Introduction
Natural environments on celestial bodies that might support life need to be protected from contamination by visiting spacecraft (Irwin and Schulze-Makuch, Reference Irwin and Schulze-Makuch2001; Rummel et al., Reference Rummel, Beaty, Jones, Bakermans, Barlow, Boston, Chevrier, Clark, de Vera, Gough, Hallsworth, Head, Hipkin, Kieft, McEwen, Mellon, Mikucki, Nicholson, Omelon, Peterson, Roden, Sherwood Lollar, Tanaka, Viola and Wray2014). The goal is to reduce the chances of confounding life detection missions due to terrestrial life carried by spacecraft. Should native life exist in another world, microbes capable of colonizing the alien environment could jeopardize microbial communities. Extensive protocols are used to reduce the bioburden on robotic spacecraft sent to Mars, the ocean worlds or any body where life is reasonably plausible. Spacecraft are assembled in clean rooms to reduce biocontamination. Air is filtered, workers don personal protective equipment and airlocks are used to limit the transport of dust and microbes into the facility. Studying the microbes in spacecraft assembly facilities (SAFs), which are by location the most likely cells to be carried by spacecraft, will help us understand the probability that relevant microbial assemblages could survive and proliferate in another world. Further, describing the lifestyles and mechanisms by which microbes are successful under environmental conditions that are analogues of Mars and the ocean worlds, suggests adaptations that might be expected from native life on these worlds.
The environments of Mars and the ocean worlds are characterized by extreme chemical and physical conditions, including very low temperatures (Mancinelli et al., Reference Mancinelli, Fahlen, Landheim and Klovstad2004; Davila et al., Reference Davila, Duport, Melchiorri, Jänchen, Valea, de los Rios, Fairén, Möhlmann, McKay, Ascaso and Wierzchos2010; Rummel et al., Reference Rummel, Beaty, Jones, Bakermans, Barlow, Boston, Chevrier, Clark, de Vera, Gough, Hallsworth, Head, Hipkin, Kieft, McEwen, Mellon, Mikucki, Nicholson, Omelon, Peterson, Roden, Sherwood Lollar, Tanaka, Viola and Wray2014). Since liquid water is necessary for canonical life, its scarcity on Mars and other bodies reduces the extent of special regions that are potentially habitable. High concentrations of salts can substantially depress the freezing point of water, thereby maintaining liquid water at temperatures as low as −70°C, expanding the extent of special regions. However, high solute concentrations can lower water activity (aw) to the point that only organisms with rare adaptations are able to survive and proliferate. Even the modest salinity of the ocean (2% NaCl; aw = 0.97) greatly limits the microbial taxa capable of colonization (Grant, Reference Grant2004). In saturated NaCl, with an aw of 0.75, only extremely halotolerant organisms survive. Driving freezing points to a range where liquid water could currently persist near the surface of Mars requires high enough concentrations of (per)chlorate salts to lower the aw below ~0.6, the known limit of life (Grant, Reference Grant2004; Schneegurt, Reference Schneegurt and Vreeland2012; Rummel et al., Reference Rummel, Beaty, Jones, Bakermans, Barlow, Boston, Chevrier, Clark, de Vera, Gough, Hallsworth, Head, Hipkin, Kieft, McEwen, Mellon, Mikucki, Nicholson, Omelon, Peterson, Roden, Sherwood Lollar, Tanaka, Viola and Wray2014; Primm et al., Reference Primm, Gough, Chevrier and Tolbert2017; Pál and Kereszturi, Reference Pál and Kereszturi2020; Rivera-Valentín et al., Reference Rivera-Valentín, Chevrier, Soto and Martínez2020). Growth at record low aw previously has been demonstrated with fungi in high sugars and salts (Pitt and Christian, Reference Pitt and Christian1968; Williams and Hallsworth, Reference Williams and Hallsworth2009).
The microbial communities in SAFs are closely monitored by planetary protection teams to understand and reduce bioburden on spacecraft. Halotolerant fungi in SAFs appear to be in greatly lower abundance than halotolerant bacteria (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, Rooney and Spry2014; Checinska et al., Reference Checinska, Probst, Vaishmpayan, 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). In one study, fungi were found to be ~2% of the culturable microbial community (Hendrickson et al., Reference Hendrickson, Urbaniak, Malli Mohan, Heidi and Venkateswaran2017), while in another, cultivable fungi on SAF dust particles were ~1 log unit lower in abundance (103–104 CFU g−1 dust) than that of bacteria (Checinska et al., Reference Checinska, Probst, Vaishmpayan, White, Kumar, Stepanov, Fox, Nilsson, Pierson, Perry and Venkateswaran2015). It is interesting to note that archaea, often associated with hypersaline environments, were nearly absent in SAFs. The bacterial assemblages in SAFs have been shown to be quite diverse, both by cultivation and molecular analyses (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, Reference La Duc, Venkateswaran and Conley2014; Moissl et al., Reference Moissl, Osman, La Duc, Dekas, Brodie, DeSantis and Venkateswaran2007, Reference Moissl, Bruckner and Venkateswaran2008; Bashir et al., Reference Bashir, Ahmed, Weinmaier, Ciobanu, Ivanova, Pieber and Vaishampayan2016; Danko et al., Reference Danko, Sierra, Benardini, Guan, Wood, Singh, Seuylemezian, Butler, Ryon, Kuchin, Meleshko, Bhattacharya, Venkateswaran and Mason2021; Hendrickson et al., Reference Hendrickson, Urbaniak, Minich, Aronson, Martino, Stepanauskas, Knight and Venkateswaran2021; Carte et al., Reference Carte, Chen, Clark and Schneegurt2024). Substantial halotolerance has been observed for SAF isolates, suggesting that these dry environments select for salinotolerant microbes (Moissl-Eichinger et al., Reference Moissl-Eichinger, Pukall, Probst, Stieglmeier, Schwendner, Mora, Barczyk, Bohmeier and Rettberg2013; Venkateswaran et al., Reference Venkateswaran, Vaishampayan, Benardini, Rooney and Spry2014; Smith et al., Reference Smith, Benardini, Anderl, Ford, Wear, Schrader, Schubert, DeVeaux, Paszczynski and Childers2017; Zanmuto et al., Reference Zanmuto, Fuchs, Fiebrandt, Stapelmann, Ulrich, Maugeri, Pukall, Gugliandolo and Moeller2018). Wipe samples from SAFs cultivated at very high salinities (50% MgSO4 or 20% NaClO3) enriched for a microbial community that appeared to retain the functional groups needed to perpetually maintain biogeochemical cycles under these analogue chemical conditions (Carte et al., Reference Carte, Chen, Clark and Schneegurt2024).
Fungi are widely found in aquatic and terrestrial hypersaline environments (Butinar et al., Reference Butinar, Santos, Spencer-Martins, Oren and Gunde-Cimerman2005; Evans et al., Reference Evans, Hansen, Stone and Schneegurt2013). Marine salterns (Butinar et al., Reference Butinar, Santos, Spencer-Martins, Oren and Gunde-Cimerman2005; Cantrell et al., Reference Cantrell, Casillas-Martínez and Molina2006; Nayak et al., Reference Nayak, Gonsalves and Nazareth2012), sabkhas (Borut, Reference Borut1960; Al-Musallam et al., Reference Al-Musallam, Al-Sammar and Al-Sané2011), salt flats (Evans et al., Reference Evans, Hansen, Stone and Schneegurt2013) and even the Dead Sea (Guiraud et al., Reference Guiraud, Steiman, Seigle-Murandi and Sage1995; Steiman et al., Reference Steiman, Guiraud, Sage, Siegle-Murandi and LaFond1995) are known to harbour populations of salinotolerant fungi. Hypersaline fungal communities are dominated by Ascomycetes, mainly Aspergillus, Eurotium and Penicillium. Alternaria, Chaetomium, Cladosporium, Eurotium, Fusarium and Hortaea are commonly observed, along with Mucor, Rhizopus and Ulocladium. Melanized xerophilic yeasts are commonly considered the most halotolerant fungi (Gunde-Cimerman et al., Reference Gunde-Cimerman, Zalar, de Hoog and Plemenitaš2000). Since cosmopolitan genera, such as Aspergillus and Penicillium, dominate hypersaline fungal assemblages, it has been suggested that these represent a halotolerant subset of common soil fungi in these locales. Ranzoni (Reference Ranzoni1968) concluded that there are no fungi characteristic of hypersaline and arid environments, a suggestion that has been echoed by other researchers (Guiraud et al., Reference Guiraud, Steiman, Seigle-Murandi and Sage1995; Grishkan et al., Reference Grishkan, Nevo and Wasser2003; Evans et al., Reference Evans, Hansen, Stone and Schneegurt2013).
Halophilic fungi (requiring high salt for growth) tend to be far less common than halophilic bacteria (Grant, Reference Grant2004; Evans et al., Reference Evans, Hansen, Stone and Schneegurt2013; Plemenitaš et al., Reference Plemenitaš, Lenassi, Konte, Kejžar, Zajc, Gostinčar and Gunde-Cimerman2014). Fungi from hypersaline environments typically are halotolerant (able to grow at high salinities) but grow better at lower salinities (Hujslová et al., Reference Hujslová, Kubátová, Chudičková and Kolařik2010; Nayak et al., Reference Nayak, Gonsalves and Nazareth2012; Evans et al., Reference Evans, Hansen, Stone and Schneegurt2013). Broad tolerance to salinity, without halophilicity, with slower growth at high salinities, supports the suggestion that fungal communities in hypersaline environments are not highly specialized. Fungi use mechanisms similar to those of bacteria to survive in brines, including tight membranes, ion efflux pumps and the accumulation of compatible solutes to balance internal osmotic potential with that of the medium (Plemenitaš et al., Reference Plemenitaš, Lenassi, Konte, Kejžar, Zajc, Gostinčar and Gunde-Cimerman2014; Leo and Onofri Reference Leo and Onofri2023; Suryanarayanan and Ravishankar, Reference Suryanarayanan and Ravishankar2023). In addition, fungi reinforce their cell wall to withstand increased turgor pressure and resist salt uptake (Gunde-Cimerman and Zalar, Reference Gunde-Cimerman and Zalar2014).
The current study examines the fungal assemblages that formed when wipe samples from SAF surfaces were enriched in medium containing either 50% MgSO4 or 20% NaClO3. Community DNA extracts, weeks and months after inoculation, were the subject of next generation sequencing of rRNA genes to identify phylogenetic groups. A companion study describing the bacterial assemblages in these enrichments reported substantial diversity (Carte et al., Reference Carte, Chen, Clark and Schneegurt2024). Here we report that the fungal assemblage was not as diverse, being dominated by yeasts and rich in common Ascomycetes.
Methods
Sampling of SAF
Sterile polyester wipes (Texwipe; Kernersville, NC) were moistened with 15 ml of sterile water and used to swab three 1-m2 surfaces of high-traffic floors in the Aseptic Assembly Facility at NASA Jet Propulsion Laboratory (JPL), a certified ISO 5 clean room, during the assembly of the Mars 2020 Sample Caching System hardware (Fig. 1). A fourth sterile wipe was used as a procedural control. During sampling, a fresh pair of sterile gloves was worn for each sample collection. 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 from the wipes. After 10 min, liquid was squeezed from the wipes within the tubes using a sterile syringe plunger. The extracts then were used to inoculate enrichment cultures and for direct DNA extractions.
Fig. 1. Map of the aseptic assembly facility in the JPL SAF showing the locations of three wipe samples of high-traffic areas of the floor.
Enrichment cultures
Enrichment cultures were maintained in selective salt plains (SP) medium containing (per liter): NaCl, 1.0 g; KCl, 2.0 g; MgSO4⋅7H2O, 1.0 g; CaCl2⋅H2O, 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 (Caton et al., Reference Caton, Witte, Nguyen, Buchheim, Buchheim and Schneegurt2004), supplemented with either 50% (w/v) MgSO4 (2.0 M; aw = 0.94) or 20% (w/v) NaClO3 (1.9 M; aw = 0.91). While not specific for fungi, as it contains less sugar than the fungal media of Emmons (Reference Emmons, Emmons, Binford and Utz1963) or Sabouraud (Reference Sabouraud1892), SP medium is eutrophic and rich in amino acids. Flasks (100 ml) were inoculated with aliquots (1 ml) of the fluids extracted from SAF wipes and maintained on an Innova rotary shaker (125 rpm; 1-in stroke dia; New Brunswick Scientific, Edison, NJ) at room temperature for a month. Samples (6 ml) were taken weekly during this incubation for community analyses. The enrichment cultures were then removed from the shaker, wrapped with parafilm to limit evaporation and stored static for an additional five months, before sampling for community analyses.
DNA extraction and molecular analyses
Crude DNA extracts were made from aliquots (6 ml) of samples, isolates and enrichment cultures 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 six 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 heated for 5 min at 80°C. Extracts were stored at −20°C before PCR amplification. Direct extracts from wipe samples before enrichment yielded insufficient DNA for reliable PCR amplification and community analyses as expected, since samples with extremely low biomass typically require specialized extraction methods.
Crude community DNA extracts from enrichment culture samples were used for Illumina sequencing (miSeq v2 Nona PE-250bp) of 18S rRNA genes by a commercial vendor (University of Kansas Center for Genomics). Each sample in the multiplex reactions produced ~ 30 000 reads and the FASTQ files were demultiplexed for forward and reverse reads. Metagenomic 18S sequence libraries were analysed on the Galaxy platform (Afgan et al., Reference Afgan, Baker, Batut, van den Beek, Bouvier, Čech, Chilton, Clements, Coraor, Grüning, Guerler, Hillman-Jackson, Hiltemann, Jalili, Rasche, Soranzo, Goecks, Taylor, Nekrutenko and Blankenberg2018) following the microbial analysis package workflow, incorporating tools designed for MOTHUR (Schloss et al., Reference Schloss, Westcott, Ryabin, Hall, Hartmann, Hollister, Lesniewski, Oakley, Parks, Robinson, Sahl, Stres, Thallinger, Van Horn and Weber2009). Forward and reverse reads were combined to create contigs, generating an average read length of 334 nucleotides. Chimeras were found using chimera.vsearch and removed using remove.seqs in MOTHUR on the Galaxy platform. These were aligned using SILVA alignment v.138 and classified in reference to SILVA taxonomy v.138.
Results
Succession of SAF fungal communities enriched in 20% NaClO3
Three wipes from SAF floor sampling were used to inoculate microbial enrichments in SP medium supplemented with 20% (w/v) NaClO3. Fungi were detected by Illumina sequencing of PCR-amplified 18S rRNA genes for all wipes at all time points. Prior to enrichment, direct extraction of wipe samples did not yield sufficient DNA for amplification of fungal sequences, as observed previously for bacterial analyses (Highlander et al., Reference Highlander, Wood, Gillece, Folkerts, Fofanov, Furstenau, Singh, Guan, Seuylemezian, Benardini, Engelthaler, Venkateswaran and Keim2023; Carte et al., Reference Carte, Chen, Clark and Schneegurt2024). The overall number of sequences obtained in the fungal libraries from the wipe samples were far fewer (<1%) than those obtained from the analysis of bacteria in these enrichment cultures (Carte et al., Reference Carte, Chen, Clark and Schneegurt2024). The greatest number of fungal sequences were obtained during the first three weeks of enrichment in 20% NaClO3 (Fig. 2). After six months of enrichment, ~4- to 10-fold fewer sequences were obtained than after three weeks of enrichment.
Fig. 2. Abundance of fungal families observed by pyrosequencing and phylogenetic analyses of 18S rRNA genes from metagenomic extracts of SAF wipe assemblages after 1 wk, 2 wk, 3 wk, 4 wk, and 6 mo of enrichment in SP medium supplemented with 20% (w/v; ~1.9 M) NaClO3.
The families of fungi obtained are shown as a percentage of the total assemblage, to illustrate successional changes over time for each wipe (Fig. 3). Certain features of the assemblage were relatively consistent among the three wipes. For the first month of enrichment at 20% NaClO3, Saccharomycetaceae within the Ascomycota were dominant. The remaining assemblage was mainly other Ascomycota. Basidiomycota were practically absent in these enrichments. Non-fungal Eukarya, including Kinetoplastida in the Euglenozoa, were more numerous. The assemblages remaining after six months of enrichment in 20% NaClO3 exhibited a decidedly different structure. Ascomycota were greatly reduced, particularly the Saccharomycetaceae that were so dominant during the first month of enrichment. Overall numbers of fungal sequences were greatly reduced, but libraries were enriched for non-fungal Eukarya including Euglenozoans, although these were still in low abundance.
Fig. 3. Relative abundance of fungal families observed by pyrosequencing and phylogenetic analyses of 18S rRNA genes from metagenomic extracts of SAF wipe assemblages after 1 wk, 2 wk, 3 wk, 4 wk, and 6 mo of enrichment in SP medium supplemented with 20% (w/v; ~1.9 M) NaClO3.
The number of OTUs observed and the Chao estimates of OTUs decreased during the 6-mo enrichment period (Table 1). The reductions in OTUs were between 68 and 86%, with the subspecies level being most impacted. Good's coverage was relatively low at the highest sequence identity level (subspecies at 99%) but was high at the family level (88% identity). Diversity based on the Non-parametric Shannon Index was greatest at the 99% identity level and was above 3.0 through the genus level of identity (94%). The Shannon Evenness Index showed small increases during the enrichment period, with the family level of identity (88%) exhibiting the greatest increase in evenness.
Table 1. Diversity indices for the enrichment cultures in 20% NaClO3
Succession of SAF fungal communities enriched in 50% MgSO4
Three wipes from SAF floor sampling were used to inoculate microbial enrichments in SP medium supplemented with 50% (w/v) MgSO4 and fungi were again detected by Illumina sequencing of PCR-amplified 18S rRNA genes. Very few fungal sequences were obtained overall, with only a few samples (mainly from Wipe 3) showing substantial amplification (Fig. 4). The observed fungal assemblages were noticeably less consistent between wipes and time points than those observed from the 20% NaClO3 enrichments. There were apparently large shifts in the fungal sequences present at different time points during the first month of enrichment (Fig. 5). Wipe 3 was nearly entirely Basidiomycota for the first three weeks of enrichment. There were shifts within the Basidiomycota among Microbotryomycetes, Tremellomycetes and others during this time. The other wipes also showed greater contributions from Basidiomycota in the fungal assemblages of the MgSO4 enrichments than in those of the NaClO3 enrichments. After six months of enrichment in 50% MgSO4, few fungal sequences were detected. These libraries were mainly composed of Ascomycota and non-fungal unclassified Eukarya, although these were in low abundance.
Fig. 4. Abundance of fungal families observed by pyrosequencing and phylogenetic analyses of 18S rRNA genes from metagenomic extracts of SAF wipe assemblages after 1 wk, 2 wk, 3 wk, 4wk, and 6 mo of enrichment in SP medium supplemented with 50% (w/v; ~2.0 M) MgSO4.
Fig. 5. Relative abundance of fungal families observed by pyrosequencing and phylogenetic analyses of 18S rRNA genes from metagenomic extracts of SAF wipe assemblages after 1 wk, 2 wk, 3 wk, 4wk, and 6 mo of enrichment in SP medium supplemented with 50% (w/v; ~2.0 M) MgSO4.
Far fewer fungal OTUs were detected (~5 to 20%) in the 50% MgSO4 enrichments (Table 2) than in the 20% NaClO3 enrichments. There was less speciation, with the number of OTUs at the level of subspecies being close to the number of OTUs at the family level. Good's coverage also was generally lower but showed more variability. The number of OTUs detected decreased over time in the 20% NaClO3 enrichments, but this trend was not observed in the 50% MgSO4 enrichments. The greatest number of OTUs were observed after 4 weeks, especially at the higher levels of sequence identity. The Non-parametric Shannon Diversity Index showed a dip at 4 weeks as well. Overall, the assemblage was less diverse in the 50% MgSO4 enrichments than in the 20% NaClO3 enrichments. Shannon evenness was high indicating that the OTUs represented were in approximately equal abundance, substantially more even than the fungal assemblage enriched at 20% NaClO3.
Table 2. Diversity indices for the enrichment cultures in 50% MgSO4
Discussion
Fungi exhibit characteristics that increase their potential to survive or proliferate on celestial bodies (Selbmann et al., Reference Selbmann, de Hoog, Mazzaglia, Friedmann and Onofri2005; Evans et al., Reference Evans, Hansen, Stone and Schneegurt2013; Blachowicz et al., Reference Blachowicz, Chiang, Elsaesser, Kalkum, Ehrenfreund, Stajich, Torok, Wang and Venkateswaran2019). Fungal spores are remarkably tough, vegetative cells tend to ferment under anaerobic conditions and many fungi exhibit growth tolerances to high salinity and low temperatures. Growth was observed previously with fungi at near-record low aw in high sugar syrups (Pitt and Christian, Reference Pitt and Christian1968). Fungi are found in low abundance in SAFs, seemingly insignificant (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, Rooney and Spry2014; Checinska et al., Reference Checinska, Probst, Vaishmpayan, 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). However, their resiliency suggests that fungi remain a particular concern in regard to forward planetary protection (Leo and Onofri, Reference Leo and Onofri2023).
The diversity of the fungal assemblage in SAFs appears to be very much lower than that of the bacterial assemblage. Fungal spores and hyphae have been observed on fallout dust particles in SAFs (Checinska et al., Reference Checinska, Probst, Vaishmpayan, White, Kumar, Stepanov, Fox, Nilsson, Pierson, Perry and Venkateswaran2015; Yuan et al., Reference Yuan, Hong, Biao, Lantao, Xi and Kanyan2017; Mohan et al., Reference Mohan, Stricker and Venkateswaran2019). The isolates tended to be limited to common moulds and yeasts of the Ascomycota, such as Aspergillus, Cladosporium, Penicillium and Saccharomyces, and the Basidiomycota in the Tremellomycetes. Of 26 genomes sequenced from SAF fungal isolates, 9 genera were detected comprising 13 species, with Aspergillus and Penicillium being most common (Chander et al., Reference Chander, Singh, Simpson, Seuylemezian, Mason and Venkateswaran2022). From a Chinese SAF, 13 families of fungi were isolated, with Tremellomycetes being most common (Yuan et al., Reference Yuan, Hong, Biao, Lantao, Xi and Kanyan2017). Analyses of SAF fungal assemblages using molecular techniques gave similar results but also uncovered greater diversity. An analysis of dust microbiomes from SAF and International Space Station (ISS) samples detected 153 OTUs by ITS sequencing of metagenomic extracts, with Ascomycetes being most prominent, including Dothideomycetes, Eurotiomycetes and Saccharomycetes (Checinska et al., Reference Checinska, Probst, Vaishmpayan, White, Kumar, Stepanov, Fox, Nilsson, Pierson, Perry and Venkateswaran2015). Basidiomycetes also were detected, including Cytobasidiomycetes, Microbotryomycetes and Tremellomycetes. A study of an SAF in Japan and the ISS by ITS sequencing suggested that ~50 OTUs of fungi were present in the SAF, with fewer than 10 detected for the ISS, including Cladosporium (90% of sequences), Alternaria and Aspergillus (Satoh et al., Reference Satoh, Nishiyama, Yamazaki, Sugita, Tsukii, Takatori, Benno and Makimura2011). The molecular analysis of samples from a Chinese SAF detected ~50 different species, including Debaryomyces and Pichia (Yuan et al., Reference Yuan, Hong, Biao, Lantao, Xi and Kanyan2017). Fungal assemblages in SAFs appear to be dominated by common Ascomycetes with relatively limited diversity. It does not seem that the SAF assemblages are rich in fungi found only in specialized environments.
The environment of clean rooms used as SAFs is maintained in a similar way as clean rooms used for surgery and pharmaceutical production. Bacteria dominate the microbial communities of these clean rooms, with fungi accounting for less than 1% of the microbes detected (Sandle, Reference Sandle2011). In a vaccine preparation facility, fungi were detected at 300–800 CFU m−3 of air (Utescher et al., Reference Utescher, Franzolin, Trabulsi and Gambale2007), comparable to levels reported in SAFs (Checinska et al., Reference Checinska, Probst, Vaishmpayan, White, Kumar, Stepanov, Fox, Nilsson, Pierson, Perry and Venkateswaran2015; Hendrickson et al., Reference Hendrickson, Urbaniak, Malli Mohan, Heidi and Venkateswaran2017). Hospital buildings had somewhat lower counts of airborne fungi with <100 CFU m−3 (Ekhaise et al., Reference Ekhaise, Ighosewe and Ajakpovi2008; Saadoun et al., Reference Saadoun, Al Tayyar and Elnasser2008; Kim et al., Reference Kim, Kim and Kim2010). Aspergillus, Cladosporium, Penicillium and Saccharomycetes were most common among these clean rooms, not unlike the assemblages observed in SAFs (Utescher et al., Reference Utescher, Franzolin, Trabulsi and Gambale2007; Ekhaise et al., Reference Ekhaise, Ighosewe and Ajakpovi2008; Saadoun et al., Reference Saadoun, Al Tayyar and Elnasser2008; Nagano et al., Reference Nagano, Walker, Loughrey, Millar, Goldsmith, Rooney, Elborn and Moore2009; Kim et al., Reference Kim, Kim and Kim2010; Sandle, Reference Sandle2011). A global survey of indoor fungi by ITS sequencing found that the fungal genera observed were mainly cosmopolitan, with few differences at the class level (Amend et al., Reference Amend, Seifert, Samson and Burns2010). Diversity was correlated with latitude and building function did not have a significant effect on the fungal assemblages. Ascomycetes dominated the indoor environments detected by ITS sequencing and by cultivation. This supports the suggestion that the assemblage of fungi in clean rooms is not specialized, as observed in the current report for SAFs and as previously suggested for fungal assemblages in natural hypersaline environments (Ranzoni, Reference Ranzoni1968; Guiraud et al., Reference Guiraud, Steiman, Seigle-Murandi and Sage1995; Grishkan et al., Reference Grishkan, Nevo and Wasser2003).
The Ascomycota detected in SAF enrichment cultures of the current report were common Saccharomycetaceae and Trichomaceae (Aspergillus, Cladosporium, Eurotium and Penicillium), which appear to exhibit high halotolerance (Grishkan et al., Reference Grishkan, Nevo and Wasser2003; Gostinčar et al., Reference Gostinčar, Grube, de Hoog, Zalar and Gunde-Cimerman2010; Gonsalves et al., Reference Gonsalves, Nayak and Nazareth2012; Zajc et al., Reference Zajc, Zalar, Gunde-Cimerman, Buzzini, Lachance and Yurkov2017). Ascomycetes are reported to be the dominant fungi in the harshest environments of the Antarctic dry valleys (Coleine et al., Reference Coleine, Pombubpa, Zucconi, Onofri, Stajich and Selbmann2020). Halotolerance and xerophilicity of Eurotium have been reported for isolates from salterns and the Dead Sea (Andrews and Pitt, Reference Andrews and Pitt1987; Abdel-hafez et al., Reference Abdel-Hafez, Mohawed and El-Said1989; Kis-Papo et al., Reference Kis-Papo, Oren, Wasser and Nevo2003; Butinar et al., Reference Butinar, Santos, Spencer-Martins, Oren and Gunde-Cimerman2005). Yeasts in the Saccharomycetaceae and Basidiomycota (Microbotryomycetes and Tremellomycetes) were the most abundant fungi in SAF enrichment cultures. These groups are commonly reported from marine, saltern and other hypersaline environments, often exhibiting high salinity tolerances in culture (Singh and Raghukumar, Reference Singh and Raghukumar2014; Zajc et al., Reference Zajc, Zalar, Gunde-Cimerman, Buzzini, Lachance and Yurkov2017). Many yeasts from natural hypersaline environments are melanized, being especially halotolerant (Gunde-Cimerman et al., Reference Gunde-Cimerman, Zalar, de Hoog and Plemenitaš2000; Zajc et al., Reference Zajc, Zalar, Gunde-Cimerman, Buzzini, Lachance and Yurkov2017), but those were in low abundance in our SAF enrichment cultures. Less work has been done with athalassohaline fungal cultures like those of the current study, which are not predominantly NaCl. An extensive study of yeasts from diverse environments demonstrated high tolerances to salts including MgCl2, MgSO4, KCl, and NaBr (Zajc et al., Reference Zajc, Džeroski, Kocev, Oren, Sonjak, Tkavc and Gunde-Cimerman2014). Tolerance to 0.5 M MgClO3 has been reported for a strain of Penicillium (Espeso et al., Reference Espeso, Villarino, Carreras, Alonso-Guirado, Alonso, Melgarejo and Larena2019). Both MgSO4 and NaClO3 were used for the enrichments in the current study.
The fungal assemblages in the SAF enrichments of the current study changed over time and differed between brines and wipe samples. Saccharomycetes were dominant in the eukaryotic assemblage in early stages of the enrichment but were succeeded mainly by non-fungal eukaryotes in low abundance at 6 months of enrichment. This may be interpreted in terms of the success strategies of these organisms. The Saccharomycete yeasts tend to be fast growing when nutrients of high quality are present and abundant, a trait that would be expected for r-strategists, which are successful due to their rapid growth rate. After 1 month of aeration by rotary shaking, the culture flasks were left static for the next 5 month, without adding nutrients. This change should favour organisms that are characteristically K-strategists, being permanent members of a community, relying on adaptability and versatile nutritional requirements for success. In the bacterial community of these SAF enrichments, actinobacteria were most successful after 6 mo, as these are generally K-strategists (Carte et al., Reference Carte, Chen, Clark and Schneegurt2024). Initially, there were dominating blooms of staphylococci, which are fast-growing r-strategists. We might expect that the harsh, relatively low-nutrient environment of Mars would select K-strategists, in the same way that these enrichment cultures did.
For the 50% MgSO4 enrichment culture of Wipe 3, there was a bloom of Basidiomycete yeasts, also r-strategists, which was succeeded by non-fungal Eukaryotes in low abundance. It is interesting to note that during this yeast bloom from Wipe 3 in weeks 2 and 3 of enrichment, bacterial numbers fell dramatically (Carte et al., Reference Carte, Chen, Clark and Schneegurt2024). By 6 mo, the bacterial community had recovered its former diversity and abundance. Changes to the fungal assemblage in the Wipe 3 enrichment culture were different than those observed in the enrichment cultures from the other two wipes. Since the wipe samples were taken from the same SAF environment, only meters apart, the suggestion is that the successional process had a substantial stochastic element, as was observed for the bacterial community. There were far fewer fungi detected in the enrichment cultures at 50% MgSO4 than in those at 20% NaClO3. The molarities of ions were similar as was the aw of the two brines. The reason for the difference between salts is not clear and this was not observed for the bacterial community. During the SAF enrichments, both bacterial and fungal community members were followed by design. The base SP medium used was not as rich in sugar as typical fungal media (Sabouraud, Reference Sabouraud1892; Emmons, Reference Emmons, Emmons, Binford and Utz1963). With higher concentrations of simple sugars, the bloom of r-strategist yeasts may have been greater and lasted longer, but ultimately the K-strategists would be expected to succeed them. Given the limited abundance and diversity of fungi after 6 month of enrichment, it seems that the SAF assemblage was not rich in K-strategists, being succeeded mainly by non-fungal Eukaryotes and actinobacteria.
Fungi exhibit remarkable tolerances to harsh environmental conditions that are present on solar system bodies such as hypersalinity. Their resistant perennating structures give them additional survivability under conditions non-permissive for growth. Fungi currently hold the record for growth at the lowest aw (Pitt and Christian, Reference Pitt and Christian1968; Williams and Hallsworth, Reference Williams and Hallsworth2009). Therefore, discussions of planetary protection need to include salinotolerant fungi (Leo and Onofri, Reference Leo and Onofri2023). Finding fungi capable of long-term survival and proliferation in concentrated MgSO4 and NaClO3 brines, demonstrates that fungi may be carried by spacecraft and further have the potential to influence life detection missions or native microbial communities. This concern should be tempered by the observation that fungal K-strategists were not successful end members of SAF enrichments in brines relevant to Mars. Further, heterotrophic fungal metabolism is relatively limited and fungi do not fill critical roles in biogeochemical cycles. The end members of the bacterial community in these SAF enrichments included representatives of each functional group needed to maintain biogeochemical cycles for the microbial community (Carte et al., Reference Carte, Chen, Clark and Schneegurt2024), and therefore pose a greater threat to forward planetary protection than fungi.
Acknowledgements
The authors are grateful for the technical assistance of James Beck, Fawn Beckman, Sreenavya Gandikota, Jennifer Hackett (University of Kansas), Gregory Houseman, Ebsen Kjaer, Mark Lindemann (Purdue University), and Bin Shuai. Preliminary accounts of this work previously have been presented and abstracted (Carte et al., Reference Carte, Gandikota, Chen, Clark and Schneegurt2020, Reference Carte, Chen, Clark and Schneegurt2021).
Financial support
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, 22-PPR22-0012) and University of Kansas Center for Genomics. Additional student support was provided by Kansas INBRE, National Institute of General Medical Sciences (NIGMS), National Institutes of Health (NIH) (P20 GM103418). Part of the research was carried out at JPL, California Institute of Technology, under a contract with NASA (80NM0018D004).
Conflict of interest
The authors report no conflict of interest.
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