Survival of Deinococcus radiodurans Against Laboratory-Simulated Solar Wind Charged Particles

Abstract

In this experimental study, cells of the radiation-resistant bacterium Deinococcus radiodurans were exposed to several different sources of radiation chosen to replicate the charged particles found in the solar wind. Naked cells or cells mixed with dust grains (basalt or sandstone) differing in elemental composition were exposed to electrons, protons, and ions to determine the probability of cell survival after irradiation. Doses necessary to reduce the viability of cell population to 10% (LD₁₀) were determined under different experimental conditions. The results of this study indicate that low-energy particle radiation (2–4 keV), typically present in the slow component of the solar wind, had no effect on dehydrated cells, even if exposed at fluences only reached in more than 1000 years at Sun-Earth distance (1 AU). Higher-energy ions (200 keV) found in solar flares would inactivate 90% of exposed cells after several events in less than 1 year at 1 AU. When mixed with dust grains, LD₁₀ increases about 10-fold. These results show that, compared to the highly deleterious effects of UV radiation, solar wind charged particles are relatively benign, and organisms protected under grains from UV radiation would also be protected from the charged particles considered in this study. Key Words: Laboratory simulation experiments—Interplanetary dust—Radiation physics—Extremophilic microorganisms. Astrobiology 11, 875–882.

1. Introduction

L ow-energy cosmic radiation in the interplanetary medium is essentially generated by the solar wind. Solar wind ions, mostly protons and electrons (∼92%, in roughly equal numbers), and alpha particles (∼8%), with only traces of ionic heavier elements, are produced by a plasma expansion with the He/H ratio soaring from 2% to 4.5% in solar maxima. Its velocity becomes supersonic at distances of just a few solar radii, roughly 400 km · s⁻¹ corresponding to energies of ∼1 keV/amu (Gosling, 2007). It has to be stressed that such solar wind particle radiation will only penetrate a thin surface layer (of the order of 2000 Å) of any planetary, cometary, or dusty material. At 1 AU, the solar wind density is of the order of 5 protons · cm⁻³, corresponding to a flux of ∼2×10⁸ protons · cm⁻² · s⁻¹ (Strazzulla et al., 1995). This flux drops with the inverse of the square distance from the Sun, and some 10¹⁷ protons · cm⁻² reach a hypothetical average-sized asteroidal surface at 3 AU in only 100 years. Such high flux is expected to produce sputtering (Thiel et al., 1982) and ion implantation (Strazzulla et al., 1995). The electron distribution functions in the solar wind display an approximately Maxwellian energy distribution with average energies on the order of 10 eV (Veselovsky, 2006) and a similar density to protons of around 5 electrons · cm⁻³ (Salem et al., 2003), corresponding to a flux of 2×10⁸ electrons · cm⁻² · s⁻¹ at 1 AU.

It is expected that radiation damage induced in deeper layers of any rocky material will demand more energetic ions. These may be produced during some solar flares where solar protons that have energies on the order of 100 keV, corresponding to a penetration depth of about 1 μm, are produced. Current estimates about the flux of these ions vary from 2.8×10¹⁰ protons·cm⁻²·year⁻¹, based on the Ulysses spacecraft measurements averaged for more than 1 solar cycle (1990–2004) (Denker et al., 2007), to 4×10¹⁰ protons·cm⁻²·year⁻¹, as calculated by the Space Environment Information System from the European Space Agency (ESA, 2011) with a cumulative solar proton event model (Xapsos et al., 2000) that averages over 30 years, from the solar maximum of 1981 to 2011.

Biological effects of low-energy charged particle irradiation on plants and microbes, especially with energies in the range of several to hundreds of kiloelectronvolts, have been the subject of several studies (Yang et al., 1991; Yu, 1998). Low-energy ion irradiation has been implemented in many important biotechnology applications such as the production of higher rates of mutation, more diverse mutation phenotypes, and less cell death than other forms of irradiation on cotton, rice, wheat, and rye (Huang and Yu, 2007). Low-energy ion irradiation is also being investigated for potential application in cancer treatment (Kamada et al., 2002; Matsushita et al., 2006), and it has been adopted as a sterilization procedure by the medical industry (Raballand et al., 2008).

In the present study, we explored the effects of low-energy ions on microbial cells in an effort to experimentally test the panspermia hypothesis (Nicholson, 2009; Wesson, 2010; Wickramasinghe, 2010). According to the panspermia hypothesis, bacterial cells can be transferred across large distances of interplanetary space and thus “seed” planetary bodies. Any viable life-form putatively traveling from one inhabited planet to another would, therefore, have to cope with the potential heavy particle bombardment inflicted en route, in addition to the UV and X-ray irradiation, which has been considered elsewhere (Horneck et al., 2010; Olsson-Francis and Cockell, 2010; Paulino-Lima et al., 2010).

The physical conditions necessary to ensure microbial survival in the space environment have been extensively simulated since the 1930s (reviewed by Olsson-Francis and Cockell, 2010). However, the effects of low-energy particle radiation on microorganisms have not been fully investigated under simulated space conditions, although as a general result low-energy ion bombardment (10–100 keV) has been shown to result in less cell death than that observed for other forms of radiation (e.g., X-rays and UV irradiation), albeit at the cost of higher mutation rates (Yu, 1993). Existing data show that the biological hazards induced by such irradiation are related to their highly localized energy deposition, with cell inactivation being restricted to those cells placed directly within the radiation path (Horneck, 1994), depending on the radiation linear energy transfer (LET) (Kozubek et al., 1995). Low-energy ion bombardment may, however, cause etching on microbial cells and form microchannels, which thus increases their deleterious effects when irradiated with fluences as high as 10¹⁶ ions·cm⁻² (Song and Yu, 2000; Raballand et al., 2008). It is also known that low-energy electrons appear to interact with particular DNA targets by resonance mechanisms (McKoy and Winstead, 2008), and the error-prone non-homologous end joining has been demonstrated by Moeller et al. (2008) to be the key mechanism that repairs DNA breaks induced by particle bombardment in Bacillus subtilis.

In the present study, we performed a series of experiments in which exposure to interplanetary space travel conditions was simulated to better understand the resilience of the ionizing radiation–resistant bacteria Deinococcus radiodurans to low-energy charged particles. Cells of this microorganism were desiccated and irradiated in vacuum with high fluences of charged particles that differed in energy and nature. Scanning electron microscope images were produced in order to check whether such irradiation caused any physical damage to the cell surface. We then embedded this same bacterium into dust layers to explore whether they could shield the bacterium against the radiation (Secker et al., 1994).

2. Experimental Methodology

2.1. Sample preparation and sample analysis after irradiation

Cultures of Deinococcus radiodurans (R1 wild-type strain) were obtained from a stock kept at the Instituto de Radioproteção e Dosimetria, Rio de Janeiro, Brazil. They were cultivated in tryptone-glucose-yeast extract (TGY) culture medium (0.5% tryptone, 0.3% yeast extract, 0.1% glucose) at 32°C, while being spun at 200 rpm for 18 h. Basalt or sandstone grains smaller than 20 μm were then added to the culture (10⁸ cells·mL⁻¹), as described by Rettberg et al. (2002), for a final concentration of 1.25% (w/v) (Fig. 1). The cell/grain mixture (1 μL) was deposited on black polycarbonate filters (Millipore GTTP02500), resulting in circular samples (∼2 mm diameter). Cells that were previously washed in water were used as controls to prepare cell monolayers on the filters, thus avoiding possible biological shielding from dead cells or organic molecules from the culture medium, which could interfere with the actual radiation resistance of the microorganism (Paulino-Lima et al., 2010).

FIG. 1.

Optical micrographs (a, c) and electron micrographs (b, d) showing Deinococcus radiodurans cells (10⁸ mL⁻¹) mixed with basalt (a, b) and sandstone (c, d) grains (12.5 μg · μL⁻¹).

After irradiation, the filters that contained cells of D. radiodurans were removed from the sample holder and put into 1.5 mL microcentrifuge tubes (Eppendorf) that contained 100 μL TGY. After gently mixing, the cells were resuspended as a homogeneous mixture, with an expected cell concentration of 10⁵ cells · 100 μL⁻¹ TGY. Serial dilutions (10⁻¹, 10⁻², 10⁻³, 10⁻⁴) and a volume of 10 μL were taken from each tube and plated on agar-solidified TGY-containing Petri dishes, which was followed by incubation at 32°C for up to 48 h. Colony-forming units were then counted and multiplied by the corresponding dilution factor to estimate the average number of survivors (N). This number was divided by the value corresponding to the non-irradiated control (N ₀), which yielded a survival rate (N/N ₀) for each dose. Survival curves were plotted on log-log graphs with survival rates plotted on the ordinates versus radiation doses plotted on the abscissas.

2.2. Irradiation sources

Three different radiation beams were used in these experiments: protons, carbon ions, and electrons.

Proton beam irradiation was performed at the INAF–Osservatorio Astrofisico di Catania, Italy. A vacuum chamber capable of maintaining the samples at pressures under 10⁻⁴ Pa was coupled to an ion source that generated 200 keV protons in a 16 mm diameter beam spot.

Experiments with carbon ions were performed at Queens University Belfast, Northern Ireland, UK, with an electron cyclotron resonance source to generate a 4 mm diameter beam spot of 4 keV single-charged carbon ions. Once again samples were placed in a vacuum chamber capable of maintaining a vacuum of 10⁻⁵ Pa.

Electron irradiation experiments were performed at the Open University, UK, with an electron gun model ELG-2/EGPS-1022 (Kimball Physics, Wilton, NH, USA) to generate a 9 mm diameter beam spot of 2 keV electrons.

In all these experiments, our samples were deposited on polycarbonate filters (Millipore GTTP02500), placed into a sample holder and affixed with double-sided carbon tape, and positioned normal to the beam direction. Samples of both naked cells and cells mixed with basalt or sandstone grains were exposed to the doses indicated in the survival graphs. To reduce statistical errors, each experiment was performed with three samples, and the average survival among them was calculated. The statistical analysis was performed by using a two-sample t test under 0.95 confidence and the free software MYSTAT (Cranes Software International Ltd., Bangalore, Karnataka, India).

In a pilot experiment, samples of D. radiodurans were also deposited on substrates made of cosmic dust analogues, namely, forsterite (Mg₂SiO₄) or fayalite (Fe₂SiO₄), to investigate whether they could influence cell inactivation by ion irradiation. These minerals are abundant in circumstellar environments and are found naturally in silicate-bearing meteoritic materials. They have also been detected in interplanetary dust particles.

3. Results

Considering the average elemental composition of biological material, the estimated atomic percentages of bacterial dry cells are 31% carbon, 49% hydrogen, 13% oxygen, and 7% nitrogen (Salton, 1964; Hiragi, 1972). These values were used to evaluate the LET to the target (estimated as a single cell 2 μm in diameter) by using the SRIM code for ions (Ziegler, 2010), which also allowed the estimation of the target density as 0.9392 g · cm⁻³, and CASINO v2.42 (Drouin et al., 2007) for electrons. These values were then used to infer the deposited doses on the cells. To compare with the actual solar wind, the measured fluxes (particles · cm⁻¹ · s⁻¹) from the different irradiation sources were used, which, when integrated in time, allowed the fluence (particles · cm⁻²) to be calculated. Fluences and doses are considered here to be linearly proportional to the particle energy and exposure times. It is also assumed that biological response does not vary with the dose rate, since the cells are metabolically inactive under vacuum.

For all experiments, there were no statistical differences (0.95 confidence) between the external controls (original samples kept in the dark at room pressure and room temperature, outside the vacuum chamber) and the internal controls (non-irradiated controls submitted to all experimental conditions except the irradiation itself). This high survival rate shown by control samples was probably due to the short time of the treatments, which were a few hours.

For practical purposes, we used acronyms to designate the different sample types: NC for naked cells and SST for cells mixed with sandstone, as well as B for cells mixed with basalt grains.

3.1. Proton irradiation

For 200 keV protons, the calculated LET is 6.24 eV · Å⁻¹, with a penetration path estimated to be 2.86 μm. From Fig. 2, it can be seen that the absorbed dose necessary to inactivate 90% of the population of naked cells (LD₁₀) is about 1 kGy, which corresponds to an ion fluence of about 10¹⁰ protons · cm⁻². It is important to note that LD₁₀ values are specific to a particular organism (i.e., Deinococcus radiodurans) under particular irradiation conditions (i.e., humidity, pressure, etc.) because these conditions can affect microbial survival (Bauermeister et al., 2011). All samples used in this study were irradiated under room temperature and at 10⁻⁵ Pa. The maximum fluence that was tested that generated a detectable survival rate was 2.75×10¹³ protons · cm⁻², which corresponds to more than 100 years at the Sun-Earth distance (1 AU). In this case, the survival rate of cells mixed with both grain types was shown to average 2% (±0.4%) (Fig. 2).

FIG. 2.

Survival curves of D. radiodurans after irradiation by a proton beam at 200 keV. NC, naked cells; SST, cells mixed with grains of sandstone; B, cells mixed with grains of basalt. Mg₂SiO₄ represents cells deposited on forsterite substrate. Cells deposited on a fayalite substrate (Fe₂SiO₄) could not be recovered.

Many cells appeared to be protected by overlapped grains and therefore survived even the highest dose tested (Fig. 2). There was no detectable difference in the protection afforded by sandstone or basalt grains for the two fluences tested (t<1.203, p>0.275). No statistical comparison was performed between naked cells and cells mixed with grains because the proton fluences tested were not identical. However, as shown in Fig. 2, the survival rates in the presence of such granular material scored orders of magnitude above that observed for naked cells.

Cells appeared to suffer some radiation damage when deposited on cosmic dust analogues. The survival rates of cells deposited on forsterite (Mg₂SiO₄) were very similar to the survival rates of naked cells deposited on a polycarbonate filter (Millipore). Cells deposited on fayalite (Fe₂SiO₄) could not be recovered at all, which indicates that survival rates were below the detection limit of the method (10⁻⁵). Considering that the only difference between forsterite and fayalite is the replacement of magnesium in forsterite by iron in fayalite, it is possible that iron atoms somehow contribute to inactivating cells when irradiated with 200 keV protons. Further studies would be needed, however, to confirm this.

Scanning electron microscope images were performed for non-irradiated controls and for samples of D. radiodurans mixed with both types of grains. For the fluences tested, there was no detectable difference between the controls and irradiated samples with respect to the physical appearance of cell surfaces, even after microscopic inspection of naked cells irradiated at the maximum fluence (2.75×10¹³ protons · cm⁻²) (Fig. 3).

FIG. 3.

Images from electron microscopy (scanning electron microscope) of cells of D. radiodurans irradiated (a, c, e) or not (b, d, f) with 2.75×10¹³ protons · cm⁻². Naked cells (a, b) and cells mixed with grains of sandstone (c, d) and basalt (e, f) are shown.

3.2. Carbon ions

The calculated LET when using the SRIM code for 4 keV carbon ions on the target (single cell 2 μm in diameter, 0.9392 g · cm⁻³ density) was 3.57 eV · Å⁻¹, with a penetration path of 30 nm. Assuming that the dose necessary to inactivate 90% of a naked cell population (LD₁₀) was 1 kGy, as observed for 200 keV protons, the corresponding required fluence of carbon ions would be 2.65×10¹¹ ions · cm⁻². However, no cell inactivation was observed even for fluences 10,000 times above this value (Fig. 4), which corresponds to roughly 1000 years in space. This is probably due to the penetration of the 4 keV being restricted to the cell wall, which leaves the cellular DNA mostly intact.

FIG. 4.

Survival curves of D. radiodurans in the absence (NC) or presence of grains of sandstone or basalt (SST or B) irradiated with 4 keV carbon ions.

Most fluences resulted in survival rates that showed no significant differences between sample types (Table 1). There was no significant difference upon comparison of survival rates when using the maximum fluence tested (3.33×10¹⁵ ions·cm⁻²) for all sample types with the corresponding non-exposed controls (t<0.988, p>0.394).

Table 1.

Comparison between Average Survival Rates of Cells of Deinococcus radiodurans, Mixed or Not Mixed with Grains of Basalt or Sandstone, after Irradiation with Several Fluences of 4 keV Carbon Ions

	Fluences (×10¹³ ions·cm⁻²)
	0		3.33		10		33.3		100		333
Comparison	t	p	t	p	t	p	t	p	t	p	t	p
NC vs. SST	0.000	1.000	0.390	0.722	0.032	0.976	0.598	0.574	3.449	0.011	0.094	0.929
NC vs. B	0.000	1.000	0.405	0.704	5.424	0.005	0.800	0.457	1.840	0.153	0.258	0.805
SST vs. B	0.000	1.000	0.194	0.864	4.603	0.010	0.191	0.858	0.198	0.858	0.186	0.862

NC, naked cells; SST, cells mixed with sandstone grains; B, cells mixed with basalt grains.

3.3. Electrons

Little cell inactivation occurred after irradiation with 2 keV (Fig. 5). This is again probably due to the low penetration path estimated for 2 keV electrons, with 90% of the total energy deposited 20 nm deep into the cell wall.

FIG. 5.

Survival curves of D. radiodurans in the absence (NC) or presence of grains of sandstone or basalt (SST or B) irradiated with 2 keV electrons.

Most fluences resulted in survival rates that showed no significant differences between grain types (Table 2). Perhaps the expected protective effects of grains are only clearly observable for fluences higher than 6.97×10¹⁶ electrons·cm⁻², but this explanation needs to be further investigated.

Table 2.

Comparison between Average Survival Rates of Cells of Deinococcus radiodurans, Mixed or Not Mixed with Grains of Basalt or Sandstone, after Irradiation with Several Fluences of 2 keV Electrons

	Fluences (×10¹⁴ electrons·cm⁻²)
	0		8.61		25.82		77.45		232.34		697.00
Comparison	t	p	t	p	t	p	t	p	t	p	t	p
NC vs. SST	0.000	1.000	1.184	0.265	0.295	0.781	0.364	0.732	0.511	0.622	3.045	0.030
NC vs. B	0.000	1.000	1.981	0.086	0.296	0.773	3.220	0.015	0.727	0.490	4.886	0.009
SST vs. B	0.000	1.000	3.012	0.021	0.480	0.653	5.852	0.002	1.579	0.160	0.011	0.991

NC, naked cells: SST, cells mixed with sandstone grains; B, cells mixed with basalt grains.

4. Discussion

We measured the survival of the non–spore-forming radiation-resistant bacterium Deinococcus radiodurans against charged particle irradiation by simulating both the slow solar wind (∼1 keV/amu) and more energetic solar ions (hundreds of keV/amu). We used conditions in our experiments that mimicked those found in space with respect to vacuum and the energy of charged particles and fluences microorganisms would be subjected to while in transit between Mars and Earth, (Warren, 1994; Gladman, 1997). The cells were either mixed with rocky grains (basalt or sandstone) or deposited on asteroidal/cometary dust analog substrates, forsterite and fayalite, which were chosen because these minerals are considered good analogues of meteoritic metallic blends due to both their morphology and elemental composition. However, this is a simplification, because asteroids contain many other types of minerals. Nevertheless, we used forsterite and fayalite to acquire preliminary data on how silicate minerals could protect microorganisms from radiation and to investigate whether the different elemental compositions of silicates can affect the efficacy of radiation protection.

The slow component of the slow solar wind ions was simulated by 4 keV C ions. The relative abundance of C/H ions is of the order of 10⁻³, which corresponds to a flux of the order of 10⁵ ions · cm⁻² · s⁻¹ at 1 AU. Thus, the time necessary to irradiate a target at 1 AU with the maximum fluence used in the laboratory (2.65×10¹⁵ ions · cm⁻²) was equivalent to that absorbed over about 1000 years of exposure. Using a low-energy proton beam (3.5 keV) to irradiate monolayers of B. subtilis spores, Tuleta et al. (2005) showed that the spore survival fraction remains at about 10% for fluences between 10¹⁷ and 10¹⁸ protons·cm⁻², which corresponds to 10–100 years of exposure to the solar wind. Regarding electrons, the maximum fluence tested in our study (about 10¹⁷ electrons · cm⁻²) corresponds to an exposure time of about 20 years at 1 AU.

The shielding afforded by micrometric rocky grains was not demonstrated for low-energy C ions or electrons, even with fluences comparable to exposure of tens or hundreds of years in space. Considering the low penetration path of these particles and taking into account that cell inactivation by charged particle is much more dependent on the energy than the fluence, we suggest that a drop in survival rates would only be observed if cells were irradiated at much higher fluences. This is reinforced by the fact that we did not observe the formation of any microchannels on the cell surfaces as a result of sputtered atoms (Fig. 3), as was observed by Song and Wu (2000) and Raballand et al. (2008). However, Song and Wu (2000) and Raballand et al. (2008) used heavier ions, which suggests that the nature of ions also influences the formation of such microchannels. As already mentioned, the relative abundance of heavy ions in the solar wind is very low, and damage would only be induced over much longer periods of time.

In the case of more energetic protons produced during solar flares with an average energy on the order of 100 keV, current estimates vary from 2.8×10¹⁰ protons·cm⁻²·year⁻¹ (Denker et al., 2007) to 4×10¹⁰ protons·cm⁻²·year⁻¹ (Xapsos et al., 2000; ESA, 2011) with a penetration path of the order of a few microns. The maximum fluence tested here with 200 keV protons (2.75×10¹³ protons · cm⁻²) corresponds to that delivered over more than 100 years at Earth orbit (1 AU). However, solar energetic particles are accelerated to far greater energies than the 200 keV protons considered experimentally here, and these results are applicable only to the background solar wind flux and not to more energetic solar events integrated over 100 years.

The shielding afforded by microparticles against 200 keV protons was clearly evident (Fig. 2), albeit with no detectable difference between the two types of rocky grains. Given that the survival rates of cells mixed with both types of grains are similar, there is no reason to believe that differential survival rates would be observed for higher doses, which suggests that both grains can equally protect microbial cells. This was probably caused by the size of the grains. As seen in Fig.1, many of the grains are much larger than the penetration path of 200 keV protons (2.86 μm). Perhaps cells beneath those grains were not affected by the protons and remained viable for much longer periods of time. Thus, only cells exposed on the very surface of larger grains or beneath the smaller ones are expected to be inactivated. Furthermore, considering the increase in cell survival when mixed with grains as compared with naked cells, viable microbial cells (2% survival) could persist for 100 years under accumulated solar flares. Our data also show that solar wind charged particles would contribute to the death of cells on the surface of grains in addition to any UV radiation that they receive. However, UV radiation in space, having a flux greater than 100 W·m⁻² (Paulino-Lima et al., 2010), is much more deleterious. If cells are protected under grains, as they would need to be to survive UV radiation for even a period of minutes (Horneck et al., 2010), they would also survive the solar wind charged particles.

With respect to the exposure time in space, Mileikowsky et al. (2000) estimated that the number of martian meteorites that experienced temperatures below 100°C and arrived on Earth within 8 million years from launch during the past 4 billion years is on the order of 10⁸. Gladman et al. (1996) determined through Monte Carlo trajectory analysis that some 99.9% of the martian meteorite trajectories that reach Earth are slow transfers of between 10,000 and 100 million years. For approximately 0.1% of objects, however, it has been predicted that the transit times are less than 10,000 years. In addition, it has been estimated that some 10⁻⁷ of the objects might actually reach Earth in 1 year or even less (Gladman and Burns, 1996; Gladman, 1997). Thus, our results show that, during short interplanetary transfers, solar wind charged particles when considered alone would not be sufficient to account for the inactivation of microbial cells.

Footnotes

Acknowledgments

We thank Dr. Carlos Eduardo Bonacossa de Almeida (IRD/RJ) for having kindly provided the bacterial wild-type strain and Dr. Gordon Imlach from the Department of Life Sciences, Open University, Milton Keynes, United Kingdom for producing the scanning electron microscope images. We thank Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) for providing I.G. Paulino-Lima's PhD student fellowship abroad. We also thank the two anonymous reviewers who provided helpful feedback on an earlier version of this article. This work was also supported by MIUR PRIN-2008.

Author Disclosure Statement

No competing financial interests exist.

Abbreviations

LET, linear energy transfer; TGY, tryptone-glucose-yeast extract.

References

Bauermeister

, Moeller

, Reitz

, Sommer

, Rettberg

2011. Effect of relative humidity on Deinococcus radiodurans' resistance to prolonged desiccation, heat, ionizing, germicidal, and environmentally relevant UV radiation. Microb Ecol, 61:715–722.

Denker

, Reza

J.Z.

, Nelson

A.J.

, Patterson

J.D.

, Amstrong

T.P.

, Maclennan

C.G.

, Lanzerotti

L.J.

2007. Statistical study of low-energy heliosphere particle fluxes from 1.4 to 5 AU over a solar cycle. Space Weather, 5. 10.1029/2006SW000274.

Drouin

, Couture

A.R.

, Joly

, Tastet

, Aimez

, Gauvin

2007. CASINO V2.42—a fast and easy-to-use modeling tool for scanning electron microscopy and microanalysis users. Scanning, 29:92–101.

ESA. 2011. SPENVIS: The Space Environment Information System. European Space Agency: Paris http://www.spenvis.oma.be.

Gladman

1997. Destination Earth: martian meteorite delivery. Icarus, 130:228–246.

Gladman

B.J.

, Burns

J.A.

1996. Mars meteorite transfer: simulation (Letters) Science, 274:161–162.

Gladman

B.J.

, Burns

J.A.

, Duncan

, Lee

, Levison

H.F.

1996. The exchange of impact ejecta between terrestrial planets. Science, 271:1387–1392.

Gosling

J.T.

2007. The solar wind. Encyclopedia of the Solar System. McFadden

, Weissman

P.R.

, Johnson

T.V.

Academic Press: Amsterdam, 99–116.

Hiragi

1972. Physical, chemical and morphological studies of spore coat of Bacillus subtillis. J Gen Microbiol, 72:87–99.

10.

Horneck

1994. HZE particle effects in space. Acta Astronaut, 32:749–755.

11.

Horneck

, Klaus

D.M.

, Mancinelli

R.L.

2010. Space microbiology. Microbiol Mol Biol Rev, 74:121–156.

12.

Huang

W.D.

, Yu

Z.L.

2007. A dose-survival model for low energy ion irradiation. Int J Radiat Biol, 83:133–139.

13.

Kamada

, Tsujii

, Tsuji

, Yanagi

, Mizoe

, Miyamoto

, Kato

, Yamada

, Morita

, Yoshikawa

, Kandatsu

, Tateishi

Working Group for the Bone and Soft Tissue Sarcomas. 2002. Efficacy and safety of carbon ion radiotherapy in bone and soft tissue sarcomas. J Clin Oncol, 20:4466–4471.

14.

Kozubek

, Horneck

, Krasavin

E.A.

, Rýznar

1995. Interpretation of mutation induction by accelerated heavy ions in bacteria. Radiat Res, 141:199–207.

15.

Matsushita

, Ochiai

, Shimada

, Kato

, Ohno

, Nikaido

, Yamada

, Okazumi

, Matsubara

, Takayama

, Ishikura

, Tsujii

2006. The effects of carbon ion irradiation revealed by excised perforated intestines as a late morbidity for uterine cancer treatment. Surg Today, 36:692–700.

16.

McKoy

, Winstead

2008. Electron collisions with biomolecules. J Phys Conf Ser, 115:1–6.

17.

Mileikowsky

, Cucinotta

F.A.

, Wilson

J.W.

, Gladman

, Horneck

, Lindegren

, Melosh

, Rickman

, Valtonen

, Zheng

J.Q.L.

2000. Natural transfer of viable microbes in space—1. From Mars to Earth and Earth to Mars. Icarus, 145:391–427.

18.

Moeller

, Setlow

, Horneck

, Berger

, Reitz

, Rettberg

, Doherty

A.J.

, Okayasu

, Nicholson

W.L.

2008. Roles of the major, small, acid-soluble spore proteins and spore-specific and universal DNA repair mechanisms in resistance of Bacillus subtilis spores to ionizing radiation from X rays and high-energy charged-particle bombardment. J Bacteriol, 190:1134–1140.

19.

Nicholson

W.L.

2009. Ancient micronauts: interplanetary transport of microbes by cosmic impacts. Trends Microbiol, 17:243–250.

20.

Olsson-Francis

, Cockell

C.S.

2010. Experimental methods for studying microbial survival in extraterrestrial environments. J Microbiol Methods, 80:1–13.

21.

Paulino-Lima

I.G.

, Pilling

, Janot-Pacheco

, Brito

A.N.

, Barbosa

J.A.R.G.

, Leitão

A.A.C.

, Lage

C.A.S.

2010. Laboratory simulation of interplanetary ultraviolet radiation (broad spectrum) and its effects on Deinococcus radiodurans. Planet Space Sci, 58:1180–1187.

22.

Raballand

, Benedikt

, Wunderlich

, von Keudell

2008. Inactivation of Bacillus atrophaeus and of Aspergillus niger using beams of argon ions, of oxygen molecules and of oxygen atoms. J Phys D Appl Phys, 41. 10.1088/0022-3727/41/11/115207.

23.

Rettberg

, Eschweiler

, Strauch

, Reitz

, Horneck

, Wanke

, Brack

, Barbier

2002. Survival of microorganisms in space protected by meteorite material: results of the experiment “Exobiologie” of the Perseu mission. Adv Space Res, 30:1539–1545.

24.

Salem

, Hoang

, Issautier

, Maksimovic

, Perche

2003. Wind-Ulysses in situ thermal noise measurements of solar wind electron density and core temperature at solar maximum and minimum. Heliosphere at Solar Maximum, 32:491–496.

25.

Salton

M.R.J.

1964. The Bacterial Cell Wall. Elsevier: Amsterdam.

26.

Secker

, Lepock

, Wesson

1994. Damage due to ultraviolet and ionizing radiation during the ejection of shielded micro-organisms from the vicinity of 1 M main sequence and red giant stars. Astrophys Space Sci, 219:1–28.

27.

Song

, Yu

2000. Etching and damage action on microbes' cells by low energy N⁺ beam. Plasma Science and Technology, 2:415–421.

28.

Strazzulla

, Brucato

J.R.

, Cimino

, Leto

, Spinella

1995. Interaction of solar wind ions with planetary surfaces. Adv Space Res, 15:13–17.

29.

Thiel

, Sassmannshausen

, Kulzer

, Herr

1982. Ion sputtering of minerals and glasses—a 1^st step to the simulation of solar-wind erosion. Radiation Effects and Defects in Solids, 64:83–88.

30.

Tuleta

, Gabla

, Szkarlat

2005. Low-energy ion bombardment of frozen bacterial spores and its relevance to interplanetary space. Europhys Lett, 70:123–128.

31.

Veselovsky

I.S.

2006. Solar wind and solar energetic particles: origins and effects. Solar Activity and its Magnetic OriginProceedings IAU Symposium, No. 233 Bothmer

, Hady

A.A.

International Astronomical Union: Paris, 489–494.

32.

Warren

P.H

. 1994. Lunar and martian meteorite delivery services. Icarus, 111:338–363.

33.

Wesson

P.S.

2010. Panspermia, past and present: astrophysical and biophysical conditions for the dissemination of life in space. Space Sci Rev, 156:239–252.

34.

Wickramasinghe

2010. The astrobiological case for our cosmic ancestry. International Journal of Astrobiology, 9:119–129.

35.

Xapsos

M.A.

, Summers

G.P.

, Barth

J.L.

, Stassinopoulos

E.G.

, Burke

E.A.

2000. Probability model for cumulative solar proton event fluences. IEEE Trans Nucl Sci, 47:486–490.

36.

Yang

, Gan

, Lin

1991. The mutagenic effect on plant growth by ion implantation on wheat. Anhui Agriculture University Acta, 18:282–288.

37.

Z.L.

1993. Studying of ion implantation effect on biology in China. In China Nuclear Science and Technology Report(in Chinese), CNIC-00746China Nuclear Information Centre: China.

38.

Z.L.

1998. Introduction to Ion Beam Biotechnology Anhui Science and Technology Publishing Company, Hefei, ChinaEnglish version translated Liangdeng

, Vilaithong

, Brown

2006. Springer: New York.

39.

Ziegler

2010. SRIM—The Stopping and Range of Ions in Matter. http://www.srim.org.