Introduction
Spacecraft assembly facilities (SAFs) are clean rooms with stringent protocols to prevent dust and microbes from contaminating robotic probes as much as possible. The microbes found in SAFs are the most likely to be carried to celestial bodies, having the potential for contamination that may confound the search for life (Favero et al., Reference Favero, Puleo, Marshall and Oxborrow1966; Favero, Reference Favero1971; Puleo et al., Reference Puleo, Fields, Bergstrom, Oxborrow, Stabekis and Koukol1977). Diverse assemblages of microbes have been detected in SAFs and described through cultivation campaigns and molecular genetic analyses (Favero et al., Reference Favero, Puleo, Marshall and Oxborrow1966; Favero, Reference Favero1971; Foster and Winans Reference Foster and Winans1975; Puleo et al., Reference Puleo, Fields, Bergstrom, Oxborrow, Stabekis and Koukol1977; Moissl et al., Reference Moissl, Bruckner and Venkateswaran2008; Probst et al., Reference Probst, Vaishampayan, Osman, Moissl, Anderson and Venkateswaran2010; La Duc et al., Reference La Duc, Osman, Vaishampayan, Piceno, Andersen, Spry and Venkateswaran2009; Stieglmeier et al., Reference Stieglmeier, Wirth, Kminek and Moissl-Eichinger2009; Ghosh et al., Reference Ghosh, Osman, Vaishampayan and Venkateswaran2010; Bashir et al., Reference Bashir, Ahmed, Weinmaier, Ciobanu, Ivanova, Pieber and Vaishampayan2016; Hendrickson et al., Reference Hendrickson, Urbaniak, Malli Mohan, Heidi and Venkateswaran2017, Reference Hendrickson, Urbaniak, Minich, Aronson, Martino, Stepanauskas, Knight and Venkateswaran2021; Probst and Vaishampayan Reference Probst and Vaishampayan2020; Danko et al., Reference Danko, Sierra, Benardini, Guan, Wood, Singh, Seuylemezian, Butler, Ryon, Kuchin, Meleshko, Bhattacharya, Venkateswaran and Mason2021; Carte et al., Reference Carte, Chen, Clark and Schneegurt2024a,b). The SAF bacterial assemblage has been reported to be rich in Arthrobacter, Bacillus, Exiguobacterium, Filibacter, Oceanobacillus, Sporosarcina, Staphylococcus and Streptococcus, all associated with the human microbiome or soils (La Duc et al., Reference La Duc, Nicholson, Kern and Venkateswaran2003, Reference La Duc, Osman, Vaishampayan, Piceno, Andersen, Spry and Venkateswaran2009, Reference La Duc, Vaishampayan, Nilsson, Torok and Venkateswaran2012; Link et al., Reference Link, Sawyer, Venkateswaran and Nicholson2003; Venkateswaran et al., Reference Venkateswaran, Hattori, La Duc and Kern2003; Kempf et al., Reference Kempf, Chen, Kern and Venkateswaran2005; Satomi et al., Reference Satomi, La Duc and Venkateswaran2006). Greater diversity is apparent in genetic libraries, including anaerobes that are not typically cultivated (Moissl et al., Reference Moissl, Bruckner and Venkateswaran2008; Stieglmeier et al., Reference Stieglmeier, Wirth, Kminek and Moissl-Eichinger2009; La Duc et al., Reference La Duc, Venkateswaran and Conley2014; Carte et al., Reference Carte, Chen, Clark and Schneegurt2024a). Actinobacteria, Firmicutes and Gammaproteobacteria were observed, including the genera Bacillus, Clostridium, Enterococcus, Paenibacillus and Staphylococcus.
Clean rooms apply selective pressures on microbial assemblages, with low humidity, low nutrient availability and extremely low total biomass. The dry conditions in clean rooms may enrich the microbial assemblage for salinotolerant microbes, since aerosols when dried can leave salt evaporites. Bacterial isolates from SAFs have been shown to be salinotolerant, with substantial radiation resistance and tolerance to oxidants (Venkateswaran et al., Reference Venkateswaran, Satomi, Chung, Kern, Koukol, Basic and White2001, Reference Venkateswaran, Hattori, La Duc and Kern2003, 2014; La Duc et al., Reference La Duc, Nicholson, Kern and Venkateswaran2003; Link et al., Reference Link, Sawyer, Venkateswaran and Nicholson2003; Kempf et al., Reference Kempf, Chen, Kern and Venkateswaran2005; Smith et al., Reference Smith, Benardini III, Anderl, Ford, Wear, Schrader, Schubert, DeVeaux, Paszczynski and Childers2017; Zanmuto et al., Reference Zanmuto, Fuchs, Fiebrandt, Stapelmann, Ulrich, Maugeri, Pukall, Gugliandolo and Moeller2018; Carte et al., Reference Carte, Chen, Clark and Schneegurt2024a). Long-term enrichment cultures at high salinity resulted in a diverse bacterial community from SAF wipes that included representatives of each biogeochemical functional guild required for the C, N and S cycles (Carte et al., Reference Carte, Chen, Clark and Schneegurt2024a). Salinotolerant microbial communities, capable of biogeochemical cycling, are more likely to persist on celestial bodies than individual polyextremophile isolates. This becomes important in the context of the chemical conditions on Mars. All of the most attractive environments to search for life on Mars are (potentially) hypersaline, including ices and their brine channels, caves, subsurface and evaporite minerals (Carrier et al., Reference Carrier, Beaty, Meyer, Blank, Chou, DasSarma, Des Marais, Eigenbrode, Grefenstette, Lanza, Schuerger, Schwendner, Smith, Stoker, Tarnas, Webster, Bakermans, Baxter, Bell, Benner, Bolivar Torres, Boston, Bruner, Clark, DasSarma, Engelhart, Gallegos, Garvin, Gasda, Green, Harris, Hoffman, Kieft, Koeppel, Lee, Li, Lynch, Mackelprang, Mahaffy, Matthies, Nellessen, Newsom, Northup, O'Connor, Perl, Quinn, Rowe, Sauterey, Schneegurt, Schulze-Makuch, Scuderi, Spilde, Stamenković, Torres Celis, Viola, Wade, Walker, Wiens, Williams, Williams and and Xu2020). Environments on ocean worlds also may be salty, from their oceans to briny sills in their icy crusts, to evaporite deposits on their surfaces. There is practical value in understanding the salinotolerant microbial assemblages in SAFs, which are most likely to be transported to the hypersaline environments of solar system bodies.
The current study collected wipe samples of SAF surfaces to measure the abundance of viable salinotolerant microbes using most probable number (MPN) analyses. Enrichment cultures at high salinity were used to isolate microbes from the SAF wipes. Only Gram-positive bacteria were recovered, and these were characterized with respect to their growth tolerances to NaCl, MgSO4 and sucrose at high concentrations and their range of growth tolerances to pH and temperature.
Methods
Sampling of SAFs
Sterile polyester wipes (Texwipe; Kernersville, NC), moistened with 15 ml of sterile water, were used to swab 1-m2 surfaces of high-traffic floors of the NASA Jet Propulsion Laboratory (JPL) Aseptic Assembly Facility or the main assembly bay, certified ISO 5 clean rooms. All entrants into the ISO 5 clean rooms donned sterile gowning and gloves. The environment was monitored for biological cleanliness by surface sampling, air sampling and utilization of an instantaneous detection system for airborne particles (microbial and inert).
Several wipe samples and a procedural blank were taken using fresh pairs of sterile gloves. The wipes were packaged in sterile polypropylene tubes with screw caps and shipped overnight in a cool container from JPL to Wichita State University. Upon arrival, the wipes were wetted with 30 ml of a sterile chaotropic solution (0.1% Na pyrophosphate) to dislodge microbes. After 10 min, the liquid was squeezed from the wipes in the tubes with a sterile syringe plunger.
Abundance of salinotolerant microbes by MPN
Liquids from SAF wipe samples were used directly to inoculate tubes for most probable number (MPN) analyses. The arrays were designed with five repetitions of six fivefold or tenfold serial dilutions in 13 × 100-mm culture tubes with a volume of 2 ml and then maintained on an orbital shaker (150 rpm) at room temperature. MPN analyses were performed in Salt Plains (SP) medium containing (per liter): NaCl, 1 g; KCl, 2.0 g; MgSO4·7H2O, 1.0 g; CaCl2·2H2O, 0.36 g; NaBr, 0.23 g; FeCl3·6H2O, 1.0 mg; trace minerals, 0.5 ml; yeast extract, 10.0 g; tryptone, 5.0 g; glucose, 1.0 g; and brought to a final pH of 7.0 (Caton et al., Reference Caton, Witte, Nguyen, Buchheim, Buchheim and Schneegurt2004), unsupplemented or supplemented (all w/v) with 10% NaCl (1.7 M; a w = 0.92), 50% MgSO4 (2.0 M as epsomite; a w = 0.94), 5% NaClO3 (0.5 M; a w = 0.98), or 5% NaClO4 (0.4 M; a w = 0.98). Positive tubes were scored after 2 wk, visually or by measuring turbidity at 600 nm using a Genesys 10S spectrophotometer (ThermoFisher). A threshold value of 0.2 OD units was used to score positive growth. Scores were compared to a statistical table to determine the MPN and the 95% confidence interval factor was 3.3 (Woomer Reference Woomer and Weaver1994).
Bacterial isolation and characterization
Positive MPN cultures containing 50% MgSO4 were spread plated on SP medium supplemented with 25% MgSO4. Colonies were haphazardly collected and re-streaked on SP medium supplemented with 10% NaCl, selecting isolated colonies, six times to purify bacterial strains. The isolates were maintained on SP medium supplemented with 10% NaCl and their physiology and biochemistry were characterized.
Gram stain was performed using Harleco reagents (Sigma-Aldrich) following the manufacturer’s instructions. The endospore stain (Thermo Scientific) was performed following the manufacturer’s instructions. The presence of catalase was determined by applying 3% hydrogen peroxide solution to smears of culture on microscope slides. The presence of oxidase was determined using DrySlides (BBL). Starch agar plates (Difco) were inoculated and incubated at 37 °C and then flooded with iodine solution once grown to observe hydrolysis by amylase.
Solute tolerance was measured in SP medium supplemented with various concentrations (all w/v) of NaCl (1, 10, 20 and 30%), MgSO4 (20, 30, 40 and 50%) and sucrose (30, 50 and 70%). Shake-tubes (2 ml in 13 × 100-mm tubes) were lightly inoculated (to below 0.05 OD units at 600 nm) and incubated at room temperature for 2 wk. Growth was measured by absorbance spectrophotometry at 600 nm using a Genesys 10S instrument (ThermoFisher) at 1, 3, 7 and 14 d after inoculation. Similarly, growth was measured in shake-tubes using SP medium with 10% NaCl at various pHs (4 to 9) and temperatures (4 to 60 °C). The threshold for positive growth was 0.2 OD units.
DNA extraction and molecular analyses
Crude DNA extracts were made from aliquots (6 ml) of a liquid culture of each isolate using a freeze-thaw technique (Caton et al., Reference Caton, Witte, Nguyen, Buchheim, Buchheim and Schneegurt2004). Cells were collected by serial microcentrifugation for 5 min at 14,000 × g. Pellets were resuspended in 300 µL of sterile water before 6 cycles of freezing in liquid N2 and thawing at 80 °C, with vigorous vortex mixing every other cycle. Homogenates were clarified by microcentrifugation for 10 min at 14,000 × g and the final supernatant was heated for 5 min at 80 °C. Extracts were stored at –20 °C before PCR amplification.
Gene sequences encoding 16S rRNA from bacterial isolates were amplified using universal bacterial primers (EUBpA: 5’-AGAGTTTGATCCTGGCTCAF-3’ and EUBpH: 5’AAGGAGGTGATCCAGCCGCA-3’) (Edwards et al., Reference Edwards, Rogall, Blöcker, Emde and Böttger1989). Each of the 25-µL reactions contained 2.5 µL of each primer (0.2 µM), 1 U of DreamTaq DNA polymerase in master mix (Thermo Scientific) and 5 µL of DNA extract. A thermal cycler (Eppendorf Mastercycler) denatured the DNA at 95 °C for 2 min, followed by 40 cycles of 95 °C for 1 min, 58 °C for 1 min and 72 °C for 1 min, with a final 5-min extension at 72 °C. PCR amplicons were visualized under ultraviolet light with ethidium bromide stain after electrophoresis on a 2% agarose gel to confirm amplicon size and purity. Single-pass Sanger sequencing was performed by a commercial vendor (Eurofins Genomics, Louisville, KY) using the EUBpA primer. Isolate sequences (≥634 bp) appear in GenBank with accession numbers PQ895309 to PQ895335. Phylogenetic trees were constructed by maximum-likelihood analyses using Jukes-Cantor rules and 100 bootstrap repetitions in MEGAX (Kumar et al., Reference Kumar, Stecher and Tamura2016), from alignments made using SINA v1.2.12 and the SILVA v.138 database, with control sequences selected from GenBank using BLAST.
Results
Microbial abundance in SAFs by MPN
The abundance of microbes tolerant to high and low salinity was measured in washes from wipe samples of the JPL SAFs on two occasions. Microbial abundances by MPN analyses in media supplemented with 50% MgSO4 (2.0 M; a w = 0.94) or 10% NaCl (1.7 M; a w = 0.92) are presented in Table 1 for five wipe samples of SAF surfaces in the main assembly bay. Unfortunately data from the assays of low-salt control cultures were lost. The highest values were from an area near a trash can, reaching 3.6 × 105 cells m−2 for both brines. The floor entrance showed particularly high abundance of microbes tolerant to MgSO4 (2.4 × 105 cells ml−2), much more than the abundance of microbes tolerant to NaCl. The remaining samples ranged from 3.3 × 103 to 4.2 × 104 cells m−2.
Table 1. Microbial abundance by MPN of wipes from surfaces in the JPL SAF main assembly bay.
A second experiment (Table 2) used the same wipe samples from the Aseptic Assembly Facility as our previous study on long-term hypersaline cultivation, microbial isolations and molecular community analyses of the end members of the enrichment (Carte et al., Reference Carte, Chen, Clark and Schneegurt2024a,b). A low-salt control medium was included to determine the relative abundance of tolerant microbes in the SAF assemblage. The abundance of microbes tolerant to 50% MgSO4 was a log unit lower than those tolerant of 5% (per)chlorates and somewhat lower than the abundance of epsotolerant microbes observed in the main assembly bay (Table 1). The microbial abundance of microbes tolerant to 5% NaClO3 (0.5 M; a w = 0.98) (3.4 × 105 cells m−2) was ∼ 5-fold greater than the abundance of microbes tolerant to 5% NaClO4 (0.4 M; a w = 0.98) (7.1 × 104 cells m−2). Overall growth tolerance to perchlorates has been observed previously to be substantially lower than tolerance to chlorates (and chlorides and sulfates), so this result is not unexpected (Al Soudi et al., Reference Al Soudi, Farhat, Chen, Clark and Schneegurt2016). The high percentage of microbes tolerant to NaClO3 at 5% is not surprising given that bacteria in our previous study were shown to tolerate >25% (2.75 M; a w = 0.89).
Table 2. Microbial abundance by MPN of wipes from surfaces in the JPL SAF Aseptic Assembly Facility.
*The locations of these floor samples within the room are shown in Figure 1 of Carte et al., Reference Carte, Chen, Clark and Schneegurt2024a,b.
Note that the MPN technique does not distinguish between bacterial, archaeal and fungal growth; however, these enrichment cultures are likely to be dominated by bacteria, as observed previously (Moissl et al., Reference Moissl, Osman, La Duc, Dekas, Brodie, DeSantis and Venkateswaran2007; Plemenitaš et al., Reference Plemenitaš, Lenassi, Konte, Kejžar, Zajc, Gostinčar and Gunde-Cimerman2014; Venkateswaran et al., Reference Venkateswaran, Vaishampayan, Benardini III, Rooney and Spry2014; Checinska et al., Reference Checinska, Probst, Vaishampayan, White, Kumar, Stepanov, Fox, Nilsson, Pierson, Perry and Venkateswaran2015; Weinmaier et al., Reference Weinmaier, Probst, La Duc, Ciobanu, Cheng, Ivanova, Rattei and Vaishampayan2015; Hendrickson et al., Reference Hendrickson, Urbaniak, Malli Mohan, Heidi and Venkateswaran2017; Carte et al., Reference Carte, Chen, Clark and Schneegurt2024a,b). Archaea and fungi are nearly absent in SAFs. The media used here are designed for bacteria, having salinities too low for haloarchaea, which typically contain >20% NaCl. Furthermore, the cultures were not grown at 37 °C, which is the best choice for haloarchaea. The vast majority of archaea would not grow under the conditions used in the current study. Similarly, the media did not contain enough sugar to support strong growth of common fungi, which grow best with a 10-fold higher concentration of sugar, as in Sabouraud medium (Sabouraud Reference Sabouraud1892; Emmons Reference Emmons, Emmons, Binford and Utz1963). In addition, we did not observe the heavy pellicle or clump of mycellium common for molds, nor did we notice the common odor of yeasts. It is possible that a portion of the turbidity in certain tubes was attributable to yeasts, molds, or haloarchaea, but the growth conditions selected against these, while favoring bacteria. Note that the medium selected for aerobic heterotrophic bacteria that could grow at moderately high salinity and mesophilic temperatures. Therefore, anaerobes and lithotrophs, for instance, would not have substantially contributed to the turbidity observed.
Characterization of SAF isolates
Microbial isolates were obtained by repetitive streak-plating from the first set of SAF samples after a short enrichment in saline media. Identification of the isolates by 16S rRNA sequence analysis showed that all of the isolates were Gram-positive in the low G + C group (Figure 1). Most of the 38 isolates were in the Bacillaceae, with representatives clustering with the genera Bacillus, Cytobacillus, Halobacillus and Virgibacillus. The other cluster included Staphylococcus and the related Jeotgalicoccus. All of these are known from previous studies as having members that are salinotolerant.
Figure 1. Phylogenetic tree based on 16S rRNA gene sequences from SAF bacterial isolates obtained by repetitive streak-plating of saline enrichment cultures of wipe eluates from the main assembly bay.
Halotolerance of the isolates was measured, with all growing in medium supplemented with 10% NaCl (Figure 2). Ten of the isolates (JPL 4, 7, 15, 23, 24, 31-35) spread across the taxa (Figure 1) did not appear to grow at 1% NaCl (0.17 M; a w = 0.95) and may be halophilic, requiring high salt for growth. Measurements were not made in media containing between 1 and 10% NaCl. The majority of isolates (26) grew at 20% NaCl (3.4 M; a w = 0.85) and 9 isolates (JPL 2, 3, 6, 8, 13, 22, 24, 25, 26 and 36) grew in 30% NaCl (5.2 M; a w = 0.76), near saturation. Epsotolerance was prevalent among the isolates, with all but two isolates, growing at 40% MgSO4 (1.6 M; a w = 0.95). The majority of isolates (29) grew at 50% MgSO4, the highest concentration tested. Substantial sucretolerance was observed, using a nonionic solute. More than half of the isolates (21) grew at 50% sucrose (1.5 M; a w = 0.91), while 8 isolates (JPL 2, 3, 6, 13, 24, 25, 26 and 36) grew at 70% sucrose (2.0 M; a w = 0.90), near saturation. Note that all of the isolates grew at 1% sucrose, despite a few not exhibiting growth at 1% NaCl. All of the isolates that grew at 70% sucrose also grew at 30% NaCl and 50% MgSO4. All of the isolates that grew at 30% NaCl also grew at 50% MgSO4.
Figure 2. Growth tolerances of SAF bacterial isolates to NaCl, MgSO4 and sucrose.
The isolates exhibited a wide range of tolerances to temperature (Figure 3). All of the isolates grew at 37 °C, with all but three growing at 45 °C. More than half (23) grew at 60 °C and 80% (30) grew at 4 °C. There were 15 isolates that grew at both 4 and 60 °C. The isolates preferred neutral and basic pH media (Figure 3). All of the isolates grew at pH 8, with 80% (30) growing at pH 9. Eleven isolates grew at pH 10. Only 15 isolates grew at pH 5, while none grew at pH 4. All but one of the isolates that grew at pH 10 also grew at pH 5. All of the isolates were catalase positive but only eight were positive for oxidase (Figure 4). Nine of the isolates positively stained for endospores under the conditions tested. Only two isolates (JPL 9 and 21) were positive for amylase, a characteristic observed to be in low prevalence in previous studies of salinotolerant bacteria (Caton et al., Reference Caton, Witte, Nguyen, Buchheim, Buchheim and Schneegurt2004; Litzner et al., Reference Litzner, Cato and Schneegurt2006).
Figure 3. Growth tolerances of SAF bacterial isolates to pH and temperature.
Figure 4. Occurrence of enzyme activities in SAF bacterial isolates.
Discussion
Concern about microbial bioburden on spacecraft and their assembly facilities has continued since the early days of spaceflight (Favero et al., Reference Favero, Puleo, Marshall and Oxborrow1966; Favero, Reference Favero1971; Foster and Winans Reference Foster and Winans1975; Puleo et al., Reference Puleo, Fields, Bergstrom, Oxborrow, Stabekis and Koukol1977). Over time, the cleanliness of assembly facilities has improved, and the techniques used for spacecraft sterilization are more effective today. The measurement of bioburden in SAFs has taken several approaches. A number of studies have examined airborne microbes in assembly facilities (Newcombe et al., Reference Newcombe, La Duc, Vaishampayan and Venkateswaran2008; Checinska et al., Reference Checinska, Probst, Vaishampayan, White, Kumar, Stepanov, Fox, Nilsson, Pierson, Perry and Venkateswaran2015; Lu et al., Reference Lu, Yang, Zhang, Chen, Han and and Fu2023). Fallout particles from the air are particulate materials that have been shown to carry microbes such as Bacillus spp. (Mohan et al., Reference Mohan, Stricker and Venkateswaran2019). There are several published studies using cultivation to measure microbial abundance from wipe samples of SAF surfaces (v.i.). Other studies have used alternative estimators of microbial abundance such as the prevalence of rRNA genes. The results consistently show that the microbial assemblage in SAFs is nearly entirely bacteria, often with fewer than 1% being fungi or archaea (Moissl et al., Reference Moissl, Osman, La Duc, Dekas, Brodie, DeSantis and Venkateswaran2007; Plemenitaš et al., Reference Plemenitaš, Lenassi, Konte, Kejžar, Zajc, Gostinčar and Gunde-Cimerman2014; Venkateswaran et al., Reference Venkateswaran, Vaishampayan, Benardini III, Rooney and Spry2014; Checinska et al., Reference Checinska, Probst, Vaishampayan, White, Kumar, Stepanov, Fox, Nilsson, Pierson, Perry and Venkateswaran2015; Weinmaier et al., Reference Weinmaier, Probst, La Duc, Ciobanu, Cheng, Ivanova, Rattei and Vaishampayan2015; Hendrickson et al., Reference Hendrickson, Urbaniak, Malli Mohan, Heidi and Venkateswaran2017; Carte et al., Reference Carte, Chen, Clark and Schneegurt2024b). It is not unusual to find the assemblage enriched for organisms that exhibit high tolerance to environmental factors such as salinity, pH, or temperature.
An extensive study of 26 SAF clean room environments determined microbial abundance from surface wipe samples using ATP luminescence, cultivation and qPCR. Cultivable bacteria (in a single heterotrophic medium) ranged over three orders of magnitude from 103 to 106 CFU m−2 (La Duc et al., 2007). Total bacteria determined by qPCR of 16S rRNA genes also ranged greatly, from 106 to 108 copies m−2. There was no agreement across the three methods on microbial abundance, due to factors such as ATP content per cell, gene copies per cell and cultivable vegetative cells versus spores. The cultivated bacterial collection was predominantly Gram-positive Bacillus and Staphylococcus. Proteobacteria were isolated in lower numbers. In another study, cultivable bacteria from ∼ 100 SAF wipe samples averaged 1.2 × 101 to 6.6 × 103 CFU m−2 (mean of 4.4 × 102), while another set of samples assayed by ATP content averaged 2.8 × 104 CFU m−2 (Hendrickson et al., Reference Hendrickson, Urbaniak, Malli Mohan, Heidi and Venkateswaran2017). Heat-shocked isolates were nearly all Bacilli, with Virgibacillus being most abundant, and related Brevibacillus, Oceanobacillus, and Paenibacillus, and Streptomyces were observed. Vaishampayan et al. (Reference Vaishampayan, Probst, La Duc, Bargoma, Benardini, Andersen and Venkateswaran2013) estimated bacterial abundance from floor swabs of an active SAF by qPCR of 16S rRNA genes. The observed abundance ranged from 5 × 104 to 2 × 106 copies m-2. Phylochip analyses found as few as 3 genera in the least populated samples and as many as 411 genera in the most populated samples. Viable microbes in another study, as measured by cultivation from wipe samples, found mesophile abundance ranged from 3 × 102 to 8 × 104 CFU m−2, with most samples towards the lower end of the range (Ghosh et al., Reference Ghosh, Osman, Vaishampayan and Venkateswaran2010). Bacillaceae were observed, but Proteobacteria were more common, and a few Actinomycetes were seen. An early study (Puelo et al., Reference Puleo, Fields, Bergstrom, Oxborrow, Stabekis and Koukol1977) reported that the abundance of cultivable aerobic microbes detected on surfaces of the Viking spacecraft were low (mainly 102, but as high as 104 CFU m−2) relative to floor swabs in previous studies and consisted mainly of Bacillus, Micrococcus and Staphylococcus.
Previous studies have measured the abundance of mesophilic heterotrophic aerobic bacteria. The low-salt controls in the current study, estimating microbial abundance to be between 4.9 × 104 to 6.9 × 106 cells m−2, certainly fall within the wide range observed in previous studies. Our experiments were primarily directed at organisms with greater salinotolerance, which are generally 1 to 10% of the total cultivable bacteria. A previous study reported that SAF floor swabs yielded as many at 1.4 × 103 CFU (3.9 × 103 CFU m−2; calculated based on 0.36 m−2 area, as in Stieglmeier et al., Reference Stieglmeier, Rettberg, Barczyk, Bohmeier, Pukall, Wirth and Moissl-Eichinger2012) on medium supplemented with 10% NaCl, with as many as 8.1 × 103 CFU (2.25 × 104 CFU m−2) at 3.5% NaCl (Moissl et al., 2013). These levels are similar to the abundances observed in the current study. At 4 °C, a range of 5.6 × 102 to >2.0 × 104 CFU (1.6 × 103 to >5.6 × 104 CFU m−2) were observed in different SAFs, and a similar range was observed at pH 11. A lower abundance of 0.0 to 1.9 × 102 CFU (0.0 to 5.3 × 102 CFU m−2) was observed for microbes tolerant to 50 °C. Bacillus, Micrococcus, Paenibacillus and Staphylococcus were isolated that grew at 10% NaCl and pH 11, with some also growing at 50 °C. In contrast, none of the isolates from the largest study SAF microbial abundance were substantially halotolerant (La Duc et al., 2007). In the current study, for instance, Bacillus cereus str. JPL2 and Bacillus stratosphericus str. JPL3 showed growth from pH 4 to 10, from 4 to 60 °C, and over a wide range of salt and sugar concentrations. Overall, more than half of our isolates were tolerant to 20% NaCl (69%) or 50% sucrose (55%), and nearly all (76%) were tolerant to 50% MgSO4. Nearly all (84%) of the collection was tolerant to pH 9, with 39% growing at pH 5. Nearly all (79%) of our isolates grew at 4 °C, with 61% growing at 60 °C. Taken together, these results demonstrate that the SAF isolates from the current study are remarkably capable polyextremophiles.
It is not surprising that SAF isolate collections tend to be dominated by Gram-positive bacteria, particularly those that form endospores, given the dry, oligotrophic conditions in clean rooms. We previously isolated only Gram-positive bacteria from long-term hypersaline enrichment cultures from SAF floor swabs (Carte et al., Reference Carte, Chen, Clark and Schneegurt2024a). While isolates from genera known to produce endospores predominated the collection characterized in the current study, some isolates do not produce endospores, such as Jeotgalicoccus and Staphylococcus. Our cultures were not specifically designed to promote sporulation, however, 9 isolates were positive for endospores by staining. The growth observed in MPN experiments may well have arisen from endospores in the floor wipe samples, since the SAF environment is so dry and oligotrophic, as to be unfavorable for vegetative cells. It is interesting to note our related study, where we measured the abundance of salinotolerant microbes in common soils, with subsequent isolation and characterization (Howell et al., Reference Howell, Kilmer, Porazka and Schneegurt2022). The major isolates were similar to those of the current study, namely, Bacillus, Halobacillus, Staphylococcus and Virgibacillus. When in these natural soils, the organisms were found as vegetative cells, with ∼ 0.1% of viable cells being endospores that survived boiling. Surprisingly, neither study recovered Gram-negative bacteria, despite species such as Halomonas being in high relative abundance in natural hypersaline environments (Caton et al., Reference Caton, Witte, Nguyen, Buchheim, Buchheim and Schneegurt2004).
Our finding that salinotolerant microbes represent a substantial portion of the total microbial community on surfaces in SAFs has implications for planetary protection and the search for life, since the microbes within SAFs are the most likely to be transported to another world by spacecraft. Observing a substantial abundance of microbes tolerant to high concentrations of sulfate and (per)chlorate salts has relevance to Mars regolith, which is richer in sulfate and (per)chlorate salts than soils on Earth. Note that the 5% (per)chlorate used in the current study is much greater, and more inhibitory to microbial growth, than the 0.6% (per)chlorate salts detected on Mars (Hecht et al., Reference Hecht, Kounaves, Quinn, West, Young, Ming, Catling, Clark, Boynton, Hoffman, Deflores, Gospodinova, Kapit and Smith2009; Kounaves et al., 2010; Clark and Kounaves, Reference Clark and Kounaves2015). While (per)chlorate salts would only be relevant to arid worlds, sulfate and chloride salts are found on both arid and ocean worlds.
Acknowledgments
The authors are thankful for the technical assistance of Fawn Beckman and Jonathan Wilks. A preliminary account of this work previously has been presented and abstracted (Carte et al., Reference Carte, Gandikota, Chen, Clark and Schneegurt2020).
Funding statement
This project was supported by awards from National Aeronautics and Space Administration (NASA), Research Opportunities in Space and Earth Science (ROSES), Planetary Protection Research (09-PPR09-0004, 14-PPR14-2-0002 and 22-PPR22-012). Additional student support was from Kansas IDeA (Institutional Development Award) Networks of Biomedical Research Excellence (K-INBRE), National Institute of General Medical Sciences (NIGMS), National Institutes of Health (NIH) (P20 GM103418). Part of this work was conducted at the Jet Propulsion Laboratory, California Institute of Technology, under a contract with NASA (80NM0018D0004).
Competing interests
The authors report no conflict of interest.
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