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".     

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.     

Thymine photoproduct formation and inactivation of intact spores of Bacillus subtilis irradiated with short wavelength UV (200-300nm) at atmospheric pressure and in vacuo.

Vacuum exposure renders the survival of spores of Bacillus subtilis approximately five times more sensitive to ultraviolet light irradiation than exposure under atmospheric conditions. The photoproduct formation in spores irradiated under ultrahigh vacuum (UHV) conditions is compared to the photoproduct formation in spores irradiated at atmospheric pressure. Compared to irradiation at atmospheric pressure, where only the "spore photoproduct" 5-thyminyl-5,6-dihydrothymine (TDHT) can be detected, two additional photoproducts, known as the c,s and t,s isomers of thymine dimer (T<>T) are produced in vacuo. The spectral efficiencies for photoproduct formation in spores under atmospheric and vacuum conditions are compared. Since there is no increased formation of TDHT after irradiation in vacuum, TDHT cannot be made responsible for the observed vacuum effect. "Vacuum specific" photoproducts may cause a synergistic response of spores to the simultaneous action of ultraviolet light (UV) and UHV. Three different mechanisms are discussed for the enhanced sensitivity of B. subtilis spores to UV radiation in vacuum. The experiments described contribute valuable research information on the chance for survival of microorganisms in outer space.     

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.     

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.     

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.     

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.     

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.     

The effects of vacuum-UV radiation (50-190nm) on microorganisms and DNA.

Early difficulties with the light source in the study of vacuum-UV effects on biological materials were overcome largely by the introduction of the synchrotron radiation source in 1977. Highly monochromatic vacuum-UV radiation has been used to obtain action spectra of various biological targets in the range 50-190 nm. Bacillus subtilis spores were particularly suitable because of their inherent tolerance to vacuum, and action spectra for inactivation were measured down to 50 nm. Action spectra for DNA strand breaks were also measured in the same range with isolated plasmid DNA. Studies are in progress of vacuum-UV induced molecular changes in nucleic acids and their model compounds, especially of chain scission and base modification.     

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.     

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.     

Survival in extreme dryness and DNA-single-strand breaks.

A wide variety of organisms (the so-called "anhydrobiotes') is able to survive long periods of time in a state of utmost dehydration and can thus survive in extremely dry environments including artificially imposed or space vacuum. Known strategies of survival include the accumulation of certain polyols, especially disaccharides, which help prevent damage to membranes and proteins. Here we report that DNA in vacuum-dried spores is damaged to a very substantial degree by processes leading to DNA strand breaks. Most of these lesions are obviously repaired during germination, but extensive damage to DNA and enzymes after long exposure times (months to years) finally diminish the chances of survival.     

The influence of radiation quality on the formation of DNA breaks.

We have aimed to present a comprehensive review of our understanding to date of the formation of DNA strand breaks induced by high LET radiation. We have discussed data obtained from DNA in solution as well as from the formation and "repair" of strand breaks in cell DNA. There is good agreement, qualitatively, between these two systems. Results were evaluated for two parameters: (1) effectivity per particle, the cross section (sigma) in micrometers 2/particle; and (2) the strand break induction frequency as number of breaks per Gy per unit DNA (bp or dalton). A series of biological effects curves (one for each Z-number) is obtained in effectivity versus LET plots. The relationships between induction frequencies of single-strand breaks, or double-strand breaks, or the residual "irrepairable" breaks and LET-values have been evaluated and discussed for a wide spectrum of heavy ions, both for DNA in solution and for DNA in the cell. For radiation induced total breaks in cell DNA, the RBE is less than one, while the RBE for the induction of DSBs can be greater than one in the 100-200 keV/micrometers range. The level of irrepairable strand breaks is highest in this same LET range and may reach 25 percent of the initial break yield. The data presented cover results obtained for helium to uranium particles, covering a particle incident energy range of about 2 to 900 MeV/u with a corresponding LET range of near 16 to 16000 keV/micrometers.     

Induction and repair of DNA strand breaks in bovine lens epithelial cells after high LET irradiation.

The lens epithelium is the initiation site for the development of radiation induced cataracts. Radiation in the cortex and nucleus interacts with proteins, while in the epithelium, experimental results reveal mutagenic and cytotoxic effects. It is suggested that incorrectly repaired DNA damage may be lethal in terms of cellular reproduction and also may initiate the development of mutations or transformations in surviving cells. The occurrence of such genetically modified cells may lead to lens opacification. For a quantitative risk estimation for astronauts and space travelers it is necessary to know the relative biological effectiveness (RBE), because the spacial and temporal distribution of initial physical damage induced by cosmic radiation differ significantly from that of X-rays. RBEs for the induction of DNA strand breaks and the efficiency of repair of these breaks were measured in cultured diploid bovine lens epithelial cells exposed to different LET irradiation to either 300 kV X-rays or to heavy ions at the UNILAC accelerator at GSI. Accelerated ions from Z=8 (O) to Z=92 (U) were used. Strand breaks were measured by hydroxyapatite chromatography of alkaline unwound DNA (overall strand breaks). Results showed that DNA damage occurs as a function of dose, of kinetic energy and of LET. For particles having the same LET the severity of the DNA damage increases with dose. For a given particle dose, as the LET rises, the numbers of DNA strand breaks increase to a maximum and then reach a plateau or decrease. Repair kinetics depend on the fluence (irradiation dose). At any LET value, repair is much slower after heavy ion exposure than after X-irradiation. For ions with an LET of less than 10,000 keV micrometers-1 more than 90 percent of the strand breaks induced are repaired within 24 hours. At higher particle fluences, especially for low energetic particles with a very high local density of energy deposition within the particle track, a higher proportion of non-rejoined breaks is found, even after prolonged periods of incubation. At the highest LET value (16,300 keV micrometers-1) no significant repair is observed. These LET-dependencies are consistent with the current mechanistic model for radiation induced cataractogenesis which postulates that genomic damage to the surviving fraction of epithelial cells is responsible for lens opacification.     

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.     

New approaches to biochemical radioprotection: antioxidants and DNA repair enhancement.

Chemical repair may be provided by radioprotective compounds present during exposure to ionizing radiation. Considering DNA as the most sensitive target it is feasible to biochemically improve protection by enhancing DNA repair mechanisms. Protection of DNA by reducing the amount of damage (by radical scavenging and chemical repair) followed by enhanced repair of DNA will provide much improved protection and recovery. Furthermore, in cases of prolonged exposure, such as is possible in prolonged space missions, or of unexpected variations in the intensity of radiation, as is possible when encountering solar flares, it is important to provide long-acting protection, and this may be provided by antioxidants and well functioning DNA repair systems. It has also become important to provide protection from the potentially damaging action of long-lived clastogenic factors which have been found in plasma of exposed persons from Hiroshima & Nagasaki, radiation accidents, radiotherapy patients and recently in "liquidators"--persons involved in salvage operations at the Chernobyl reactor. The clastogenic factor, which causes chromatid breaks in non-exposed plasma, might account for late effects and is posing a potential carcinogenic hazard. The enzyme superoxide dismutase (SOD) has been shown to eliminate the breakage factor from cultured plasma of exposed persons. Several compounds have been shown to enhance DNA repair: WR-2721, nicotinamide, glutathione monoester (Riklis et al., unpublished) and others. The right combination of such compounds may prove effective in providing protection from a wide range of radiation exposures over a long period of time.     

Degradation of thermal control materials under a simulated radiative space environment

A spacecraft with a passive thermal control system utilizes various thermal control materials to maintain temperatures within safe operating limits. Materials used for spacecraft applications are exposed to harsh space environments such as ultraviolet (UV) and particle (electron, proton) irradiation and atomic oxygen (AO), undergo physical damage and thermal degradation, which must be considered for spacecraft thermal design optimization and cost effectiveness. This paper describes the effect of synergistic radiation on some of the important thermal control materials to verify the assumptions of beginning-of-life (BOL) and end-of-life (EOL) properties. Studies on the degradation in the optical properties (solar absorptance and infrared emittance) of some important thermal control materials exposed to simulated radiative geostationary space environment are discussed. The current studies are purely related to the influence of radiation on the degradation of the materials; other environmental aspects (e.g., thermal cycling) are not discussed. The thermal control materials investigated herein include different kind of second-surface mirrors, white anodizing, white paints, black paints, multilayer insulation materials, varnish coated aluminized polyimide, germanium coated polyimide, polyether ether ketone (PEEK) and poly tetra fluoro ethylene (PTFE). For this purpose, a test in the constant vacuum was performed reproducing a three year radiative space environment exposure, including ultraviolet and charged particle effects on North/South panels of a geostationary three-axis stabilized spacecraft. Reflectance spectra were measured in situ in the solar range (250–2500 nm) and the corresponding solar absorptance values were calculated. The test methodology and the degradations of the materials are discussed. The most important degradations among the low solar absorptance materials were found in the white paints whereas the rigid optical solar reflectors remained quite stable. Among the high solar absorptance elements, as such the change in the solar absorptance was very low, in particular the germanium coated polyimide was found highly stable.     

DNA-lesion and cell death by alpha-particles and nitrogen ions.

When the natural logarithm of the surviving fraction is plotted against the dose of radiation, curves with shoulders at relatively high survival levels are obtained after gamma-rays. The curves were practically linear in case of HMV-I and HA-1 cells irradiated by charged particle beams. These cells were derived from human malignant melanoma and Chinese hamster cells, respectively. The amount of DNA single strand breaks (ssb) by gamma-rays or nitrogen-ions (LET=530KeV/micrometers) in HMV-I cells increases linearly with increment in dose, when the ssb is detected using the alkaline elution technique. There is no close relationship between the dose-response curve of the ssb and the dose-survival curves after gamma-rays or N-ions. The amount of DNA double strand breaks (dsb) by gamma-rays increases quadratically with increment of dose, in both HMV-I cells and HA-1 cells, when the dsb is detected using the neutral elution technique. The survival fraction for HA-1 cells is slightly higher than that for HMV-I cells, at the same dose, and the amount of dsb for HA-1 cells is considerably greater than that for HMV-I cells. These results suggest that the radiosensitivities to gamma-rays in different cell lines do not correspond to the number of DNA strand breaks. The amount of both non-repairable ssb and dsb also increases quadratically with increment of dose for gamma-rays and almost linearly with increment of dose for N-ions and alpha-particles (LET=36keV/micrometers for HA-1 cells and LET=77keV/micrometers for HMV-I cells). The dose-response curves for non-repairable dsb in case of these radiations seemed to mirror image the dose-survival curves for these radiations, in both cell lines. The number of non-repairable DNA strand breaks in the two cell lines, at the same level of survival was much the same. These results show the close relationship between the induction of non-repairable DNA strand breaks and cell killing.     

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.