Interaction of a mixed yeast culture in an "autotroph-heterotroph" system with a closed atmosphere cycle and spatially separated components.

The study considers an experimental model of the "autotroph-heterotroph" system with a closed atmosphere cycle, in which the heterotrophic link is a mixed yeast population. The autotrophic link is represented by the algae Chlorella vulgaris and the heterotrophic link by the yeasts Candida utilis and Candida guilliermondii. The controls are populations of Chlorella and the same yeasts isolated from the atmosphere. It has been shown that the outcome of competition in the heterotrophic link depends on the strategy of the yeast population towards the substrate and oxygen. The C. utilis population quickly utilizes the substrate as it is an r-strategist and is less sensitive to oxygen deficiency. The C. guilliermondii population consumes low concentrations of the substrate because it is a K-strategist, but it is more sensitive to oxygen deficiency. That is why, in the "autotroph-heterotroph" system with a closed gas cycle, after a considerable amount of the substrate has been consumed, the C. guilliermondii population becomes more competitive that the C. utilis population. In the culture of yeasts, isolated from the atmosphere, the C. utilis population finds itself in more favorable conditions due to oxygen deficiency. The system with a complex heterotrophic component survive longer than a system whose heterotrophic component is represented by only one yeast species. This is explained for by the positive metabolite interaction of yeasts and a more complete utilization of the substrate by a mixed culture of yeasts featuring different strategies towards the substrate.     

Competition of the natural and manmade biotic cycles in the closed aquatic system.

This study addresses competition between the Paramecium bursaria and zoochlorella-endosymbiosis and the infusoria Paramecium caudatum in a closed aquatic system. The system is a natural model of a simple biotic cycle. P. bursaria consumes glucose and oxygen released by its zoochlorella and releases nitrogenous compounds and carbon dioxide necessary for algal photosynthesis. P. caudatum was fed on bacteria. It was shown that the infusoria P. bursaria united in one cycle with Chlorella had a higher competitive ability than P. caudatum. With any initial percentage of the infusoria in the mixed culture, the end portion of P. bursaria reached 90-99%, which was significantly higher than the end potion of the P. caudatum population. It is assumed that the sustenance expenditures of P. caudatum were greater than those of the endosymbiotic paramecium, i.e. the closing of the components into a biotic cycle leads to a decrease in sustenance expenditures.     

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.     

Self-restoration as fundamental property of CES providing their sustainability.

Sustainability is one of the most important criteria in the creation and evaluation of human life support systems intended for use during long space flights. The common feature of biological and physicochemical life support systems is that basically they are both catalytic. But there are two fundamental properties distinguishing biological systems: 1) they are auto-catalytic: their catalysts--enzymes of protein nature--are continuously reproduced when the system functions; 2) the program of every process performed by enzymes and the program of their reproduction are inherent in the biological system itself--in the totality of genomes of the species involved in the functioning of the ecosystem. Actually, one cell with the genome capable of the phenotypic realization is enough for the self-restoration of the function performed by the cells of this species in the ecosystem. The continuous microalgal culture of Chlorella vulgaris was taken to investigate quantitatively the process of self-restoration in unicellular algae population. Based on the data obtained, we proposed a mathematical model of the restoration process in a cell population that has suffered an acute radiation damage.     

Experimental and mathematical model of the interactions in the mixed culture of links in the “producer–consumer” cycle

The paper presents a experimental and mathematical model of interactions between invertebrates (the ciliates Paramecium caudatum and the rotifers Brachionus plicatilis) in the “producer–consumer” aquatic biotic cycle with spatially separated components. The model describes the dynamics of the mixed culture of ciliates and rotifers in the “consumer” component, feeding on the mixed algal culture of the “producer” component. It has been found that metabolites of the algae Scenedesmus produce an adverse effect on the reproduction of the ciliates P. caudatum. Taking into account this effect, the results of investigation of the mathematical model were in qualitative agreement with the experimental results. In the “producer–consumer” biotic cycle it was shown that coexistence is impossible in the mixed culture of invertebrates of the “consumer” component. The ciliates P. caudatum are driven out by the rotifers B. plicatilis.     

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.     

Catalase activity as a potential indicator of the reducer component of small closed ecosystems.

Dynamics of catalase activity has been shown to reflect the growth curve of microorganisms in batch cultivation (celluloselythic bacteria Bacillus acidocaldarius and bacteria of the associated microflora Chlorella vulgaris). Gas and substrate closure of the three component ecosystems with spatially separated components "producer-consumer-reducer" (Chl. vulgaris-Paramecium caudatum-B. acidocaldarius, two bacterial strains isolated from the associated microflora Chl. vulgaris) demonstrated that the functioning of the reducer component can be estimated by the catalase activity of mciroorganisms of this component.     

Energetic metabolism response in algae and higher plant species from simulation experiments with the clinostat.

Adenylate state is acknowledged to be among the most convenient approaches in the study of physiological changes in plant cells under simulation of altered gravity condition with the clinostat. Adenylate levels and the ATP/ADP ratio in cytoplasmic and mitochondrial extracts of cultivated cells of Haplopappus gracilis and algae cells of Chlorella vulgaris under initial stages of the fast-rotating and slow-rotating clinorotation, as well as the long-term clinorotation, have been investigated. For analysis of ATP and ADP levels in the plant cells under the clinorotation, we applied a high-sensitive bioluminescence method using the luciferase and piruvate kinase enzyme systems. It has been shown that the adenylate ratio is already increased during at the start of clinorotation with the different speed of rotation in the biological material tested. The considerable changes in mitochondrial ultrastructure of Chlorella cells, as well as the rising ATP level and dropping of the ATP/ADP ratio appear after long-duration clinorotation if compared to control material. It is probably connected with the distinctions in ATP-synthetase functioning in mitochondria of the cells under the clinorotation conditions.     

Peculiarities of the submicroscopic organization of Chlorella cells cultivated on a solid medium in microgravity.

The submicroscopic organization of Chlorella vulgaris cells (strain LARG-1) growing over 30 days on a solid agarized medium aboard the orbital station "Mir" was studied. A number of differences in the ultrastructure of cells of the experimental population compared to the control has been revealed. Thus, changes in the membrane system of plastids, in particular, appearance of numerous vesicles of different diameter and outgrowths of the plastids and their contact with the plasmalemma as well as a considerable decrease of reserve polysaccharide number in the plastids. Moreover, an increase in the size of mitochondria, their cristae and lipid drops in cytoplasm, the formation of more complicated configuration folding of plasmalemma and appearance of small-granular material of mean electron density in the periplasmic space of Chlorella cells grown during space flight, are demonstrated. Comparative cytological analysis has revealed general regularities of rearrangements of the submicroscopic organization in Chlorella cells cultivated on both solid and semiliquid agarized nutrient media.     

Operation of an experimental algal gas exchanger for use in a CELSS.

Concepts of a CELSS anticipate the use of photosynthetic organisms (higher plants and algae) for air revitalization. The rates of production and uptake of carbon dioxide and oxygen between the crew and the photosynthetic organisms are mismatched. An algal [correction of aglal] system used for gas exchange only will have the difficulty of an accumulation or depletion of these gases beyond physiologically tolerable limits (in a materially closed system the mismatch between assimilatory quotient (AQ) and respiratory quotient (RQ) will be balanced by the operation of the waste processor). We report the results of a study designed to test the feasibility of using environmental manipulations to maintain physiologically appropriate atmospheres for algae (Chlorella pyrenoidosa) and mice (Mus musculus strain DW/J) in a gas-closed system. Specifically, we consider the atmosphere behavior of this system with Chlorella grown on nitrate or urea and at different light intensities and optical densities. Manipulation of both the photosynthetic rate and AQ of the alga has been found to reduce the mismatch of gas requirements and allow operation of the system in a gas-stable manner. Operation of such a system in a CELSS may be useful for reduction of buffer sizes, as a backup system for higher plant air revitalization and to supply extra oxygen to the waste processor or during crew changes. In addition, mass balance for components of the system (mouse, algae and a waste processor) are presented.     

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

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

Design and operation of an algal photobioreactor system.

A photobioreactor system has been designed, constructed, and implemented to achieve efficient oxygen production for a closed ecological life support system (CELSS). The special features of this system are the optical transmission system, uniform light distribution, continuous cycling of cells, gravity-independent gas exchange, and an ultrafiltration unit. The fiber optic based optical transmission system illuminates the reactor internally and includes a light source which is external to the reactor, preventing heat generation problems. Uniform light distribution is achieved throughout the reactor without interfering with the turbulent regime inside. The ultrafiltration unit exchanges spent with fresh media and its use results in very high cell densities, up to 10(9) cells/ml for Chlorella vulgaris. The prototype photobioreactor system was operated in a batch and continuous mode for over two months. The oxygen production rate measured at 4-6 mmoles per liter of the culture per hour under continuous operation, is consistent with the expected performance of the unit for the provided light intensity.     

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.     

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.     

Behavioural changes in Paramecium and Didinium exposed to short-term microgravity and hypergravity.

The swimming behaviour of two ciliate species, Paramecium caudatum and Didinium nasutum was analyzed under microgravity and hypergravity. In Paramecium the differences between former upward and downward swimming rates disappeared under weightlessness. At microgravity the swimming rates equalled those of horizontally swimming cells at 1g. In contrast, the swimming rates of Didinium increased under microgravity conditions, being larger than horizontal swimming rates at 1g. These findings are in accordance with a hypothesis of gravireception in ciliates based on electrophysiological data, which considers the different topology of mechanoreceptor channels in theses species. The hypothesis received further support by data recorded under hypergravity conditions.     

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.     

Modelling of genetically engineered microorganisms introduction in closed artificial microcosms.

The possibility of introducing genetically engineered microorganisms (GEM) into simple biotic cycles of laboratory water microcosms was investigated. The survival of the recombinant strain Escherichia coli Z905 (Apr, Lux+) in microcosms depends on the type of model ecosystems. During the absence of algae blooming in the model ecosystem, the part of plasmid-containing cells E. coli decreased fast, and the structure of the plasmid was also modified. In conditions of algae blooming (Ankistrodesmus sp.) an almost total maintenance of plasmid-containing cells was observed in E. coli population. A mathematics model of GEM's behavior in water ecosystems with different level of complexity has been formulated. Mechanisms causing the difference in luminescent exhibition of different species are discussed, and attempts are made to forecast the GEM's behavior in water ecosystems.     

Experimental and mathematical models for small aqueous closed ecosystems with spatially separated components.

Experimental and theoretical models of closed "autotroph-heterotroph" (chlorella-yeast, chlorella-protozoa) ecosystems with spatially separated components have been created and studied. The chart of flows and interaction of components of gas-closed "chlorella-yeast" system have formed the basis describe mathematically the functioning of the given system, experimental results have been found to agree with computer solution of the problem in terms of quality. Investigation of the experimental model of the "producer-consumer" trophic chain demonstrated the role of protozoa in nitrogen turnover. "Production-decomposition" and "production-grazing-decomposition" cycle models has been theoretically analyzed and compared. The predator has been shown to be a more intensive mineralizer than the reducer component.     

Non-conventional approaches to food processing in CELSS. I--Algal proteins; characterization and process optimization.

Protein isolate obtained from green algae (Scenedesmus obliquus) cultivated under controlled conditions was characterized. Molecular weight determination of fractionated algal proteins using SDS-polyacrylamide gel electrophoresis revealed a wide spectrum of molecular weights ranging from 15,000 to 220,000. Isoelectric points of dissociated proteins were in the range of 3.95 to 6.20. Amino acid composition of protein isolate compared favorably with FAO standards. High content of essential amino acids leucine, valine, phenylalanine and lysine makes algal protein isolate a high quality component of CELSS diets. To optimize the removal of algal lipids and pigments supercritical carbon dioxide extraction (with and without ethanol as a co-solvent) was used. Addition of ethanol to supercritical CO2 resulted in more efficient removal of algal lipids and produced protein isolate with a good yield and protein recovery. The protein isolate extracted by the above mixture had an improved water solubility.     

Closed and continuous algae cultivation system for food production and gas exchange in CELSS.

In CELSS (Controlled Ecological Life Support System), utilization of photosynthetic algae is an effective means for obtaining food and oxygen at the same time. We have chosen Spirulina, a blue-green alga, and have studied possibilities of algae utilization. We have developed an advanced algae cultivation system, which is able to produce algae continuously in a closed condition. Major features of the new system are as follows. (1) In order to maintain homogeneous culture conditions, the cultivator was designed so as to cause a swirl on medium circulation. (2) Oxygen gas separation and carbon dioxide supply are conducted by a newly designed membrane module. (3) Algae mass and medium are separated by a specially designed harvester. (4) Cultivation conditions, such as pH, temperature, algae growth rate, light intensity and quantity of generated oxygen gas are controlled by a computer system and the data are automatically recorded. This equipment is a primary model for ground experiments in order to obtain some design data for space use. A feasibility of algae cultivation in a closed condition is discussed on the basis of data obtained by use of this new system.