effects have been studied. This paper, however, considers in addition four pivotal events in early evolution that would seem to have been significant for the later success and diversification of animal life. These are evolution of the cytoskeleton, cell motility (flagellae and cilia), gravity detecting devices (accelerometers), and biomineralization. All are functionally calcium dependent in eukaryotes and all occurred or were foreshadowed in prokaryotes. A major question is why calcium was selected as an ion of great importance to the structure and function of living matter; another is whether gravity played a role in its selection.     

MELISSA: global control strategy of the artificial ecosystem by using first principles models of the compartments.

MELISSA is a micro-organisms based ecosystem conceived as a tool for understanding the behaviour of artificial ecosystems, and developing the technology for a future biological life support system for long term space mission. The driving element of MELISSA is the recovering of oxygen and edible biomass from waste (faeces, urea). Due to its intrinsic instability and the safety requirements of manned missions, an important control strategy is developed to pilot this system and to optimize its recycling performance. This is a hierarchical control strategy. Each MELISSA compartment has its local control system, and taking into account the states of other compartments and a global desired functioning point, the upper level determines the setpoints for each compartment. The developed approach is based on first principles models of each compartment (physico chemical equations, stoichiometries, kinetic rates, ...). Those models are used to develop a global simulator of the system (in order to study the global functioning). They are also used in the control strategy, which is a non linear predictive model based strategy. This paper presents the general approach of the control strategy of the loop from the compartment level up to the overall loop. At the end, some simulation and experimental results are presented.     

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.     

A combined modeling and experimental approach for achieving a simplified closed ecosystem.

In CELSS both biological and physico-chemical processes have to be used to support the main needs of the crews and to minimize the re-supply of food and air from Earth. The basic idea is to create a complete food chain (an artificial ecosystem), beginning from the crew, with its wastes, and returning to the crew to supply it with food and air. Two main other steps of this food chain are a waste treatment process and a biomass production including higher plants. We set up the connection of these key modules, which we called ECLAS (Ecosysteme Clos Artificiel Simplifie). A growth chamber containing higher plants is connected to a continuous supercritical water oxidation reactor, that converts the harvested biomass into carbon dioxide and enables the photosynthesis of the canopy. To achieve a stable coupling through optimized regulations between the modules, we programmed a modular numerical simulation of the system, in order to assess the involved fluxes and to constrain the last degrees of freedom of the experimental system already built. Simulation results and the first experimental results are here compared.     

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.     

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.     

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.