Every spacecraft sent to Mars is allowed to land viable microbial bioburden, including hardy endospore-forming bacteria resistant to environmental extremes. Earth's stratosphere is severely cold, dry, irradiated, and oligotrophic; it can be used as a stand-in location for predicting how stowaway microbes might respond to the martian surface. We launched E-MIST, a high-altitude NASA balloon payload on 10 October 2015 carrying known quantities of viable Bacillus pumilus SAFR-032 (4.07 × 107 spores per sample), a radiation-tolerant strain collected from a spacecraft assembly facility. The payload spent 8 h at ∼31 km above sea level, exposing bacterial spores to the stratosphere. We found that within 120 and 240 min, spore viability was significantly reduced by 2 and 4 orders of magnitude, respectively. By 480 min, <0.001% of spores carried to the stratosphere remained viable. Our balloon flight results predict that most terrestrial bacteria would be inactivated within the first sol on Mars if contaminated spacecraft surfaces receive direct sunlight. Unfortunately, an instrument malfunction prevented the acquisition of UV light measurements during our balloon mission. To make up for the absence of radiometer data, we calculated a stratosphere UV model and conducted ground tests with a 271.1 nm UVC light source (0.5 W/m2), observing a similarly rapid inactivation rate when using a lower number of contaminants (640 spores per sample). The starting concentration of spores and microconfiguration on hardware surfaces appeared to influence survivability outcomes in both experiments. With the relatively few spores that survived the stratosphere, we performed a resequencing analysis and identified three single nucleotide polymorphisms compared to unexposed controls. It is therefore plausible that bacteria enduring radiation-rich environments (e.g., Earth's upper atmosphere, interplanetary space, or the surface of Mars) may be pushed in evolutionarily consequential directions. Key Words: Planetary protection—Stratosphere—Balloon—Mars analog environment—E-MIST payload—Bacillus pumilus SAFR-032. Astrobiology 17, 337–350.

Whether terrestrial life can withstand the martian environment is of paramount interest for planetary protection measures and space exploration. To understand microbial survival potential in Mars-like conditions, several fungal and bacterial samples were launched in September 2019 on a large NASA scientific balloon flight to the middle stratosphere (∼38 km altitude) where radiation levels resembled values at the equatorial Mars surface. Fungal spores of Aspergillus niger and bacterial cells of Salinisphaera shabanensis, Staphylococcus capitis subsp. capitis, and Buttiauxella sp. MASE-IM-9 were launched inside the MARSBOx (Microbes in Atmosphere for Radiation, Survival, and Biological Outcomes Experiment) payload filled with an artificial martian atmosphere and pressure throughout the mission profile. The dried microorganisms were either exposed to full UV-VIS radiation (UV dose = 1148 kJ m−2) or were shielded from radiation. After the 5-h stratospheric exposure, samples were assayed for survival and metabolic changes. Spores from the fungus A. niger and cells from the Gram-(–) bacterium S. shabanensis were the most resistant with a 2- and 4-log reduction, respectively. Exposed Buttiauxella sp. MASE-IM-9 was completely inactivated (both with and without UV exposure) and S. capitis subsp. capitis only survived the UV shielded experimental condition (3-log reduction). Our results underscore a wide variation in survival phenotypes of spacecraft associated microorganisms and support the hypothesis that pigmented fungi may be resistant to the martian surface if inadvertently delivered by spacecraft missions.

The survival and transit of microorganisms in Earth's upper atmosphere is relevant to terrestrial ecology and astrobiology, but the topic is understudied due to a scarcity of suitable flight systems. We designed, built, and flew a self-contained payload, Exposing Microorganisms in the Stratosphere (E-MIST), on a large scientific balloon launched from New Mexico on 24 August 2014. The payload carried Bacillus pumilus SAFR-032, a highly-resilient spore-forming bacterial strain originally isolated from a NASA spacecraft assembly facility. Our test flight evaluated E-MIST functionality in the stratosphere, including microbiological procedures and overall instrument performance. Herein, we summarize features of the E-MIST payload, protocols, and preliminary results that indicate it is possible to conduct a tightly-controlled microbiological experiment in the stratosphere while collecting pertinent environmental data. Additional studies of this nature may permit survival models for microbes traveling through Earth's harsh upper atmosphere. Moreover, measuring the endurance of spacecraft-associated microbes at extreme altitudes may help predict their response on the surface of Mars.

 Radiation dose values, fluence and energy distribution spectra were measured for two distinct stratospheric balloon flights  Long duration balloon flight observations occurred over New Mexico (7 hours) and over Antarctica (28 days)  The Regener maximum was identified and described for both missions Abstract Remarkably, we know more about the radiation environment onboard the International Space Station than we do about radiation values at altitudes between 30-40 km in the middle stratosphere. Within this work, we provide data about the radiation dose measured during two consecutive balloon flights flown within a 4-month timeframe over New Mexico and Antarctica. Data were measured with the M-42 radiation detector. On each flight, the M-42 was installed as part of a larger research payload: MARSBOx (New Mexico, 23 September 2019); and E-MIST (Antarctica, 15 December 2019-12 January 2020). The temporal proximity of the flightsprovided similar prevailing space weather conditions and solar activity (minimal during each mission). Against that common backdrop, the main differences between flights, including mission duration and geomagnetic shielding could be readily compared. Near identical space weather conditions provided a window of opportunity for studying the influence of altitude and geomagnetic shielding on dose and fluence rate of galactic cosmic radiation under maximum intensity conditions. Herein, we report relevant count-and dose rates for the missions, alongside Geant4 Monte Carlo calculations; this included crossings of the Regener maximum during the ascent and descent flights over New Mexico and the absence of a distinct maximum in dose rates at zero geomagnetic shielding for the polar flight. While dose rates in silicon at float altitudes (≈35 km-39 km) were a maximum of 2.5±0.4 µGy/h over New Mexico, we reached values of up to 8.4±0.3 µGy/h over Antarctica, thereby approaching dose rates similar to the surface of Mars.