RETRACTED: Twenty-Three Species of Hypobarophilic Bacteria Recovered from Diverse Ecosystems Exhibit Growth under Simulated Martian Conditions at 0.7 kPa

2.1. Sample sources

Soils were collected by several individuals from nine locations including Arctic, Antarctic, desert, alpine, and Kennedy Space Center sites (Table 1). Soils were shipped and stored at −15°C (most soils) or −70°C (permafrost soils only) until processed. All soils were collected from surface profiles to a depth of 1–2 cm, except the Siberian permafrost soils, which were collected from permafrost boreholes (Nicholson et al., 2013).

Non-soil samples were collected from diverse ecological settings. Several local volunteers provided saliva, hand-rinsate, forearm hair, or scalp hair for assays. In addition, we collected plant leaf imprints on selective media, seawater samples from the surf along the Florida coast at the Merritt Island National Refuge, and swabs of bench tops in one of our labs (ACS). Saliva samples were collected as 1 mL aliquots in 15 mL sterile polystyrene tubes. Saliva samples (100 μL/plate) were then plated directly onto 0.5× trypticase soy agar (TSA). Hand rinsates were collected by submerging hands in 500 mL of sterile deionized water for 60 s (with agitation), the water filtered through sterile 0.45 μm nitrocellulose filters (Whatman, 7190-004, GE Healthcare Life Sciences, Kent, England), filters inverted on TSA, and incubated as described below. Hair was collected aseptically in the lab from volunteers and placed directly on selective media. Seawater was collected, and then three 500 mL aliquots were filtered through separate sterile 0.45 μm nitrocellulose filters, the filters inverted on selective media, and incubated at 0.7 kPa, as described below. After 7 days, the filters from both hand rinsate and seawater samples were removed and discarded. The filter cultures were then returned to the low-pressure conditions. Leaf imprints were created by randomly selecting fully expanded true leaves from a coral bean plant (Erythrina herbacea L.) adjacent to the Space Life Sciences Lab (SLSL; Merritt Island, FL). The leaves were inverted and the adaxial surfaces pressed into agar surfaces for 30 s. The leaf imprints were then incubated at 0.7 kPa, as described below.

2.2. Soil dilution plate assays

All soils were removed from cold storage and allowed to partially thaw for sample removal. One gram of each soil was added to separate 125 mL flasks containing 25 mL of autoclaved 0.1% water agar, and vigorously mixed with magnetic stir bars for 10 min. The 0.1% water-agar mixtures were semiliquid when stirred and acted to keep soil particles in suspension. The soil/agar suspensions (200 μL/plate) were plated directly onto double-thick (30 mL per plate) 0.5× TSA (BD Difco, Becton, Dickinson, and Company, Sparks, MD, USA) or R2A agar (BD Difco) plates.

2.3. Hypobaric conditions

After plating, all samples were immediately placed under low-pressure (0.7 kPa), low-temperature (0°C), and CO₂-enriched anoxic (low ppO₂) conditions (henceforth called low-PTA conditions), as described previously (Schuerger et al., 2013). The hypobaric assays were conducted in 4-L polycarbonate desiccators (model 08-642-7, Fisher Scientific, Pittsburg, PA, USA) fitted with 0.45 μm HEPA filters (Whatman, model 6723-5000) on the top and bottom sections for sterile operation of the vacuum and repressurization processes (Schuerger et al., 2013). The desiccators were connected to an external pump and controller (model PU-842, KNF Neuberger, Trenton, NJ, USA) capable of holding pressure at 0.7 kPa (±0.1 kPa).

Anoxic conditions within the desiccators were achieved by first placing four anaerobic sachets (Remel AnerobicPack, Fisher Scientific) into the vacuum chambers, closing the desiccators, and flushing them with research-grade CO₂ for 5 min. The samples were then held in the low-PTA conditions for 28 days, with the anaerobic sachets changed every 7 days. After 4 weeks, the plates were inspected visually for observable colonies (Round 1 hypobaric assay). Visible colonies were picked, streaked-purified on the same media (0.5× TSA or R2A) used in the Round 1 assays, and incubated at lab conditions of 101.3 kPa, 24°C, and Earth-normal O₂/N₂ atmosphere. The pure cultures were then applied to fresh media with heat-sterilized loops and incubated under low-PTA conditions for 14 days (Round 2). Single isolated colonies from Round 2 were picked, re-streaked onto fresh plates of the same medium, and incubated a third time under low-PTA conditions for 14 days (Round 3). Isolates that grew through Rounds 1–3 were given strain designations (Table 2), stored at −70°C as frozen glycerol stocks, and characterized further.

The Siberian permafrost isolates were then processed for a separate study on the effects of low temperature on colony morphology and pigmentation. Permafrost hypobarophiles were grown on 0.5× TSA for 7 days at 15°C under either an anoxic atmosphere (CO₂-enriched anaerobic chambers) or a lab-normal oxygenated atmosphere (O₂/N₂; 21%/78%, respectively). Bacterial colonies were imaged at identical magnifications and lighting conditions.

If either 0.5× TSA or R2A media were placed in the desiccators and pumped directly from 101.3 to 0.7 kPa, the media would often bubble, crack, and deform. Thus, to permit the outgassing of dissolved lab air from the agar, a three step pump-down procedure was developed. Media in Petri dishes were placed in the 4-L desiccators as described above, set in the 0°C incubator, pumped to a range of 7.5–10.0 kPa, and allowed to equilibrate for 30 min. Then the vacuum chamber was pumped to 2.5 kPa for an additional 30 min. After a total elapsed time of 1 h for outgassing, the vacuum chambers were then allowed to equilibrate at 0.7 kPa.

2.4. 16S rRNA sequencing

Bacteria that demonstrated growth at 0.7 kPa through Rounds 1–3 were labeled as hypobarophiles, and the 16S rRNA genes were sequenced from purified strains. DNA was extracted from each strain with the UltraClean Microbial DNA Isolation Kit (12224-50, MoBio Laboratories, Inc., Carlsbad, CA, USA) and PCR amplified according to the protocols of Benardini et al. (2003). All PCR amplicons were then cleaned with the QIAquick PCR Purification Kit (model 28104, QIAGEN Sciences, Valencia, CA, USA). Universal bacterial primers for the 16S rRNA region were B27F (5′-GAGTTTGATCMTGGCTCAG-3′) and B1512R (5′-AAGGAGGTGATCCANCCRCA-3′) (Lueders et al., 2004). Purified PCR amplicons were Sanger sequenced at the Interdisciplinary Center for Biotechnology Research (ICBR) at the University of Florida (Gainesville, FL, USA).

Strains were assigned taxonomic affiliations based on ≥97.5% similarities (Stackebrandt et al., 2002) to published entries in the Ribosomal Database Project (RDP) library (Release 10, Update 31) (Cole et al. 2009) [http://rdp.cme.msu.edu]. Nucleotide sequences have been deposited in GenBank [http://ncbi.nlm.nih.gov/genbank; National Center for Biotechnology Information, USA] under the accession numbers given in Table 2.

2.5. Growth of type-strains for the genus Serratia under low-PTA conditions

Schuerger et al. (2013) demonstrated that the type-strain Serratia liquefaciens (ATCC 25792) was capable of growth under low-PTA conditions at 0.7 kPa, and Nicholson et al. (2013) demonstrated the same capability for nine type-strains in the genus Carnobacterium. To determine whether a genus-wide capability for low-pressure growth similar to Carnobacterium existed for Serratia, eight type-strains in the genus Serratia were incubated under low-PTA conditions.

Cultures of Serratia type-strains (Table 3) were grown under low-PTA conditions on 0.5× TSA for 35 days. Four additional bacterial strains were grown at 0.7 kPa as either positive controls (Carnobacterium inhibens subsp. gilichinskyi, strain WN1359; Nicholson et al., 2015) or as negative controls (Bacillus subtilis, Escherichia coli, and Sporosarcina aquamarina; Schuerger et al., 2013). Growth for each strain was recorded using a simple rating system that ranked growth relative to the size and morphology of the control strains at Earth-normal lab conditions of 101.3 kPa, 30°C, and Earth-normal O₂/N₂ atmosphere (21%/78%, respectively). Ratings in Table 3 are given as [+] indicating weak growth to [++++] indicating vigorous growth (see Fig. 1); [−] = no growth. All bacteria with a recorded [−] growth response after 35 days were returned to lab conditions for 48 h. In all cases, the strains exhibiting negative growth at 0.7 kPa responded to the lab conditions and grew normally (data not shown).

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FIG. 1.

Cultures of soil dilution plates incubated under lab (A) or martian (B) conditions. Soil dilution plates of soils were incubated on 0.5× TSA or R2A media under lab (101.3 kPa, 25°C, and 21% ppO₂) or martian (0.7 kPa, 0°C, and CO₂-enriched anoxic atmosphere) conditions. (A) Bacterial (b) and fungal (f) colonies were abundant on agar surfaces when maintained 28 days under lab conditions. (B) In contrast, only bacterial colonies (arrows) were observed at low numbers on soil dilution plates incubated under martian conditions. Compare the microbial growth on lab-incubated soil dilutions with Colour Lake soils (Fig. 1A; lower left) to the growth of only hypobarophiles (arrows) from the Colour Lake soils incubated at 0.7 kPa (Fig. 1B). (Color graphics available at www.liebertonline.com/ast)

3.1. Isolation of hypobarophilic bacteria from soils

Most soil samples grown under lab conditions of 101.3 kPa, 20°C, and Earth-normal O₂/N₂ conditions exhibited high diversities of bacteria and fungi on the agar surfaces of the soil-dilution plates. Figure 1A is an example of the high microbial diversity observed in four soils from Río Tinto, Spain; Atacama Desert, Chile; Colour Lake, Axel Heiberg Island, Canada; and Mt. Baker, Washington, USA. Both bacterial and fungal colonies were observed with multiple colony morphologies and pigmentations. In contrast, when hypobarophilic bacteria were recovered from soil-dilution plates maintained at 0.7 kPa, only white, translucent, and smooth-margined colonies were observed (Fig. 1B).

Total culturable bacteria in soils were estimated to range between 5.8 × 10¹ colony-forming units (cfu)/g for the Antarctica soils from Battleship Promontory and 1.5 × 10⁸ cfu/g of soil for the permafrost soils (Table 1). The total culturable fungi were estimated to range between zero for three cold-temperature soils from the Arctic or Antarctica and 3.7 × 10⁵ cfu/g for the Florida soils collected around the Payload Hazardous Servicing Facility at the Kennedy Space Center.

Hypobarophilic bacteria were recovered from soils in a range between zero cfu/g for one-half of the soils tested and 5.1 × 10⁴ cfu/g of soils from Colour Lake (Table 1). Although fungal colonies were abundant in most soils grown under lab conditions (Fig. 1A), no fungi were observed to grow on either 0.5× TSA or R2A when plates were incubated at low-PTA conditions. In addition, all bacteria-like isolates recovered from TSA or R2A agar surfaces incubated under low-PTA conditions (Fig. 1B) were initially sequenced with 16S primers. In all cases, 16S amplicons were recovered and identified to species in the domain Bacteria. Thus, no archaea were recovered from TSA or R2A cultures incubated at low-PTA conditions.

Two hypobarophilic bacteria, Streptomyces aureus and S. vinaceus (Table 2), were isolated on three separate iterations of Round 1 assays with the Colour Lake soils but failed to grow under low-PTA conditions when streak-purified and tested in Round 2 assays. Although it is unknown whether the failure to grow in Round 2 assays for S. aureus and S. vinaceus was due to missing geochemical or biological components in the soil matrix, the Streptomyces species were consistently isolated in three separate Round 1 assays in the presence of the soil particles and undescribed biological communities. Thus, we list them here as presumptive hypobarophiles. All other bacteria that maintained growth at low-PTA conditions for Rounds 2 and 3 are listed as confirmed hypobarophiles (Table 2).

The focus for the current study was to recover and identify only culturable hypobarophilic species; thus no further work was attempted to isolate or identify culturable or nonculturable bacteria, archaea, and fungi that may have been concomitantly present in the soils. Furthermore, all hypobarophiles that grew under anoxic conditions at 0.7 kPa were also able to grow under Earth-normal ppO₂ at 101.3 kPa, except two strains of the obligate anaerobe Clostridium sp. (Table 2).

3.2. Isolation of hypobarophiles from non-soil samples

After Round 1 assays, hypobarophilic bacterial colonies were observed on agar surfaces used to process human saliva and hand rinsate (volunteer #1 only) and seawater (coastal surf along Florida on the Merritt Island National Refuge). The hypobarophiles from saliva and hand rinsate were identified as Leuconostoc gasicomitatum, L. gelidum, and L. inhae (Table 2). In contrast, the two colonies observed at low-PTA conditions from seawater were not recovered during Round 2 subculturing assays (Table 1). Other samples from coral bean plants, forearm hair, lab benches, and scalp hair were negative for the presence of hypobarophilic bacteria. Fungi were not observed on any of the non-soil samples incubated at low-PTA conditions.

Several of the non-soil sample assays were conducted in a nonquantitative manner, so no estimates of background populations of culturable bacteria or fungi were possible. However, coral bean surfaces (10 cm² basis) and lab benches (100 cm² basis) yielded low numbers of bacteria and fungi per unit areas of the assays (Table 1). In contrast, human saliva yielded no culturable fungi and high numbers of culturable bacteria (6.4 × 10⁶ cfu/mL) from all three volunteers.

3.3. 16S rRNA sequencing for confirmed hypobarophiles

Table 2 lists 76 strains of hypobarophilic bacteria from all sources and is arranged in the following order of source materials: soils, non-soil samples, and type-strains of Serratia with the soils, and then hypobarophiles listed in alphabetical order within each category. Although Table 2 represents primarily confirmed hypobarophiles that successfully completed three rounds of incubation under low-PTA conditions, five strains of the presumptive hypobarophiles Streptomyces aureus and S. vinaceus are included for completeness. Assigning taxonomic affiliation at the species level was based on the closest matches being at ≥97.5% 16S sequence identity (Stackebrandt et al., 2002). If the closest matches were either below 97.5% in the RDP database or the taxonomic affiliations listed in the RDP database lacked species-level identities, the taxonomic affiliations were kept at the genus level.

Hypobarophilic bacteria were identified representing 3 phyla, 10 families, and 11 genera (Table 2), demonstrating the presence of hypobarophiles in diverse and globally distributed ecosystems. The highest numbers of hypobarophiles, with the greatest diversity, were recovered from Colour Lake and Siberian permafrost soils and human saliva (volunteer #1), representing the genera Carnobacterium, Exiguobacterium, Leuconostoc, Paenibacillus, and Trichococcus. However, many of the hypobarophiles remain to be characterized at the species level. Hypobarophiles are now identified in the families Bacillaceae, Bacillales_Incertae Sedis XII, Carnobacteriaceae, Clostridiaceae, Enterobacteriaceae, Leuconostocaceae, Microbacteriaceae, Norcardiaceae, Paenibacillaceae, and Streptomycetaceae.

3.4. Growth of type-strains in the genus Serratia under low-PTA conditions

Six of eight type-strains in the genus Serratia exhibited growth under low-PTA conditions, including S. ficaria, S. fonticola, S. grimesii, S. liquefaciens, S. plymuthica, and S. quinivorans (Fig. 2; Table 3). In contrast, S. marcescens and S. rubidaea failed to grow under all low-temperature and low-pressure conditions; thus the effect of low pressure alone could not be separated from the effect of low temperature. The growth of Carnobacterium inhibens subsp. gilichinskyi, strain WN1359 at low-PTA conditions confirms earlier work on growth of nine type-strains at 0.7 kPa for the genus Carnobacterium, including the observation that WN1359 grew slightly better at the low-PTA conditions at 0.7 kPa when compared to growth under aerobic lab conditions at 101.3 kPa (Nicholson et al., 2013). The growth or lack of growth for the positive and negative controls, respectively (Table 3), matched the growth under low-PTA conditions previously reported for these strains (Nicholson et al., 2013; Schuerger et al., 2013).

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FIG. 2.

Growth of Serratia spp. on TSA under diverse conditions of pressure, temperature, and gas composition. Cultures were incubated at (A) Earth-lab conditions of 101.3 kPa, 30°C, and O₂/N₂ atmospheres, (B) low-temperature conditions of 101.3 kPa, 0°C, and O₂/N₂ atmospheres, (C) low-temperature and high-CO₂ conditions of 101.3 kPa, 0°C, and CO₂-enriched anoxic atmospheres, and (D) Mars conditons of 0.7 kPa, 0°C, and CO₂-enriched anoxic atmospheres. Control bacterial strains included the following (left to right; bottom row): Bacillus subtilis 168, Carnobacterium inhibens subsp. gilichinskyi WN1359, Escherichia coli ATCC 35218, and Sporosarcina aquamarina SAFN-008. The numbering system goes left to right and from top to bottom (1 through 12) and coincides with the order in Table 3 (from top to bottom). (Color graphics available at www.liebertonline.com/ast)

Hypobarophilic bacteria picked from 0.5× TSA or R2A media maintained under low-PTA conditions were, in general, white and translucent (Fig. 1B). Similarly, six of eight Serratia type-strains that grew under low-PTA conditions lost the pigmentation observed under lab conditions of 101.3 kPa, 30°C, and Earth-normal O₂/N₂ atmosphere (Fig. 2). In addition, the pigmentation of two strains, Serratia plymuthica and Sporosarcina aquamarina, exhibited increased red pigmentation under a lab pressure of 101.3 kPa when incubated at 0°C in a O₂/N₂ atmosphere, but then returned to a nonpigmented condition when S. plymuthica was grown at 101.3 kPa, 0°C, and CO₂-enriched anoxic atmosphere (Fig. 2). Results suggest that both temperature and low ppO₂ are responsible for the loss of pigmentation in bacterial cells of Serratia spp. tested under low-PTA conditions. Because maintaining stable hydrated media at 0.7 kPa requires low temperatures at or below 0°C (i.e., near the triple-point of water; Haberle et al., 2001), the effects of low pressure alone on cell pigmentation could not be discerned.

3.5. Growth and colony morphology of permafrost strains at 15°C in the presence or absence of oxygen

Similar to the depigmentation observed at reduced temperatures for the hypobarophiles S. plymuthica and S. aquamarina, multiple strains of Cryobacterium sp. (WN1502 and WN1485) and Exiguobacterium sibiricum (WN1486, WN1488, WN1492, and WN1493) were observed to lose their yellow/orange pigmentation when grown under anoxic conditions at 15°C (Fig. 3). The rest of the permafrost hypobarophile isolates produced rather small (<1 mm diameter), cream or light tan colonies and grew either slightly better without oxygen or at approximately the same rate regardless of the presence or absence of oxygen (Fig. 3). Thus, all isolates appeared to be either facultative aerobes or facultative anaerobes. And lastly, all permafrost hypobarophiles picked from soil-dilution plates incubated at low-PTA conditions were not pigmented (similar to Fig. 1B).

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FIG. 3.

Growth and colony morphology of permafrost isolates after 7 days of growth at 15°C. Cultures were incubated on 0.5× TSA either in the presence [(+)O₂] or absence [(-)O₂] of oxygen. Numbers above each paired set of images refer to the WN strain collection numbers in Table 2. Distinctive yellow (Y) and orange (O) pigmented colonies are denoted. Photographs were all taken at the same magnification (see scale bar in lower right corner). (Color graphics available at www.liebertonline.com/ast)

4.1. Implications for growth of bacteriain Earth's atmosphere

Numerous papers have reported the recovery of terrestrial bacteria and fungi in Earth's atmosphere, with the accepted maximum altitude of detection at 41 km (see review by Smith, 2013). But none of the atmospheric sampling studies cited by Smith (2013) examined metabolic activity at the sampled altitudes, or pressure simulations of those altitudes. It remains a goal of aeromicrobiology to probe the upper limits of the active terrestrial biosphere, but such studies have relied upon sampling of probably dormant airborne cells by aircraft or balloons to predict activity. We report here on a series of experiments to recover and identify hypobarophilic bacteria capable of growth at 0.7 kPa; the pressure equivalent of 34 km in the middle stratosphere. Although the experimental conditions were originally designed to simulate the surface of Mars, the results reported here should be applicable to predicting metabolic activity and growth in the terrestrial atmosphere because all bacterial strains, except Clostridium sp., were found to be facultative anaerobes.

Figure 4 depicts the temperature profile of Earth's atmosphere from the surface to 80 km (based on NASA/NOAA, 1976). Initially the temperature decreases linearly in the troposphere from the surface to the tropopause (12–15 km) with a lapse rate of 6.5°C per kilometer. The temperature remains stable near −60°C through the tropopause, increases through the stratosphere approaching 0°C at the stratopause, and then decreases to −80°C at the top of the mesosphere. Recent work on temperature minima for metabolic activity and growth in bacteria has demonstrated that some species are capable of cell division and growth down to −18°C and maintenance metabolism down to perhaps −33°C (e.g., Rivkina et al., 2000; Panikov and Sizova, 2007; Clarke et al., 2013; see review by Rummel et al., 2014).

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FIG. 4.

Temperature (left) and pressure (right) profiles from sea level to 80 km for a standard Earth atmosphere. The box depicting microbial growth in Earth's troposphere is bounded on the left by a low-temperature threshold of −18°C and extends beyond the graph axis on the right into thermophilic conditions above 100°C. The box depicting conditions conducive for microbial growth on Mars is bounded on the left by the temperature minimum for growth (-18°C) and on the right by the stability of liquid water (0°C at 0.7 kPa). The altitudes in Earth's atmosphere that approximate the surface pressures on Mars are between 30 and 48 km (0.1–1.0 kPa); thus there is a zone in the upper stratosphere that might fall within temperature and pressure ranges that mimic a portion of the martian surface. (Graph based on the US Standard Atmosphere, 1976; NASA TM-X-74335.)

Based on these low-temperature limits for microbial activity and growth, the upper altitude threshold for microbial growth at −18°C is likely between 5 and 6 km in the troposphere (54–47 kPa) with maintenance metabolism extending perhaps to 7–8 km (41–36 kPa) (Fig. 4). Recent data (Nicholson et al., 2010; Fajardo-Cavazos et al., 2012; Schuerger et al., 2013; Waters et al., 2014) suggest that pressures down to 5 kPa are not limiting for microbial growth for most species; thus microbial activity will be constrained by other parameters in the atmosphere such as UV irradiation, temperature, desiccation effects, and access to nutrients and liquid water. Based on temperature constraints alone, the only other plausible region in Earth's atmosphere where microbial growth might resume would be in the upper stratosphere and stratopause between 40 and 50 km in which temperature once again approaches 0°C. However, the pressure range in the upper stratosphere from 40 to 50 km is ∼0.28 to 0.076 kPa (2.8 to 0.76 mbar), a pressure range that has not yet been shown capable of supporting microbial growth.

Work here with hypobarophiles at 0.7 kPa and other research with mesobarophiles down to 2.5 kPa (Schuerger and Nicholson, 2006; Schuerger et al., 2006, 2013; Nicholson et al., 2010; Fajardo-Cavazos et al., 2012; Waters et al., 2014) suggests that most airborne bacteria are capable of metabolic activity and growth up to the middle troposphere where temperature, not pressure, becomes limiting at 5–8 km. This should hold for bacteria present as individual cells, aggregates of cells, cell/aerosol assemblages, or embedded cells in ice crystals. Although the atmosphere warms up again approaching 0°C in the upper stratosphere, it has not yet been demonstrated that bacteria are capable of acquiring nutrients and water under the ultralow pressures in the upper stratosphere near 0.28 kPa to permit cellular metabolism and growth. Thus, the lower to middle troposphere may be designated an airborne microbial habitat as suggested by Diehl (2013), but higher altitudes may serve only as conduits for microbial dispersal between terrestrial and marine ecosystems. To fully characterize the upper limits of the active aerial biosphere on Earth, work must be expanded to probe the absolute minima for microbial metabolism and growth for pressures below 0.7 kPa.

4.2. Implications for growth of bacteriaon the surface of Mars

Robotic missions to potentially habitable destinations on Mars (i.e., called Special Regions; see Rummel et al., 2014) are required to comply with international guidelines of planetary protection as established by the Committee on Space Research (COSPAR) (Bruckner et al., 2009). In addition, guidelines are established to align future human missions to Mars with existing planetary protection protocols for mitigating forward contamination of the surface (Criswell et al., 2005; Race et al., 2008). Critical to planetary protection guidelines for both robotic and crewed missions to Mars is an understanding of how and to what extent terrestrial microorganisms might survive, grow, and evolve on the surface.

Here, we extend to 34 the number of bacterial species capable of growth under martian conditions at 0.7 kPa (current results; Nicholson et al., 2013; Schuerger et al., 2013) and widen the source regions for recovery of hypobarophiles to a diversity of arctic, temperate, and even human ecosystems. However, two key questions must be addressed to properly model the risk of contaminating the surface of Mars. First, are hypobarophiles present on spacecraft hardware? And second, if present, will they be able to grow on the actual surface of Mars?

Comparing the species listed in Table 2 with microbial surveys of planetary spacecraft and human missions, several of the hypobarophiles identified here may have been present on space hardware prior to launch. For example, species in the genera Bacillus, Clostridium, Exiguobacterium, Paenibacillus, Rhodococcus, Serratia (including S. liquefaciens), and Streptococcus are reported here as hypobarophiles and have been recovered from robotic spacecraft (La Duc et al., 2003), spacecraft assembly facilities (SAFs) (Moissl et al., 2007; Probst et al., 2010; Vaishampayan et al., 2010), and human missions to the International Space Station or the Mir space station (La Duc et al., 2004; Novikova, 2004). Furthermore, at least three species of Leuconostoc were recovered from human saliva and hand rinsate that exhibited growth at 0.7 kPa. Thus, it is reasonable to suggest that hypobarophiles may have been present on some robotic and human spacecraft prior to launch.

Next, can launched hypobarophiles plausibly grow on the surface of Mars? First, our results here and elsewhere (Nicholson et al., 2013; Schuerger et al., 2013) support the conclusion that hypobarophiles are capable of growth under low-PTA conditions near 0.7 kPa that approximate some of the environmental conditions on the surface of Mars. However, the growth rates of the hypobarophiles were extremely slow, and assays often required between 10 and 14 days of growth on continuously stable, hydrated, and nutrient-rich media for small pin-prick-sized colonies to appear on the agar surfaces. Thus, can terrestrial hypobarophiles acquire adequate liquid water and nutrients on the thermodynamically unstable surface of Mars over the course of several sols to carry out metabolism and growth?

Rummel et al. (2014) discussed the thermodynamics of liquid water on the martian surface in order to model the occurrence of Special Regions on Mars, which are defined as “a region within which terrestrial organisms are likely to replicate, or a region which is interpreted to have a high potential for the existence of extant martian life forms.” With few exceptions, the martian surface appears to be either too cold (<−18°C minimum), too dry (<0.60 water activity [a _w] minimum), or both for growth of terrestrial microorganisms. At low latitudes, surface temperatures can rise above the −18°C minimum threshold for terrestrial microbial growth, but then a _w is significantly below the 0.60 minimum for microbial activity (often <0.05 a _w on Mars). At higher latitudes, a _w may begin to approach conditions conducive for microbial activity (>0.60), but the temperature quickly falls below −18°C. Several possible exceptions were noted by Rummel et al. (2014), with recurring slope lineae (RSL; most likely caused by subsurface seepage down steep slopes by liquid brines at temperatures > −20°C), shallow subsurface ices, and partially collapsed lava caves being the most likely to provide stable hydrated niches that might support terrestrial microbial activity and growth. Thus, diurnal and seasonal fluctuations in temperature and a _w are likely to inhibit active metabolism and growth of terrestrial microorganisms on most martian terrains, with possible exceptions of hypobarophiles colonizing locations with stable hydrated brines that fall within temperature and pressure ranges conducive for growth.

One such example of a stable liquid environment near the martian surface may be active RSL recently identified with spectral signatures of hydrated perchlorate and chlorate salts consistent with liquid brines, although other salts could not be ruled out (Ojha et al., 2015). Perchlorates can be both toxic to, and utilized as an energy source by, microorganisms (Coates and Achenbach, 2004). Previously, we have demonstrated the growth of Bacillus subtilis and B. pumilus in hydrated soil solutions extracted from a Mars analog soil modeled after the Phoenix landing site with 1.5 wt % sodium perchlorate salt (Nicholson et al., 2012). And of the 23 hypobarophilic species described here, we have tested the growth of S. liquefaciens in nonperchlorate salt solutions relevant to the surface of Mars. Vegetative cells of S. liquefaciens were shown capable of growth between 5°C and 30°C in brines up to 5% for MgCl₂ and NaCl, and up to 10% for MgSO₄ (Berry et al., 2010). The presence of hydrated salts in RSL may provide liquid niches conducive for growth of terrestrial hypobarophiles delivered to the surface by spacecraft, and may provide accessible locations in which to search for extant life on Mars. If hydrated brines are present as shallow subsurface features in active RSL sites, salt tolerance becomes an additional parameter that terrestrial hypobarophiles must overcome in order to grow near the martian surface. Future research should explore interactive effects of low pressures near 0.7 kPa, low temperatures required for stable liquid brines at 0.7 kPa, and high salt concentrations that mimic surface regolith conditions on Mars.

And lastly, Fig.4 depicts a zone in Earth's upper stratosphere in which temperature is above the microbial growth minimum of −18°C and lies between the pressure range on Mars of 0.1 kPa (top of Olympus Mons) and 1.0 kPa (Hellas Basin). The Mars-like microbial growth zone in Earth's stratosphere is also bounded by 0°C at the high end of temperature due to the stability of liquid water at low pressures (Haberle et al., 2001; Rummel et al., 2014). Above 0°C at 0.7 kPa, liquid water is not stable and sublimates directly from ice to the gaseous phase. Research into microbial activity and growth of hypobarophiles in situ within the terrestrial stratospheric “Mars zone” by long-duration experiments on high-altitude balloons or in ground simulations at pressure and temperature conditions present in the stratosphere (current study) will inform planetary protection and astrobiology efforts in locating Special Regions on Mars. We demonstrate here that hypobarophiles are present in diverse terrestrial ecosystems and that there may be zones in both the terrestrial atmosphere and the surface of Mars that may support metabolism and growth of terrestrial microorganisms.