Morphological and molecular adaptation of Aspergillus niger to simulated spaceflight and Mars-like conditions

The International Space Station (ISS) is an indoor-closed environment in Low Earth Orbit (LEO). Outside of the ISS, radiation is the most challenging factor outside. In turn, inside the ISS, spaceflight microgravity is the one factor that cannot be evaded. Aspergillus niger and Penicillium rubens are two common isolates of the ISS microbiota. As filamentous fungi, they form highly resistant airborne spores that can easily spread and colonize the spacecraft habitat. Fungi surface-associated growth (or biofilm formation), can biodegrade surfaces and clog life-support systems, and their spores can potentially infect the humans on board. In contrast, on Earth filamentous fungi play an important role in biotechnology, producing a widerange of compounds of interest, from food to antibiotics. Because of this, envisioned long-term spaceflight missions going far beyond low Earth orbit, to the Moon or Mars, will require an intensification of the fungal research, not only in relation to astronaut health and spacecraft safety, but also establishing opportunities for fungal-based biotechnology in space. Thus, this thesis aims to answer three main questions: i) can A. niger spores resist space radiation, and if yes, could they endure interplanetary space travel? ii) if brought to the surface of Mars, could A. niger spores survive the martian environment? and iii) how does simulated microgravity affect A. niger colony growth and biofilm formation? In total, four strains of A. niger were analyzed in this thesis: the industrial and highly pigmented wild-type strain (N402), a strain defective in pigmentation (ΔfwnA), a strain defective in DNA repair (ΔkusA), and a strain defective in polar growth (ΔracA). To assess the level of resistance and survival limits of fungal spores in a long-term interplanetary mission scenario, A. niger spores were exposed to high radiation doses of X-rays and cosmic radiation (helium- and iron-ions) and of UV-C radiation. Results show that wild-type spores of A. niger were able to withstand high doses of the all tested types of space radiation. This suggests that A. niger spores might endure space travel, when considering the radiation factor alone. To evaluate the survival of A. niger to Mars surface conditions, dried spores were launched in a stratospheric balloon mission called MARSBOx. Throughout the mission, A. niger spores were exposed to desiccation, simulated martian atmosphere and pressure, as well as to full UV-VIS radiation. Results revealed that the highly pigmented wild-type spores would survive in a Mars-like middle stratosphere environment with radiation exposure, even as a spore monolayer (106 spores/ml), i.e. with no self-shielding. Spore survival to space radiation and martian conditions suggest that current planetary protection guidelines should be revisited integrating the high resistance of fungal spores. Furthermore, A. niger colony growth and biofilm formation under simulated microgravity was investigated. Scanning Electron Microscopy (SEM) pictures reveal never-before seen ultrastructure of A. niger colonies and biofilms (i.e. vegetative mycelium embedded in extracellular matrix). Results reveal changes in biofilm thickness, spore production and dry biomass, suggesting an increased potential for A. niger to colonize spaceflight habitats. Lastly, P. rubens was proven as a model organism for a spaceflight biofilm experiment aboard the International Space Station. Overall, this thesis highlights the extraordinary resistance of fungal spores to extraterrestrial conditions and reveals their ability to cope with spaceflight microgravity. This advocates for future research that will enable better monitoring and controlling of fungal contaminations in space habitats, and that will help establish filamentous fungi as valuable companions of human space exploration.2021-10-2

Growth and biofilm formation of Penicillium chrysogenum in simulated microgravity

Penicillium sp. are one of the main fungal genera detected on board the Russian Space Station (MIR) and the International Space Station (ISS), demonstrating its ability to grow on the space stations´ walls and to maintain growth under microgravity (1-3). As a spore-forming microorganism, Penicillium sp. poses a concern for planetary protection and to human/astronaut health, as its spores, associated with respiratory diseases, can be dispersed through the air (4). Fungal growth on the ISS has shown to promote biodegradation of the spacecraft materials, compromising their integrity. Biofilms are groups of organisms adhered to each other by self-synthesized extracellular polymeric substances, and are ubiquitous in industrial and natural environments (5). It has been reported that Penicillium sp. forms biofilms, which are associated with higher tolerance/resistance to adverse conditions (6). Therefore, biofilm formed on the ISS may have deleterious effects on astronaut’s health and/or on ISS materials. To gain valuable knowledge to control biofilm during long duration spaceflight missions, the NASA-funded project “Characterization of Biofilm Formation, Growth, and Gene Expression on Different Materials and Environmental Conditions in Microgravity” is currently being prepared. Pre-flight testing include: defining and optimizing the growth medium and culturing conditions of P. chrysogenum DSM 1075; characterizing the morphological response of P. chrysogenum growth under simulated microgravity; assessing biofilm formation by P. chrysogenum under different conditions. The study of this fungal strain represents the beginning of a new line of research on board ISS. The knowledge gained can be applicable to a) the safety and maintenance of crewed spacecraft, b) planetary protection, c) mitigation of biofilm-associated illnesses on the crew, as well as on the Earth. Besides, P. chrysogenum is of major medical and historical importance, as it presents the original and present-day industrial source of the antibiotic penicillin, and as an important producer of antifungal proteins and other relevant enzymes

Colony growth and biofilm formation of Aspergillus niger under simulated microgravity

The biotechnology- and medicine-relevant fungus Aspergillus niger is a common colonizer of indoor habitats such as the International Space Station (ISS). Being able to colonize and biodegrade a wide range of surfaces, A. niger can ultimately impact human health and habitat safety. Surface contamination relies on two key-features of the fungal colony: the fungal spores, and the vegetative mycelium, also known as biofilm. Aboard the ISS, microorganisms and astronauts are shielded from extreme temperatures and radiation, but are inevitably affected by spaceflight microgravity. Knowing how microgravity affects A. niger colony growth, in particular regarding the vegetative mycelium (biofilm) and spore production, will help prevent and control fungal contaminations in indoor habitats on Earth and in space. Because fungal colonies grown on agar can be considered analogs for surface contamination, we investigated A. niger colony growth on agar in normal gravity (Ground) and simulated microgravity (SMG) conditions by fast-clinorotation. Three strains were included: a wild-type strain, a pigmentation mutant (ΔfwnA), and a hyperbranching mutant (ΔracA). Our study presents never before seen scanning electron microscopy (SEM) images of A. niger colonies that reveal a complex ultrastructure and biofilm architecture, and provide insights into fungal colony development, both on ground and in simulated microgravity. Results show that simulated microgravity affects colony growth in a strain-dependent manner, leading to thicker biofilms (vegetative mycelium) and increased spore production. We suggest that the Rho GTPase RacA might play a role in A. niger’s adaptation to simulated microgravity, as deletion of ΔracA leads to changes in biofilm thickness, spore production and total biomass. We also propose that FwnA-mediated melanin production plays a role in A. niger’s microgravity response, as ΔfwnA mutant colonies grown under SMG conditions showed increased colony area and spore production. Taken together, our study shows that simulated microgravity does not inhibit A. niger growth, but rather indicates a potential increase in surface-colonization. Further studies addressing fungal growth and surface contaminations in spaceflight should be conducted, not only to reduce the risk of negatively impacting human health and spacecraft material safety, but also to positively utilize fungal-based biotechnology to acquire needed resources in situ

The effects of space radiation on filamentous fungi

Aspergillus was the main fungal genera detected aboard the Russian Space Station (Mir) and the International Space Station (ISS), and fungal growth has been shown to promote biodegradation of the spacecraft materials and compromise life-support systems [1-2]. Moreover, as spore formers, filamentous ungi are a threat to astronauts’ health, and their resistant spores may pose a threat to planetary protection. This makes monitoring and controlling fungal contamination a challenge to be met in the current and future space missions [3-5]. The topic of my master internship at the DLR is: Fungal spore resistance to space radiation and mechanisms by which observed resistance is mediated

Aspergillus niger Spores Are Highly Resistant to Space Radiation

The filamentous fungus Aspergillus niger is one of the main contaminants of theInternational Space Station (ISS). It forms highly pigmented, airborne spores that have thick cell walls and low metabolic activity, enabling them to withstand harsh conditions and colonize spacecraft surfaces. Whether A. niger spores are resistant to space radiation, and to what extent, is not yet known. In this study, spore suspensions of a wild-type and three mutant strains (with defects in pigmentation, DNA repair, and polar growth control) were exposed to X-rays, cosmic radiation (helium- and iron-ions) and UV-C (254 nm). To assess the level of resistance and survival limits of fungal spores in a long-term interplanetary mission scenario, we tested radiation doses up to 1000 Gy and 4000 J/m2. For comparison, a 360-day round-trip to Mars yields a dose of 0.66 0.12 Gy. Overall, wild-type spores of A. niger were able to withstand high doses of X-ray (LD90 = 360 Gy) and cosmic radiation (helium-ion LD90 = 500 Gy; and iron ion LD90 = 100 Gy). Drying the spores before irradiation made them more susceptible toward X-ray radiation. Notably, A. niger spores are highly resistant to UV-C radiation (LD90 = 1038 J/m2), which is significantly higher than that of other radiation-resistant microorganisms (e.g., Deinococcus radiodurans). In all strains, UV-C treated spores (1000 J/m2) were shown to have decreased biofilm formation (81% reduction in wild typespores). This study suggests that A. niger spores might not be easily inactivatedby exposure to space radiation alone and that current planetary protection guidelines should be revisited, considering the high resistance of fungal spores

Icy exposure of microorganisms

The most hostile place on Earth with the lowest temperature ever recorded of -89.2 °C is the Antarctic ice sheet. This cold, arid, remotely located and perennially ice covered environment has long been considered an analogue to how life might persist in the frozen landscape of the major Astrobiological targets of our solar system suchasMarsortheJupiter’sice-covered moon Europa. In the frame of the ICEXPOSE project presented here the parameters outside the Antarctic Concordia station are utilized as a testbed for performed or planned long-duration space flights and to study the survivability of selected test organisms in an extremely cold (with temperature swings) and highly variable UV environment. The most likely terrestrial organisms to endure such an excursion are extremely tolerant and/or (multi-) resistant microbesextremophiles- that have evolved mechanisms to withstand such severe conditions. The survivability of a variety of human-, space-flight and extreme-associated microorganisms from all three domains of life (plus viruses) will be investigated using a multiuser exposure facility called EXPOSE that has already been successfully flown on ISS for space exposure durations of up to 2 years. The EXPOSE Mission Ground Reference (MGR) trays are still available and will be reused to accommodate the samples for passive exposure. Microbiological response to single and combined extraterrestrial conditions including simulations of astrobiological relevant environments, like simulated Martian atmospheric conditions, will be tested. The scientific questions addressed in ICEXPOSE are: how is the survival of human-associated and Polar Regionsderived microorganisms compared to (other) environmental extremophilic microorganisms; which physiological state (i.e., cells, spores or colony/biofilms) harbors the weakest or strongest viability and/or mutagenicity detectable after exposure; what type of morphologic and molecular changes can be identified and to which extent does the exposure conditions (e.g. UV-exposed versus UV-shielded) influence the microbial physiology (e.g. pathogenicity, antibiotic resistance, and metabolism) of the exposed species. The results of the ICEXPOSE experiment will provide valuable information on the definition of the physicalchemical limits of life as well as the potential habitability of other planetary bodies; the assessment of the risk of microbial contamination inside human inhabited confined areas and consequent challenges for human health; how to better monitor and control microbial contamination in spaceflight environments, as a key-factor for the success of future space exploration missions; whether specific microorganisms pose possible forward contamination risks that could impact planetary protection policy; and will provide complementary results for the two selected future ESA space experiments MEXEM and IceCold

MARSBOX: FUNGAL SPORES SURVIVE MARS-LIKE CONDITIONS ABOARD STRATOSPHERIC BALLOON FLIGHT

The ability of terrestrial life to survive in the Martian environment is of particular interest for both planetary protection measures and for future colonization endeavors. Many studies have examined bacterial spore survival and decontamination, however, little is known about fungal spore resistance properties. To understand the survival potential of fungal spores to Mars conditions, Aspergillus niger spores were sent on a 6.5-hour balloon flight to Earth’s stratosphere ( 38 km), where UV radiation and temperature conditions were similar to levels typical for equatorial Mars. Spores were carried inside the TREX unit – a sealed aluminum container filled with a Mars gas mixture – and flown aboard the MARSBOx balloon payload. Two different spore concentrations were tested on the TREX, exposed as dried samples in small quartz discs (20 µL). Discs were set in two different layers: a top layer exposed to direct UV radiation [M(+)UV, in a total dose of 1500 J/m²], and a bottom layer that was shielded from radiation [M(-)UV]. After the flight, fungal spore survival was determined by plating on agar and determining colony forming units (CFU/ml). A germination rate was calculated based on light microscopy analysis and revival metabolism assay was completed with resazurin dye. Results show that A. niger spores can survive Mars-like conditions [M(+)UV] for the 6.5 htime period tested in the middle stratosphere with only a 2-log reduction and slight delays in germination and revival metabolism compared to unflown lab controls. When shielded from UV, but exposed to Mars gas, pressure and temperature [M(-)UV] spore survival and germination were not affected. This study provides valuable insights on whether fungal spores could survive on Mars, and underscores the need for longer-duration exposure studies in Earth’s stratosphere to better characterize microbial resistance to space-related conditions

Fungi in space: Implications for astronaut health and planetary protection

Aspergillus and Penicillium were the predominant fungal genera detected aboard the Russian Space Station (Mir) as well as the International Space Station (ISS), and fungal growth has been shown to promote biodegradation of spacecraft materials which might compromise life-support systems [1-2]. Moreover, as spore formers, filamentous fungiareathreattoastronauts’health,andtheirresistantsporesmayposeathreattoplanetaryprotection.This, together with their ability to form biofilms, makes monitoring and controlling fungal populations a challenge when it comes to meeting the medical and operation requirements for the current and future space missions [3-5]. The doctoral study work here presented focuses on i) understanding fungal growth and biofilm formation in the space environment, ii) searching for spaceflight-relevant antimicrobial surfaces; iii) assessing fungal radiation resistance, and iv) identifying the potential of these fungi in space biotechnology