A physical and chemical characterization of Martian permafrost as a possible habitat for viable microorganisms.

Data from experiments with model samples show that ion transfer coefficients in the water-rich permafrost on Mars must be three orders of magnitude less than those of terrestrial permafrost. The effects of low temperatures and of carbon dioxide have been accounted for. Exchange between cells and the environment is impeded in Martian permafrost. The microscopic distributional heterogeneity of concentration, pH, Eh, and other physicochemical parameters may be more pronounced in the permafrost of Mars. We present a classification of unfrozen water types in the permafrost that is based on the structures of unfrozen water films and on their functions with respect to cells. Any viable microorganisms on Mars probably exist with minimum metabolism in compact zones with energy carriers and high transfer coefficients. These zones may be microvolumes of unfrozen water in which cells accumulate.     

Preservation of cell structures in permafrost: a model for exobiology.

The present report is the first contribution toward a comprehensive fine-structural study of microbial cells from permafrost. Prokaryotes with a variety of cell wall types demonstrate high stability of cell structure after long-term cryopreservation in frozen soils and sediments of the Arctic. The surface capsular layers that were a salient feature of the cells both in situ and on nutrient media may be an adaptation to low temperature. To the extent that permafrost regions on Earth approximate Martian conditions, preservation of cell structure there can serve as the basis for predictions about preservation in Martian permafrost sediments.     

Bacterial survival in response to desiccation and high humidity at above zero and subzero temperatures

Earthly microorganisms might have contaminated Mars for millions of years by intellectual activities or natural transfer. Knowledge on the preservation of microorganisms may help our searching for life on outer planets, particularly Mars-contaminated earthly microorganisms at ancient time. Extreme dryness is one of the current Mars characteristics. However, a humid or watery Mars at earlier time was suggested by evidence accumulated in recent decades. It raises the question that whether water helps preservation of the microorganisms or not, particularly those with high possibility of interplanetary transfer like spores and Deinococci. In this study, we examined the effects of desiccation and high humidity on survival and DNA double strand breaks (DSB) of Escherichia coli, Deinococcus radiodurans and spores of Bacillus pumilus at 25, 4 and −70 °C. They exhibited different survival rates and DSB patterns under desiccation and high humidity. Higher survival and less DSB occurred at lower temperature. We suggest that some Mars-contaminated bacteria might have been viably preserved on cold Mars regions for long periods, regardless of water availability. It is more likely to find ancient spores than ancient Deinococci on Mars. In our search for preserved extraterrestrial life, priority should be given to the Mars Polar Regions.     

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.     

Survival strategies of microorganisms in extreme saline environments

Halophilic representatives are found in all main lines of evolutionary descendence of microbes: in archaebacteria, Gram-negative and Gram-positive eubacteria, and also in eucaryotes. In principe all halophilic microorganisms have to adapt their surface and membrane structures to their highly ionic environments. Concerning their intracellular compartment two different strategies have been developed: Inorganic ions are largely excluded in some microorganisms while such ions are actively accumulated in others. In particular the second group of organisms has to adapt the whole metabolic machinery to the highly ionic conditions of several molar salts, whereas in the first group only the outer surface of the cytoplasmic membrane and the extracytoplasmic structures are in contact with high concentrations of inorganic ions. In this latter group, a variety of organic solutes is accumulated in response to increases of the salinity of the environment.     

High temperature effect on microflora of radish root-inhabited zone and nutrient solutions for radish growth.

The effect of high temperatures (35 and 45 degrees C) on microflora of the root zone of radish plants grown in phytotron was evaluated by the response of microorganisms from 9 indicator groups. Phytotron air temperature elevated to 35 degrees C for 20 hours caused no significant changes in qualitative and quantitative composition of the root microflora in experimental plants. By the end of the experiment, the species diversity of microflora had changed. The amount of phytopathogenic microorganisms decreased which can be interpreted as more stable co-existence of microflora with plants. The numbers of microbes from other indicator groups was in dynamic equilibrium. The plants' condition did not deteriorate either. Exposure to the temperature of 45 degrees C for 7 hours have been found to change the numbers and species diversity in the radish root zone microflora. The microorganisms were observed to increase their total numbers at the expense of certain indicator groups. Bacteria increased spore forms at the stage of spores. Colon bacillus bacteria of increased their numbers by the end of experiment by an order. By the end of experiment the roots of experiment plants had microscopic fungi from Mucor, Aspergillus, Trichoderma, Cladosporium genera. The observed changes in the microbial complex seem to be associated with the changes of root emissions and general deterioration of the plants' condition. It is suggested that the response of the microorganisms can be indicative of the condition of plants under investigation.     

Role of cell differentiation in high tolerance by prokaryotes of long-term preservation in permafrost

The effect of low temperature on the cell structure of bacteria isolated from permafrost results in structural changes leading to cell differentiation into types of resting cells rangign from spores showing a high endogenous dormancy to typical “dormant” cells of non-spore-forming bacteria showing exogenous dormancy, which is considered to be less highly resistant to extreme conditions in laboratory experiments. In permafrost, dormant cells of non-spore-forming bacteria may demonstrate considerable resistance to long-term freezing and as a result a higher survival level than spore-forming bacteria.     

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.     

A mathematical model of "plants-microorganisms" interaction on complete mineral medium and under nitrogen limitation.

A mathematical model concerning the interaction of plants and rhizospheric microorganisms on complete mineral medium and under nitrogen limitation has been constructed. The model takes into account the closeness of plants and microorganisms in terms of the matter released by the plant and consumed by the microorganisms. The effect of rhizospheric microorganisms on plant growth with normal carbon dioxide and complete mineral medium has been demonstrated. Plants interacting with microorganisms have a greater biomass than plants growing without microorganisms. Wheat growth stimulation by metabolites of rhizospheric microorganisms under laboratory conditions on artificial soil has been experimentally demonstrated (Pechurkin, 1997). Under nitrogen limitation, the biomass of plants, with or without microorganisms, is identical, and is substantially reduced as compared with the medium with standard nitrogen.     

Estimation and assessment of Mars contamination.

Since the beginning of the exploration of Mars, more than fourty years ago, thirty-six missions have been launched, including fifty-nine different space systems such as fly-by spacecraft, orbiters, cruise modules, landing or penetrating systems. Taking into account failures at launch, about three missions out of four have been successfully sent toward the Red Planet. The fact today is that Mars orbital environment includes orbiters and perhaps debris, and that its atmosphere and its surface include terrestrial compounds and dormant microorganisms. Coming from the UN Outer Space Treaty [United Nations Treaty on Principles Governing the Activities of States in the Exploration and Use of Outer Space, including the Moon and Other Celestial Bodies (the "Outer Space Treaty") referenced 610 UNTS 205 - resolution 2222(XXI) of December 1966] and according to the COSPAR planetary protection policy recommendations [COSPAR Planetary Protection Policy (20 October 2002), accepted by the Council and Bureau, as moved for adoption by SC F and PPP, prepared by the COSPAR/IAU Workshop on Planetary Protection, 4/02 with updates 10/0, 2002], Mars environment has to be preserved so as not to jeopardize the scientific investigations, and the level of terrestrial material brought on and around Mars theoretically has to comply with this policy. It is useful to evaluate what and how many materials, compounds and microorganisms are on Mars, to list what is in orbit and to identify where all these items are. Considering assumptions about materials, spores and gas location and dispersion on Mars, average contamination levels can be estimated. It is clear now that as long as missions are sent to other extraterrestrial bodies, it is not possible to keep them perfectly clean. Mars is one of the most concerned body, and the large number of missions achieved, on-going and planned now raise the question about its possible contamination, not necessarily from a biological point of view, but with respect to all types of contamination. Answering this question, will help to assess the potential effects of such contamination on scientific results and will address concerns relative to any ethical considerations about the contamination of other planets.     

Fundamental studies concerning planetary quarantine in space.

If there is a possibility that the organisms carried from Earth to space can live for a significant period on planets, the contamination of planets should be prevented for the purpose of future life-detection experiments. In connection with quarantine for interplanetary missions, we have examined the survivabilities of terrestrial microorganisms under simulated space conditions. In this study, examined the survivabilities of terrestrial organisms under simulated Mars conditions. The Mars conditions were simulated by ultraviolet (UV) and proton irradiation under low temperature, high vacuum, and simulated gaseous conditions. After exposure to the simulated Mars condition, the survivabilities of the organisms were examined. The spores of Bacillus subtilis and Aspergillus niger, some anaerobic bacterias and algaes, showed considerably high survivabilities even after UV and proton irradiation corresponding to 200 years on Mars. This subject is not restricted to academic curiosity but concerns problems involving the contamination of Mars with terrestrial organisms carried by space-probes.     

ERA-experiment "Space Biochemistry".

The general goal of the experiment was to study the response of anhydrobiotic (metabolically dormant) microorganisms (spores of Bacillus subtilis, cells of Deinococcus radiodurans, conidia of Aspergillus species) and cellular constituents (plasmid DNA, proteins, purple membranes, amino acids, urea) to the extremely dehydrating conditions of open space, in some cases in combination with irradiation by solar UV-light. Methods of investigation included viability tests, analysis of DNA damages (strand breaks, DNA-protein cross-links) and analysis of chemical effects by spectroscopic, electrophoretic and chromatographic methods. The decrease in viability of the microorganisms was as expected from simulation experiments in the laboratory. Accordingly, it could be correlated with the increase in DNA damages. The purple membranes, amino acids and urea were not measurably effected by the dehydrating condition of open space (in the dark). Plasmid DNA, however, suffered a significant amount of strand breaks under these conditions. The response of these biomolecules to high fluences of short wavelength solar UV-light is very complex. Only a brief survey can be given in this paper. The data on the relatively good survival of some of the microorganisms call for strict observance of COSPAR Planetary Protection Regulations during interplanetary space missions.     

Simulation of the environmental climate conditions on martian surface and its effect on Deinococcus radiodurans

The resistance of terrestrial microorganisms under the thermo-physical conditions of Mars (diurnal temperature variations, UV climate, atmospheric pressure and gas composition) at mid-latitudes was studied for the understanding and assessment of potential life processes on Mars. In order to accomplish a targeted search for life on other planets, e.g. Mars, it is necessary to know the limiting physical and chemical parameters of terrestrial life. Therefore the polyextremophile bacterium Deinococcus radiodurans was chosen as test organism for these investigations. For the simulation studies at the Planetary and Space Simulation Facilities (PSI) at DLR, Cologne, Germany, conditions that are present during the southern summer at latitude of 60° on Mars were applied.We could simulate several environmental parameters of Mars in one single experiment: vacuum/low pressure, anoxic atmosphere and diurnal cycles in temperature and relative humidity, energy-rich ultraviolet (UV) radiation as well as shielding by different martian soil analogue materials. These parameters have been applied both single and in different combinations in laboratory experiments. Astonishingly the diurnal Mars-like cycles in temperature and relative humidity affected the viability of D. radiodurans cells quite severely. But the martian UV climate turned out to be the most deleterious factor, though D. radiodurans is red-pigmented due to carotenoids incorporated in its cell wall, which have been assigned not only a possible role as free radical scavenger but also as a UV-protectant. An additional UV-protection was accomplished by mixing the bacteria with nano-sized hematite.     

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.     

Planetary protection program for Mars 94/96 mission.

Mars surface in-situ exploration started in 1975 with the American VIKING mission. Two probes landed on the northern hemisphere and provided, for the first time, detailed information on the martian terrain, atmosphere and meteorology. The current goal is to undertake larger surface investigations and many projects are being planned by the major Space Agencies with this objective. Among these projects, the Mars 94/96 mission will make a major contributor toward generating significant information about the martian surface on a large scale. Since the beginning of the Solar System exploration, planets where life could exist have been subject to planetary protection requirements. Those requirements accord with the COSPAR Policy and have two main goals: the protection of the planetary environment from influence or contamination by terrestrial microorganisms, the protection of life science, and particularly of life detection experiments searching extra-terrestrial life, and not life carried by probes and spacecrafts. As the conditions for life and survival for terrestrial microorganisms in the Mars environment became known, COSPAR recommendations were updated. This paper will describe the decontamination requirements which will be applied for the MARS 94/96 mission, the techniques and the procedures which are and will be used to realize and control the decontamination of probes and spacecrafts.     

Microorganisms and enzyme activity in permafrost after removal of long-term cold stress

Associations of immobilized microbial cells and organic-mineral complexes containing active enzymes are resistant to long-term (from tens of thousands to millions of years) effects of extremely low temperatures. This association enables the cells to restore their metabolic activity during permafrost thawing, because interactions with the heterogenous medium is made possible by the availability of active immobilized enzymes. The long-term effect of the cold probably favors an adaptational change of microbial metabolism that activates enzymes and cells during thawing.     

A new enzymatic technique to estimate the efficiency of microbial degradation of pollutants.

Dynamics of active sludge microorganism activity in aerotanks under chemostat conditions has been studied. Dependence of microorganism catalase activity has been found to depend on residual substrate concentration in proportion to the biomass of microorganisms. Experimental data and field observations has formed the basis to develop a technique to evaluate in relative units the amount of the substrate consumed by biocenosis of the active sludge in the air tanks of purification facilities.