A Balloon-Based Payload for Exposing Microorganisms in the Stratosphere (E-MIST)

Microorganisms in the upper atmosphere emanate from surface and marine ecosystems (Burrows et al., 2009), and are capable of reaching high altitudes by strong uplifting forces or mixing between the troposphere and stratosphere (Homeyer et al., 2011; Randel and Jensen, 2013). In addition to microbes naturally lofted, landfills, wastewater treatment plants, slash-and-burn agriculture, air traffic, and desertification also contribute to the total amount of bioaerosols in the atmosphere (Smith, 2013). Prevailing winds can connect distant biomes on Earth (Creamean et al., 2013; Smith et al., 2013), and residence time in the upper atmosphere exerts a harsh combination of stresses on microbes outside the range of conditions normally encountered on the surface (e.g., lower pressure, higher irradiation, desiccation, and oxidation). Thus, airborne transport might provoke exceptional types of cellular damage or mutation. Some bacteria found in previous atmospheric surveys (reviewed by Griffin (2007) and Polymenakou (2012)) have traits that would improve persistence aloft – including cell pigmentation, DNA repair, and the ability to form endospores (hereafter referred to as ‘spores’). While microbial survival has been examined using environmental simulation chambers (Smith et al., 2011) and small meteorological balloons (Beck-Winchatz and Bramble, 2014), more comprehensive platforms for controlled, long-duration experiments in the upper atmosphere are needed. Earth's middle stratosphere, about 25 to 40 km above sea level (ASL), resembles the surface conditions of Mars (Kaplan, 1988); thus, the planetary protection community could obtain an improved understanding of the survival of terrestrial microbes on Mars rovers and landers by flying stratospheric experiments. Furthermore, data from survival experiments could contribute to models for bioaerosols carried on globally-circulated winds (Smith et al., 2013; Yu et al., 2013), which is vitally important to global food security (Brown and Hovmøller, 2002). For example, aerially-dispersed wheat fungi threaten a staple calorie and protein source for 4.5 billion people across 94 developing nations (Singh et al., 2011).

Studying microbial survival in the upper atmosphere presents two fundamental challenges: first, removing potential influences from pre-flight, ascent, descent, and landing so as to limit the experiment to targeted altitudes; second, maintaining aseptic conditions within a closed payload system to preserve the integrity of test samples. Large scientific balloons provide unique access to the stratosphere and careful control over exposure experiments. Our aim in this study was to design, construct, and fly a self-contained payload (autonomous avionics, power, environmental sensors) that could attach to the exterior of large balloon gondolas, permitting a survival-based microbiology experiment at desired altitudes. A secondary aim was to collect baseline data and establish microbiology procedures (including ground and negative controls) for enabling future science flights. We worked with Bacillus pumilus SAFR-032, originally isolated from a spacecraft assembly facility at the Jet Propulsion Laboratory (Kempf et al., 2005). The strain was a suitable model microorganism for our study because: (1) the resistance of its spore to environmental extremes is well documented (Gioia et al., 2007; Vaishampayan et al., 2012 and references therein); (2) the full genome is available for transcriptomic and proteomic analysis (Gioia et al., 2007; Tirumalai et al., 2013); (3) the species has no exosporium or extraneous layer associated with spores (Link et al., 2004); and (4) Bacillus sp. are commonly found in the upper atmosphere (Smith et al., 2012).

Our test flight demonstrated the functionality and reliability of a new payload for exposing microbes in the stratosphere. Baseline data collected can better prepare future research teams using this system. Table 1 summarizes upper and lower limits of key environmental data from the balloon launch, ascent, and float. Internal payload heaters performed nominally, keeping hardware components and sensors within operating limits – particularly in the upper troposphere during the coldest part of the flight. For instance, at 16.4 km ASL when the free air temperature was −67.5°C (measured by an independent, CSBF gondola sensor), the avionics board inside E-MIST remained at −4.10°C. However, a few instruments and components did not function properly. First, the altimeter failed, possibly due to radio frequency interference with other gondola instruments. Second, UV measurements were lost because the stand-alone radiometer was not powered off during payload recovery (forcing the instrument to eventually overwrite the data stored during float). Obtaining UV data on future experiments will be critical since other survival studies have shown a relationship between irradiation and bacterial inactivation (Smith et al., 2011). Looking forward, modifying the radiometer to store data on the payload flight computer will resolve this issue, and stratospheric UV measurements from other balloon missions (e.g., McPeters et al., 1984) can be used to establish an expected range of irradiation. Finally, the rotation of the sample skewers was suboptimal. In principle, end-to-end bacterial coupons will receive near-identical sunlight if the skewers open evenly (on the same plane) and no shadows cross the payload face – yet, neither condition was observed in flight. Intermittent gondola shadows passed over the skewers but were transitory since the balloon was constantly rotating. We will build a smart switch system that communicates with the motor controller to ensure skewers open evenly on the next flight opportunity.

Concerns about contamination (e.g., external microbes penetrating the payload) or inactivation (e.g., outside biocidal factors besides stratospheric conditions) motivated this methods-focused study. Including Bacillus pumilus SAFR-032 on the test flight allowed us to evaluate the success of payload containment, microbiological procedures, and protocols. A properly-controlled stratospheric microbiology survival experiment should only expose samples to the targeted region of the upper atmosphere. Our experimental design assessed unknowns associated with sample transportation, balloon gondola installation, balloon ascent/descent, and time lingering in the hot, dusty New Mexico desert awaiting launch and recovery. We created a batch of experimental control coupons (each containing approximately 1 × 10⁶ spores) used throughout the investigation for ground and flight test purposes. Several treatment categories were evaluated: Lab Ground Coupons (kept in the KSC laboratory); Transported Ground Coupons (traveled to New Mexico and back, but not installed in payload); Face-Up Flight Coupons (flown, experienced internal payload conditions and exposed to stratospheric sunlight for 2 s); Inverted Flight Coupons (flown, experienced internal payload conditions and exposed to stratospheric conditions other than sunlight for 2 s). A subset of coupons from each treatment category was processed, resulting in MPN values reported in Table 2. Averages between coupon groups were compared using one-way permutations (Kruskal-Wallis rank-sum test) at a 95% confidence level, and the differences in the means were not statistically significant. Notably, the brief (2 s) skewer rotation in the lower stratosphere did not reduce the number of spores on the Face-Up Coupons. Also, time spent on the ground did not impact survival numbers. Temperatures ranging from 80°C to 110°C are generally capable of inactivating spores (Ghosh et al., 2009), but the highest temperatures recorded on the ground (32.4°C for Payload Internal Temperature; 40.1°C for Proxy Coupon) and in flight (46.1°C for Proxy Coupon) were below the threatening range. Taken together, nearly identical numbers from all coupons indicate that balloon flight operations and payload procedures did not influence spore survival.

If future flights deployed the same experimental design and exposed microbes for hours at float, we would expect to see a rapid inactivation. Smith et al. (2011) simulated stratospheric conditions and measured a 99.9% loss of viable Bacillus subtilis spores after only 6 hours of direct UV irradiation. Beyond investigating viability alone, transcriptome sequencing and differential expression analysis of RNA could provide valuable insight into the functional effects of stratospheric transport on surviving microorganisms. For instance, this approach could help identify genes associated with repairing damage to the cell envelope, genome, and/or core metabolic proteins. Even with complete survival, our coupons yielded only between 0.53 to 0.97 μg total RNA. Generally, about 1 μg RNA per sample is needed for preparing a transcript library from germinated survivors. Recent developments in library preparation may soon allow reproducible single-cell transcriptomics using as little as 10 pg of starting total RNA (Sasagawa et al., 2013). Nevertheless, follow-on stratosphere experiments may require an order of magnitude increase in the number of spores doped onto coupon surfaces since longer exposures will inactivate a greater portion of samples. Boosting biomass to harvest enough RNA may generate misleading survival data, resulting in a layer of spores that block irradiation effects. Our coupon preparation method using a 100 μl aliquot generated a layer of 1 to 3 spores (Figure 4). Larger coupons (accommodating more spores across the surface) or flying more replicates (to pool samples) may yield enough recoverable RNA without creating unwanted spore layers. The simplicity of the skewer base plate lends itself to customization. Biological requirements can guide the optimal engineering of the skewer base plate. Future investigators can easily reconfigure the base plate to allow for the desired number of replicates and microbe concentrations, or accommodate other categories of microorganisms incompatible with the aluminum coupons.

Earth's stratosphere is extremely dry, cold, irradiated, and hypobaric, and it may be useful for the archive of microorganisms isolated from NASA spacecraft assembly facilities (e.g., Benardini III et al., 2014) to be evaluated in an environment analogous to Mars. Survival-based studies were recently deployed outside the International Space Station (ISS) (Horneck et al., 2012) and the same category of experiments can be conducted in the stratosphere. We hope the baseline data, procedures, and controls discussed herein can provide a pathway for future investigations using E-MIST. While spore-forming bacteria have remarkable resistance to atmospheric extremes, non-spore-forming bacteria, archaea, fungi, algae, and viruses should also be examined. A species-specific inactivation model that predicts the persistence of microbes in Earth's upper atmosphere (e.g., pathogenic cereal rusts), or even on the surface of Mars, is one of many possible outcomes from stratospheric microbiology research.