Biological responses to space: results of the experiment "Exobiological Unit" of ERA on EURECA I.

Spores of different strains of Bacillus subtilis and the Escherichia coli plasmid pUC19 were exposed to selected conditions of space (space vacuum and/or defined wavebands and intensities of solar ultraviolet radiation) in the experiment ER 161 "Exobiological Unit" of the Exobiology Radiation Assembly (ERA) on board of the European Retrievable Carrier (EURECA). After the approximately 11 months lasting mission, their responses were studied in terms of survival, mutagenesis in the his (B. subtilis) or lac locus (pUC19), induction of DNA strand breaks, efficiency of DNA repair systems, and the role of external protective agents. The data were compared with those of a simultaneously running ground control experiment. The survival of spores treated with the vacuum of space, however shielded against solar radiation, is substantially increased, if they are exposed in multilayers and/or in the presence of glucose as protective, whereas all spores in "artificial meteorites", i.e. embedded in clays or simulated Martian soil, are killed. Vacuum treatment leads to an increase of mutation frequency in spores, but not in plasmid DNA. Extraterrestrial solar ultraviolet radiation is mutagenic, induces strand breaks in the DNA and reduces survival substantially; however, even at the highest fluences, i.e. 3 x 10(8) J m-2, a small but significant fraction of spores survives the insolation. Action spectroscopy confirms results of previous space experiments of a synergistic action of space vacuum and solar UV radiation with DNA being the critical target.     

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.     

Survival rates of some terrestrial microorganisms under simulated space conditions.

In connection with planetary quarantine, we have been studying the survival rates of nine species of terrestrial microorganisms (viruses, bacteria, yeasts, fungi, etc.) under simulated interstellar conditions. If common terrestrial microorganisms cannot survive in space even for short periods, we can greatly reduce expenditure for sterilizing space probes. The interstellar environment in the solar system has been simulated by low temperature, high vacuum (77 k, 4 x 10(-6) torr), and protons irradiation from a Van de Graaff generator. After exposure to a barrage of protons corresponding to about 250 years of irradiation in solar space, Tobacco mosaic virus, Bacillus subtilis spores, Aspergillus niger spores and Clostridiun mangenoti spores showed survival rates of 82%, 45%, 28%, and 25%, respectively. Furthermore. pathogenic Candida albicans showed 7% survival after irradiation corresponding to about 60 years in space.     

UV photobiochemistry under space conditions

The response of spores of Bacillus subtilis, cells of Deinococcus radiodurans and conidia of Aspergillus ochraceus to actual and simulated space conditions (UV in combination with long-term exposure to extremely dry conditions, including vacuum) has been studied: The following effects have been analyzed: decrease of viability, occurrence of DNA double strand breaks, formation of DNA-protein cross-links and DNA-DNA cross-links. All organisms show an increased sensitivity to UV light in extreme dryness (dry argon or vacuum) compared to an irradiation in aqueous suspension. The UV irradiation leads in all cases to a variety of DNA lesions. Very conspicuous is the occurrence of double strand breaks. Most of these double strand breaks are produced by incomplete repair of other lesions, especially base damages. The increase in DNA lesions can be correlated to the loss in viability. The specific response of the chromosomal DNA to UV irradiation in extreme dryness, however, varies from species to species and depends on the state of dehydration. The formation of DNA double strand breaks and DNA-protein cross-links prevails in the case of B. subtilis spores. In cells of Deinococcus radiodurans DNA-DNA cross-links often predominate, in conidia of Aspergillus ochraceus double strand breaks. The results obtained by direct exposure to space conditions (EURECA mission and D2 mission) largely agree with the laboratory data.     

Survival of microorganisms in space: A review

Spores of $\text{Bacillus subtilis}$ were exposed to selected factors of space (vacuum, solar UV radiation, heavy ions of cosmic radiation), and their response was studied after recovery. These investigations were supplemented by ground-based studies under simulated space conditions. The vacuum of space did not inactivate the spores. However, vacuum-induced structural changes in the DNA, and probably in the proteins, caused a supersensitivity to solar UV radiation. This phenomenon is caused by the production of specific photoproducts in DNA and protein, which cannot be removed by normal cellular repair processes. In vegetative bacterial cells, exposed to vacuum, cell dehydration led to damage of the cell membrane, which could be partly repaired during subsequent incubation. The high local effectiveness of the cosmic heavy ions further decreases the chance that spores can survive for any length of time in space. Nonetheless, a spore travelling through space and protected from ultraviolet radiation could possibly survive an interplanetary journey. Such a situation favors panspermia as a possible explanation for the origin of life.     

Microorganisms and biomolecules in space environment experiment ES 029 on Spacelab-1.

Bacterial spores are proper test organisms for studying problems of space biology and exobiology. During the Spacelab 1 mission, studies on the limiting factors for survival of Bacillus subtilis spores in free space have been performed. An exposure tray on the pallet of Spacelab 1 accomodated 316 samples of dry spores for treatment with space vacuum and/or the following selected wavelengths of solar UV: > 170 nm, 220 nm, 240nm, 260nm and 280 nm. After recovery, inactivation, mutation induction, reparability, and photochemical damages in DNA and protein have been studied. The results contribute to the understanding of the mechanisms of increased UV sensitivity of bacterial spores in vacuo and to a better assessment of the chance of survival of resistant forms in space and of interplanetary transfer of life.     

Photoproducts in DNA irradiated in vitro and in vivo under extreme environmental conditions.

UV-irradiated DNA forms different photoproducts in accordance with its state of hydration, and the environment in which the irradiation takes place. Photoproducts in addition to the well-known thymine dimer are produced, some of which probably not recognized due to being heat or acid labile, and milder methods for DNA hydrolysis are needed. The isolation, structure and properties of photoproducts which are formed in UV-irradiated frozen thymine solutions are described. Urea, n-propylurea and dihydrothymine are obtained as photolytic products by high radiation doses in low concentrations of thymine. The cyclobutane cis-anti thymine dimer is obtained at high concentrations of thymine, following several cycles of freezing, thawing and irradiations. A trimer is obtained with 290 nm UV light filtered through Pyrex. It reverts back to thymine dimer and thymine when reirradiated in solution. The cis-syn dimer is obtained at all concentrations of frozen thymine and in a dose dependent form. The adduct 5-hydroxy-6-4' (5'-methylpyrimid 2'-1) dihydrothymine is also obtained. In vacuum-dried thymine or DNA, other photoproducts are formed, including the spore-product, TDHT. Several solvent systems were used to develop chromatograms that allow separation of photoproducts.     

Biological space experiments for the simulation of Martian conditions: UV radiation and Martian soil analogues.

The survivability of resistant terrestrial microbes, bacterial spores of Bacillus subtilis, was investigated in the BIOPAN facility of the European Space Agency onboard of Russian Earth-orbiting FOTON satellites (BIOPAN I -III missions). The spores were exposed to different subsets of the extreme environmental parameters in space (vacuum, extraterrestrial solar UV, shielding by protecting materials like artificial meteorites). The results of the three space experiments confirmed the deleterious effects of extraterrestrial solar UV radiation which, in contrast to the UV radiation reaching the surface of the Earth, also contains the very energy-rich, short wavelength UVB and UVC radiation. Thin layers of clay, rock or meteorite material were shown to be only successful in UV-shielding, if they are in direct contact with the spores. On Mars the UV radiation climate is similar to that of the early Earth before the development of a protective ozone layer in the atmosphere by the appearance of the first aerobic photosynthetic bacteria. The interference of Martian soil components and the intense and nearly unfiltered Martian solar UV radiation with spores of B. subtilis will be tested with a new BIOPAN experiment, MARSTOX. Different types of Mars soil analogues will be used to determine on one hand their potential toxicity alone or in combination with solar UV (phototoxicity) and on the other hand their UV protection capability. Two sets of samples will be placed under different cut-off filters used to simulate the UV radiation climate of Mars and Earth. After exposure in space the survival of and mutation induction in the spores will be analyzed at the DLR, together with parallel samples from the corresponding ground control experiment performed in the laboratory. This experiment will provide new insights into the principal limits of life and its adaptation to environmental extremes on Earth or other planets which and will also have implications for the potential for the evolution and distribution of life.     

Long-term survival of bacterial spores in space.

On board of the NASA Long Duration Exposure Facility (LDEF), spores of Bacillus subtilis in monolayers (10(6)/sample) or multilayers (10(8)/sample) were exposed to the space environment for nearly six years and their survival was analyzed after retrieval. The response to space parameters, such as vacuum (10(-6) Pa), solar electromagnetic radiation up to the highly energetic vacuum-ultraviolet range (10(9) J/m2) and/or cosmic radiation (4.8 Gy), was studied and compared to the results of a simultaneously running ground control experiment. If shielded against solar ultraviolet (UV)-radiation, up to 80 % of spores in multilayers survive in space. Solar UV-radiation, being the most deleterious parameter of space, reduces survival by 4 orders of magnitude or more. However, up to 10(4) viable spores were still recovered, even in completely unprotected samples. Substances, such as glucose or buffer salts serve as chemical protectants. With this 6 year study in space, experimental data are provided to the discussion on the likelihood of "Panspermia".     

Spore dosimetry of solar UV radiation: applications to monitoring of daily irradiance and personal exposure.

Environmental UV radiation can be quantified using spore dosimetry, which measures the inactivation of repair-deficient Bacillus subtilis spores dried on a membrane filter. The system exhibits highly selective sensitivity to UV radiation, not being affected by various environmental adversities, such as high and low temperature and humidity. Biologically-effective dose rate and cumulative dose of ambient radiation are measurable under various conditions at various places on the earth, including tropical, temperate, and polar sites. Applications to monitor the exposure at the surface of organisms including humans and plants have also been advanced.     

The potential of the lichen symbiosis to cope with the extreme conditions of outer space II: germination capacity of lichen ascospores in response to simulated space conditions.

Complementary to the already well-studied microorganisms, lichens, symbiotic organisms of the mycobiont (fungi) and the photobiont (algae), were used as "model systems" in which to examine the ecological potential to resist to extreme environments of outer space. Ascospores (sexual propagules of the mycobiont) of the lichens Fulgensia bracteata, Xanthoria elegans and Xanthoria parietina were exposed to selected space-simulating conditions (up to 16 h of space vacuum at 10(-3) Pa and UV radiation at 160 nm < or = lambda < or = 400 nm), while embedded in the lichen fruiting bodies. After exposure, the ascospores were discharged and their viability was tested as germination capacity on different culture media including those containing Mars regolith simulant. It was found that (i) the germination rate on media containing Mars regolith simulant was as high as on other mineral-containing media, (ii) if enclosed in the ascocarps, the ascospores survived the vacuum exposure, the UV-irradiation as well as the combined treatment of vacuum and UV to a high degree. In general, 50 % or more viable spores were recovered, with ascospores of X. elegans showing the highest survival. It is suggested that ascospores inside the ascocarps are well protected by the anatomical structure, the gelatinous layer and the pigments (parietin and carotene) against the space parameters tested.     

Chemical and biological evolution in space

Astronomical infrared spectra are used to confirm the existence of complex organic molecules produced by ultraviolet photoprocessing of interstellar grain mantles. This material is shown to be the major component of the interstellar grains between the sun and the galactic center and, by inference, constitutes more than 10 million solar masses — or close to one part in a thousand of the entire mass of the milky way galaxy. It may be demonstrated that the primitive chemistry of the earth's surface was dominated by these extraterrestrial molecules after aggregated into comets if the rate of comet impacts with the earth was comparable with that required to account for the extinction of species over the past 300 million years.

Ultraviolet irradiation of bacterial spores has been studied for the first time under simulated interstellar conditions. The inactivation time predicted for the less dense regions of space is at most several hundred years. Within molecukar clouds it is shown on theoretical and experimental grounds that this t the estimated cloud. However survival of spores during their initial exposure to the solar ultraviolet presents a problem for panspermia because it requires that in the process of ejection from the earth's surface they must be enclosed within a cocoon (or mantle) of ultraviolet absorbing material of 0.6 μm thickness. Thus, although panspermia can not be rejected on the basis of lack of interstellar survival there may remain insurmountable obstacles to its occuring because of the very special protective shield requirements during ejection from its planetary source.     

Extreme dryness and DNA-protein cross-links.

Exposure of fungal conidia (Aspergillus ochraceus) or spores of Bacillus subtilis to extreme dryness or vacuum induces DNA lesions, including strand breaks and the formation of DNA-protein cross-links. In wet cells only a small amount of protein is bound to DNA, but exposure to conditions of lowered water activity results in an increasing number of cross-links between DNA and proteins. In fungal conidia these cross-links are detected after selective iodination (125 J) of the DNA-bound proteins followed by gel electrophoresis and subsequent autoradiography. Another approach is the labelling of DNA with 32P by means of nick translation and the detection of differences in the electrophoretic mobility of DNA before and after digestion with proteinase K of proteins bound to DNA.     

Physical determinants of radiation sensitivity in bacterial spores.

Several factors modifying radiation sensitivity in dry bacterial spores are described and discussed. Vacuum inducing the loss of critical structural water, very low dose rates of radiation from which the cell may recover, radiations of high linear energy transfer, and the action of temperature over long periods of time on previously irradiated cells are recognized from extensive laboratory work as important in determining survival of spores exposed to low radiation doses at low temperatures for long periods of time. Some extensions of laboratory work are proposed.     

Stability of nucleic acid under the effect of UV radiation.

Nucleic acids (combined with protein molecules) are essential constituents of the living systems playing an important role in the early evolution of life as well. A specific feature of these molecules has been found and directly confirmed recently: under the influence of short-wavelength UV radiation bipyrimidine photoproducts (cyclobutane dimers and 6-4 bipyrimidines) are induced and the reversion of them can be provoked by the same photons. However, reversion is preferred by the shorter wavelengths. With increasing ratio of the longer wavelength components of the radiation (using artificial UV sources and solar light on the Earth's surface) the impact of the reversible photoproducts in the harmful biological effect decreases and other photoproducts are dominant. Assuming the photoinduced reactions (dimerisation and reversion) are statistical events, during the irradiation the chance for a number of nucleoprotein molecules to survive the radiation damage can be reality. The theoretical and experimental basis of these assumptions will be discussed in the case of bacteriophage T7 nucleoprotein.     

Studies in the search for life on Mars.

The ability of living organisms to survive extraterrestrial conditions has implications for the origins of life in the solar system. We have therefore studied the survival of viruses, bacteria, yeast, and fungi under simulated Martian conditions. The environment on Mars was simulated by low temperature, proton irradiation, ultraviolet irradiation, and simulated Martian atmosphere (CO2 95.46%, N2 2.7%, water vapor 0.03%) in a special cryostat. After exposure to these conditions, tobacco mosaic virus and spores of Bacillus, Aspergillus, Clostridium, and some species of coccus showed significant survival.     

Survival of microorganisms in space protected by meteorite material: results of the experiment 'EXOBIOLOGIE' of the PERSEUS mission.

During the early evolution of life on Earth, before the formation of a protective ozone layer in the atmosphere, high intensities of solar UV radiation of short wavelengths could reach the surface of the Earth. Today the full spectrum of solar UV radiation is only experienced in space, where other important space parameters influence survival and genetic stability additionally, like vacuum, cosmic radiation, temperature extremes, microgravity. To reach a better understanding of the processes leading to the origin, evolution and distribution of life we have performed space experiments with microorganisms. The ability of resistant life forms like bacterial spores to survive high doses of extraterrestrial solar UV alone or in combination with other space parameters, e.g. vacuum, was investigated. Extraterrestrial solar UV was found to have a thousand times higher biological effectiveness than UV radiation filtered by stratospheric ozone concentrations found today on Earth. The protective effects of anorganic substances like artificial or real meteorites were determined on the MIR station. In the experiment EXOBIOLOGIE of the French PERSEUS mission (1999) it was found that very thin layers of anorganic material did not protect spores against the deleterious effects of energy-rich UV radiation in space to the expected amount, but that layers of UV radiation inactivated spores serve as a UV-shield by themselves, so that a hypothetical interplanetary transfer of life by the transport of microorganisms inside rocks through the solar system cannot be excluded, but requires the shielding of a substantial mass of anorganic substances.     

Biologically weighted measurement of UV radiation in space and on Earth with the biofilm technique.

Biological dosimetry has provided experimental proof of the high sensitivity of the biologically effective UVB doses to changes in atmospheric ozone and has thereby confirmed the predictions from model calculations. The biological UV dosimeter 'biofilm' whose sensitivity is based on dried spores of B. subtilis as UV target weights the incident UV radiation according to its DNA damaging potential. Biofilm dosimetry was applicated in space experiments as well as in use in remote areas on Earth. Examples are long-term UV measurements in Antarctica, measurements of diurnal UV profiles parallel in time at different locations in Europe, continuous UV measurements in the frame of the German UV measurement network and personal UV dosimetry. In space biofilms were used to determine the biological efficiency of the extraterrestrial solar UV, to simulate the effects of decreasing ozone concentrations and to determine the interaction of UVB and vitamin D production of cosmonauts in the MIR station.     

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.     

Photo-alteration of hydantoins against UV light and its relevance to prebiotic chemistry

Aqueous solutions of 5-substituted hydantoins were irradiated with ultraviolet (UV) light, to investigate their structural stability against UV radiation as well as the possible photolysis products. The photolysis products were identified and the degree of photolysis was measured using reversed-phase and ion-exchange high-performance liquid chromatography. Hydantoin (2,4-imidazolidinedione) was dominantly detected as a photolysis product of 5-substituted hydantoins. With hydrolysis of UV-irradiated 5-substituted hydantoins, glycine and alanine were dominantly detected. These experimental results are important for the prebiotic photochemistry of 5-substituted hydantoins in the formation of hydantoin since they have been detected in Solar System materials.