Introduction
Filamentous fungi are widely distributed eukaryotic organisms that can thrive in extreme environments (Zhdanova et al., Reference Zhdanova, Tugay, Dighton, Zheltonozhsky and McDermott2004; Godinho et al., Reference Godinho, Gonçalves, Santiago, Figueredo, Vitoreli, Schaefer, Barbosa, Oliveira, Alves, Zani, Junior, Murta, Romanha, Kroon, Cantrell, Wedge, Duke, Ali, Rosa and Rosa2015; Ameen et al., Reference Ameen, AlNAdhari, Yassin, Al-Sabri, Almansob, Alqahtani and Stephenson2022). Their ability to form highly resistant spores and develop biofilms allows them to withstand harsh conditions, including those found in space habitats (Dijksterhuis, Reference Dijksterhuis2019). Indeed, Penicillium spp. and Aspergillus spp. are among the most abundant fungal species isolated from the former Mir space station and the International Space Station (ISS) (Makimura et al., Reference Makimura, Hanazawa, Takatori, Tamura, Fujisaki, Nishiyama, Abe, Uchida, Kawamura, Ezaki and Yamaguchi2001; Vesper et al., Reference Vesper, Wong, Kuo and Pierson2008; Satoh et al., Reference Satoh, Yamazaki, Nakayama, Umeda, Alshahni, Makimura and Makimura2016).
The space habitat environment of the ISS, in Low Earth Orbit (LEO), is primarily characterised by microgravity and radiation as key stress factors, influencing all life onboard (Horneck et al., Reference Horneck, Klaus and Mancinelli2010). As space travel becomes more accessible, research is essential to study microbial adaptations under these conditions. Fungal spores are of particular interest when studying extreme environmental resistance, as their specialised structures, including thick cell walls and protective pigmentation, enhance their ability to withstand harsh conditions such as radiation, desiccation and oxidative stress, making them highly relevant in astrobiology (Beauvais et al., Reference Beauvais, Fontaine, Aimanianda and Latgé2014; Cordero and Casadevall, Reference Cordero and Casadevall2017; Mota et al., Reference Mota, Koch, Matthiae, Santos and Cortesão2025).
Gravity has been the only constant during the evolution of life on Earth, shaping the adaptations of all organisms to a gravitational force of 1 × g (9.81 m s−2) (Najrana and Sanchez-Esteban, Reference Najrana and Sanchez-Esteban2016). Therefore, the microgravity environment onboard the ISS (10−4–10−6 g) presents a unique environment that influences every living organism (Stenzel, Reference Stenzel2016). When it comes to radiation, the ISS is largely shielded from the sun’s non-ionising ultraviolet radiation and ionising X-rays. This radiation protection is granted not only by the ISS’s engineered walls but also by the Earth’s magnetosphere which still reaches LEO. However, cosmic events generate ionising galactic cosmic radiation (GCR), composed of highly charged, energetic heavy ions (HZE) that can penetrate the currently used spaceflight materials, making efficient protection particularly challenging (Horneck et al, Reference Horneck, Klaus and Mancinelli2010; Chancellor et al., Reference Chancellor, Blue, Cengel, Auñón-Chancellor, Rubins, Katzgraber and Kennedy2018). These charged particles are emitted from cosmic events that generate ionising GCR consisting primarily of protons (87%), helium (He) (12%) and heavy ions (1–2%) such as iron (Fe). Radiation is the most challenging factor for life in the Universe, as it induces damage to cells and DNA (Chancellor et al., Reference Chancellor, Scott and Sutton2014, Reference Chancellor, Blue, Cengel, Auñón-Chancellor, Rubins, Katzgraber and Kennedy2018). However, several microorganisms are known to withstand high levels of different types of radiation, as well as other extreme environmental conditions.
Fungi Penicillium spp. are commonly isolated from extreme environments, exhibiting adaptability to a wide range of harsh conditions, including extreme temperatures, high pressure, and radiation (Zhdanova et al., Reference Zhdanova, Tugay, Dighton, Zheltonozhsky and McDermott2004; Liu et al., Reference Liu, Wang, Zhou, Xue and Liu2024). Studies have demonstrated biocorrosive, pathogenic, and biomining tendencies, as well as the ability to form biofilms that enhance resistance to stress conditions (Klintworth et al., Reference Klintworth, Reher, Viktorov and Bohle1999; Oshikata et al., Reference Oshikata, Tsurikisawa, Saito, Watanabe, Kamata, Tanaka, Tsuburai, Mitomi, Takatori, Yasueda and Akiyama2013; Satishkumar et al., Reference Sathishkumar, Krishnaraj, Rajagopal, Sen and Lee2016; Omotayo et al., Reference Omotayo, Omotayo, Mwanza and Babalola2019; Shah et al., Reference Shah, Palmieri, Sponchiado and Bevilaqua2022). Studies on Penicillium spp. in simulated space environments have reported increased spore germination, pathogenicity and antimicrobial activity in microgravity and high resistance to radiation (Sathishkumar et al., Reference Sathishkumar, Krishnaraj, Rajagopal, Sen and Lee2016; Sheet et al., Reference Sheet, Sathishkumar, Sivakumar, Shim and Lee2017; Hupka et al., Reference Hupka, Kedia, Schauer, Shepard, Granados-Presa, vande Hei, Flores and Zea2023; Dorbani et al., Reference Dorbani, Berberian, Riedel, Duport and Carlin2024).
The production of secondary metabolites plays a crucial role in fungi ecological interactions and survival and is often driven by environmental stressors (Keller, Reference Keller2015). While many fungal metabolites have industrial applications, Penicillium spp. secreted metabolites can also contribute to surface biodegradation and structural damage of built material (Gutarowska, Reference Gutarowska2010; Li et al., Reference Li, Zhou, Du, Chen, Takahashi and Liu2020; Correia et al., Reference Correia, Borges, Simões and Simões2023). For instance, P. rubens was identified as the cause of surface degradation onboard the Mir space station (Klintworth et al., Reference Klintworth, Reher, Viktorov and Bohle1999). However, filamentous fungi can not only contaminate and biofoul surfaces but can also cause diseases in humans. Toxigenic species produce and secrete mycotoxins, allergens and volatile organic compounds, which can have either mutagenic or carcinogenic effects, that contribute to serious human diseases or cause other symptoms relating to “sick-building-syndrome” (Omotayo et al., Reference Omotayo, Omotayo, Mwanza and Babalola2019; Sayan and Dülger Reference Sayan and Dülger2021). Moreover, fungal pathogens can physically invade and colonise host organisms, causing fungal biofilms on mucous membranes with varying severity, particularly in immunocompromised individuals (Bongomin et al., Reference Bongomin, Gago, Oladele and Denning2017). This especially poses a serious threat to astronauts, whose immune responses are impaired during space travel due to changes caused by altered gravity, radiation and confinement in spaceflight environments (Cervantes and Hong, Reference Cervantes and Hong2016; de Middeleer et al., Reference de Middeleer, Leys, Sas and de Saeger2019). The space habitat environment, such as the ISS itself, has the potential to increase the virulence of pathogens and support the growth of fungal colonies (Wilson et al., Reference Wilson, Ott, Hö Ner Zu Bentrup, Ramamurthy, Quick, Porwollik, Cheng, Mcclelland, Tsaprailis, Radabaugh, Hunt, Fernandez, Richter, Shah, Kilcoyne, Joshi, Nelman-Gonzalez, Hing, Parra and Nickerson2007; Gilbert et al., Reference Gilbert, Torres, Clemens, Hateley, Hosamani, Wade and Bhattacharya2020; Cortesão et al., Reference Cortesão, Holland, Schütze, Laue, Moeller and Meyer2022). Consequently, microbial monitoring and regular cleaning of the spacecraft during all missions are critical to prevent infections.
Nevertheless, beyond these challenges, filamentous fungi also offer promising applications for space biotechnology, which could play an essential role in advancing sustainable space exploration. Their ability to produce valuable compounds of interest facilitates in situ resource utilisation (ISRU), enabling sustainable long-term missions and the development of self-sufficient space habitats (Cortesão et al., Reference Cortesão, Schütze, Marx, Moeller and Meyer2020). For instance, Penicillium remains the only industrial source of penicillin, the most used antibiotic in medical history (Haque et al., Reference Haque, Nath, Johnston, Haruna, Ahn, Ovissipour and Ku2024). Additionally, Penicillium and Aspergillus spp. produce several metabolites of biotechnological and pharmaceutically importance (Guzmán-Chávez et al., Reference Guzmán-Chávez, Zwahlen, Bovenberg and Driessen2018). This altogether makes species of filamentous fungi, especially their distribution units (spores), relevant microorganisms to look into as astrobiological model organisms (Simões et al., Reference Simões, Cortesão, Azua-Bustos, Bai, Canini, Casadevall, Cassaro, Cordero, Fairén, González-Silva, Gunde-Cimerman, Koch, Liu, Onofri, Pacelli, Selbmann, Tesei, Waghmode, Wang and Antunes2023).
To explore potential adaptive changes that enhance survival in space environments, this study investigated fungal responses to space conditions, by comparing a wild-type strain of P. rubens with an ISS isolate. This study reports colony growth under simulated microgravity (SMG) by clinorotation, and spore survival to cosmic radiation (helium- and iron-ions). Understanding the expected differences in growth and resistance influenced by the space environment, in particular microgravity and radiation, is essential to create and improve safety measures and strengthen risk assessment of future space missions.
Materials and methods
Strains, media and culture conditions
Two strains of Penicillium rubens (formerly Penicillium chrysogenum) were selected for the experiments: a wild-type strain (DSM 1075), which has a fully sequenced genome, and an ISS isolate (IF2SW-F4), obtained from a surface inside the waste and hygiene compartment of the ISS during NASA JPL’s second microbial tracking study flight (Blachowicz et al., Reference Blachowicz, Singh, Wood, Debieu, O’Hara, Mason and Venkateswaran2021). P. rubens spores were harvested after five days of incubation at room temperature (22°C) on potato dextrose agar (PDA) by flooding the plates with 10 mL of saline solution (0.9% NaCl) and collecting spores using a sterile cotton swab. The spore suspensions were retrieved from the agar plates and filtered through a Miracloth filter (Millipore) to remove any residual hyphal fragments. All experiments were performed with fresh spore suspensions that were less than two weeks old and stored at 4°C.
Colony area determination using Orbis
To compare the growth of both strains, 10 µL of a 106 spores/mL suspension of each strain was spotted onto the middle of PDA plates, which was then incubated on the clinostat at 22°C (n = 12). Was documented over a period of 11 days. Images were captured on days 1, 3, 7, 8 and 11 of incubation, using a Sony a500 APS-C camera equipped with a macro lens (E 3.5/30) and mounted on a fixed tripod to maintain a consistent height throughout the experiment. The colony pigmentation was analysed with ImageJ by analysing the colour intensity and the data was parsed as described in the “Data analysis” section.
For the colony area analysis, colony photographs were processed with Orbis (v1.0.0). Orbis is a Fiji/ImageJ macro developed for automated colony area measurement. To validate Orbis, the macro was tested with 146 images from various experiments, including mostly fungal but also bacterial colonies (Figure 1). Orbis’ output showed strong correlation (Pearson R 2 > 0.99) with manual measurements, with average and median absolute errors of <0.5%, confirming high accuracy for different image styles (Figure 1A). For further validation, seven novice ImageJ users were given 18 fungal colony images, divided into three sets of six photos. Each set consisted of images of a species visually distinct from the other sets. Users were tasked with measuring colony areas with Fiji/ImageJ manually and then with Orbis. The colony area values were collected from each user by both methods and compared between each other as well as to the reference (“correct”) measurements (Figure 1B). No significant differences were found between the reference areas and the users’ measured values, both with manual techniques and using Orbis (one-way ANOVA, p = 0.92).
Figure 1. Validation of colony area measurements. (A) Brand–Altman plot showing measurement differences between manual and Orbis measurements. The mean difference is negligible (−0.01 cm2), and most measurements do not differ by more than 5%, as seen by the 95% limit lines in green. (B) Multiuser validation with box plots shows colony area (cm2) for three different samples, measured manually by users (green), by Orbis (purple), and the reference values (blue). Orbis yields colony area values in close agreement with manual measurements from all users, which are also in agreement with the reference value. No statistically significant differences observed between methods (one-way ANOVA, p = 0.92).
Additionally, time efficiency of Orbis was assessed (Figure 2). Orbis substantially reduced measurement time by around 40% per sample when compared to manual techniques (Figure 2A). Manual measurements took about 16 minutes per batch, whereas, using Orbis, users took around 9 minutes for the same task. A paired t-test demonstrated that this time saving was statistically significant (p < 0.001). Furthermore, Orbis provided more consistent timing between users, reducing time gaps between more and less proficient ImageJ users while maintaining measurement accuracy. This efficiency increases compounds over time and becomes increasingly beneficial as sample sizes increase (Figure 2B).
Figure 2. Time and efficiency validation of Orbis. (A) Distribution of measurement times for manual vs. Orbis methods. The manual method (green) spans higher times, while Orbis (purple) shows shorter durations. (B) Comparison of total measurement time per sample set (in minutes) for manual vs. Orbis measurements. Orbis greatly reduced the time required for colony measurements for all users.
Clinostat cultivation
Due to limited accessibility and high costs of real microgravity platforms, like the ISS, satellites or sounding rockets, most studies rely on ground-based microgravity simulations. Exposure of microorganisms under SMG is commonly induced by cultivation on clinostats, and the effects have been studied in Deinococcus radiodurans and Aspergillus niger (Ott et al., Reference Ott, Fuchs, Moeller, Hemmersbach, Kawaguchi, Yamagishi, Weckwerth and Milojevic2019; Cortesão et al., Reference Cortesão, Holland, Schütze, Laue, Moeller and Meyer2022). Clinorotation simulates microgravity by continuously rotating around a horizontal axis, averaging the gravity vector to near zero for samples positioned at the exact centre of the rotational axis, which correlates in this case to the centre of the petri dish (Figure 3A). This provides a functional simulation of the effects of microgravity on the cells, as it prevents particle sedimentation; however, it lacks the persistent free-fall state of real microgravity (Herranz et al., Reference Herranz, Anken, Boonstra, Braun, Christianen, de Geest, Hauslage, Hilbig, Hill, Lebert, Javier Medina, Vagt, Ullrich, van Loon and Hemmersbach2013).
Figure 3. Microgravity simulation using a 2D-clinostat. (A) Clinorotation prevents the particle sedimentation inside of the cells (B) Colonies were grown in petri dishes fit into the clinostat, which simulates microgravity by constant fast rotation at 60 rpm.
In this study, P. rubens was cultivated on a fast-rotating 2D clinostat (Figure 3B) at a rotating speed of 60 rpm to investigate the effect of microgravity on the growth of both strains (Cortesão et al., Reference Cortesão, Holland, Schütze, Laue, Moeller and Meyer2022). Clinorotation can achieve high-quality microgravity conditions, ranging between 4.02 × 10−3 and 4.02 × 10−2 g, if the colony area does not exceed a radius of 1 cm. If the colony exceeds these limitations, additional shear forces may occur of which the impact requires further investigation (Anken, Reference Anken2013; Hauslage et al., Reference Hauslage, Cevik and Hemmersbach2017). To examine the effect of clinorotation on P. rubens, 10 µL of a 106 spores/mL suspension was spotted onto the middle of a PDA plate, which was then incubated on the clinostat at 22°C. Similarly, samples were prepared and incubated in same location (at 22°C) without clinorotation to serve as a ground control in normal gravity conditions (1 × g). The experiment was performed in biological triplicates, each with four replicates per condition (n = 12).
To assess the effect of SMG on P. rubens, colony growth was measured in the samples incubated in the clinostat, as well as the samples incubated in normal gravity conditions (1 × g). Colony areas were measured after two and five days of incubation at 22°C, following the same method as described above. After five days, the colony areas exceeded the requirements for high-quality clinostat cultivation and thus were not further analysed (Anken, Reference Anken2013).
Cosmic radiation exposure
Cosmic radiation (GCR) is one of the main sources of radiation in space. Exposure of spores to helium- and iron-ions, two important components of GCR, was performed at the heavy ion medical accelerator (HIMAC) facility at the National Institute of Radiological Sciences (NIRS) in Chiba, Japan. Spore suspensions at a concentration of 5 × 107 spores/ml were prepared in biological triplicates (n = 3), of which 100 µL aliquots were distributed into individual PCR tubes (Brand).
For radiation exposure, the PCR tubes were placed into stacked petri dishes that were stored in plastic bags. The samples were directly facing the ion beam and were exposed to 10, 100, 250 and 500 Gy. Non-irradiated lab controls were kept at 22°C at the German Aerospace Centre (DLR), while non-irradiated transport controls were stored at room temperature at the HIMAC facility. The energy of the helium-ion beam was constantly held at 150 MeV/n, with a linear energy transfer (LET) of 2.2 keV/µm, whereas the energy of the iron-ion beam was maintained at 500 MeV/n with an LET of 200 keV/µM. Exposure times for each dose were adjusted according to the determined energy and LET values.
Spore viability after radiation
Spore viability after radiation exposure was determined by testing their ability to form colonies. Serial dilutions were prepared up to 10−8 for each sample in a 96-well plate, where each well contained a total volume of 300 µL. Out of every dilution, 20 µL was plated onto 1/8 of a PDA plate supplemented with 0.05% Triton X-100 to facilitate colony counting. The plates were incubated at 22°C for two days before colony forming units (CFU) were counted for each dilution step. Based on the spore viability, the survival fractions of exposed samples (N/N0, where N equals the CFU of treated samples and N0 equals the CFU of transport control samples) were calculated.
Data analysis
Displayed data are presented as arithmetic means, with error bars representing the standard error. A two-sample t-test was performed to assess significance, where p ≤ 0.05 indicates statistical significance (visually highlighted with *). Lethal doses for 90% cell death (LD90) were determined by applying linear regression to the survival fraction data.
Results
In this study, the effect of SMG on the colony area and cosmic radiation resistance of fungal spores was comparatively analysed in a wild-type strain and an ISS isolate of the filamentous fungus P. rubens. Biological triplicates of both strains were cultivated on agar under normal gravity (1 × g) and SMG conditions using a fast-rotating 2D clinostat, and colony growth was analysed. Furthermore, biological triplicates of both strains were irradiated with helium- and iron-ions at the HIMAC facility, and survival rates as well as the lethal dose (LD90) were determined. This study investigated the resistances of P. rubens toward extreme space conditions and provides insight into the adaptations of a strain that was originally isolated from an environment with prevalent space-related stressors.
Colony growth of P. rubens strains in normal gravity conditions (1 × g)
When incubated under normal gravity conditions, the wild-type strain displayed a radial extension rate of 0.163 cm/day over the time span of 11 days, whereas the ISS isolate displayed a radial extension rate of 0.353 cm/day (217% increase) (Figure 4, Table 1 and Supplemental Figure 1). Additionally, a significant difference (p = 0.0014) in spore pigmentation was observed between the two strains. High pigmentation was clearly visible after three days of incubation in the IF2SW-F4 strain, whereas the DSM 1075 strain exhibited only slight spore pigmentation after 7 days, with no profound increase even after 11 days of incubation.
Figure 4. Colony growth under normal gravity conditions (1 × g). Colonies of the wild-type strain and the ISS isolate comparing morphology after 1, 3, 7, 8 and 11 days of incubation at room temperature (scale = 1 cm).
Table 1. Colony areas under normal gravity conditions (1 × g). Colonies of both strains comparing colony size (cm2) after 1, 3, 7, 8 and 11 days of incubation at room temperature
The observed colony areas of the ISS isolate increased by 680% from day 1 to day 3 of incubation, growing from 0.40 to 3.12 cm2. The wild-type strain exhibited a colony area of 0.26 cm2, which then expanded to 1.34 cm2, corresponding to a 415% increase. Overall, the difference in colony size between the two strains was 54% and 133%, respectively.
After seven days, the colony area of the ISS isolate measured 16.65 cm2 (434% increase), while the wild-type strain reached 4.97 cm2 (270% increase), resulting in an approximate difference of 235% between the two strains. After 8 days, the ISS isolate expanded to 22.39 cm2 (35% increase), while the wild-type strain grew to 5.92 cm² (19% increase), leading to a 278% difference between them.
At the final measurement point, after 11 days, the ISS isolate further increased by 111%, reaching a colony area of 47.26 cm2, while the wild-type strain grew by 70%, reaching 10.11 cm2. This resulted in an overall 367% difference between both strains.
Colony growth of P. rubens strains in SMG conditions
The colony areas of both P. rubens strains were analysed after two and five days of incubation, under both normal gravity and SMG conditions. The results indicated that the ISS isolate not only exhibited a generally faster colony growth than the wild-type strain but also showed a significant increase (p < 0.0001) of 14% when cultivated in SMG conditions after five days, with an average colony area of 4.13 cm2, compared to cultivation under normal gravity conditions (Figure 5). However, after two days of incubation, the ISS strain demonstrated significantly faster outgrowth (p = 0.0192) under normal gravity conditions.
Figure 5. Effect of SMG on colony areas of P. rubens strains. Data shown as mean and SE, p ≤ 0.05 was considered significant (*).
In contrast, the colony areas of the wild-type strain did not significantly differ between cultivation under normal gravity and SMG conditions. Growth was not analysed further, as the ISS isolate exceeded the limitations for high-quality microgravity conditions on a clinostat (r = 1 cm) after five days of cultivation under SMG.
The radial extension rates of the wild-type strain did not differ significantly when incubated under normal gravity conditions (0.177 cm/day) or SMG (0.174 cm/day). However, the difference for the ISS isolate was significant (p < 0.0001) when comparing the radial extension rate under normal gravity conditions (0.209 cm/day) to the rate under SMG conditions (0.229 cm/day) (Table 2).
Table 2. Radial extension rates of colonies grown under normal gravity conditions (1 × g) and SMG conditions for the wild-type strain and the ISS isolate. The values represent the mean and SE
P. rubens spore resistances toward cosmic radiation
To assess the resistance of P. rubens spores to cosmic radiation, spores were exposed to doses of up to 500 Gy of helium- and iron-ions. Overall, the ISS isolate showed higher resistances toward cosmic radiation than the wild-type strain.
At 500 Gy, spores of both strains showed lower survival rates after irradiation with iron-ions (0.030 ± 0.004% for the wild-type strain and 0.345 ± 0.027% for the ISS isolate) compared to irradiation with helium-ions (2.165 ± 0.390% for the wild-type strain and 9.681 ± 0.092% for the ISS isolate) (Figure 6).
Figure 6. Survival fraction of P. rubens strains after exposure to cosmic radiation (He-ions left, Fe-ions right). Survival was standardised to the transport controls.
Based on the survival fraction data, the lethal doses (LD90) required to kill 90% of the cells were calculated (Table 3). For the wild-type strain, a dose of 306 Gy of helium-ions was necessary to achieve 90% cell death, whereas only 90 Gy of iron-ions was efficient to achieve the same effect.
Table 3. Lethal dose (LD90) values for P. rubens spores irradiated with different components of cosmic radiation
For the ISS isolate, the lethal doses were calculated to be 540 Gy for helium-ions and 245 Gy for iron-ions.
Discussion
This study aimed to comparatively analyse the resilience of spores of a wild-type strain and an ISS isolate of P. rubens under space conditions. For that, the growth of both strains was examined in microgravity, and their resistance toward cosmic radiation was assessed.
Comparing the growth of both P. rubens strains under normal gravity conditions (1 × g) showed distinct colony morphologies. The ISS isolate exhibited a significantly higher radial extension rate (0.352 cm/day) compared to the wild-type strain (0.163 cm/day), resulting in a 367% difference in colony size between both strains after 11 days. Additionally, spore pigmentation of the ISS isolate was significantly more pronounced and extensive, even after only three days of incubation, at which stage no pigmentation was observed in the wild-type strain. These morphological differences may be a result of the adaptations the ISS isolate underwent in the space environment. However, it is also possible that these differences arise from the fact that the ISS isolate does not share the same origin as the wild-type strain, thus potentially exhibiting distinct phenotypic or genotypic characteristics from the start.
Previous studies have demonstrated that fungal spore germination is enhanced in SMG, leading to increased colony area, spore production and biofilm formation (Cortesão et al., Reference Cortesão, Holland, Schütze, Laue, Moeller and Meyer2022). However, Hupka et al. (Reference Hupka, Kedia, Schauer, Shepard, Granados-Presa, vande Hei, Flores and Zea2023) reported no significant differences in biofilm surface area and thickness between samples grown on the ISS and samples grown on Earth over a 20-day time span. Conflicting results in these regard highlight the need for further investigation to determine the extent to which microgravity influences fungal growth dynamics and spore viability over time.
When comparing the colony areas and radial extension rate of the wild-type strain under normal gravity and SMG conditions, no significant differences were detected. Nonetheless, these findings indicate that P. rubens growth was neither negatively affected nor inhibited by SMG on a fast-rotating 2D-clinostat. In contrast, the colony areas of the ISS isolate were significantly increased when cultivated in SMG after 5 days and the radial extension rate under SMG conditions significantly differed from that under normal gravity conditions, suggesting a growth advantage under these conditions. However, after only two days of incubation, the ISS isolate exhibited a larger colony area in normal gravity conditions. The observed change in growth dynamics suggests that, although microgravity conditions may not induce immediate advantages, prolonged exposure could lead to adaptations that enable the ISS isolate to thrive under these conditions. The difference in growth patterns at two and five for the ISS isolate may indicate an initial period of adaptation to microgravity, followed by the development of a distinct growth strategy in response to the sustained exposure. These observations suggest that the interaction between the duration of exposure and the environmental conditions may be more significant than previously considered.
The precise mechanism of fungal gravitropism remains not yet fully understood. Several studies have attempted to describe fungal gravity-sensing. One study highlighted the sedimentation of vacuolar protein crystals and floating lipid globules in the hyphal apices as essential for gravity-sensing, while another study suggested the involvement of Rho GTPase RacA, a key regulator for actin polymerisation, in the adaptation to microgravity (Schimek et al., Reference Schimek, Eibel, Grolig, Horie, Ootaki and Galland1999; Cortesão et al., Reference Cortesão, Holland, Schütze, Laue, Moeller and Meyer2022). Notably, Rho GTPases have already been linked to gravity adaptation in mammalian cells (Louis et al., Reference Louis, Deroanne, Nusgens, Vico and Guignandon2015).
Additionally, secondary metabolites have been proposed to be involved in microgravity adaptation, as they play vital important roles in environmental responses and adaptations. Studies have reported differentially expressed production of secondary metabolites in filamentous fungi grown under microgravity conditions (Huang et al., Reference Huang, Li, Huang and Liu2018; Jiang et al., Reference Jiang, Guo, Li, Lei, Shi and Shaoa2019). Moreover, increased responses to environmental stimuli and stress tolerances, accompanied with an upregulation of genes involved in pathogenicity, were found in P. chrysogenum cultivated in microgravity conditions (Sathishkumar et al., Reference Sathishkumar, Krishnaraj, Rajagopal, Sen and Lee2016).
This study suggests that extended exposure to microgravity conditions enables fungi to adapt and thrive, potentially due to differentially expressed metabolites, such as the intensified pigmentation observed in the ISS isolate. These adaptations may support fungal colony growth even upon return to environments with normal gravity. This could lead to challenges and complications in future long-duration human spaceflight missions, as increased fungal surface contaminations due to biofouling of built materials not only risks deteriorating spacecraft materials but poses potential health threats to the astronauts, given their ability to act as opportunistic pathogens, especially in immunocompromised humans (Klintworth et al., Reference Klintworth, Reher, Viktorov and Bohle1999; Oshitaka et al., Reference Oshikata, Tsurikisawa, Saito, Watanabe, Kamata, Tanaka, Tsuburai, Mitomi, Takatori, Yasueda and Akiyama2013; Ghajari et al., Reference Ghajari, Lotfali, Azari, Fateh and Kalantary2015; Stratis et al., Reference Stratis, Trudel, Rocheleau, Pelchat and Laneuville2023).
This highlights the need for novel decontamination strategies with focus on fungicidal activity to reduce the risks associated with fungal contamination in space environments.
Radiation can influence microbial cells either directly by DNA damage or indirectly through the production of radicals, including reactive oxygen species (ROS). The biological effects of equal radiation doses can vary, as they are mainly dependent on the LET (Moeller et al., Reference Moeller, Raguse, Leuko, Berger, Hellweg, Fujimori, Okayasu and Horneck2017).
Previous studies have demonstrated that direct biological damage plays a significant role in high-LET radiation, such as iron-ion radiation, as it induces complex DNA, like double- or single-strand breaks. In contrast, indirect damage is the predominant cause of mutations in low-LET radiation, like helium-ion radiation and X-ray radiation. Nonetheless, low-LET radiation can also contribute to DNA damage, as the ROS generated by radiation exposure can negatively impact DNA stability. As a result, radiation with heavy iron-ions causes greater cellular damage than lighter helium-irons (Cucinotta and Durante, Reference Cucinotta and Durante2006; Hirayama et al., Reference Hirayama, Ito, Tomita, Tsukada, Yatagai, Noguchi, Matsumoto, Kase, Ando, Okayasu and Furusawa2009; Cortesão et al., Reference Cortesão, Schütze, Marx, Moeller and Meyer2020; Juan et al., Reference Juan, Pérez de la Lastra, Plou and Pérez-Lebeña2021).
These findings align with previous research, as the results of this study also indicate that iron-ions caused greater cellular damage at lower doses compared to helium-ions. The wild-type strain exhibited lower resistance towards cosmic radiation, with LD90 values of 306 Gy for the helium-ions and 90 Gy for the iron-ions, compared to the ISS isolate, which depicted LD90 values of 540 Gy for helium-ions and 245 Gy for iron-ions. These results suggest that the ISS isolate of P. rubens may have undergone adaptation to radiation exposure. Since radiation levels in LEO are not fungicidal, this hypothesis is further supported by Berger et al. (Reference Berger, Burmeister, Matthiä, Przybyla, Reitz, Bilski, Hajek, Sihver, Szabo, Ambrozova, Vanhavere, Gaza, Semones, Yukihara, Benton, Uchihori, Kodaira, Kitamura and Boehme2017), Cortesão et al. (Reference Cortesão, Schütze, Marx, Moeller and Meyer2020), and Straube et al. (2023).
Moreover, secondary metabolites are involved in the adaptations to environments, and pigments such as melanins exhibit radiation-shielding properties, particularly against UV-C radiation, which is one of the most prevalent conditions in LEO (Dadachova and Casadevall, Reference Dadachova and Casadevall2008; Horneck et al., Reference Horneck, Klaus and Mancinelli2010; Keller, Reference Keller2015; Koch et al., Reference Koch, Freidank-Pohl, Siontas, Cortesao, Mota, Runzheimer, Jung, Rebrosova, Siler, Moeller and Meyer2023). A DHN-melanin biosynthesis pathway has been identified in Penicillium spp., and its regulation may contribute to the adaptation mechanisms of the ISS isolate, as evidenced by its pronounced pigmentation even after a short time of cultivation period (Cleere et al., Reference Cleere, Novodvorska, Geib, Whittaker, Dalton, Salih, Hewitt, Kokolski, Brock and Dyer2024). Various melanin-containing Penicillium spp. have previously been isolated from the Chernobyl Nuclear Power Plant that depicted positive radiotropism (Zhdanova et al., Reference Zhdanova, Tugay, Dighton, Zheltonozhsky and McDermott2004). The energy absorbing properties of the melanin, along with several DNA repair mechanisms of fungal spores, contribute to the enhanced resistance of fungi to radiation exposure (Goldman et al., Reference Goldman, McGuire and Harris2002; Koch et al., Reference Koch, Freidank-Pohl, Siontas, Cortesao, Mota, Runzheimer, Jung, Rebrosova, Siler, Moeller and Meyer2023).
This study suggests that the prolonged exposure to fungi to radiation in LEO may lead to adaptive changes that enhance their resistance, with pigmentation playing a critical role in this process. This highlights the potential for fungal strains to develop mechanisms for withstanding cosmic radiation, which could also be beneficial in space exploration, for example, in biomining minerals from regolith or as radiation shielding (Cockell et al., Reference Cockell, Santomartino, Finster, Waajen, Eades, Moeller, Rettberg, Fuchs, van Houdt, Leys, Coninx, Hatton, Parmitano, Krause, Koehler, Caplin, Zuijderduijn, Mariani, Pellari and Demets2020; Shah et al., Reference Shah, Palmieri, Sponchiado and Bevilaqua2022; Figueira et al., Reference Figueira, Koch, Müller, Slawik, Cowley, Moeller and Cortesao2023; Koch et al., Reference Koch, Freidank-Pohl, Siontas, Cortesao, Mota, Runzheimer, Jung, Rebrosova, Siler, Moeller and Meyer2023; Koehle et al., Reference Koehle, Brumwell, Seto, Lynch and Urbaniak2023). Research on the specific molecular pathways involved in these adaptations, with a focus on the role of pigmentation, could help to develop strategies for microbial monitoring and planetary protection in space missions.
Overall, the ISS isolate exhibited higher resistances to the tested conditions, suggesting adaptations to the space environments upon prolonged exposure. These adaptations appear to selectively enhance radiation resistance while also altering the growth dynamics in microgravity. Nonetheless, the exact adaptation mechanisms of fungi require further investigation, especially with a focus on genetic and metabolic differences.
Further analysis of molecular responses and secondary metabolism of both strains could provide deeper insights into fundamental fungal mechanisms, with implications in relation to astronaut health, microbial monitoring, planetary protection policies and biotechnological applications in space. Understanding these mechanisms could enhance the safety of future space travel by assessing the risks posed by opportunistic pathogens and surface degrading fungi in spaceflight environments. At the same time, it allows for determining possible utilisation of fungi in sustainable life-support systems for long-term space missions.
Supplementary material
The supplementary material for this article can be found at https://doi.org/10.1017/S147355042510013X
Funding statement
AS was supported by the German Space Agency at the German Aerospace Centre (Grant 50WB2230). SMT and AM were supported by the DLR grant FuE-Projekt ISS LIFE (Programm RF-FuW, Teilprogramm 475). MC was supported by the DLR/DAAD Research Fellowship Doctoral Studies in Germany, 2017 (57370122).
Competing interests
The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.
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