Bacterial spore morphology remains highly recognizable after exposure to simulated enceladus and europa surface conditions

ABSTRACT The subsurface oceans of Enceladus and Europa are thought to be some of the best candidate environments for finding life beyond Earth. Realistically, the first missions aimed at

searching for life on these worlds will likely be restricted to the shallow subsurface. Here, we investigated whether indicators of life, or _biosignatures_, deposited near the surface could

persist long enough to be detected, given that the extremely harsh conditions there would tend to degrade them. We exposed _Bacillus subtilis_ spores to Ocean World surface conditions and

used electron microscopy combined with spectroscopic approaches to assess the preservation potential of structural and morphological biosignatures derived from spores. Our results show that

spore structure is highly resilient in the face of extreme conditions long after they have been inactivated, suggesting that methods targeting cell morphology would be valuable components in

a suite of life detection strategies used in future missions to Ocean Worlds. SIMILAR CONTENT BEING VIEWED BY OTHERS AEROBIC MICROBIAL LIFE PERSISTS IN OXIC MARINE SEDIMENT AS OLD AS 101.5

MILLION YEARS Article Open access 28 July 2020 THE POLAR NIGHT SHIFT: SEASONAL DYNAMICS AND DRIVERS OF ARCTIC OCEAN MICROBIOMES REVEALED BY AUTONOMOUS SAMPLING Article Open access 11

December 2021 SEA SPRAY ALLOWS FOR THE GROWTH OF SUBAERIAL MICROBIALITES AT THE DRIEST DESERT ON EARTH Article Open access 28 August 2024 INTRODUCTION The frozen moons of the outer Solar

System, especially Europa and Enceladus, are some of the most enticing targets in the search for life beyond Earth. This stems primarily from the potential for rock-water interactions

occurring at their seafloors, which may produce environments that resemble hydrothermal vent ecosystems here on Earth1,2,3,4,5,6. Observations made by the Galileo and Cassini spacecraft

provided evidence for the presence of a salt-rich ocean hidden below the icy surfaces of these moons, suggesting that conditions favorable for the development of life may exist there7.

Upcoming missions including Europa Clipper and JUICE are likely to yield further insights into the subsurface composition of Europa and may provide important guidance for future lander

missions to the surfaces of icy moons8,9,10. However, the thick ice shells covering these bodies present considerable challenges for direct sampling of their subsurface oceans, where

conditions are most compatible with the chemistry of life11. While robotic solutions for subsurface exploration are in development, initial attempts at detecting life on these worlds may

have to rely on geological processes, such as plumes or cryovolcanism and ice sheet tectonics, to transport subsurface ocean material to accessible depths closer to the surface. Fortunately,

such processes appear to exist in the Southern Polar region of Enceladus, and possibly on Europa12,13,14,15. While the subsurface oceans of Europa and Enceladus may be hospitable, their

surface environments are hostile towards the chemistry of life as we know it. The surfaces of these moons are characterized by extremely low temperatures ranging from 60 to 150 Kelvin, and

are continually bombarded by ultraviolet (UV) photons from the Sun and high-energy charged particles accelerated by the magnetic fields of the giant planets16,17,18,19,20. These harsh

conditions would pose significant challenges to the survival of biological organisms if they exist on these worlds and were expressed on the surface. Ionizing and non-ionizing sources of

radiation rapidly degrade organic material, as demonstrated by the numerous studies on the degradation of bare organic molecules21,22,23,24,25,26,27,28,29,30,31. While laboratory studies

have demonstrated the effects of radiation on organics in ices, the specific effects of radiation at exposure levels relevant to Ocean World surface ages on biosignature preservation from

material that started as a cell remains relatively unexplored. Understanding the preservation of biosignatures originating from the most resilient life forms to these conditions is crucial

for informing future missions aimed at searching for life in such extreme environments. Bacterial spores are among the most resilient forms of life on Earth, with _Bacillus subtilis_ serving

as a model organism for studying the responses of biological systems to extreme conditions. In addition to being well studied, straightforward to culture, and amenable to genetic

manipulation, _B. subtilis_ spores exhibit remarkable resistance to many stressors, including exposure to space vacuum, temperature extremes, and radiation32. Because of this, they are

regularly used to test the efficacy of sterilization techniques and verify spacecraft cleanliness for planetary protection purposes33,34,35,36,37. As a result, the molecular and

physiological responses of spores under conditions replicating aspects of several planetary environments, including low-Earth orbit and the Martian surface, have been well characterized, as

have the specific mechanisms leading to spore inactivation38,39,40,41. Experiments exposing _B. subtilis_ spores to UV, microwave, gamma ray, and electron irradiation under different vacuum

and temperature conditions have collectively shown that while survival under these conditions is enhanced relative to less resilient microbial cells, prolonged exposure to both ionizing and

non-ionizing radiation results in rapid loss of viability42,43,44,45,46,47,48,49,50,51,52,53. In particular, when exposed to solar UV radiation at Ocean World surface temperatures, the

majority of spores are inactivated within minutes, with a slight enhancement in survival at lower temperatures54,55. This suggests that viable spores are unlikely to persist long enough to

be recovered at the surface of Ocean Worlds. However, whether spores leave behind biosignatures that can be detected long after they are inactivated remains an open question. The broad goal

of this work was to experimentally determine whether biosignatures derived from microbial spores persist under conditions representative of Ocean World surfaces over geologically relevant

timescales. Specifically, the objective was to use microscopic and spectroscopic methods to survey how spore morphology and cellular structure change in response to radiation and temperature

extremes, as well as compare the effects of ionizing and non-ionizing radiation. By simulating exposures relevant to the surface ages of Europa and Enceladus and their geological processes,

the results of this work may provide important insights into the likelihood of detecting biological material on these worlds and whether structural and morphological biosignatures are

preserved over timescales that would enable detection by future astrobiology missions. The temperature, vacuum, and radiation conditions of Europa and Enceladus were simulated in a

high-vacuum chamber equipped with a cryostat, onto which stainless steel tabs containing a sub-monolayer of _B. subtilis_ spores were mounted and subjected to either electron radiation to

simulate the Europan surface, or a UV photon source to replicate conditions at Enceladus (Fig. 1). Current crater count-based estimates place the surface age of Europa at 40–90 million years

old, which suggests that any organic material that might be present at the surface could be heavily processed by multiple types of radiation bombarding the surface for extended periods of

time56. To reach less processed material, sampling would need to occur below this heavily processed layer where the effects of deeper penetrating electrons (e-) originating from Jupiter’s

magnetosphere would be dominant over other forms of radiation16. Therefore, our experiments simulating the shallow subsurface of Europa focused on electron radiation produced by an electron

flood gun aimed at spore tabs in our high-vacuum chamber set-up. Electron irradiation times were selected to reach a cumulative dose of 6 × 108 Grays (600 MGy), a community accepted standard

for experiments involving the irradiation of water ice at which all chemical bonds are presumed to have been broken at least once, equivalent to 100 eV per oxygen atom16,18,26,57,58. We

also performed an experiment simulating a cumulative dose of 9000 MGy, allowing us insight into even more heavily processed (i.e., shallower) depths near the surface of Europa. Models

constructed from surface bombardment patterns and particle tracing have provided dose-depth estimates for energetic electrons at Europa58. Based on these models, the time to reach a

cumulative dose of 600 MGy at the Europan surface is estimated to be about 10 years, while it would take 107 years to reach the same dose 10 cm below the surface, roughly equivalent to the

mean surface age of Europa56. Given these dose estimates at a sampling depth of 10 cm, our experiments therefore simulate timescales on the order of 107–108 years of electron radiation

exposure. Future lander missions to Enceladus are likely to target the South polar region, where plumes are actively depositing material originating from the deeper subsurface, and possibly

from the ocean itself, onto the surface7,11. This freshly deposited material is likely to be relatively unprocessed by ionizing radiation. However, a major concern in this case are the

effects of strong UV radiation from the sun, which is also highly damaging to biological molecules. Therefore, we simulated the Enceladus radiation environment by irradiating spores with

solar-like UV produced by a xenon arc lamp in the 200–300 nm range. We chose to simulate 1 year, 10 years, and 29 years at Enceladus, with the longest exposure time replicating one Saturnian

orbit around the Sun. We selected this value as it represents a highly conservative scenario in which material deposited from plumes remains at the surface without burial for an extended

period, resulting in more pronounced degradation. RESULTS All UV and electron irradiation experiments were carried out at room temperature with no temperature control, and at 100 K with and

without a 375 nm layer of amorphous water ice. This ice thickness was selected based on the stopping power of electrons in ice and represents a thickness that allows electrons to reach the

spore layer rather than being fully absorbed by the ice while shielding the underlying stainless-steel substrate to suppress the production of substrate-generated X-rays. In the UV case,

water ice below 1 micron in thickness is effectively transparent to UV photons but may act as a desorption barrier and therefore influence spore responses to irradiation. To visually inspect

spores for morphological changes in response to irradiation in our experimental set-up, we observed tab-mounted spores directly using a scanning electron microscope (SEM). Representative

micrographs for each radiation type can be seen in Fig. 2. Normal spores appear plump and cylindrical in shape. However, even in samples that were not exposed to radiation, a significant

number of spores (~20–30%) appear to have a deflated appearance. This fraction of spores is likely the result of spores becoming dehydrated under the high-vacuum conditions of the experiment

chamber and/or the SEM chamber. Examples of spore damage induced by electron and UV radiation in our experiments are highlighted in panels A–D of Fig. 2 and include distinct holes in the

outer-most visible layer of spores (likely the spore coat), signs of lysis, deflation, and scattered material surrounding spores. After counting the number of visibly damaged spores for each

treatment, we found that even the most extreme of exposures for both UV and electron experiments yielded marginal visual evidence of increased spore damage (Fig. 3). For reference, spores

specifically inactivated by lysis through exposure to wet heat in an autoclave can be seen in Supplementary Fig. 1, and in this case all spores visible are either heavily shriveled or fully

deflated. For UV irradiations at room temperature, we observed a slight increase in the fraction of damaged spores as a function of simulated exposure time, but no significant

time-dependence at low temperature both in the presence and absence of ice (ANOVA; _F_(14) = 1.22, _p_ = 0.3 and _F_(14) = 0.99, _p_ = 0.34, respectively). Temperature did not play a

significant role during electron irradiation (ANOVA; _F_(14) = 1.49, _p_ = 0.37), nor did the electron energy at 100 K with or without ice, although a slight but significant increase in

spore damage occurred at room temperature using 10 keV electrons (ANOVA; _F_(14) = 5.17, _p_ = 0.0098). At 100 K, increasing the cumulative dose from 600 MGy to 9000 MGy increased the

percentage of damaged spores by ~15% (Fig. 3) (_t_(14) = −2.52, _p_ = 0.012). Viability assays performed on spores recovered from experiments that simulated the shortest and least aggressive

UV and electron irradiations (1 year and 600 MGy with 3 keV electrons, respectively) revealed that all spores had been inactivated, despite appearing structurally intact by SEM (Fig. 4).

Elemental analysis was carried out alongside SEM imaging with energy-dispersive X-ray spectroscopy (EDS) to determine whether an elemental enrichment suggestive of material originating from

spores could still be detected after irradiation (Fig. 5). We found that intact spores were enriched in C and Ca2+ relative to the stainless-steel background in the majority of fields, and

that damaged spores retained the C enrichment while Ca2+ was not consistently detectable. We then analyzed tab-mounted spores by Fourier transform infrared spectroscopy (FTIR) to assess

whether chemical changes could be detected spectroscopically. Spore IR spectral features have previously been investigated to distinguish between _Bacillus_ species and between vegetative

cells and spores55,59,60,61. Thanks to this work, we were able to identify signals corresponding to amides, sugars, fatty acids, and dipicolinic acid, a compound specific to bacterial

endospores involved in their resistance to multiple stressors38 (Fig. 6). All of these features were consistently detected in control samples. In UV experiments, spores irradiated for the

equivalent of 1 year at Enceladus retained the majority of these features, with some decreases in intensity. A broad peak appeared around 3500 cm−1, which is likely to be an artifact

(perhaps due to irregularities on the tab surface) based on the peak shape. Extending the UV exposure time to the equivalent of 10 and 29 years decreased the intensity of all IR spectral

features, with peaks at around 2900 cm−1 and 1600 cm−1 remaining discernable. In the electron case, both doses resulted in the loss of most spectral features, with the most pronounced change

occurring at the highest dose of 9000 MGy. Irradiation with both sources produced color changes in the spore layer, with the electron-irradiated spores appearing more orange than the

UV-irradiated layer, which looked to be more yellow. To further assess the stability of the spore material observed by electron microscopy and simulate a generic liquid handling protocol for

chemical analysis as part of a hypothetical life detection mission, electron-irradiated and control spores were recovered from tabs by suspending them in water and re-depositing the

suspension on new tabs with the same membrane filtering method used to initially mount the spores for irradiation. SEM analysis of these re-deposited spores revealed that spore morphology

was still clearly recognizable and appeared similar in control and irradiated samples (Supplementary Fig. 2). Finally, to qualitatively determine if the spore form has behavior completely

different from standard cells, _B. subtilis_ vegetative cells were exposed to the shortest exposures of UV and electron irradiation (1 year and 600 MGy with 3 keV electrons, respectively) at

room temperature. These cells also did not show catastrophic signs of damage or morphological changes when observed by SEM, although the UV-irradiated cells specifically did display some

damage (Supplementary Fig. 3). DISCUSSION The effects of radiation on biological material and the resilience of _Bacillus subtilis_ spores under extreme conditions have been well documented,

but little work has been done to characterize how spores respond to Ocean World surface conditions over geologically relevant timescales to assess the preservation potential of the

resulting biosignatures. In this work, we exposed spores to radiation, vacuum, and temperature conditions representative of Europa and Enceladus surfaces and found that surprisingly, spore

structure and morphology remained recognizable even after the most extreme of exposures, despite all spores being rapidly inactivated. Neither the physical alteration of biomolecules or

indirect damage through the production of reactive oxygen species (ROS), which both cause irreparable damage to vital cellular components and prevent spores from germinating, lead to enough

destruction in the outermost spore layers to be readily discernable by microscopy. While we did observe a small increase in damage in spores exposed to UV at room temperature, this is more

likely due to the effects of heat generated by the lamp than to the UV photons alone. Without temperature control, the tabs reached ~60 °C during the irradiation experiments, which is

expected to sensitize the spores to chemical damage. The lack of observable damage to the outer spore layers in response to irradiation is consistent with prior work showing that exposing

spores to damaging electrical discharge in water, which produces some UV light, does not result in morphological damage as determined by scanning and transmission electron microscopy62.

While previous studies have demonstrated that the likelihood of finding viable spores on the surfaces of Europa and Enceladus is low, these results indicate that cellular morphology would

still be detectable long after spores have been inactivated even under extreme conditions. The qualitatively similar response of _B. subtilis_ vegetative cells irradiated as part of this

study, also hints at the fact that the high preservation potential of cellular morphology is not unique to spores, but further work is needed to generalize these observations. Elemental

analysis also revealed that the Ca2+ enrichment expected in visually intact spores in the control sample, due to the high intracellular concentrations of Ca2+ characteristic of spores in the

_Bacillus_ genus32, was also present in intact spores of irradiated samples. The loss of Ca2+ signal in damaged spores may be due both to the loss of intracellular Ca2+ as a result of

damage and to a reduction in spore thickness upon deflation and therefore a change in incident electron penetration depth through the spore. In contrast, most of the characteristic

vibrational spectral features have been significantly reduced in strength or eliminated completely, especially for the most intense radiation conditions. In both radiation environments, the

most long-lived of the observed spectral signatures correspond to stretching and bending of amide bonds. This persistence may be simply related to the fact that proteins are the largest

class of compounds by mass in any given cell, or it may also be that proteins degrade more slowly than other molecules under the conditions we tested. The spore coat is responsible for

conferring resistance to damage induced by heat, desiccation, chemical agents, and radiation, and is composed of more than 70 spore coat-specific proteins63, further supporting the idea that

the proteins themselves may generally outlast other biomolecules under these challenging conditions. Interestingly, both UV and electron irradiation led to similar changes in the spectra,

e.g., comparing the 600 MGy electron spectra to the 29-year UV spectra in Fig. 6. Given the differences in energy profiles for UV photons and electrons, it is somewhat surprising that a more

target- or radiation-specific change in spectra was not observed. This may be attributed to the fact that much of the cellular damage is caused by reactive photolysis products. Follow-on

work will recover the spores to extract and analyze specific molecular biosignatures such as fatty acids, amino acids, and chromosomal DNA to more quantitatively understand if some of the

differences in molecular-specific destruction are lost with the less quantitative IR analysis performed here. In this work, we were limited in the length of exposure times, radiation

energies, and doses we could feasibly execute in our experimental set-up. Despite these limitations, we were able to simulate timescales equivalent to or exceeding the mean surface age of

Europa (~107 years) assuming a 10-cm sampling depth. In our experiments, the electron source and safety considerations associated with our vacuum chamber set-up placed significant

constraints. To eliminate the risk of harmful X-ray production, experiments were carried out with a maximum electron energy of 10 keV. However, spore damage after irradiation with MeV

electrons has previously been characterized by SEM and while there are clear signs of damage, spores appear to largely retain their morphology as seen in our own investigations45. There are

always caveats with using discrete e- energies and then extrapolating to the entire spectrum of energies present at Europa. However, comparing 3 keV and 10 keV in our experiments, along with

the MeV electrons of the previous study does not reveal any discernable qualitative differences suggesting that maintaining morphological structure is a reliable feature under electron

irradiation across a wide range of energies. The specific energy of incident electrons is also unlikely to result in significantly different damage profiles, as it is secondary electrons

produced by substrate ionization that are responsible for inducing the majority of chemical change64. In the UV experiments, the conditions in our set-up may be more extreme than what we

would expect to encounter at the surface of Enceladus, where burial by plume material would provide additional shielding from UV radiation and presumably enhance biosignature preservation.

Recent estimates of Enceladus UV reflectance spectra from the Hubble telescope and resurfacing rates suggest that organic material would be buried to a UV-shielded depth of 100 microns

within several years65. Because our experiments involved depositing sub-monolayers of spores on stainless steel tabs, the effects of shielding were virtually eliminated, and spores were,

therefore, more vulnerable to damage than they would be in a plausible planetary scenario based on these estimates. Taken together, our results indicate that spore morphology is a highly

resilient biosignature that may withstand the harsh conditions found at the surfaces of Europa and Enceladus long enough to be reliably detected with the appropriate instrumentation. This

work also shows that the likely first step of any sample handling system, which would be to melt the sample and move it fluidically, does not disrupt this morphological biosignature, a

critical issue when thinking about how to design a mission and instruments for maximum life detection science return. Cellular morphology is readily discernable using various forms of

microscopy, and both the proposed Europa Lander and the Enceladus Orbilander mission concepts included microscopes as part of their life detection instrument suites8,10. While cellular

morphology by itself would not be a convincing biosignature given the high probability of false-positives, it would be a powerful complement to other measurements, and successful life

detection beyond Earth will almost certainly require different lines of evidence targeting distinct biosignature types66,67. The ability to identify cell morphology in addition to the

detection of complex organics and other definitive indicators of life would increase the certainty in a positive life detection scenario. Overall, the results of this work suggest that

including the capacity to identify cell-like structures would be especially important in the search for signs of life on the surfaces of Ocean Worlds. METHODS VACUUM CHAMBER SET-UP All

irradiation experiments were carried out in a high-vacuum chamber (_P_ < 5 × 10−7 Torr at room temperature) that allows for sample temperature control between 11 and 350 K using a

closed-cycle He cryostat, and a resistive element which are mounted on a rotary platform with 360° rotation and can be moved to face any of the chamber ports (Fig. 1). Eight tabs were

prepared for each irradiation experiment and attached to a copper mounting block, which was affixed to the end the cryostat. This cryostat was then placed in the chamber. With this setup,

the tabs were either irradiated at room temperature (without temperature control) or maintained at a constant temperature of 100 K, which was monitored with a Si diode sensor attached to the

copper mounting block. For experiments performed with water ice, a thin layer was deposited onto the spores by allowing HPLC-grade water vapor at 10−6 Torr to condense onto the chilled

tabs, with the ice thickness monitored using laser interferometry with a 633 nm laser and photodiode (Thorlabs CPS635F, PDA100A2). This technique allowed for the deposition of amorphous

compact water ice with a thickness of 375 nm in this study. For each irradiation experiment, four tabs were exposed to the radiation source, and the remaining four tabs were shielded with a

stainless-steel cover. For Europa experiments, spores were exposed to an electron gun (Kimball Physics) with beam washing to simultaneously irradiate the four exposed tabs with a uniform

electron flux. For Enceladus experiments, tabs were irradiated with a Xenon arc lamp (Oriel; model # 6266) with a solar-like UV spectrum. SPORE STOCK PREPARATION _Bacillus subtilis_ spores

(ATCC 27370) were prepared from the stock used in ref. 55. To begin, spores were plated onto tryptic soy agar (TSA) plates to stimulate germination at 37 °C for 2 days until vegetative cells

reached an exponential growth stage. The cells were then inoculated onto a sporulation medium composed of 1.6% nutrient broth, 1.7% agar, 0.2% KCl, 0.05% MgSO4, 1 mM Ca(NO3)2, 100 μM

MnCl2·4H2O, 1 μM FeSO4, and 1% glucose and incubated at 37 °C for a total of ~2.5 days, until sporulation reached 80%, which was verified by phase contrast microscopy. The bacterial spores

were separated from the remaining vegetative cells and debris by cycles of suspension, sonication, and centrifugation in the following solutions: sterile water (H2O, Molecular Biology Grade,

Fisher BioReagents), 1 M NaCl, sterile water, 0.5 M NaCl+, 1 M KCl, sterile water, 0.2 mg/mL lysozyme and 10 mM tris-HCl solution under constant shaking for 2 h at 37 °C and an additional

seven times with sterile water until no more cellular debris was visible. The concentration of spores was determined by plating on TSA and counting colonies. The resulting stock was used to

make solutions of fixed CFU ml−1 to use in irradiation experiments. SPORE MOUNTING ON STAINLESS STEEL TABS To prepare spores for irradiation, spores were deposited onto 14 mm diameter

stainless steel tabs by filtering an aqueous solution containing 1 × 108 CFU mL−1 onto a membrane filter (0.2 μm pore diameter, GE Healthcare Whatman Nucleopore Black Polycarbonate #110656)

and then wetting it by placing on a water-soaked filter paper (GE Healthcare Whatman Grade 93 Qualitative Filter Paper Wet-Strengthened #1093-110) to keep the membrane wet, which is

necessary for spore transfer, without disturbing the spore layer. The membrane was then placed onto the stainless-steel surface and dried under a slow flow of N2. This procedure ensures a

low-density distribution of the spores across the surface with sub-monolayer coverage to limit the risk of self-shielding68,69. UV FLUX AND ENCELADUS EXPOSURE TIME CALCULATIONS We used a

Xenon arc lamp (Oriel model # 6266) as a source of solar-like UV used to simulate the Enceladus surface environment. To determine the UV flux at the spore tabs, a photodiode (International

Radiation Detectors Inc. SXUV-100; model #09−718) was placed at the tab location and connected to a picoammeter outside the chamber to measure the current. The UV beam was passed through a

280 nm bandpass filter (Edmund Optics #67-880). We measured a current of 952 nA, which corresponds to a flux of 5.85 × 1018 photons s−1 m−2 based on the filter transmission and photodiode

quantum efficiency. This value and the published spectrum for the lamp were used to generate an interpolated UV spectrum and compared to the solar flux at Enceladus (9.6 a.u.), the data for

which were obtained from Woods et al.70 (Solar spectrum at 1 a.u.; Supplementary Fig. 4) with appropriate inverse square law scaling. The ratio between integrated areas was then used to

determine exposure time equivalencies used in our experiments. ELECTRON DOSE AND EUROPA EXPOSURE TIME CALCULATIONS The electron flux was calculated from current measurements obtained by

mounting a Faraday cup (Kimball Physics model # FC-72) fitted with a molybdenum mesh connected to a picoammeter and affixed the tab location in the vacuum chamber. The cup was positively

biased with 120 V supplied by batteries placed in series with the cup. Calibration curves were generated using both 3 keV and 10 keV electrons at room temperature and recording the average

current at the Faraday cup at different gun emission currents ranging from 1 μA to 80 μA (Supplementary Fig. 5). The resulting curves were used to determine the tab current obtained with the

33.2 μA gun emission current used in the spore experiments, which produced a current value of 130 nA cm−2 with 3 keV electrons. This value was then converted to Grays by multiplying the

recorded current by the estimated stopping power _S_ generated by the National Institutes of Standards and Technology ESTAR program with the following spore elemental composition: H 52.5%, O

17.5% C 25%, N 3.5%, Ca 1.5% (_S_ = 51 meV cm2 g−1). Based on these calculations, we found that using 3 keV electrons, a 33.2 μA emission current, and 30% spot size, a second of irradiation

in our chamber produces 8.6 kGy s−1. With 10 keV electrons, a second of irradiation in the chamber produces 21 kGy s−1. Based on these values, reaching the selected cumulative dose of 600

MGy requires 19 h in our chamber with 3 keV electrons, or 8 h with 10 keV electrons. We also carried out a more aggressive irradiation to reach 9000 MGy with 10 keV electrons, which required

120 h of irradiation. VIABILITY ASSAYS Spores were recovered from stainless steel tabs by dropping 100 μL of sterile nanopure water onto the tabs and scraping the spores with a sterile

silicone cell scraper. The spore suspension was transferred to a tube and the process was repeated 5 times yielding a total of 0.5 mL of spore suspension, which was added to 0.5 mL of

sterile water for a total volume of 1 mL. The solution was diluted 1/1000 and plated in triplicate on TSA plates, which were incubated at 37 °C overnight. Colonies were counted and used to

determine the recovered CFU mL−1, which corresponds to CFU recovered for each tab. SCANNING ELECTRON MICROSCOPY (SEM) AND ENERGY-DISPERSIVE X-RAY SPECTROSCOPY (EDS) Spores were coated with a

7 nm layer of platinum using a sputter coater and loaded into a high-resolution analytical SEM (Zeiss 1550 VP). Imaging was performed with the in-lens secondary electron detector. Three

tabs were analyzed at 15 kV for each treatment and five random fields at 1000x magnification were imaged per tab for a total of 15 fields per treatment. Spores were then counted and the

number of intact and damaged spores determined visually. For an example of how spores were scored and percent damage calculated, see Supplementary Fig. 6. The elemental composition of the

stainless-steel substrate, intact spores, and damaged spores was determined by EDS connected to the SEM (Oxford X-Max SDD EDS system). EDS spectra were obtained using a 10-kV accelerating

voltage and 30 s acquisition time. SEM fields used for EDS analysis were scored for the presence of a carbon enrichment relative to the stainless-steel background and for the presence of

absence of calcium. All raw SEM images can be accessed publicly via the JPL Open Repository71. FOURIER-TRANSFORM INFRARED SPECTROSCOPY (FTIR) Infrared spectra of tab-mounted spores were

collected directly with Fourier transform infrared spectrometer (Thermo Nicolet 6700) in reflectance using a PIKE Technologies DIFFUSIR reflection accessory. A blank stainless-steel tab was

used as a background. To ensure a good signal, a thicker layer of spores was deposited onto the tabs (5 × 108 CFU mL−1). Baseline correction was performed in the OMNIC software. A drop of

water was placed on a blank tab and the resulting spectrum was subtracted from the experimental spectra. DATA AVAILABILITY All raw data generated from this work, including electron

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edn https://doi.org/10.48577/jpl.98PC5B (2024). Download references ACKNOWLEDGEMENTS We would like to thank Chi Ma and the Caltech Geology and Planetary Science Analytical Facility for

providing access and support to the SEM and EDS. This work was funded by the NASA Exobiology Program and the NASA Postdoctoral Program. The research was carried out at the Jet Propulsion

Laboratory, California Institute of Technology, under a contract with the National Aeronautics and Space Administration (80NM0018D0004). AUTHOR INFORMATION AUTHORS AND AFFILIATIONS * Jet

Propulsion Laboratory, California Institute of Technology, Pasadena, CA, USA Lena N. Vincent, Edith C. Fayolle, Robert Hodyss, Paul V. Johnson & Aaron C. Noell Authors * Lena N. Vincent

View author publications You can also search for this author inPubMed Google Scholar * Edith C. Fayolle View author publications You can also search for this author inPubMed Google Scholar *

Robert Hodyss View author publications You can also search for this author inPubMed Google Scholar * Paul V. Johnson View author publications You can also search for this author inPubMed 

Google Scholar * Aaron C. Noell View author publications You can also search for this author inPubMed Google Scholar CONTRIBUTIONS L.V. performed the experiments, analyzed the data, and

wrote the manuscript, E.F., A.N., R.H., and P.J. contributed to the design of the experiments and the final version of the manuscript, E.F. and A.N. supervised the project, and E.F., A.N.,

R.H., and P.J. conceived of the original idea. All authors provided critical feedback to help guide the research, analyses, and manuscript. CORRESPONDING AUTHOR Correspondence to Aaron C.

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