Nanosatellites for Biology in Space: In Situ Measurement of Bacillus subtilis Spore Germination and Growth after 6 Months in Low Earth Orbit on the O/OREOS Mission

Abstract

We report here complete 6-month results from the orbiting Space Environment Survivability of Living Organisms (SESLO) experiment. The world's first and only long-duration live-biology cubesat experiment, SESLO was executed by one of two 10-cm cube-format payloads aboard the 5.5-kg O/OREOS (Organism/Organic Exposure to Orbital Stresses) free-flying nanosatellite, which launched to a 72 • -inclination, 650-km Earth orbit in 2010. The SESLO experiment measured the long-term survival, germination, metabolic, and growth responses of Bacillus subtilis spores exposed to microgravity and ionizing radiation including heavy-ion bombardment. A pair of radiation dosimeters (RadFETs, i.e., radiation-sensitive field-effect transistors) within the SESLO payload provided an in-situ dose rate estimate of 6-7.6 mGy/day throughout the mission. Microwells containing samples of dried spores of a wild-type B. subtilis strain and a radiation-sensitive mutant deficient in Non-Homologoous End Joining (NHEJ) were rehydrated after 14, 91, and 181 days in space with nutrient medium containing with the redox dye alamarBlue (aB), which changes color upon reaction with cellular metabolites. Three-color transmitted light intensity measurements of all microwells were telemetered to Earth within days of each 24-hour growth experiment. At 14 and 91 days, spaceflight samples germinated, grew, and metabolized significantly more slowly than matching ground-control samples, as measured both by aB reduction and optical density changes; these rate differences notwithstanding, the final optical density attained was the same in both flight and ground samples. After 181 days in space, spore germination and growth appeared hindered and abnormal. We attribute the differences not to an effect of the space environment per se, as both spaceflight and ground-control samples exhibited the same behavior, but to a pair of~15-day thermal excursions, after the 91-day measurement and before the 181-day experiment, that peaked above 46 • C in the SESLO payload. Because the payload hardware operated nominally at 181 days, the growth issues point to heat damage, most likely to component(s) of the growth medium (RPMI 1640 containing aB) or to biocompatibility issues caused by heat-accelerated outgassing or leaching of harmful compounds from components of the SESLO hardware and electronics.

Rocket Science: The Effect of Spaceflight on Germination Physiology, Ageing, and Transcriptome of Eruca sativa Seeds

“…A number of studies have investigated seed viability during long-term exposure to the harsh space environment without and with shielding against space radiation [1,5,[28][29][30][31]. Arabidopsis thaliana (Arabidopsis) and tobacco seed lots exposed for >>1 year to the outside space of the ISS (LEO~450 km altitude, temperatures fluctuating between −25 and 50 • C, vacuum 10 −4 to 10 −7 Pa, space radiation absorbed dose 350-400 mGy) showed delayed germination (reduced germination speed), but with~90% viability when shielded from short-wavelength ultraviolet (UV) radiation (>1 GJ m −2 UV 110-400 nm ) [30,31].…”

No Effect of Microgravity and Simulated Mars Gravity on Final Bacterial Cell Concentrations on the International Space Station: Applications to Space Bioproduction

“…A third explanation is that, although the different physical environment in each gravity regime could have led to different growth rates and responses, by the end of the experiment all cells had effectively reached stationary phase, resulting in similar cell numbers within each bacterial species. Other space experiments reported comparable final cell concentrations with respect to their ground controls, despite differences in early growth phases ( Klaus et al, 1997 ; Nicholson and Ricco, 2020 ), and after several days of growth in simulated microgravity compared to normal gravity control ( Mastroleo et al, 2013 ). In our experiment, the bacterial cultures were grown for 21 days (which allowed us to maximize the bioleaching of elements from the rock).…”

“…(ii) Bacillus subtilis NCIB 3610 (DSM 10) [German Aerospace Center (DLR) Cologne, Germany] ( Nye et al, 2017 ), a Gram-positive, motile and spore-forming bacillus (phylum Firmicutes). B. subtilis is a very well characterized model organism and has been widely used in space experiments ( Kacena et al, 1999b ; Fajardo-Cavazos et al, 2018 ; Morrison et al, 2019 ; Nicholson and Ricco, 2020 ). It can survive in harsh environments and grow on rock substrates ( Song et al, 2007 ).…”

“…The majority of the data on bacterial growth in microgravity are the results of separate projects with necessarily diverse experimental designs and a limited number of replicate samples, and overall they addressed various endpoints for distinct scientific questions ( Pollard, 1965 ; Bouloc and D’Ari, 1991 ; Gasset et al, 1994 ; Kacena et al, 1997 , 1999b ; Kacena and Todd, 1997 ; Klaus et al, 1997 ; Baker et al, 2004 ; Nickerson et al, 2004 ; Horneck et al, 2010 ; Mulders et al, 2011 ; Kim et al, 2013a ; Zea et al, 2016 , 2017 ; Huang et al, 2018 ; Milojevic and Weckwerth, 2020 ; Nicholson and Ricco, 2020 ). Consequently, it is difficult to draw generalizable conclusions on the effects of gravity on bacterial cell growth.…”

“…Nevertheless, other studies using different E. coli strains did not confirm this observation after space flight ( Bouloc and D’Ari, 1991 ; Gasset et al, 1994 ), while an experiment in simulated microgravity showed medium-dependent changes ( Baker et al, 2004 ). Growth of Bacillus subtilis in microgravity produced inconclusive results as well ( Kacena et al, 1997 , 1999b ; Kacena and Todd, 1997 ; Nicholson and Ricco, 2020 ). Similar discrepancies were reported for other bacterial species ( Kim et al, 2013a ) and for eukaryotic microorganisms ( Mulders et al, 2011 ).…”