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Astrobiology Manuscript Central: http://mc.manuscriptcentral.com/astrobiology

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

Journal: Astrobiology

Manuscript ID: AST-2011-0649.R2

Manuscript Type: Research Articles (Papers)

Date Submitted by the Author:

03-Jul-2011

Complete List of Authors: Paulino-Lima, Ivan; Universidade Federal do Rio de Janeiro, Instituto de Biofísica Carlos Chagas Filho; Open University, Department of Physics and Astronomy; Universidade Estadual de Londrina, Departamento de Bioquímica e Biotecnologia Pacheco, Eduardo; Universidade de Sao Paulo, Astronomia e Geofisica Galante, Douglas; Universidade de São Paulo, Instituto de Astronomia, Geofisica e Ciencias Atmosfericas Cockell, Charles; Open University, Planetary and Space Science Research Institute Olsson-Francis, Karen; Open University, Planetary and Space Science Research Institute Brucato, John Robert; Instituto Nazionale di Astrofisica, Osservatorio Astrofísico di Arcetri Baratta, Giuseppe; Instituto Nazionale di Astrofisica, Osservatorio Astrofísico di Catania Strazzulla, Giovanni; Instituto Nazionale di Astrofisica, Osservatorio Astrofísico di Catania Merrigan, Tony; Queens University Belfast, School of Mathematics and Physics McCullough, Robert; Queens University Belfast, School of Mathematics and Physics Mason, Nigel; Open University, Department of Physics and Astronomy LAGE, Claudia; Universidade Federal do Rio de Janeiro, Structural & Molecular Biology

Keyword: Laboratory Simulation Experiments, Interplanetary Dust, Radiation Physics, Extremophilic microorganisms

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Survival of Deinococcus radiodurans against Laboratory-Simulated Solar

Wind Charged Particles

Authorship:

Ivan Gláucio Paulino-Lima1,7,*

Eduardo Janot-Pacheco2,7

Douglas Galante2

Charles Cockell3

Karen Olsson-Francis3

John Robert Brucato4

Giuseppe Antonio Baratta5

Giovanni Strazzulla5

Tony Merrigan6

Robert McCullough6

Nigel Mason7

Claudia Lage1, 7

* Corresponding author

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Affiliations:

1- Instituto de Biofísica Carlos Chagas Filho, Universidade Federal do Rio de

Janeiro, Brazil.

2- Instituto de Astronomia, Geofisica e Ciencias Atmosféricas, Universidade de

São Paulo, Brazil.

3- Planetary and Space Science Research Institute, Open University, UK.

4- INAF / Osservatorio Astrofisico di Arcetri, Italy.

5- INAF / Osservatorio Astrofisico di Catania, Italy.

6- Centre for Plasma Physics, School of Mathematics and Physics, Queens

University Belfast, UK.

7- Department of Physics and Astronomy, The Open University, UK.

Correspondence:

Ivan Gláucio Paulino-Lima

Present address: Departamento de Bioquímica e Biotecnologia, Centro de

Ciências Exatas, Universidade Estadual de Londrina, Brazil. Rodovia Celso

Garcia Cid, PR 445 - Km 380, Jardim Portal Versalhes, 86055-900.

Running title: Deinococcus radiodurans and solar wind

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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 keV to 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 a thousand 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 one 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.

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1. Introduction

Low 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 400km·sec-1 corresponding to energies

of ~1keV/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 Angstroms)

of any planetary, cometary, or dusty material. At 1 AU (Astronomical Unit), the

solar wind density is of the order of 5 protons·cm-3, corresponding to a flux of

~2x108 protons·cm-2·sec-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 in the order of 10eV (Veselovsky, 2006) and a similar density to protons

of around 5 electrons·cm-3 (Salem et al., 2003), corresponding to a flux of 2 x 108

electrons·cm-2·s-1 at 1 AU.

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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 in the order of 100keV,

corresponding to a penetration depth of about 1 micrometer, are produced.

Current estimates about the flux of these ions vary from 2.8 x 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 x 1010 protons·cm-2·year-1, as

calculated by the Space Environment Information System (SPENVIS) from the

European Space Agency (ESA, 2011) with a cumulative Solar Proton Event

(SPE) 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 keV,

have been the subject of several studies (Yang et al., 1991; Yu, 1998). Low

energy ion irradiation had 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 (Matsushita et

al., 2006; Kamada et al., 2002), and it has been adopted as a sterilization

procedure by the medical industry (Raballand et al., 2008).

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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 et al., 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

keV - 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,

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however, cause etching on microbial cells and form micro-channels, which thus

increases their deleterious effects when irradiated with fluences as high as 1016

ions·cm-2 (Raballand et al., 2008; Song and Yu, 2000). 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 (NHEJ) 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 were 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 Microscopy (SEM) 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).

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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 hours. 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).

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·100µl-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

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dishes, which was followed by incubation at 32 °C for up to 48 hours. Colony-

forming units (CFU) 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 (N0), which

yielded a survival rate (N/N0) for each dose. Survival curves were plotted on log-

log graphs with survival rates plotted on the ordinates vs 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 beamspot.

Experiments with carbon ions were performed at Queens University

Belfast, Northern Ireland, UK with an electron cyclotron resonance (ECR) source

to generate a 4 mm diameter beamspot of 4 keV single charged carbon ions. Once

again samples were placed in a vacuum chamber capable of maintaining vacuum

of 10-5 Pa.

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Electron irradiation experiments were performed at the Open University,

UK with an electron gun model ELG-2/EGPS-1022 (Kimball Physics, Wilton,

New Hampshire, USA) to generate a 9 mm-diameter beamspot 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 (CDAs), 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 (IDPs).

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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 linear energy transfer (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.

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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 figure 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 x 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).

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

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(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 CDAs.

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.

SEM 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 x 1013

protons·cm-2) (Fig. 3).

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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 cells population (LD10) was 1 kGy, as observed for 200 keV

protons, the corresponding required fluence of carbon ions would be 2.65 x 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 a thousand 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.

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

x 1015 ions·cm-2) for all sample types with the corresponding non-exposed

controls (t < 0.988, p > 0.394).

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.

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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 x 1016

electrons·cm-2, but this explanation needs to be further investigated.

4. Discussion

We measured the survival of the non-sporeforming 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, (Gladman, 1997; Warren, 1994). The cells were either mixed with

rocky grains (basalt or sandstone) or deposited on asteroidal/cometary dust

analogue 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.

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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 sec-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 x 1015 ions·cm-2) was equivalent to that absorbed over about

1,000 years of exposure. Using 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 micro-

channels 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

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the nature of ions also influences the formation of such micro-channels. 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 x 1010

protons·cm-2·year-1 (Denker et al. 2007) to 4 x 1010 protons·cm-2

·year-1 (ESA,

2011, Xapsos et al., 2000) with a penetration path of the order of a few microns.

The maximum fluence tested here with 200 keV protons (2.75 x 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 micro-particles 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

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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.

In 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 thousand 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 one year or even less

(Gladman and Burns, 1996; Gladman, 1997). Thus, our results show that, during

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short interplanetary transfers, solar wind charged particles when considered alone

would not be sufficient to account for the inactivation of microbial cells.

Acknowledgements

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 Microscopy 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 have 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.

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Table 1. Comparison between average survival rates of cells of Deinococcus radiodurans, mixed or not with grains of

basalt or sandstone, after irradiation with several fluences of 4 keV carbon ions.

Fluences (x1013 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

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Table 2. Comparison between average survival rates of cells of Deinococcus radiodurans, mixed or not with grains of

basalt or sandstone, after irradiation with several fluences of 2 keV electrons.

Fluences (x1014 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

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Figure Legends

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 (1.25 g·µl-1).

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.

FIG. 3. Images from electron microscopy (SEM) of cells of D. radiodurans

irradiated (a, c, e) or not (b, d, f) with 2.75 x 1013 protons·cm-2. Naked cells (a, b)

and cells mixed with grains of sandstone (c, d) and basalt (e, f) are shown.

FIG. 4. Survival curves of D. radiodurans in the absence (NC) or presence of

grains of sandstone or basalt (B or SST) irradiated with 4 keV carbon ions.

FIG. 5. Survival curves of D. radiodurans in the absence (NC) or presence of

grains of sandstone or basalt (B or SST) irradiated with 2 keV electrons.

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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 (1.25 g•µl-1).

160x119mm (300 x 300 DPI)

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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.

85x85mm (600 x 600 DPI)

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FIG. 3. Images from electron microscopy (SEM) of cells of D. radiodurans irradiated (a, c, e) or not (b, d, f) with 2.75 x 1013 protons•cm-2. Naked cells (a, b) and cells mixed with grains of sandstone

(c, d) and basalt (e, f) are shown. 179x201mm (300 x 300 DPI)

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

85x85mm (600 x 600 DPI)

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FIG. 5. Survival curves of D. radiodurans in the absence (NC) or presence of grains of sandstone or basalt (B or SST) irradiated with 2 keV electrons.

86x93mm (600 x 600 DPI)

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