Aquatic modules for bioregenerative life support systems based on the C.E.B.A.S. biotechnology

Most concepts for bioregenerative life support systems are based on edible higher land plants which create some problems with growth and seed generation under space conditions. Animal protein production is mostly neglected because of the tremendous waste management problems with tetrapods under reduced weightlessness. Therefore, the “Closed Equilibrated Biological Aquatic System” (C.E.B.A.S.) was developed which represents an artificial aquatic ecosystem containing aquatic organisms which are adpated at all to “near weightlessness conditions” (fishes Xiphophorus helleri, water snails Biomphalaria glabrata, ammonia oxidizing bacteria and the rootless non-gravitropic edible water plant Ceratophyllum demersum). Basically the C.E.B.A.S. consists of 4 subsystems: a ZOOLOGICASL COMPONENT (animal aquarium), a BOTANICAL COMPONENT (aquatic plant bioreactor), a MICROBIAL COMPONENT (bacteria filter) and an ELECTRONICAL COMPONENT (data acquisition and control unit). Superficially, the function principle appears simple: the plants convert light energy into chemical energy via photosynthesis thus producing biomass and oxygen. The animals and microorganisms use the oxygen for respiration and produce the carbon dioxide which is essential for plant photosynthesis. The ammonia ions excreted by the animals are converted by the bacteria to nitrite and then to nitrate ions which serve as a nitrogen source for the plants. Other essential ions derive from biological degradation of animal waste products and dead organic matter. The C.E.B.A.S. exists in 2 basic versions: the original C.E.B.A.S. with a volume of 150 liters and a self-sustaining standing time of more than 13 month and the so-called C.E.B.A.S. MINI MODULE with a volume of about 8.5 liters. In the latter there is no closed food loop by reasons of available space so that animal food has to be provided via an automated feeder. This device was flown already successfully on the STS-89 and STS-90 spaceshuttle missions and the working hypothesis was verified that aquatic organisms are nearly not affected at all by space conditions, i . e. that the plants exhibited biomass production rates identical to the ground controls and that as well the reproductive, and the immune system as the the embryonic and ontogenic development of the animals remained undisturbed. Currently the C.E.B.A.S. MINI MODLULE is prepared for a third spaceshuttle fligt (STS-107) in spring 2001. Based on the results of the space experiments a series of prototypes of aquatic food production modules for the implementation into BLSS were developed. This paper describes the scientific disposition of the STS-107 experiments and of open and closed aquaculture systems based on another aquatic plant species, the Lemnacean Wolffia arrhiza which is cultured as a vegetable in Southeastern Asia. This plant can be grown in suspension culture and several special bioreactors were developed for this purpose. W. arrhiza reproduces mainly vegetatively by buds but also sexually from time to time and is therefore especially suitable for genetic engineering, too. Therefore it was used, in addition, to optimize the C.E.B.A.S. MINI MODULE to allow experiments with a duration of 4 month in the International Space Station the basic principle of which will be explained. In the context of aquaculture systems for BLSS the continuous replacement of removed fish biomass is an essential demand. Although fish reproduction seems not to be affected in the short-term space experiments with the C.E.B.A.S. MIMI MODULE a functional and reliable hatchery for the production of siblings under reduced weightlessness is connected with some serious problems. Therefore an automated “reproduction module” for the herbivorous fish Tilapia rendalli was developed as a laboratory prototype. It is concluded that aquatic modules of different degrees of complexity can optimize the productivity of BLSS based on higher land plants and that they offer an unique opportunity for the production of animal protein in lunar or planetary bases.     

Novel aquatic modules for bioregenerative life-support systems based on the closed equilibrated biological aquatic system (C.E.B.A.S.)

The closed equilibrated biological aquatic system (C.E.B.A.S) is a man-made aquatic ecosystem which consists of four subcomponents: an aquatic animal habitat, an aquatic plant bioreactor, an ammonia oxidizing bacteria filter and a data acquisition/control unit. It is a precursor for different types of fish and aquatic plant production sites which are disposed for the integration into bioregenerative life-support systems. The results of two successful spaceflights of a miniaturized C.E.B.A.S version (the C.E.B.A.S. MINI MODULE) allow the optimization of aquatic food production systems which are already developed in the ground laboratory and open new aspects for their utilization as aquatic modules in space bioregenerative life support systems. The total disposition offers different stages of complexity of such aquatic modules starting with simple but efficient aquatic plant cultivators which can be implemented into water recycling systems and ending up in combined plant/fish aquaculture in connection with reproduction modules and hydroponics applications for higher land plants. In principle, aquaculture of fishes and/or other aquatic animals edible for humans offers optimal animal protein production under lowered gravity conditions without the tremendous waste management problems connected with tetrapod breeding and maintenance. The paper presents details of conducted experimental work and of future dispositions which demonstrate clearly that aquaculture is an additional possibility to combine efficient and simple food production in space with water recycling utilizing safe and performable biotechnologies. Moreover, it explains how these systems may contribute to more variable diets to fulfill the needs of multicultural crews.     

Novel laboratory approaches to multi-purpose aquatic bioregenerative closed-loop food production systems

Based on the construction principle of the Closed Equilibrated Biological Aquatic System (C.E.B.A.S.) two novel combined animal-plant production systems were developed in laboratory scale the first of which is dedicated to mid-term operation in closed state up to two years. In principle both consist of the "classic" C.E.B.A.S. subcomponents: animal tank (Zoological Component), plant cultivators (Botanical Component), ammonia converting bacteria filter (Microbial Component) and data acquisition/control unit (Electronical Component). The innovative approach in the first system is the utilization of minimally three aquatic plant cultivators for different species. In this one the animal tank has a volume of about 160 liters and is constructed as an "endless-way system" surrounding a central unit containing the heat exchanger and the bacteria filter with volumes of about 1.5 liters each. A suspension plant cultivator (1 liter) for the edible duckweed Wolffia arrhiza is externally connected. The second plant cultivator is a meandric microalgal bioreactor for filamentous green algae. The third plant growth facility is a chamber with about 2.5 liters volume for cultivation of the "traditional" C.E.B.A.S. plant species, the rootless buoyant Ceratophyllum demersum. Both latter units are illuminated with 9 W fluorescent lamps. In the current experiment the animal tank contains the live-bearing teleost fish Xiphophorus helleri and the small pulmonate water snail Biomphalaria glabrata because their physiological adaptation to the closed system conditions is well known from many previous C.E.B.A.S. experiments. The water temperature is maintained at 25 degrees C and the oxygen level is regulated between 4 and 7 mg/l by switching on and off the plant cultivator illuminations according to a suitable pattern thus utilizing solely the oxygen produced by photosynthesis. The animals and the microorganisms of filter and biofilm provide the plants with a sufficient amount of carbon dioxide. Oxygen concentration, pH value, temperature and redox potential are on-line recorded. Ion concentrations and numbers of living germs in the system water are determined twice monthly in the laboratory from samples taken from a special "sample removal module"; the sample volume is automatically replaced from an reservoir container. A rotatory pump produces a water flow of about 38 l/min. For a similar smaller test system with approx. 10 l volume developed from the C.E.B.A.S.-MINI-MODULE a novel indirect solar energy supply is tested which has a buffer capacity to maintain the system for 7 days in darkness under central European climate conditions also in winter. It contains only a single plant cultivator which is operated with Wollfia arrhiza. This lemnacean plant is able to produce large amounts of plant biomass in a short time by vegetative reproduction via daughter fronds. This easy-to-handle apparatus is dedicated to be operative more than 4 month. The experimental animals and microorganisms are the same as in the large system. The paper provides detailed information on the system construction principles and the biological, physical and chemical data of the initial phase of the test runs of both systems with the main focus on the large one.     

Animal protein production modules in biological life support systems: novel combined aquaculture techniques based on the Closed Equilibrated Biological Aquatic System (C.E.B.A.S.)

Based on the experiences made with the Closed Equilibrated Biological Aquatic System (C.E.B.A.S.) which was primarily deveoloped for long-term and multi-generation experiments with aquatic animals and plants in a space station highly effective fresh water recycling modules were elaborated utilizing a combination of ammonia oxidizing bacteria filters and higher plants. These exhibit a high effectivity to eliminate phosphate and anorganic nitrogen compounds and arc. in addidition. able to contribute to the oxygen supply of the aquatic animals. The C.E.B.A.S. filter system is able to keep a closed artificial aquatic ecosystem containing teleost fishes and water snails biologically stable for several month and to eliminate waste products deriving from degraded dead fishes without a decrease of the oxygen concentration down to less than 3.5 mg/l at 25 °C. More advanced C.E.B.A.S. filter systems, the BIOCURE filters, were also developed for utilization in semiintensive and intensive aquaculture systems for fishes. In fact such combined animal-plant aquaculture systems represent highly effective productions sites for human food if proper plant and fish species are selected The present papers elucidates ways to novel aquaculture systems in which herbivorous fishes are raised by feeding them with plant biomass produced in the BIOCURE filters and presents the scheme of a modification which utilizes a plant species suitable also for human nutrition. Special attention is paid to the benefits of closed aquaculture system modules which may be integrated into bioregenerative life support systems of a higher complexity for, e. g.. lunar or planetary bases including some psychologiccal aspects of the introduction of animal protein production into plant-based life support systems. Moreover, the basic reproductive biological problems of aquatic animal breeding under reduced gravity are explained leading to a disposition of essential research programs in this context.     

Perspectives of different type biological life support systems (BLSS) usage in space missions

In the paper an attempt is made to combine three important criteria of LSS comparison: minimum mass, maximum safety and maximum quality of life. Well-known types of BLSS were considered: with higher plant, higher plants and mushrooms, microalgae, and hydrogen-oxidizing bacteria. These BLSSs were compared in terms of "integrated" mass for the case of a vegetarian diet and a "normal" one (with animal proteins and fats). It was shown that the BLSS with higher plants and incineration of wastes becomes the best when the exploitation period is more than 1 yr. The dependence of higher plants' LSS structure on operation time was found. Comparison of BLSSs in terms of integral reliability (this criterion includes mass and quality of life criteria) for a lunar base scenario showed that BLSSs with higher plants are advantageous in reliability and comfort. This comparison was made for achieved level of technology of closing and for perspective one.     

Response of Phaseolus vulgaris L. plants to low-let ionizing radiation: Growth and oxidative stress

The scenarios for the long-term habitation of space platforms and planetary stations involve plants as fundamental part of Bioregenerative Life Support Systems (BLSS) to support the crew needs. Several constraints may limit plant growth in space: among them ionizing radiation is recognized to severely affect plant cell at morphological, physiological and biochemical level. In this work, plants of Phaseolus vulgaris L. were subjected to four different doses of X-rays (0.3, 10, 50 and 100 Gy) in order to assess the effects of ionizing radiation on this species and to analyze possible mechanisms carried out to overcome the radiation injuries. The effects of X-rays on plant growth were assessed by measuring stem elongation, number of internodes and leaf dry weight. The integrity of photosynthetic apparatus was evaluated by photosynthetic pigment composition and ribulose 1,5-bisphosphate carboxylase (Rubisco) activity, whereas changes in total antioxidant pool and glutathione S transferase activity (GST) were utilized as markers of oxidative stress. The distribution of phenolic compounds in leaf tissues as natural shielding against radiation was also determined.Irradiation of plants at 0.3 and 10 Gy did not determine differences in all considered parameters as compared to control. On the contrary, at 50 and 100 Gy a reduction of plant growth and a decrease in photosynthetic pigment content, as well as an increase in phenolic compounds and a decrease in total antioxidant content and GST activity were found. Only a slight reduction of Rubisco activity in leaves irradiated at 50 and 100 Gy was found. The overall results indicate P. vulgaris as a species with a good potential to face ionizing radiation and suggest its suitability for utilization in BLSSs.     

Effect of weightlessness on sympathetic-adrenomedullary activity of rats.

Three cosmic experiments were performed in which rats spent 18-20 days in space on board the biosatellites "COSMOS 782", "COSMOS 936" and "COSMOS 1129". The following indicators of the sympathetic-adrenomedullary system (SAS) activity were measured: tissue and plasma catecholamines (CA), CA-synthesizing enzymes--tyrosine hydroxylase (TH), dopamine-beta-hydroxylase (DBH), phenylethanolamine-N-methyltransferase (PNMT)--as well as CA-degrading enzymes-monoamine oxidase (MAO) and catechol-O-methyltransferase (COMT). Adrenal epinephrine (EPI) and norepinephrine (NE) as well as CA-synthesizing and degrading enzymes were not significantly changed in the animals after flight on COSMOS 782. On the other hand, a significant increase was found in heart CA, the indicator which is usually decreased after stress. 26 days after landing all values were at control levels. The results obtained, compared to our previous stress experiments on Earth, suggest that prolonged weightlessness does not appear to be a pronounced stressful stimulus for the SAS. Heart and plasma CA, mainly NE, were increased both in the group living in the state of weightlessness and the group living in a centrifuge and exposed to artificial gravitation 1 g (COSMOS 936), suggesting again that prolonged weightlessness is not an intensive stressful stimulus for the SAS. The animals exposed after space flight on COSMOS 1129 to repeated immobilization stress on Earth showed a significant decrease of adrenal EPI and an expressive increase of adrenal TH activity compared to stressed animals which were not in space. Thus, the results corroborate that prolonged state of weightlessness during space flight though not representing by itself an intensive stressful stimulus for the sympathetic-adrenomedullary system, was found to potentiate the response of "cosmic rats" to stress exposure after return to Earth.     

Summary of medical investigations in the U.S.S.R. manned space missions.

The results of biomedical investigations carried out in the U.S.S.R. manned space missions are discussed. Their basic result is well-documented evidence that man can perform space flights of long duration. The investigations have demonstrated no direct correlation between inflight or postflight physiological reactions of crewmembers and flight duration. In all likelihood, this can be attributed to the fact that special exercises done inflight efficiently prevented adverse effects of weightlessness. However, human reactions to weightlessness need further study. They include negative calcium balance and anemia as well as vestibulo-autonomic disorders shown by crewmembers at early stages of weightlessness. Attention should be given to psychological, social-psychological and ethical problems that may also limit further increase in flight duration.     

Assessment of the possibility of establishing material cycling in an experimental model of the bio-technical life support system with plant and human wastes included in mass exchange

A pilot model of a bio-technical life support system (BTLSS) including human and plant wastes has been developed at the Institute of Biophysics SB RAS (Krasnoyarsk, Russia). This paper describes the structure of the photosynthesizing unit of the system, which includes wheat, chufa and vegetables. The study substantiates the simultaneous use of neutral and biological substrates for cultivating plants. A novel physicochemical method for the involvement of human wastes in the cycling has been employed, which enables the use of recycled products as nutrients for plants. Inedible plant biomass was subjected to biological combustion in the soil-like substrate (SLS) and was thus involved in the system mass exchange; NaCl contained in native urine was returned to the human through the consumption of Salicornia europaea, an edible salt-concentrating plant. Mass transfer processes in the studied BLSS have been examined for different chemical components.     

Six-month space greenhouse experiments--a step to creation of future biological life support systems

SVET Space Greenhouse (SG)--the first automated facility for growing of higher plants in microgravity was designed in the eighty years to be used for the future BLSS. The first successful experiment with vegetables was carried out in 1990 on the MIR Space Station (SS). The experiments in SVET SG were resumed in 1995, when an American Gas Exchange Measurement System (GEMS) was added. A three-month wheat experiment was carried out as part of MIR-SHUTTLE'95 program. SVET-2 SG Bulgarian equipment of a new generation with optimised characteristics was developed (financed by NASA). The new SVET-GEMS equipment was launched on board the MIR SS and a successful six-month experiments for growing up of two crops of wheat were conducted in 1996 - 97 as part of MIR-NASA-3 program. The first of these "Greenhouse" experiments (123 days) with the goal to grow wheat through a complete life cycle is described. Nearly 300 heads developed but no seeds were produced. A second crop of wheat was planted and after 42 days the plants were frozen for biochemical investigations. The main environmental parameters during the six-month experiments in SVET (substrate moisture and lighting period) are given. The results and the contribution to BLSS are discussed.     

U.S. prebreathe protocol

This paper identifies and describes the prebreathe protocol currently used by the U.S. Space Shuttle Program to provide astronauts the capability to safely perform extravehicular activity. A comparison of planned vs actual prebreathe experience through the STS-37 Mission is also provided.     

The first "space" vegetables have been grown in the "SVET" greenhouse using controlled environmental conditions

The paper describes the "SVET" project--a new generation of space greenhouse with small dimensions. Through the use of a minicomputer, "SVET" is fully capable of automatically operating and controlling environmental systems for higher plant growth. A number of preliminary studies have shown the radish and cabbage to be potentially important crops for CELSS (Closed Environmental Life Support System). The "SVET" space greenhouse was mounted on the "CRYSTAL" technological module docked to the Mir orbital space station on 10 June 1990. Soviet cosmonauts Balandin and Solovyov started the first experiments with the greenhouse on 15 June 1990. Preliminary results of seed cultivation over an initial 54-day period in "SVET" are presented. Morphometrical characteristics of plants brought back to Earth are given. Alteration in plant characteristics, such as growth and developmental changes, or morphological contents were noted. A crop of radish plants was harvested under microgravity conditions. Characteristics of plant environmental control parameters and an estimation of functional properties of control and regulation systems of the "SVET" greenhouse in space flight as received via telemetry data is reported.     

Changes in the loco-regional Cerebral Blood Flow (r.C.B.F.) during a simulation of weightlessness.

Experiments of prolonged bedrest in antiorthostatic position are conducted in order to simulated cardio-circulatory modifications observed in weightlessness. Until now, no studies of r.C.B.F. have been effected in these conditions. Six young, healthy volunteers (average age 23.8) were placed in strict bedrest and in antiorthostatic position -4 degrees for 7 days. The r.C.B.F. measurements were studied by 133Xe inhalation method using a 32 detectors system. Studies were made first in basal conditions, then between the 6th and 12th hr, and finally between the 72nd and the 78th hr after the beginning of the experiment. Three of the subjects received 0.450 mg of Clonidine daily during the experiment. In the subjects having taken no Clonidine, we observed a constant increase in r.C.B.F. (12, 17 and 16% respectively) in the first 12 hr; at the 72nd hour, all values had returned to basal state. This findings agrees with the well known notion of a rapid correction of hemodynamic disturbances observed in the first days of weightlessness. In the subjects treated with Clonidine, the increase of r.C.B.F. did not occur. Several mechanisms of action are possible; the Clonidine affecting either the heart by inhibiting volemic atrial receptors or the brain by direct vasoconstriction.     

Animals in biomedical space research

The use of experimental animals has been a major component of biomedical research progress. Using animals in space presents special problems, but also provides special opportunities. Rat and squirrel monkeys experiments have been planned in concert with human experiments to help answer fundamental questions concerning the effect of weightlessness on mammalian function. For the most part, these experiments focus on identified changes noted in humans during space flight. Utilizing space laboratory facilities, manipulative experiments can be completed while animals are still in orbit. Other experiments are designed to study changes in gravity receptor structure and function and the effect of weightlessness on early vertebrate development. Following these preliminary animals experiments on Spacelab Shuttle flights, longer term programs of animal investigation will be conducted on Space Station.     

航天飞机G417载荷研制及油滴与水滴接触微重力实验

文章介绍了曾经由航天飞机STS-64飞行任务搭载的编号为G417载荷的研制经验以及利用该载荷在航天飞机上开展的油滴与水滴接触微重力实验。北京卫星环境工程研究所承担了该载荷全部研制任务,其研制经验和飞行实验可为我国将来空间站开展微重力实验提供参考。     

Future investigations onboard Soviet biosatellites of the Cosmos series.

Many rat experiments onboard Cosmos biosatellites have furnished information concerning the effects of weightlessness, artificial gravity, and ionizing radiation combined with weightlessness on structural and biochemical parameters of the animal body. The necessity to expand the scope of physiological investigations has led to the project of flight primate studies. It is planned to carry out the first primate experiments onboard the Cosmos biosatellite in 1982. At present investigations of weightlessness effects on the cardiovascular and vestibular systems, higher nervous activity, skeletal muscles and biorhythms of two rhesus monkeys are being developed and tested. It is also planned to conduct a study of weightlessness effects on embryogenesis of rats and bioenergetics of living systems onboard the same biosatellite. Further experiments onboard Cosmos biosatellites are planned.     

Pathophysiology of motor functions in prolonged manned space flights.

The influence of weightlessness on different parts of the motor system have been studied in crew members of 140 and 175 days space flights. It has been shown that weightlessness affects all parts of the motor system including (i) the leg and trunk muscles, in which severe atonia, a decrease of strength and an increase of electromyographic cost of contraction have been observed, (ii) the proprioceptive elements and the spinal reflex mechanisms in which decreased thresholds accompanied by decreases of maximal amplitude of reflexes and disturbances in cross reflex mechanisms have been found. and (iii) the central mechanisms that control characteristics of postural and locomotor activities. The intensities and durations of disturbances of different parts of the motor system did not correlate to each other, but did correlate with prophylactic activity during space flight. The data suggest a different nature of disturbances caused by weightlessness in different parts of the motor system.     

Natriuretic peptide-sensitive guanylyl cyclase expression is down-regulated in human melanoma cells at simulated weightlessness

The membrane-bound guanylyl cyclases A and B (GC-A/B), which are receptors for natriuretic peptides, are expressed in cancer cells including melanomas and may represent new anticancer targets. Here, we report down-regulation of GC-A/B expression in human metastatic melanoma cells at simulated weightlessness in comparison to 1g conditions, suggesting attenuation of metastatic potential in weightlessness.     

Changes in the vestibular function during space flight

An analysis of observations and investigations carried out in space flight has shown that some cosmonauts and astronauts have experienced vestibular disorders during the transition to weightlessness. Vestibular-sensory disorders include: Spatial illusions (the feelings of falling down, being in an upside-down position, the sensations of rotation of the craft or the body) and vertigo occurring during the onset of the orbital flight and head movements; Feelings, similar to those experienced in response to Coriolis accelerations on the Earth, which occasionally develop in weightlessness during the spacecraft rotation upon abrupt head and body movements and restrained feet; Feelings "of the load on the vestibular analyser which is unlike any Earth-bound effects" upon abrupt head movements during the first hours of an orbital flight and "a prolonged movement" during the switch-off of thrusters in weightlessness. Vestibular-vegetative disorders comprise a complex of symptoms similar to those of motion sickness: loss of appetite, stomach awareness (12%), hypersalination, nausea (9.6%) and vomiting (4.8%). Soviet studies suggest that the vestibular tolerance to the flight effects depends on the natural stability and training to the cumulative effect of adequate vestibular stimuli. This has been used in the development of the system of vestibular selection. Changes in the vestibular function seem to play the major role in the development of motion sickness in weightlessness, extra-labyrinthine factors being contributory. The current hypotheses have not yet been adequately confirmed in experiments. A detailed physiological analysis allows the conclusion that the decisive factor in the development of motion sickness may be the disturbance of the function of analysers responsible for spatial orientation which take the form of sensory conflicts as well as an altered reactivity of the organism due to the hemodynamic rearrangement.