Experimental evaluation of the processes resulting from the introduction of the transgenic microorganism Escherichia coli Z905/pPHL7 (lux+) into aquatic microcosms.

The processes resulting from the introduction of the tranagenic microorganism (TM) E. coli Z905/pPHL7 into aquatic microcosms have been modeled experimentally. It has been shown that the TM E. coli is able to adapt to a long co-existence with indigenous heterotrophic microflora in variously structured microcosms. In more complex microcosms the numerical dynamics of the introduced E. coli Z905/pPHL7 population is more stable. In the TM populations staying in the microcosms for a prolonged time, changes are recorded in the phenotypic expression of plasmid genes (ampicillin resistance and the luminescence level) and chromosome genes (morphological and physiological traits). However, in our study microcosms, the recombinant plasmid persisted in the TM cells for 6 years after the introduction, and as the population adapts to the conditions of the microcosms, the efficiency of the cloned gene expression in the cells is restored. In the microcosms with high microalgal counts (10(7) cells/ml), cells with a high threshold of sensitivity to ampicillin dominate in the population of the TM E. coli Z905/pPHL7.     

Survival and alteration of the plasmid-containing microorganism Escherichia coli Z905/pPHL7 introduced into manmade closed aquatic microcosms.

It has been demonstrated that the transgenic microorganism Escherichia coli Z905/pPHL7 (AprLux+) can exist for a long time at an elevated concentration of mineral salts. The microorganism was introduced into microcosms with sterile brackish water (salinity variable from 21 to 22 g l-1) taken from Lake Shira (Khakasia, Russia). The survival of the microorganism was estimated both by measuring the growth of the colonies on solid nutrient media and by the bioluminescence exhibited by the transgenic strain in samples from the microcosms and in the enrichment culture with the added selective factor-ampicillin (50 micrograms/ml). In the enrichment culture, the bioluminescent signal was registered through the 160-day experiment. It has been shown that in the closed microcosms with brackish water the E. coli strain becomes heterogeneous in its ampicillin resistance. The populations of the transgenic strain were mainly represented by isolates able to persist in the medium containing 50 micrograms/ml, but there were also the cells (about 10%) with the threshold of ampicillin resistance not more than 0.05 micrograms/ml. Thus, it was shown that in the microcosms with brackish water and in the absence of the selective factor the transgenic strain survives and retails the recombinant plasmid.     

Population dynamics of transgenic strain Escherichia coli Z905/pPHL7 in freshwater and saline lake water microcosms with differing microbial community structures.

Populations of Escherichia coli Z905/pPHL7, a transgenic microorganism, were heterogenic in the expression of plasmid genes when adapting to the conditions of water microcosms of various mineralization levels and structure of microbial community. This TM has formed two subpopulations (ampicillin-resistant and ampicillin-sensitive) in every microcosm. Irrespective of mineralization level of a microcosm, when E. coli Z905/pPHL7 alone was introduced, the ampicillin-resistant subpopulation prevailed, while introduction of the TM together with indigenous bacteria led to the dominance of the ampicillin-sensitive subpopulation. A high level of lux gene expression maintained longer in the freshwater microcosms than in sterile saline lake water microcosms. A horizontal gene transfer has been revealed between the jointly introduced TM and Micrococcus sp. 9/pSH1 in microcosms with the Lake Shira sterile water.     

Expression of lux-genes as an indicator of metabolic activity of cells in model ecosystem studies.

Quick response to different impacts and easy measurement make the luminescent systems of luminous bacteria an object convenient for application in various fields. Cloning of gene luminescence in different organisms is currently used to study both the survival of microbial cells and the effect of different factors on their metabolic activity, including the environment. A primary test-object in estimating bacteriological contamination of water bodies, Escherichia coli, can be conveniently used as an indicator of bactericidal properties of aquatic ecosystems. The application of Escherichia coli Z905/pPHL7 (lux+) as a marker microorganism can facilitate monitoring the microbiological status of closed biocenoses, including systems with higher organisms. The investigation of various parameters of microecosystems (carbon nutrition type, concentrations of inorganic ions and toxic compounds) shows that the recombinant strain E. coli Z905/pPHL7 can be effectively used as a marker.     

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.     

Expression of cloned genes of transgenic microorganisms introduced into man-made ecosystems.

Modeling of transgenic microorganism introduction into small man-made ecosystems can help forecast changes in expression of cloned genes under different conditions of existence. Introduction of the E. coli Z905/pPHL7 strain containing a plasmid with luminescent system genes of luminous bacteria led to changes in cell and colony morphology, reduction in metabolic activity of cells, and, as a result, a lower level of expression of cloned gene. A low concentration of nutrients has been shown to favor greatly the phenotypic change of cells of the recombinant strain. Expression of cloned genes changed due to: a lower concentration of plasmid DNA, a change in regulation of cloned genes, and a change in cells of biosynthesis of substrates needed for expression of luminescent genes. The conducted investigations can provide a basis for the use of marker transgenic microorganisms in closed ecosystems of different types. Grant numbers: 99-04-96017, 00-07-9011.     

The use of microcosms as an experimental approach to understanding terrestrial ecosystem functioning.

Since 1986, a series of microcosm experiments has been conducted at the Unit of Comparative Plant Ecology (UCPE) in an attempt to test our understanding of the principles controlling the structure and dynamics of plant communities and ecosystems. In each experiment microcosms have been seeded with a common pool of organisms, and systems have been allowed to assemble under replicated controlled conditions. Experiment variables have included mineral nutrient supply, temperature, moisture supply, soil depth, carbon dioxide concentration, mycorrhizas, rhizobia, herbivores and carnivores. Results from these experiments are presented to illustrate the value of synthesised ecosystems in ecological research.     

Mathematical modeling of response of ecosystems with different structure to external impact.

A mathematical model was used to study the response of ecosystems of different structures to external impact. The response was measured as a sensitivity coefficient: the magnitude of the system's response vs. the change of the factor in the inflow. The formula has been obtained to calculate the sensitivity coefficient for ecosystems containing different numbers of trophic links. The derived sensitivity coefficients demonstrate that the degree of compensation for the external impact can differ depending on the type of system regulation and the length of the trophic chain. E. g. the sensitivity coefficient decreases with complexity of trophic links in an ecosystem for top-down controlled systems and impact of degree of openness on sensitivity e.g. closed ecosystems show higher sensitivity then fully open ecosystem to impacts also bottom-up control system show less sensitivity then top-down. Grant numbers: N99-04-96017, N25.     

Mutagenic effects of heavy ions in bacteria.

The peculiarities and mechanisms of the mutagenic action of gamma-rays and heavy ions on bacterial cells have been investigated. Direct mutations in the lac-operon of E. coli in wild type cells and repair deficient strains have been detected. Furthermore, the induction of revertants in Salmonella tester strains was measured. It was found that the mutation rate was a linear-quadratic function of dose in the case of both gamma-rays and heavy ions with LET up to 200 keV/micrometer. The relative biological effectiveness (RBE) increased with LET up to 20 keV/micrometer. Low mutation rates were observed in repair deficient mutants with a block of SOS-induction. The induction of SOS-repair by ionizing radiation has been investigated by means of the "SOS-chromotest" and lambda-prophage induction. It was shown that the intensity of the SOS-induction in E. coli increased with increasing LET up to 40-60 keV/micrometer.     

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.     

The biological component of the life support system for a Martian expedition.

Ground-based experiments at RF SSC-IBMP RAS (State Science Center of Russian Federation--Institute of Biomedical Problems of Russian Academia of Science) were aimed at overall studies of a human-unicellular algae-mineralization LSS (life support system) model. The system was 15 m3 in volume. It contained 45 L of algal suspension with a dry substance density of 10-12 g per liter; water volume, including the algal suspension, was 59 L. More sophisticated model systems with partial substitution of unicellular algae with higher plates (crop area of 15 m2) were tested in three experiments from 1.5 to 2 months in duration. The experiments demonstrated that LSS employing the unicellular algae play not only a macrofunction (regeneration of atmosphere and water) but also carry some other functions (purification of atmosphere, formation of microbial cenosis etc.) providing an adequate human environment. It is also important that functional reliability of the algal regenerative subsystem is secured by a huge number of cells able, in the event of death of a part of population, to recover in the shortest possible time the size of population and, hence, functionality of the LSS autotrophic component. For a long period of time a Martian crew will be detached from Earth's biosphere and for this reason LSS of their vehicle must be highly reliable, robust and redundant. One of the approaches to LSS redundancy is installation of two systems with different but equally efficient regeneration technologies, i.e. physical-chemical and biological. At best, these two systems should operate in parallel sharing the function of regeneration of the human environment. In case of failure or a sharp deterioration in performance of one system the other will, by way of redundancy, increase its throughput to make up for the loss. This LSS design will enable simultaneous handling of a number of critical problems including adequate satisfaction of human environmental needs.     

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.     

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.     

The effects of microgravity on induced mutation in Escherichia coli and Saccharomyces cerevisiae.

We examined whether microgravity influences the induced-mutation frequencies through in vivo experiments during space flight aboard the space shuttle Discovery (STS-91). We prepared dried samples of repair-deficient strains and parental strains of Escherichia (E.) coli and Saccharomyces (S.) cerevisiae given DNA damage treatment. After culture in space, we measured the induced-mutation frequencies and SOS-responses under microgravity. The experimental findings indicate that almost the same induced-mutation frequencies and SOS-responses of space samples were observed in both strains compared with the ground control samples. It is suggested that microgravity might not influence induced-mutation frequencies and SOS-responses at the stages of DNA replication and/or DNA repair. In addition, we developed a new experimental apparatus for space experiments to culture and freeze stocks of E. coli and S. cerevisiae cells.     

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.     

Earth life support for aquatic organisms, system and technical aspects.

The importance of the research on Bioregenerative Life Support has increased dramatically in the last decade not only with regard to possible space flight application but also as a way to obtain a better understanding of our Earth's ecology. A major goal was to reach long-term stability of artificial model systems. Preliminary data are presented on the development of an improved aquatic system, currently dedicated for ground-based research. Closed aquatic ecosystems require reliability of the key parameters of pH, O2 and CO2 concentration and stability of sensors for monitoring. Besides the integration of an artificial lung (holofiber system and air pump with valves, allowing controlled oxygen uptake of air), in parallel to the oxygen producing water plants. Our new approach is to implement opto-chemical sensors, for such environmental monitoring. One major advantage of the new sensor technique is their better long-term reliability as compared to the electrochemical sensors. Our experiment with the new sensor technique has demonstrated satisfactory performance in closed aquatic ecosystems.     

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.     

Effects of solar UV-B radiation on aquatic ecosystems.

Solar UV degrades dissolved organic carbon photolytically so that they can readily be taken up by bacterioplankton. On the other hand solar UV radiation inhibits bacterioplankton activity. Bacterioplankton productivity is far greater than previously thought and is comparable to phytoplankton primary productivity. According to the "microbial loop hypothesis," bacterioplankton is seen in the center of a food web, having a similar function to phytoplankton and protists. The penetration of UV and PAR into the water column can be measured. Marine waters show large temporal and regional differences in their concentrations of dissolved and particulate absorbing substances. A network of dosimeters (ELDONET) has been installed in Europe ranging from Abisko in Northern Sweden to Gran Canaria. Cyanobacteria are capable of fixing atmospheric nitrogen which is then made available to higher plants. The agricultural potential of cyanobacteria has been recognized as a biological fertilizer for wet soils such as in rice paddies. UV-B is known to impair processes such as growth, survival, pigmentation, motility, as well as the enzymes of nitrogen metabolism and CO2 fixation. The marine phytoplankton represents the single most important ecosystem on our planet and produces about the same biomass as all terrestrial ecosystems taken together. It is the base of the aquatic food chain and any changes in the size and composition of phytoplankton communities will directly affect food production for humans from marine sources. Another important role of marine phytoplankton is to serve as a sink for atmospheric carbon dioxide. Recent investigations have shown a large sensitivity of most phytoplankton organisms toward solar short-wavelength ultraviolet radiation (UV-B); even at ambient levels of UV-B radiation many organisms seem to be under UV stress. Because of their requirement for solar energy, the phytoplankton dwell in the top layers of the water column. In this near-surface position phytoplankton will be exposed to solar ultraviolet radiation. This radiation has been shown to affect growth, photosynthesis, nitrogen incorporation and enzyme activity. Other targets of solar UV irradiation are proteins and pigments involved in photosynthesis. Whether or not screening pigments can be induced in phytoplankton to effectively shield the organisms from excessive UV irradiation needs to be determined. Macroalgae show a distinct pattern of vertical distribution in their habitat. They have developed mechanisms to regulate their photosynthetic activity to adapt to the changing light regime and protect themselves from excessive radiation. A broad survey was carried out to understand photosynthesis in aquatic ecosystems and the different adaptation strategies to solar radiation of ecologically important species of green, red and brown algae from the North Sea, Baltic Sea, Mediterranean, Atlantic, polar and tropical oceans. Photoinhibition was quantified by oxygen exchange and by PAM (pulse amplitude modulated) fluorescence measurements based on transient changes of chlorophyll fluorescence.     

Diurnal and seasonal variations of F2 layer characteristics over Irkutsk during the decrease in solar activity in 2003–2006: Observations and IRI-2001 model predictions

The diurnal and seasonal variations of F2 layer characteristics (critical frequency, peak height and bottomside thickness) over Irkutsk, Russia (52.3 N and 104.3 E) are studied by the method of running medians. The comparison with the IRI-2001 model during the decrease in solar activity in 2003–2006 revealed cases of both close agreement and systematic differences between predictions and observations. The systematic difference is not the only reason for disagreement between IRI and observations; there are also intrayear variations which are not associated with seasonal behavior. The period of observation was too short to make conclusions about solar activity dependence of the noon bottomside thickness and the modification of its diurnal behavior with decreasing solar activity.     

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.